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EVALUATION OF THE EFFECT OF P53 IN
CELLULAR RESPONSE FROM ELECTRON
MICROSCOPY IMAGES
ANA CATARINA FREITAS DA SILVA DE JESUS
JUNHO 2010
EVALUATION OF THE EFFECT OF P53 IN CELLULAR
RESPONSE FROM ELECTRON MICROSCOPY IMAGES
Report of the Course Practical Works, Master Course in Biomedical
Engineering Program, Faculty of Engineering of University of Porto
Ana Catarina Freitas da Silva de Jesus
Graduated in Biochemistry (2000)
Faculty of Science of University of Porto
Graduated in Nuclear Medicine (2006)
Superior School of Allied Health Sciences
Polytechnic Institute of Porto
Supervisor:
João Manuel R. S. Tavares
Assistant Professor of the Mechanical Engineering Department
Faculty of Engineering of University of Porto
ACKNOWLEDGEMENTS
To Professor João Manuel R. S. Tavares for the support provided throughout
this work, particularly for guidance, support and availability, essential for the proper
and constructive development of the same.
To all of those who make possible the development of this work.
SUMMARY
The theme of this practical work falls in the computational vision domain,
particularly in the area of the processing and analysis of biomedical images.
The objective of the correspondent MSc thesis is to perform the computational
analysis of cells represented in microscopic images. For that, the preprocessing of the
input images assumes particularly relevance. This first image preprocessing step is the
main concern of this practical work. In a later stage, that is, during the thesis project,
will be considered the segmentation of the input images and motion tracking and
analysis of cells submitted to irradiation.
In this practical work, it is emphasized the importance of the cell cycle
regulation, namely the cell death mechanisms. The associate checkpoints are
particularly important when the cells are irradiated. For this reason, in this work is
made a description of the harmful effect of radiation on cells and tissues. In addition,
the cell cultures and the adequate means to obtain reasonable laboratory culture of
cells, without contamination, for subsequent use to study the effect of radiation on
cells, are discussed.
The experimental results obtained through image processing and analysis
highlight the changes in intracellular content due to irradiation of cells and emphasize
the effects of the lack of cell regulation, specifically detecting the location of p53 and
changes in its content.
CONTENTS
CONTENTS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES i
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION 1
1.1 – Introduction 3
1.2 - Main Objectives 4
1.3 - Report Organization 6
1.4 - Major Contributions 8
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS 9
2.1 – Introduction 11
2.2 - Cell Life Cycle 12
2.2.1 – Interphase 12
2.2.2 - DNA Replication 13
2.2.3 - Cell Division 14
2.2.3.1 – Mitosis 14
2.2.3.2 – Cytokinesis 16
2.2.4 – Meiosis 16
2.3 - Progression of the cell cycle 19
2.4 - Growth characteristics of malignant cells 26
2.4.1 - Phenotypic Alterations in Cancer Cells 27
2.4.2 - Immortality of Transformed Cells in Culture 28
2.4.3 - Decreased Requirement for Growth Factors 29
2.4.4 - Loss of Anchorage Dependence 29
2.4.5 - Loss of Cell Cycle Control and Resistance to Apoptosis 30
2.5 - Cell Cycle Regulation 31
CONTENTS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES ii
2.5.1 - CDK Inhibitors 32
2.5.2 – Cyclins 33
2.5.3 - Cell Cycle Checkpoints 34
2.5.4 - Cell Cycle Regulatory Factors as Targets for Anticancer Agents 37
2.6 – Apoptosis 39
2.6.1 - Biochemical Mechanism of Apoptosis 41
2.6.2 – Caspases 44
2.6.3 - Bcl-2 Family 45
2.6.4 – Anoikis 45
2.7 - Resistance to Apoptosis in Cancer and Potential Targets for Therapy 47
2.8 – Summary 49
CHAPTER III – RADIATION EFFECT ON NORMAL AND NEOPLASTIC TISSUES 51
3.1 – Introduction 53
3.2 - Irradiation Carcinogenesis 54
3.2.1 - Ionizing Radiation 54
3.2.2 - Ultraviolet Radiation 55
3.3 - Cell Death in Mammalian Tissues 56
3.4 - Nature of Cell Populations in Tissue 59
3.5 - Cell Population Kinetics and Radiation Damage 60
3.5.1 - Growth Fraction and its significance 61
3.6 - Cell Kinetics in Normal Tissues and Tumors 62
3.7 - Models for Radiobiological Sensitivity of Neoplastic Tissues 63
CONTENTS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES iii
3.7.1 - Hewitt Dilution Assay 64
3.7.2 - Lung Colony Assay System 67
3.8 - Tumor Growth and Tumor “Cure” Models 68
3.8.1 - Tumor Volume Versus Time 68
3.8.2 - TCD50, Tumor Cure 70
3.9 - Radiobiological Responses of Tumors 70
3.10 - Hypoxia and Radiosensitivity in Tumor Cells 71
3.11 – Summary 74
CHAPTER IV – CELL CULTURE AND FLOW CYTOMETRY 75
4.1 – Introduction 77
4.2 - Cell-Culture Laboratory 77
4.3 - Maintaining Cultures 78
4.3.1 – Medium 79
4.3.2 - The use of medium in analysis and alternatives 83
4.4 - Cytogenetic Analysis of Cell Lines 84
4.4.1 - The Utility of Cytogenetic Characterization 84
4.5 - Methods to Induce Cell Cycle Checkpoints 85
4.6 - Methods for Synchronizing Mammalian Cells 86
4.7 - Analysis of the Mammalian Cell Cycle by Flow Cytometry 88
4.8 – Conclusion 89
CONTENTS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES iv
CHAPTER V – MATERIALS AND METHODS 92
5.1 – Introduction 94
5.2 - Materials and Methods of the paper “Lack of p53 function promotes
radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells” 94
5.2.1 - Cell Culture 94
5.2.2 - Light microscopy 94
5.2.3 - Bivariate BrdUrd-PI (bromodeoxiuridine-propidium iodide)
flow cytometry 95
5.2.4 - Bivariate cyclin B1-PI flow cytometry 95
5.2.5 - Western blotting 95
5.3 - Materials of the paper “Nuclear accumulation and activation of p53
in embryonic stem cells after DNA damage” 96
5.3.1 - Cell lines and their treatments 96
5.3.2 - Immunofluorescence staining 97
5.3.3 - RT-PCR 97
5.3.4 - Beta-Galactosidase staining 97
5.3.5 - MTT-assay 98
5.3.6 - Colony assay 98
5.3.7 - Apoptosis Assay by Annexin V staining 98
CHAPTER VI – IMAGE PROCESSING AND ANALYSIS 99
6.1 – Introduction 101
6.2 - Image processing and Analysis 102
CONTENTS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES v
6.3 - Comparison between original and processed images 112
6.4 - Summary 129
CHAPTER VII – CONCLUSIONS AND FUTURE WORKS 131
7.1 - Final Conclusions 133
7.2 - Future Works 134
REFERENCES 135
CHAPTER I
INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 3
1.1 – INTRODUCTION
One of the most widely used steps in the process of obtaining information from
images is image segmentation: dividing the input image into regions that hopefully
correspond to structural units in the scene or distinguish objects of interest (Russ,
1998).
Segmentation is often described by analogy to visual processes as a
foreground/background separation, implying that the selection procedure
concentrates on a single kind of feature and discards the rest. This is not quite true for
computer systems, which can generally deal much better than humans with scenes
containing more than one type of feature of interest (Russ, 1998).
In computational vision, segmentation refers to the process of partitioning a
digital image into multiple segments (sets of pixels, also known as superpixels). The
goal of segmentation is to simplify and/or change the representation of an image into
something that is more meaningful and easier to analyze. Image segmentation is
typically used to locate objects and boundaries (lines, curves, etc.) in images. More
precisely, image segmentation is the process of assigning a label to every pixel in an
image such that pixels with the same label share certain visual characteristics. The
result of image segmentation is a set of segments that collectively cover the entire
image, or a set of contours extracted from the image. Each of the pixels in a region is
similar with respect to some characteristic or computed property, such as color,
intensity, or texture. Adjacent regions are significantly different with respect to the
same characteristic(s) (Shapiro, 2001).
In this work, the goal is to study the morphology changes of irradiated cells,
more precisely the cellular changes in p53 content upon irradiation. Therefore, the
cells images are studied and processed using the image processing toolbox of the
MATLAB program, with the intent of highlight the cellular differences between the
control and irradiated cells in terms of the p53 quantity inside the cell.
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 4
1.2 – MAIN OBJECTIVES
The study report in the paper: “Nuclear accumulation and activation of p53 in
embryonic stem cells after DNA damage”, of Valeriya Solozobova, ALexandre
Rolletschek and Christine Blattner, BMC Cell Biology 2009 10:46, is based on the fact
that:
Cells are continuously subjected to DNA lesions arising both from
environmental conditions and from the intrinsic metabolism of a cell.
Such lesions can lead to mutations and large-scale genome alterations
that may be deleterious for cellular function. To maintain genomic
stability cell cycle checkpoints exist that can detect errors during DNA
replication. If errors are encountered, cell division is paused and repair
mechanisms and/or cell death ensues.
The p53 tumor suppressor protein plays an important role in this
process. By being part of a signal transduction process, p53 relays
information leading to cellular responses such as cell cycle arrest and
apoptosis, resulting from DNA lesions. P53 activity is regulated mainly at
the protein level. In response to DNA lesions, p53 is rescued from
targeted degradation, which leads to a strong increase in the amount of
the otherwise short lived tumor suppressor protein, and the protein is
intensively modified. Cells deficient in p53 fail to undergo apoptosis or
cell cycle arrest in response to DNA damage which increases the rates of
tumorigenicity and genomic instability in these animals.
Whereas the study report in the paper: “Lack of p53 function promotes
radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells”, of
Fiorenza Ianzini, Alessandro Bertoldo, Elizabeth A Kosmacek, Stacia L Philips and
Michael A Mackey, Cancer Cell International 2006 6:11, is based on the fact that:
Mitotic catastrophe (MC) has been observed following alterations in
specific cellular proteins, or by treatment of cells with chemicals, heat,
and/or ionizing radiation. MC is characterized by an aberrant nuclear
morphology observed following premature entry into mitosis and often
results in the generation of aneuploid and polyploidy cell progeny. The
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 5
initiating event in this process involves the premature entry of cells into
mitosis; cells undergo a spontaneous chromosome condensation that
produces chromosome morphologies very similar to those observed
when metaphase cells are fused with cells located late in the cell cycle.
Thus, one consider these abnormal mitotic figures as indicative of cells
undergoing spontaneous premature chromosome condensation (SPCC).
These cells entering into mitosis prematurely often either fail to achieve
cytokinesis or divide and fuse shortly thereafter, and later exhibit the
features of MC.
These cells almost always die; however, some studies have suggested
that a small fraction of cells might survive long enough to establish a
growing population of cells, and one study demonstrated a high
frequency of surviving clones containing an elevated incidence of MC.
These results may indicate that a small fraction of cells can survive MC.
Stress-induced SPCC and subsequent MC is observed under conditions
where cyclin B1/cdc2 kinase is activated while cells are delayed in S or
G2 phases, indicating that stress-induced MC is the result of abrogation
of cell cycle regulatory pathways, in particular G2 checkpoint pathways.
There are many proteins that play a role in the regulation of checkpoint
functions in G2, both inhibitory and stimulatory abrogation of the G2/M
checkpoint, due to over accumulation of cyclin B1 protein and
premature activation of cyclin B1/cdc2 kinase, plays a critical role in the
induction of SPCC and subsequent MC. Cyclin B1 biosynthesis
contributes to the regulation of mitotic entry, as cyclin B1 levels are cell
cycle regulated, with the gene being expressed only in S and G2 phases
in human and rodent cells.
At the later stages of mitosis, proteosome-mediated degradation of
cyclin B1 begins, and new cyclin B1 synthesis is required for entry into
the next mitosis. Thus, the cyclic rise and fall of cyclin B1 levels provides
for one level of regulation of this promitotic protein. Cells arrested late
in the cell cycle are located at that point in the cycle when cyclin B1
gene expression is at its peak value. Under these conditions it has been
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 6
shown that the p53 tumor suppressor gene product is a negative
regulator of cyclin B1 transcription, perhaps providing for negative
feedback regulation of cyclin B1 levels under abnormal conditions. If the
induction of MC in cells post-irradiation is due to cyclin B1 over
accumulation, a role for p53 in this response might be expected.
In this study one present data which describe such a role for p53 in the
induction of MC mediated by over accumulation of cyclin B1 occurring
during delay of cells late in the cell cycle.
In both studies, the role of the p53 is enhanced and, based on the results of
these studies I will withdraw information from the resulting images supported on the
image processing of them.
In the end of this practical work, the main goal is to identify, study and compare
techniques of image processing and analysis to performing the extraction of relevant
information from the images contained in the above mentioned papers, in order to
validate their results in an automate and robust manner.
1.3 – REPORT ORGANIZATION
It was intended to organize this document in a self-directed and self-regulating
approach to improve the access to various topics structured in seven chapters. So, it
will be described very succinctly what is treated in each remaining chapter:
Chapter II – Cell cycle regulation and apoptosis
In this chapter takes place a description of key concepts related to the cell cycle
checkpoints, to the behavior of the malignant cells and to the cellular death
mechanisms among other information related to the normal and malignant cells.
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 7
Chapter III – Radiation effect on normal and neoplastic tissues
In this chapter it is presented a description of the irradiated carcinogenesis as
well as the cell death mechanisms. It is also described important issues regarding the
cellular behavior upon irradiation.
Chapter IV – Cell culture and flow cytometry
In this fourth chapter it is performed an approach of some important issues
regarding the safety manipulation and maintenance of cells when performing cell
culture techniques. It is also described the methods to induce cell cycle checkpoints
and the flow cytometry technique.
Chapter V – Materials and Methods
In this fifth chapter a description on the materials and methods used in the
articles in which this work is based on.
Chapter VI – Image Processing and Analysis
In this chapter it is performed the analysis and the segmentation of the images,
using the MATLAB image processing toolbox, obtained from the papers used as based
for the execution of this practical work.
Chapter VII – Final Conclusions and Future Works
In the last chapter it is presented the final conclusions of the work performed,
as well as the future perspectives regarding the execution of the correspondent thesis.
CHAPTER I – INTRODUCTION TO THE THEME AND REPORT ORGANIZATION
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 8
1.4 – MAJOR CONTRIBUTIONS
This work has helped to highlight the contribution of techniques image
processing and analysis to obtain additional and complementary information to the
two papers used as basis. Additionally, knowledge about cell cycle regulation and
checkpoints that help to understand the behavior of cells when they are irradiated was
gained. This information will be helpful to study the electron microscopy images of
breast cancer cells submitted to brachytherapy for the thesis work.
CHAPTER II
CELL CYCLE REGULATION AND APOPTOSIS
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 11
2.1 - INTRODUCTION
The development of knowledge about the biochemistry and cell biology of
cancer comes from a number of disciplines. Some of this knowledge has come from
research initiated a century or more ago. There has been a flow of information about
genetics into a knowledge base about cancer, starting with Gregor Mendel and the
discovery of the principle of inherited traits and leading through Theodor Boveri’s work
on the chromosomal mode of heredity and chromosomal damage in malignant cells to
Avery’s discovery of DNA as the hereditary principle, Watson and Crick’s determination
of the structure of DNA, the human genome project, DNA microarrays, and
proteomics. Not only has this information provided a clearer understanding of the
carcinogenic process, it has also provided better diagnostic approaches and new
therapeutic targets for anticancer therapies (Ruddon, 2007).
Cancer cells contain many alterations, which accumulate as tumors develop.
Over the last 25 years, considerable information has been gathered on the regulation
of cell growth and proliferation leading to the identification of the proto-oncogenes
and the tumor suppressor genes. The proto-oncogenes encode proteins, which are
important in the control of cell proliferation, differentiation, cell cycle control and
apoptosis. Mutations in these genes act dominantly and lead to a gain in function. In
contrast the tumor suppressor genes inhibit cell proliferation by arresting progression
through the cell cycle and block differentiation. They are recessive at the level of the
cell although they show a dominant mode of inheritance. In addition, other genes are
also important in the development of tumors. Mutations leading to increase genomic
instability suggest defects in mismatch and excision repair pathways. Genes involved in
DNA repair, when mutated, also predispose the patient to developing cancer
(Macdonald, 2005).
A crucial decision in every proliferating cell is the decision to continue with a
further round of cell division or to exit the cell cycle and return to the stationary phase.
Similarly quiescent cells must make the decision, whether to remain in the stationary
phase (G0) or to enter into the cell cycle. Entry into the cycle occurs in response to
mitogenic signals and exit in response to withdrawal of these signals. To ensure that
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 12
DNA replication is complete and that any damaged DNA is repaired, cells must pass
through specific checkpoints. Tumor cells undergo uncontrolled proliferation either
due to mutations in the signal transduction pathways or because of mutations in the
regulatory mechanism of the cell cycle (Macdonald, 2005).
In this chapter, it is provided a detailed description of the cell cycle, its
progression and the cellular events involved in transforming normal cells into
malignant cells. For this purpose, the chapter starts with the explanation of the cell
cycle followed by the description of the progression of the cell cycle, the growth
characteristics of the malignant cells and the cell cycle regulation. After this, the
chapter focuses the importance of the apoptosis phenomena and ends referring the
resistance to apoptosis in cancer cells and potential targets for therapy.
2.2 – CELL LIFE CYCLE
The cell life cycle includes the changes a cell undergoes from the time it is
formed until it divides to produce two new cells. The life cycle of a cell has two stages,
an interphase and a cell division stage, Figure 2.1 (Seelev, 2004).
Figure 2.1 – Cell cycle (from (Seeley, 2004))
2.2.1 – Interphase
Interphase is the phase between cell divisions. Ninety percent or more of the
life cycle of a typical cell is spent in interphase and, during this time the cell carries out
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 13
the metabolic activities necessary for life and performs its specialized functions such as
secreting digestive enzymes. In addition, the cell prepares to divide which includes an
increase in cell size; because many cell components double in quantity, and a
replication of the cell’s DNA. Consequently, the centrioles within the centrosome are
also duplicated, when the cell divides, each new cell receives the organelles and DNA
necessary for continued functioning. Interphase can be divided into three subphases,
called G1, S, and G2. During G1 (the first gap phase) and G2 (the second gap phase), the
cell carries out routine metabolic activities. During the S phase (the synthesis phase),
the DNA is replicated (new DNA is synthesized) (Seelev, 2004).
Many cells in the human body do not divide for days, months, or even years.
These “resting” cells exit and enter the cell cycle that is called the G0 phase, in which
they remain, unless, stimulated to divide (Seelev, 2004).
2.2.2 - DNA Replication
DNA replication is the process by which two new strands of DNA are made,
using the two existing strands as templates. During interphase, DNA and its associated
proteins appear as dispersed chromatin threads within the nucleus. When DNA
replication begins, the two strands of each DNA molecule separate from each other for
some distance, Figure 2.2. Then, each strand functions as a template, or pattern, for
the production of a new strand of DNA, which is formed as new nucleotides pair with
the existing nucleotides of each strand of the separated DNA molecule. The production
of the new nucleotide strands is catalyzed by DNA polymerase, which adds new
nucleotides at the 3` end of the growing strands. One strand, called the leading strand,
is formed as a continuous strand, whereas the other strand, called the lagging strand,
is formed in short segments going in the opposite direction. The short segments are
then spliced by DNA ligase. As a result of DNA replication, two identical DNA molecules
are produced, each of them having one strand of nucleotides derived from the original
DNA molecule and one newly synthesized strand (Seelev, 2004).
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 14
Figure 2.2 – Replication of DNA (from (Seelev, 2004))
2.2.3 - Cell Division
New cells necessary for growth and tissue repair are produced by cell division.
A parent cell divides to form two daughter cells, each of which has the same amount
and type of DNA as the parent cell. Because DNA determines cell structure and
function, the daughter cells have identical structure and perform the same functions as
the parent cell. Cell division involves two major events: the division of the nucleus to
form two new nuclei, and the division of the cytoplasm to form two new cells. Each of
the new cells contains one of the newly formed nuclei. The division of the nucleus
occurs by mitosis, and the division of the cytoplasm is called cytokinesis (Seelev, 2004).
2.2.3.1 - Mitosis
Mitosis is the division of the nucleus into two nuclei, each of which has the
same amount and type of DNA as the original nucleus. The DNA, which was dispersed
as chromatin in interphase, condenses in mitosis to form chromosomes. All human
somatic cells, which include all cells except the sex cells, contain 46 chromosomes,
which are referred to as a diploid number of chromosomes. Sex cells have half the
number of chromosomes as somatic cells (Seelev, 2004).
The 46 chromosomes in somatic cells are organized into 23 pairs of
chromosomes. Twenty-two of these pairs are called autosomes. Each member of an
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 15
autosomal pair of chromosomes looks structurally alike, and together they are called a
homologous pair of chromosomes. One member of each autosomal pair is derived
from the person’s father, and the other is derived from the mother. The remaining pair
of chromosomes is the sex chromosomes. In females, the sex chromosomes look alike,
and each is called an X chromosome. In males, the sex chromosomes do not look
similar. One chromosome is an X chromosome, and the other is smaller and is called a
Y chromosome. One X chromosome of a female is derived from her mother and the
other is derived from her father. The X chromosome of a male is derived from his
mother and the Y chromosome is derived from his father (Seelev, 2004).
Mitosis is divided into four phases: prophase, metaphase, anaphase, and
telophase. Although each phase represents major events, mitosis is a continuous
process, and no discrete jumps occur from one phase to another. Learning the
characteristics associated with each phase is helpful, but a more important concept is
how each daughter cell obtains the same number and type of chromosomes as the
parent cell. The major events of mitosis are summarized in Figure 2.3 (Seelev, 2004).
Figure 2.3 – Mitosis. (1) Interphase; (2) Prophase; (3) Metaphase; (4) Anaphase; (5) Telophase; (6) Interphase,
Cytokinesis (from (Seelev, 2004))
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 16
2.2.3.2 - Cytokinesis
Cytokinesis is the division of the cytoplasm of the cell to produce two new cells
(Figure 2.3). Cytokinesis begins in anaphase continues through telophase and ends in
the following interphase. The first sign of cytokinesis is the formation of a cleavage
furrow, or puckering of the plasma membrane, which forms midway between the
centrioles. A contractile ring composed primarily of actin filaments pulls the plasma
membrane inward, dividing the cell into two halves. Cytokinesis is complete when the
membranes of the two halves separate at the cleavage furrow to form two separate
cells (Seelev, 2004).
2.2.4 – Meiosis
All cells of the body are formed by mitosis, except sex cells that are formed by
meiosis. In meiosis the nucleus undergoes two divisions resulting in four nuclei, each
containing half as many chromosomes as the parent cell. The daughter cells that are
produced by cytokinesis differentiate into gametes, or sex cells.
The gametes are reproductive cells—sperm cells in males and oocytes (egg
cells) in females. Each gamete not only has half the number of chromosomes found in
a somatic cell but also has one chromosome from each of the homologous pairs
verified in the parent cell. The complement of chromosomes in a gamete is referred to
as a haploid number. Oocytes contain one autosomal chromosome from each of the
22 homologous pairs and an X chromosome. Sperm cells have 22 autosomal
chromosomes and either an X or Y chromosome. During fertilization, when a sperm
cell fuses with an oocyte, the normal number of 46 chromosomes in 23 pairs is
reestablished. The sex of the baby is determined by the sperm cell that fertilizes the
oocyte. The sex is male if a Y chromosome is carried by the sperm cell that fertilizes the
oocyte and female if the sperm cell carries an X chromosome (Seelev, 2004).
The first division during meiosis is divided into four phases: prophase I,
metaphase I, anaphase I, and telophase I, Figure 2.4. As in prophase of mitosis, the
nuclear envelope degenerates, spindle fibers form, and the already duplicated
chromosomes become visible. Each chromosome consists of two chromatids joined by
a centromere. In prophase I, however, the four chromatids of a homologous pair of
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 17
chromosomes join together, or synapse, to form a tetrad. In metaphase I the tetrads
align at the equatorial plane and in anaphase I each pair of homologous chromosomes
separate and move toward opposite poles of the cell (Seelev, 2004).
For each pair of homologous chromosomes, one daughter cell receives one
member of the pair, and the other daughter cell receives the other member. Thus each
daughter cell has 23 chromosomes, each of which is composed of two chromatids.
Telophase I with cytokinesis is similar to telophase of mitosis and two daughter cells
are produced. Interkinesis is the phase between the formation of the daughter cells
and the second meiotic division. No duplication of DNA occurs during this phase. The
second division of meiosis also has four phases: prophase II, metaphase II, anaphase II,
and telophase II. These stages occur much as they do in mitosis, except that 23
chromosomes are present instead of 46 (Seelev, 2004).
The chromosomes align at the equatorial plane in metaphase II, and their
chromatids split apart in anaphase II. The chromatids then are called chromosomes,
and each new cell receives 23 chromosomes. In addition to reducing the number of
chromosomes in a cell from 46 to 23, meiosis is also responsible for genetic diversity
for two reasons:
A random distribution of the chromosomes is received from each
parent. One member of each homologous pair of chromosomes was
derived from the person’s father and the other member from the
person’s mother. The homologous chromosomes align randomly during
metaphase I when they split apart, each daughter cell receives some of
the father’s and some of the mother’s chromosomes. The number of
chromosomes each daughter cell receives from each parent is
determined by chance;
However, when tetrads are formed, some of the chromatids may break
apart, and part of one chromatid from one homologous pair may be
exchanged for part of another chromatid from the other homologous
pair, Figure 2.5. This exchange is called crossing-over; as a result,
chromatids with different DNA content are formed, Figure 2.5.
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 18
With random assortment of homologous chromosomes and crossing-over, the
possible number of gametes with different genetic makeup is practically unlimited.
When the distinct gametes of two individuals unite, it is virtually certain that the
resulting genetic makeup never has occurred before and never will occur again. The
genetic makeup of each new human being is unique (Seelev, 2004).
Figure 2.4 – Meiosis (from (Seelev, 2004))
CHAPTER II – CELL CYCLE REGULATION AND APOPTOSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 19
Figure 2.5 – Crossing-over (from (Seelev, 2004))
2.3 - PROGRESSION OF THE CELL CYCLE
The cell cycle is controlled by a complex pattern of synthesis and degradation of
regulators together with careful control of their spatial organization in specific
subcellular compartments. In addition, checkpoint controls can modulate the
progression of the cycle in response to adverse conditions such as DNA damage.
Cells either enter G1 from G0 in response to mitogenic stimulation or follow on
from cytokinesis if actively proliferating (i.e. from M to G1). Removal of mitogens
allows them to return to G0. The critical point between mitogen dependence and
independence is the restriction point or R which occurs during G1. It is here that cells
reach the ‘point of no return’ and are committed to a round of replication (Macdonald,
2005), Figure 2.6.
Figure 2.6 – Restriction point, R (from (Griffiths, 1999))
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Synthesis of the D-type cyclins begins at the G0/G1 transition and continues so
long as growth factor stimulation persists. This mitogen stimulation of cyclin D is in
part dependent on RAS activation, a role which is highlighted by the ability of anti-RAS
antibodies to block the progression of the cell cycle if added to cells prior to mitogen
stimulation. The availability of cyclin D activates CDK4 and 6 and these complexes then
drive the cell from early G1 through R to late G1; largely by regulation of RB which
exists in a phosphorylated state at the start of G1 complexed to a large number of
proteins. Cyclin D-CDK4/6 activation begins phosphorylation of Rb during early G1. This
initial phosphorylation leads to release of histone deacetylase activity from the
complex alleviating transcriptional repression. The E2F transcription factor remains
bound to Rb at this stage but can still transcribe some genes including cyclin E.
Therefore, levels of cyclin E increase and lead to activation of CDK2, which can then
complete phosphorylation of Rb. Consequently, complete phosphorylation of Rb
results in the release of E2F to activate genes required to drive cells through the G1/S
transition (Macdonald, 2005), Figure 2.7.
Figure 2.7 – Regulation of the G1 to S transition (from (Griffiths, 1999))
The CKIs also play a role in control of cell cycle progression at this stage and in
response to antimitogenic signals, oppose the activity of the CDKs and cause cell cycle
arrest. INK4 inhibitors bind to CDK4/6 to prevent cyclin D binding and CIP/KIP
inhibitors similarly inhibit the kinase activity of cyclin ECDK2, Figure 2.8. CIP/KIP
inhibitors also interact with cyclin D-CDK4/6 complexes during G1, but rather than
blocking cell cycle progression, this interaction is required for the complete function of
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the complex and allows G1 progression. This interaction sequesters CIP/KIP, preventing
its inhibition of cyclin E-CDK2 and thereby facilitating its full activation to contribute to
G1 progression. In the presence of an antimitogenic signal, levels of cyclin D-CDK4/6
are reduced, CIP/KIP is released, which can then interact with and inhibit CDK2 to
cause cell cycle arrest (Macdonald, 2005).
Cells which have suffered DNA damage are prevented from entering S phase
and are blocked at G1. This process is dependent on the tumor suppressor gene p53
and p21. Activation of p53 by DNA damage results in increased p21 levels which can
then inactivate cyclin E-CDK2 to prevent phosphorylation of Rb and inhibit the release
of E2F to promote transcription of genes involved in DNA synthesis, Figure 2.8. This
causes the cell cycle to arrest in G1. Clearly, loss or mutation of p53 will lead to loss of
this checkpoint control and cells will be able to enter S phase with damaged DNA. After
cells have entered S phase, cyclin E is rapidly degraded and CDK2 is released. In S
phase, a further set of cyclins and CDKs, cyclin A-CDK2, are required for continued DNA
replication. Two A-type cyclins have been identified to date: cyclin A1 is expressed
during meiosis and in early cleavage embryos whereas cyclin A2 is present in all
proliferating cells. Cyclin A2 is also induced by E2F and is expressed from S phase
through G2 and M until prometaphase when it is degraded by ubiquitin-dependent
proteolysis (Macdonald, 2005).
Cyclin A2 binds to two different CDKs. Initially, during S phase, it is found
complexed to CDK2 following its release from cyclin E and subsequently in G2 and M it
is found complexed to CDC2 (also known as CDK1). Cyclin A2 has a role in both
transcriptional regulation and DNA replication and its nuclear localization is crucial to
its function. Cyclin A regulates the E2F transcription factor and in S phase, when E2F
directed transcription is no longer required, cyclin A directs its phosphorylation by
CDK2 leading to its degradation. This down-regulation by cyclin A2 is required for
orderly S phase progression and in its absence apoptosis occurs. Recently, cyclin A as
well as cyclin E have been shown to be regulators of centrosome replication and are
able to do so because of their ability to shuttle between nucleus and cytoplasm, Figure
2.9 (Macdonald, 2005).
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Figure 2.8 – Cell cycle arrest at G1/S, mediated by cdk inhibitors (from (Shapiro, 1999))
Figure 2.9 – Dynamics of the DNA synthesome (from (Frouin, 2003))
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The final phase of the cycle is M phase, that comprises mitosis and cytokinesis.
The purpose of mitosis is to segregate sister chromatids into two daughter cells so that
each cell receives a complete set of chromosomes, a process that requires the
assembly of the mitotic spindle. Mitosis is split into a number of stages that includes
prophase, prometaphase, metaphase, anaphase and telophase (Macdonald, 2005).
Cytokinesis, the process of cytoplasmic cleavage, follows the end of mitosis and
its regulation is closely linked to mitotic progression. Mitosis involves the last of
cyclin/CDKs, cyclin B1 and CDC2 as well as additional mitotic kinases. These include
members of the Polo family (PLK1), the aurora family (aurora A, B and C) and the NIMA
family (NEK2) plus kinases implicated at the mitotic checkpoints (BUB1), mitotic exit
and cytokinesis (Macdonald, 2005).
Entry into the final phase of the cell cycle, mitosis, is signaled by the activation
of the cyclin B1-CDC2 complex also known as the M phase promoting factor or MPF.
This complex accumulates during S and G2, but is kept in the inactive state by
phosphorylation of tyrosine 15 and threonine 14 residues on CDC2 by two kinases,
WEE1 and MYT1. WEE1 is nuclear and phosphorylates tyrosine 15, whereas MYT1 is
cytoplasmic and phosphorylates threonine 14. At the end of G2, the CDC25
phosphatase is stimulated to dephosphorylate these residues thereby activating CDC2.
These enzymes are all controlled by DNA structure checkpoints which delay the onset
of mitosis if DNA is damaged. Regulation of cyclin B1-CDC2 is also regulated by
localization of specific subcellular compartments. It is initially localized to the
cytoplasm during G2, but is translocated to the nucleus at the beginning of mitosis. A
second cyclin B, cyclin B2, also exists in mammalian cells and is localized to the Golgi
and endoplasmic reticulum where it may play a role in disassembly of the Golgi
apparatus at mitosis (Macdonald, 2005).
A further checkpoint exists at the end of G2 which checks that DNA is not
damaged before entry into M. Once more p21 activation by p53 can arrest the cell
cycle as at the end of G1. In addition, the CHK1 kinase can phosphorylate CDC25 to
create a binding site for the 14–3–3 protein, a process which inactivates CDC25,
thereby preventing dephosphorylation of CDC2 and halting the cell cycle, Figure 2.10
(Macdonald, 2005).
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Tumor cells can enter mitosis with damaged DNA, suggesting a defect in the
G2/M checkpoint. Tumor cell lines have been shown to activate the cyclin B-CDC2
complex irrespective of the state of the DNA. Activation of cyclin B1-CDC2 leads to
phosphorylation of numerous substrates including the nuclear lamins, microtubule-
binding proteins, condensins and Golgi matrix components that are all needed for
nuclear envelope breakdown, centrosome separation, spindle assembly, chromosome
condensation and Golgi fragmentation respectively. During prophase, the
centrosomes—structures which organize the microtubules and which were duplicated
during G2—separate to define the poles of the future spindle apparatus, a process
regulated by several kinases including the NIMA family member NEK2, as well as
aurora A. At the same time centrosomes begin nucleating the microtubules which
make up the mitotic spindle (Macdonald, 2005).
Chromatin condensation also occurs accompanied by extensive histone
phosphorylation to produce well defined chromosomes. Nuclear envelope breakdown
occurs shortly after centrosome separation. The nuclear envelope is normally
stabilized by a structure known as the nuclear lamin which is composed of lamin
intermediate filament proteins. This envelope is broken down as a result of
hyperphosphorylation of lamins by cyclin B-CDC2 (Macdonald, 2005).
During prometaphase, the microtubules are captured by kinetochores, the
structure which binds to the centromere of the chromosome. Paired sister chromatids
interact with the microtubules emanating from opposite poles resulting in a stable
bipolar attachment. Chromosomes then sit on the metaphase plate where they
oscillate during metaphase. Once all bipolar attachments are complete anaphase is
triggered. This is characterized by simultaneous separation of all sister chromatids.
Each chromosome must be aligned in the center of the bipolar spindle such that its
two sister chromatids are attached to opposite poles. If this is correct, the anaphase-
promoting complex (APC) together with CDC20 is activated to control degradation of
proteins such as securin. This in turn activates the separin protease which cleaves the
cohesion molecules between the sister chromatids allowing them to separate. At this
stage, there is one final checkpoint, the spindle assembly checkpoint, at the
metaphase to anaphase transition, which checks the correct assembly of the mitotic
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apparatus and the alignment of chromosomes on the metaphase plate. The
gatekeeper at this checkpoint is the APC complex. Unaligned kinetochores are
recognized and associate with the MAD2 and BUB proteins which can prevent
activation of APC and cell arrest at metaphase preventing exit from mitosis. In tumor
cell abnormalities of spindle formation are found, suggesting that checkpoint control is
lost (Macdonald, 2005).
Mitotic exit requires that sister chromatids have separated to opposite poles.
During telophase, nuclear envelopes can begin to form around the daughter
chromosomes and chromatin decondensation occurs. The spindle is also disassembled
and cytokinesis is completed. The control of these processes requires destruction of
both the cyclins and other kinases such as NIMA and aurora family members by
ubiquitin dependent proteolysis mediated by APC. Daughter cells can now re-enter the
cell cycle (Macdonald, 2005).
Figure 2.10 – Cell cycle regulation of cyclin dependent kinase (Cdk1) Cyclin-B (CycB) complex (from (Novák, 2010))
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2.4 - GROWTH CHARACTERISTICS OF MALIGNANT CELLS
Cancer can be characterized as a disease of genetic instability, altered cellular
behavior and altered cell–extracellular matrix interactions. These alterations lead to
dysregulated cell proliferation, and ultimately to invasion and metastasis. There are
interactions between the genes involved in these steps. For example, the genes
associated with loss of control of cell proliferation may also be involved in genetic
instability (rapidly proliferating cells have less time to repair DNA damage) and tumor
vascularization that leads to dysregulated proliferation of cells, which in turn eats up
more oxygen, creates hypoxia, and turns on HIF-1 and additional angiogenesis.
Similarly, genes involved in tumor cell invasion may also be involved in loss of growth
control (invasive cells have acquired the skills to survive in ‘‘hostile’’ new
environments) and evasion of apoptosis (less cell death even in the face of a normal
rate of cell proliferation produces more cells). The molecular genetic alterations of
cancer cells lead to cells that can generate their own growth-promoting signals are less
sensitive to cell cycle checkpoint controls, evade apoptosis, and thus have almost
limitless replication potential. This redundancy makes design of effective signal
transduction-targeted chemotherapeutic drugs that target a single pathway very
difficult indeed (Ruddon, 2007).
Cancer cells can also subvert the environment in which they proliferate.
Alterations in both cell–cell and cell–extracellular matrix interactions also occur,
leading to creation of a cancer-facilitating environment. For example, a common
alteration in epithelial carcinomas is alteration of E-cadherin expression, which is a
cell–cell adhesion molecule found on all epithelial cells. Cancer cells exhibit remarkable
plasticity and have the ability to mimic some of the characteristics of other cell types
as they progress and became less well differentiated. For example, cancer cells may
assume some of the structure and function of vascular cells. As cancer cells
metastasize, they may eventually take on a new phenotype such that the tissues of
origin may become unclear—so-called cancers of unknown primary site (Ruddon,
2007).
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2.4.1 - Phenotypic Alterations in Cancer Cells
Treatment of animals or cells in culture with carcinogenic agents is a means of
studying discrete biochemical events that lead to malignant transformation, Figure
2.11. However, studies of cell transformation in vitro have many pitfalls. These ‘‘tissue
culture artifacts’’ include overgrowth of cells not characteristic of the original
population of cultured cells (e.g., overgrowth of fibroblasts in cultures that were
originally primarily epithelial cells), selection for a small population of variant cells with
continued passage in vitro, or appearance of cells with an abnormal chromosomal
number or structure (karyotype). Such changes in the characteristics of cultured cell
populations can lead to ‘‘spontaneous’’ transformation that mimics some of the
changes seen in populations of cultured cells treated with oncogenic agents. Thus, it is
often difficult to sort out the critical malignant events from the noncritical ones
(Ruddon, 2007).
Figure 2.11 – Cellular response (from (Gil, 2006))
Although closer to the carcinogenic process in humans, malignant
transformation induced in vivo by treatment of susceptible experimental animals with
carcinogenic chemicals or oncogenic viruses or by irradiation, is even more difficult
because it is hard to discriminate toxic from malignant events and to determine what
role a myriad of factors, such as the nutritional state of the animal, hormone levels, or
endogenous infections with microorganisms or parasites, might have on the in vivo
carcinogenic events. Moreover, tissues in vivo are a mixture of cell types, and it is
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difficult to determine in which cells the critical transformation events are occurring
and what role the microenvironment of the tissue plays. Thus, most studies designed
to identify discrete biochemical events occurring in cells during malignant
transformation have been done with cultured cells, since clones of relatively
homogeneous cell populations can be studied and the cellular environment defined
and manipulated. The ultimate criterion that establishes whether cells have been
transformed, however, is their ability to form a tumor in an appropriate host animal.
The generation of immortalized ‘‘normal’’ cell lines of a given differentiated phenotype
from human embryonic stem cells, has enhanced the ability to study cells of a normal
genotype from a single source. Such cell lines may also be generated by transfection of
the telomerase gene into cells to maintain chromosomal length (Ruddon, 2007).
Over the past 60 years, much scientific effort has gone into research aimed at
identifying the phenotypic characteristics of in vitro transformed cells that correlate
with the growth of a cancer in vivo. This research has tremendously increased our
knowledge of the biochemistry of cancer cells. However, many of the biochemical
characteristics initially thought to be closely associated with the malignant phenotype
of cells in culture has subsequently been found to be dissociable from the ability of
those cells to produce tumors in animals. Furthermore, individual cells of malignant
tumors growing in animals or in humans exhibit marked biochemical heterogeneity, as
reflected in their cell surface composition, enzyme levels, immunogenicity, response to
anticancer drugs, and so on. This has made it extremely difficult to identify the
essential changes that produce the malignant phenotype (Ruddon, 2007).
2.4.2 - Immortality of Transformed Cells in Culture
Most normal diploid mammalian cells have a limited life expectancy in culture.
For example, normal human fibroblast lines may live for 50 to 60 population doublings
(the ‘‘Hayflick index’’), but then viability begins to decrease rapidly, unless they
transform spontaneously or are transformed by oncogenic agents. However, malignant
cells, once they become established in culture, will generally live for an indefinite
number of population doublings, provided the right nutrients and growth factors
(Ruddon, 2007).
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It is not clear what limits the life expectancy of normal diploid cells in culture,
but it may be related to the continual shortening of chromosomal telomeres each time
cells divide. Transformed cells are known to have elevated levels of telomerase that
maintain telomere length. Transformed cells that become established in culture also
frequently undergo karyotypic changes, usually marked by an increase in
chromosomes (polyploidy), with continual passage. This suggests that cells with
increased amounts of certain growth-promoting genes are generated and/or selected
during continual passage in culture. The more undifferentiated cells from cancers of
animals or patients also often have an atypical karyology, thus the same selection
process may be going on in vivo with progression over time of malignancy from a lower
to a higher grade (Ruddon, 2007).
2.4.3 - Decreased Requirement for Growth Factors
Other properties that distinguish transformed cells from their non transformed
counterparts are decreased density-dependent inhibition of proliferation and the
requirement for growth factors for replication in culture. Cells transformed by
oncogenic viruses have lower serum growth requirements than do normal cells. Cancer
cells may also produce their own growth factors that may be secreted and activate
proliferation in neighboring cells (paracrine effect) or, if the same malignant cell type
has both the receptor for a growth factor and the means to produce the factor, self-
stimulation of cell proliferation (autocrine effect) may occur. One example of such an
autocrine loop is the production of tumor necrosis factor-alpha (TNF-α) and its
receptor TNFR1 by diffuse large cell lymphoma. Co-expression of TNF-α and its
receptor are negative prognostic indicators of survival, suggesting that autocrine loops
can be powerful stimuli for tumor aggressiveness and thus potentially important
diagnostic and therapeutic targets.
2.4.4 - Loss of Anchorage Dependence
Most freshly isolated normal animal cells and cells from cultures of normal
diploid cells do not grow well when they are suspended in fluid or a semisolid agar gel.
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However, if these cells contact with a suitable surface they attach, spread, and
proliferate. This type of growth is called anchorage-dependent growth. Many cell lines
derived from tumors and cells transformed by oncogenic agents are able to proliferate
in suspension cultures or in a semi solid medium (methylcellulose or agarose) without
attachment to a surface. This is called anchorage-independent growth and this
property of transformed cells has been used to develop clones of malignant cells. This
technique has been widely used to compare the growth properties of normal and
malignant cells. Another advantage that has been derived from the ability of malignant
cells to grow in soft agar (agarose), is the ability to grow cancer cells derived from
human tumors to test their sensitivity to chemotherapeutic agents and to screen for
potential new anticancer drugs (Ruddon, 2007).
2.4.5 - Loss of Cell Cycle Control and Resistance to Apoptosis
Normal cells respond to a variety of suboptimal growth conditions by entering a
quiescent phase in the cell division cycle, the G0 state. There appears to be a decision
point in the G1 phase of the cell cycle, at which time the cell must make a commitment
to continue into the S phase, the DNA synthesis step, or to stop in G1 and wait until
conditions are more optimal for cell replication to occur. If this waiting period is
prolonged, the cells are said to be in a G0 phase. Once cells make a commitment to
divide, they must continue through S, G2, and M to return to G1. If the cells are blocked
in S, G2, or M for any length of time, they die. The events that regulate the cell cycle
are called cell cycle checkpoints (Ruddon, 2007).
The loss of cell cycle check point control by cancer cells may contribute to their
increased susceptibility to anticancer drugs. Normal cells have mechanisms to protect
themselves from exposure to growth-limiting conditions or toxic agents by calling on
these check point control mechanisms. Cancer cells, by contrast, can continue through
these checkpoints into cell cycle phases that make them more susceptible to the
cytotoxic effects of drugs or irradiation. For example, if normal cells accrue DNA
damage due to ultraviolet (UV) or X-irradiation, they arrest in G1 so that the damaged
DNA can be repaired prior to DNA replication. Another check point in the G2 phase
allows repair of chromosome breaks before chromosomes are segregated at mitosis,
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Figure 2.12. Cancer cells, which exhibit poor or absent check point controls, proceed to
replicate the damaged DNA, thus accounting for persisting and accumulating
mutations (Ruddon, 2007).
2.5 - CELL CYCLE REGULATION
Cyclin-dependent protein kinases (CDKs), of which CDC2 is only one, are crucial
regulators of the timing and coordination of eukaryotic cell cycle events. Transient
activation of members of this family of serine/threonine kinases occurs at specific cell
cycle phases (Ruddon, 2007).
Figure 2.12 - Major pathways where Plks may play a role in intra-S-phase checkpoint in mammalian systems (from
(Suqing, 2005))
In budding yeast G1 cyclins encoded by the CLN genes, interact with and are
necessary for the activation of, the CDC2 kinase (also called p34cdc2), driving the cell
cycle through a regulatory point called START (because it is regulated by the cdc2 or
start gene) and committing cells to enter S phase. START is analogous to the G1
restriction point in mammalian cells. The CDKs work by forming active heterodimeric
complexes following binding to cyclins, their regulatory subunits. CDK2, 4, and 6, and
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possibly CDK3 cooperate to push cells through G1 into S phase. CDK4 and CDK6 form
complexes with cyclins D1, D2, and D3, and these complexes are involved in
completion of G1. Cyclin D–dependent kinases accumulate in response to mitogenic
signals and this leads to phosphorylation of the Rb protein. This process is completed
by the cyclin E1- and E2-CDK2 complexes. Once cells enter S phase, cyclin E is degraded
and A1 and A2 cyclins get involved by forming a complex with CDK2. There are a
number of regulators of CDK activities; where they act in the cell cycle is depicted in
Figure 2.13 (Ruddon, 2007).
Figure 2.13 - Restriction point control and the G1-S transition (from (Ruddon, 2007))
2.5.1 - CDK Inhibitors
The inhibitors of CDKs include the Cip/Kip and INK4 family of polypeptides. The
Cip/Kip family includes p21cip1, p27kip1, and p57kip2. The actions of these proteins
are complex. Although the Cip/Kip proteins can inhibit CDK2, they are also involved in
the sequestration of cyclin D-dependent kinases that facilitates cyclin E-CDK2
activation necessary for G1/S transition (Ruddon, 2007).
The INK4 proteins target the CDK4 and CDK6 kinases, sequester them into
binary CDKINK4 complexes, and liberate bound Cip/Kip proteins. This indirectly inhibits
cyclin E–CDK and promotes cell cycle arrest. The INK4-directed arrest of the cell cycle
in G1 keeps Rb in a hypophosphorylated state and represses the expression of S-phase
genes. Four INK4 proteins have been identified: p16INK4a, p15INK4b, p18INK4c, and
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p19INK4d. INKA4a loss of function occurs in a variety of cancers including pancreatic
and small cell lung carcinomas and glioblastomas. INK4a fulfills the criteria of a tumor
suppressor and appears to be the INK4 family member with the most active role in this
regard. The INK4a gene encodes another tumor suppressor protein called ARF
(p14ARF). Mice with a disrupted ARF gene have a high propensity to develop tumors,
including sarcomas, lymphomas, carcinomas, and CNS tumors. These animals
frequently die at less than 15 months of age. ARF and p53 act in the same pathway to
insure growth arrest and apoptosis in response to abnormal mitogenic signals such as
myc-induced carcinogenesis, Figure 2.14 (Ruddon, 2007).
Figure 2.14 - Regulation of the Rho pathway and the cytoskeleton by cyclin-dependent kinase (CDK) inhibitors (from
(Besson, 2004))
2.5.2 - Cyclins
The originally discovered cyclins, cyclin A and B, identified in sea urchins, act at
different phases of the cell cycle. Cyclin A is first detected near the G1/S transition and
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cyclin B is first synthesized during S phase and accumulates in complexes with p34cdc2
as cells approach the G2-to-M transition. Cyclin B is then abruptly degraded during
mitosis. Thus, cyclins A and B regulate S and M phase, but do not appear to play a role
in G1 control points such as the restriction point (R point), which is the point where key
factors have accumulated to commit cells to enter S phase (Ruddon, 2007).
Three more recently discovered mammalian cyclins, C, D1, and E, are the
cyclins that regulate the key G1 and G1/S transition points. Unlike cyclins A and B,
cyclins C, D1, and E are synthesized during the G1 phase in mammalian cells. Cyclin C
levels change only slightly during the cell cycle but peak in early G1. Cyclin E peaks at
the G1–S transition, suggesting that it controls entry into S. Three distinct cyclin D
forms, D1, 2, and 3, have been discovered and are differentially expressed in different
mouse cell lineages. These D cyclins all have human counterparts and cyclin D levels
are growth factor dependent in mammalian cells: when resting cells are stimulated by
growth factors, D-type cyclin levels rise earlier than cyclin E levels, implying that they
act earlier in G1 than E cyclins. Cyclin D levels drop rapidly when growth factors are
removed from the medium of cultured cells. All of these cyclins (C, D, and E) form
complexes with, and regulate the activity of various CDKs and these complexes control
the various G1, G1–S, and G2–M transition points, Figure 2.15 (Ruddon, 2007).
Interestingly, negative growth regulators also interact with the cyclin-CDK system. For
example, TGF-b1, which inhibits proliferation of epithelial cells by interfering with G1-S
transition, reduced the stable assembly of cyclin E-CDK2 complexes in mink lung
epithelial cells, and prevented the activation of CDK2 kinase activity and the
phosphorylation of Rb. This was one of the first pieces of data suggesting that the
mammalian G1 cyclin-dependent kinases are targets for negative regulators of the cell
cycle (Ruddon, 2007).
2.5.3 - Cell Cycle Checkpoints
The role of various CDKs, cyclins, and other gene products in regulating
checkpoints at G1 to S, G2 to M, and mitotic spindle segregation have been described in
detail previously. Alterations of one or more of these checkpoint controls occur in
most, if not all, human cancers at some stage in their progression to invasive cancer. A
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key player in the G1–S checkpoint system is the retinoblastoma gene Rb (Ruddon,
2007).
Figure 2.15 - Cell-cycle regulation (from (Charles, 2004))
Phosphorylation of the Rb protein by cyclin D–dependent kinase releases Rb
from the transcriptional regulator E2F and activates E2F function. Inactivation of Rb by
genetic alterations occurs in retinoblastoma and is also observed in other human
cancers, for example, small cell lung carcinomas and osteogenic sarcomas (Ruddon,
2007).
The p53 gene product is an important cell cycle checkpoint regulator at both
the G1–S and G2–M checkpoints but does not appear to be important at the mitotic
spindle checkpoint because gene knockout of p53 does not alter mitosis. The p53
tumor suppressor gene is the most frequently mutated gene in human cancer,
indicating its important role in conservation of normal cell cycle progression. One of
p53’s essential roles is to arrest cells in G1 after genotoxic damage, to allow for DNA
repair prior to DNA replication and cell division. In response to massive DNA damage,
p53 triggers the apoptotic cell death pathway. Data from short-term cell-killing assays,
using normal and minimally transformed cells, have led to the conclusion that mutated
p53 protein confers resistance to genotoxic agents (Ruddon, 2007).
The spindle assembly checkpoint machinery involves genes called bub (budding
uninhibited by benomyl) and mad (mitotic arrest deficient). There are three bub genes
and three mad genes involved in the formation of this checkpoint complex. A protein
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kinase called Mps1 also functions in this checkpoint function. The chromosomal
instability, leading to aneuploidy in many human cancers, appears to be due to
defective control of the spindle assembly checkpoint. Mutant alleles of the human
bub1 gene have been observed in colorectal tumors displaying aneuploidy. Mutations
in these spindle checkpoint genes may also result in increased sensitivity to drugs that
affect microtubule function because drug-treated cancer cells do not undergo mitotic
arrest and go on to die (Ruddon, 2007).
Maintaining the integrity of the genome is a crucial task of the cell cycle
checkpoints. Two checkpoint kinases, called Chk1 and Chk2 (also called Cds1), are
involved in checkpoint controls that affect a number of genes involved in maintenance
of genome integrity. Chk1 and Chk2 are activated by DNA damage and initiate a
number of cellular defense mechanisms that modulate DNA repair pathways and slow
down the cell division cycle to allow time for repair. If DNA is not successfully mended,
the damaged cells usually undergo cell death via apoptosis. This process prevents the
defective genome from extending its paternity into daughter cells (Ruddon, 2007).
Upstream elements activating the checkpoint signaling pathways such as those
turned on by irradiation or agents causing DNA double strand breaks include the ATM
kinase, a member of the phosphatidylinositol 3-kinase (PI3K) family, which activates
Chk2 and its relative ATR kinase that activates Chk1. There is also cross talk between
ATM and ATR that mediates these responses. Chk1 and Chk2 phosphorylate CDC25A
and C, which inactivate them. In its dephosporylated state CDC25A activates the CDK2-
cyclin E complex that promotes progression through S phase. It should be noted that
this is an example of dephosphorylation rather than phosphorylation activating a key
biological function. This is in contrast to most signal transduction pathways, where the
phosphorylated state of a protein (often a kinase) is the active state and the
dephosphorylated state is the inactive one. In addition, Chk1 renders CDC25A
unstable, which also diminishes its activity. CDC25A also binds to and activates CDK1-
cyclin B, which facilitates entry into mitosis. G2 arrest induced by DNA damage induces
CDC25A degradation and, in contrast, G2 arrest is lost when CDC25A is overexpressed.
A number of proteins are now known to act as mediators of checkpoint responses by
impinging on the Chk1 and 2 pathways. These include the BRCT domain–containing
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proteins 53BP1, BRCA1, and MDC1.These proteins are involved in activation of Chk1
and Chk2 by acting through protein–protein interactions that modulate the activity of
these checkpoint kinases. In general, these modulators are thought to be tumor
suppressors (Ruddon, 2007).
Chk1 and 2 have overlapping roles in cell cycle regulation, but different roles during
development. Chk1 but not Chk2 is essential for mammalian development, as
evidenced by the early embryonic lethality of Chk1 knockout mice. Chk2-deficient mice
are viable and fertile and do not have a tumor-prone phenotype unless exposed to
carcinogens, and this effect is more evident later in life. As illustrated in Figure 2.16,
there are interactions between the Chk kinases and the p53 pathway. Chk2
phosphorylates threonine-18 or serine-20 on p53, which attenuates p53’s interaction
with its inhibitor MDM2, thus contributing to p53 stabilization and activation.
However, Chk2 and p53 only have partially overlapping roles in checkpoint regulation
because not all DNA-damaging events activate both pathways, Figure 2.16 (Ruddon,
2007).
2.5.4 - Cell Cycle Regulatory Factors as Targets for Anticancer Agents
The commonly observed defects in cell cycle regulatory pathways in cancer
cells distinguish them from normal cells and provide potential targets for therapeutic
agents. One approach is to inhibit cell cycle checkpoints in combination with DNA-
damaging drugs or irradiation. The rationale for this is that normal cells have a full
complement of checkpoint controls, whereas tumor cells are defective in one or more
of these and thus are more subject to undergoing apoptosis in response to excessive
DNA damage. This has been accomplished by combining ATM/ATR inhibitors such as
caffeine or Chk1 inhibitors in combination with DNA-damaging drugs. So far this
approach has not been demonstrated clinically, and indeed is somewhat counter
intuitive, since p53 mutant tumor cells are more resistant to many chemotherapeutic
drugs. p53 is a key player in causing cell death in drug treated, DNA-damaged cells
(one exception to that is the microtubule inhibitor paclitaxel), and active, unmutated
p53 is needed for this response (Ruddon, 2007).
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Figure 2.16 - Simplified scheme of cell-cycle checkpoint pathways induced in response to DNA damage (here
DSBs), with highlighted tumor suppressors shown in red and proto-oncogenes shown in green (from (Kastan,
2004))
Another approach is to target the cyclin dependent kinases directly. Alteration
of the G1–S checkpoint occurs in many human cancers. Cyclin D1 gene amplification
occurs in a subset of breast, esophageal, bladder, lung, and squamous cell carcinomas.
Cyclins D2 and D3 are overexpressed in some colorectal carcinomas. In addition, the
cyclin D–associated kinases CDK4 and CDK6 are over expressed or mutated in some
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cancers. Mutations or deletions in the CDK4 and CDK6 inhibitor INK4 have been
observed in familial melanomas, and in biliary tract, esophageal, pancreatic, head and
neck, non small cell lung, and ovarian carcinomas. Inactivating mutations of CDK4
inhibitory modulators p15, p16, and p18 have been observed in a wide variety of
human cancers. Cyclin E is also amplified and overexpressed in some breast and colon
carcinomas and leukemias (Ruddon, 2007).
Human cancers have a variety of mutations in cell cycle regulatory genes. This
includes overexpression of D1 and E1 cyclins and CDKs (mainly CDK4 and CDK6) as
noted above. Loss of CDK inhibitory functions (mainly INK4a and 4b and Kip1) also
occurs, as does loss of Rb, one of the first tumor suppressor genes identified. Loss of
Kip1 function and overexpression of cyclin E1 occur frequently and are associated with
poor prognosis in breast and ovarian cancers (Ruddon, 2007).
The mitogen-stimulated proliferation of cells is mediated via a retinoblastoma
(Rb) pathway that involves phosphorylation of Rb, its dissociation form and activation
of the E2F family of transcription factors, and subsequent turn-on of genes involved in
G1–S transition and DNA synthesis. Disruption of this pathway by overexpression of
cyclin D1, loss of the INK4 inhibitor p16, mutation of CDK4 to a p16-resistant form, or
loss or mutation of Rb is frequently seen in cancer cells. The activation of CDK
inhibitory factors such as p16INK4 or p27kip1 and inhibition of cyclin dependent
kinases are, therefore, potential ways to interdict the overactive cell proliferation
pathways in cancer cells. Thus, inhibition of cyclins D1 and E and CDKs, especially CDK4
and CDK6, could be targets for inhibiting growth of cancers. As more knowledge of the
complicated steps in cell cycle regulation is gained, more potential targets become
available (Ruddon, 2007).
2.6 - APOPTOSIS
Apoptosis (sometimes called programmed cell death) is a cell suicide
mechanism that enables multicellular organisms to regulate cell number in tissues and
to eliminate unneeded or aging cells as an organism develops. The biochemistry of
apoptosis has been well studied in recent years, and the mechanisms are now
reasonably well understood (Ruddon, 2007).
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The apoptosis pathway involves a series of positive and negative regulators of
proteases called caspases, which cleave substrates, such as poly (ADP-ribose)
polymerase, actin and lamin. In addition, apoptosis is accompanied by the
intranucleosomal degradation of chromosomal DNA, producing the typical DNA ladder
seen for chromatin isolated from cells undergoing apoptosis. The endonuclease
responsible for this effect is called caspase-activated DNase, or CAD (Ruddon, 2007).
A number of ‘‘death receptors’’ have also been identified, they are cell surface
receptors that transmit apoptotic signals initiated by death ligands, Figure 2.17. The
death receptors sense signals that tell the cell that it is in an uncompromising
environment and needs to die. These receptors can activate the death caspases within
seconds of ligand binding and induce apoptosis within hours. Death receptors belong
to the tumor necrosis factor (TNF) receptor gene superfamily and have the typical
cystine rich extracellular domains and an additional cytoplasmic sequence termed the
death domain (Ruddon, 2007).
Figure 2.17 - Apoptosis signaling through death receptors (from (Frederik, 2002))
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The best-characterized death receptors are CD95 (also called Fas or Apo1) and
TNF receptor TNFR1 (also called p55 or CD120a). The importance of the apoptotic
pathway in cancer progression is seen when there are mutations that alter the ability
of the cell to undergo apoptosis and allow transformed cells to keep proliferating
rather than die. Such genetic alterations include the translocation of the bcl-2 gene in
lymphomas that prevents apoptosis and promotes resistance to cytotoxic drugs. Other
genes involved as players on the apoptosis stage include c-myc, p53, c-fos, and the
gene for interleukin-1b-converting enzyme (ICE). Various oncogene products can
suppress apoptosis, like the adenovirus protein E1b, ras, and n-abl (Ruddon, 2007).
Mitochondria plays a pivotal role in the events of apoptosis by at least three
mechanisms:
1) Release of proteins, e.g., cytochrome c, that triggers activation of caspases;
2) Alteration of cellular redox potential;
3) Production and release of reactive oxygen species after mitochondrial
membrane damage.
Another mitochondrial link to apoptosis is implied by the fact that Bcl-2, the
anti-apoptotic factor, is a mitochondrial membrane protein that appears to regulate
mitochondrial ion channels and proton pumps, Figure 2.18 (Ruddon, 2007).
2.6.1 - Biochemical Mechanism of Apoptosis
Multicellular organisms, from the lowest to the highest species, must have a
way to get rid of excess cells or cells that are damaged in order for the organism to
survive. Apoptosis is the mechanism that they use to do this. It is the way that the
organism controls cell numbers and tissue size and protects itself from ‘‘rogue’’ cells.
A simplified version of the apoptotic pathways can be visualized in Figure 2.19
(Ruddon, 2007).
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Figure 2.18 - Apoptosis signaling through mitochondria (from (Frederik, 2002))
The death receptor–mediated pathway is turned on by members of the death
receptor superfamily of receptors including Fas receptor (CD95) and TNF receptor 1,
which are activated by Fas ligand and TNF, respectively. Interaction of these ligands
with their receptors induces receptor clustering, binding of the receptor clusters to
Fas-associated death domain protein (FADD), and activation of caspase-8, Figure 2.20.
This activation step is regulated by c-FLIP. Caspase-8, in turn, activates caspase-3 and
other ‘‘executioner’’ caspases, which induce a number of apoptotic substrates. The
DNA damage–induced pathway invokes a mitochondrial-mediated cell death pathway
that involves pro-apoptotic factors like Bax (blocked by the anti-apoptotic protein Bcl-
2). This results in cytochrome c release from the mitochondria and triggering of
downstream effects facilitating caspase-3 activation, which is where the two pathways
intersect. There are both positive and negative regulators that also interact on these
pathways (Ruddon, 2007).
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Figure 2.19 - The two main apoptotic signaling pathways (from (Frederik, 2002))
Figure 2.20 - Illustration of the main TNF receptor signaling pathways (from (Dash, 2003))
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2.6.2 - Caspases
Caspases are a family of cysteine proteases that are activated specifically in
apoptotic cells. This family of proteases is highly conserved through evolution all the
way from hydra and nematodes up to humans. Over 12 caspases have been identified
and although most of them appear to function during apoptosis, the function of all of
them is not yet clear. The caspases are called cysteine-proteases because they have a
cysteine in the active site that cleaves substrates after asparagines in a sequence of
asp-X, with the four amino acids amino-terminal to the cleavage site determining a
caspase’s substrate specificity (Ruddon, 2007).
The importance of the caspases in apoptosis is demonstrated by the inhibitory
effects of mutation or drugs that inhibit their activity. Caspases can either inactivate a
protein substrate by cleaving it into an inactive form or activate a protein by cleaving a
pro-enzyme negative regulatory domain. In addition, caspases themselves are
synthesized as pro-enzymes and are activated by cleavage at asp-x sites. Thus, they can
be activated by other caspases, producing elements of the ‘‘caspase cascade’’ shown
in Figure 2.21.
Figure 2.21 – Caspase activation (from (Dash, 2003))
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Also, as illustrated in Figure 2.21, caspases are activated in a number of steps
by proteolytic cleavage by an upstream caspase or by protein–protein interactions,
such as that seen for the activation of caspase-8 and the interaction of cytochrome c
and Apaf-1 in the activation of caspase-9. A number of important substrates of
caspases have been identified, including the caspase-activated DNase (CAD), noted
above, which is the nuclease responsible for the DNA ladder of cells undergoing
apoptosis. Activation of CAD is mediated by caspase-3 cleavage of the CAD-inhibitory
subunit. Caspase-mediated cleavage of other specific substrates has been shown to be
responsible for other typical changes seen in apoptotic cells, such as the cleavage of
nuclear lamins required for nuclear shrinkage and budding, loss of overall cell shape by
cleavage of cytoskeleton proteins, and cleavage of PAK2, a member of the p21-
activated kinase family, that mediates the blebbing seen in dying cells.
2.6.3 - Bcl-2 Family
Mammalian Bcl-2 was first identified as anti-apoptotic protein in lymphomas
cells. It turned out to be a homolog of an anti-apoptotic protein called Ced-9 described
in C. elegans and protects from cell death by binding to the pro-apoptotic factor Ced-4.
Similarly, in mammalian cells, Bcl-2 binds to a number of pro-apoptotic factors such as
Bax, Figure 2.22. One concept is that pro- and anti- apoptotic members of the Bcl-2
family of proteins form heterodimers, which can be looked on as reservoirs of plus and
minus apoptotic factors waiting for the appropriate signals to be released (Ruddon,
2007).
2.6.4 - Anoikis
Anoikis is a form of apoptosis that occurs in normal cells that lose their
adhesion to the substrate or extracellular matrix (ECM) on which they are growing.
Adherence to a matrix is crucial for the survival of epithelial, endothelial, and muscle
cells. Prevention of their adhesion usually results in rapid cell death, which occurs via
apoptosis. Thus, anoikis is a specialized form of apoptosis caused by prevention of cell
adhesion (Ruddon, 2007).
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Figure 2.22 – Apoptotic pathways. Two major pathways lead to apoptosis: the intrinsic cell death pathway
controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling
(from (Zhang, 2005))
The term anoikis means ‘‘homelessness’’ in Greek and although the observation
of this phenomen occurs only with cultured cells, it is likely to occur also in vivo
because it is known that cell-cell and cell-ECM interactions are crucial to cell
proliferation, organ development, and maintenance of a differentiated state. This may
be a way that a multicellular organism protects itself from free-floating or wandering
cells (such as occurs in tumor metastasis). The basic rule for epithelial and endothelial
cells appears to be ‘‘attach or die’’. Interestedly, cells that normally circulate in the
body such as hematopoietic cells do not undergo anoikis (Ruddon, 2007).
Cell attachment is mediated by integrins, and ECM integrin interactions
transduce intracellular signaling pathways that activate genes involved in cell
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proliferation and differentiation. Although the cell death pathways induced by
disruption of these cell attachment processes are not clearly worked out, cell
detachment–induced anoikis does result in activation of caspases-8 and -3 and is
inhibited by Bcl-2 and Bcl-XL, indicating some similarities to the typical apoptosis
mechanisms. In addition, integrin-ECM interaction activates focal adhesion kinase
(FAK) and attachment-mediated activation of PI3-kinase. Both of these steps protect
cells from anoikis, whereas inhibition of the PI3-kinase pathway induces anoikis
(Ruddon, 2007).
Disruption of cell-matrix interactions also turns on the JNK /p38 pathway, a
stress-activated protein kinase. The mitogen-activated kinase system may also be
involved, since caspase mediated cleavage of MEKK-1 occurs in cells undergoing
anoikis. As stated earlier, one of the hallmarks of malignantly transformed cells
growing in culture is their ability to grow in an anchorage independent manner,
whereas normal cells do not. Thus, cancer cells may develop resistance to anoikis. This
may be a way that metastatic cancer cells can survive in the bloodstream until they
seed out in a metastatic site (Ruddon, 2007).
2.7 - RESISTANCE TO APOPTOSIS IN CANCER AND POTENTIAL TARGETS FOR THERAPY
It would be a mistake to portray apoptosis as only a mechanism to kill cells
damaged by some exogenous insult such as DNA-damaging toxins, drugs, or
irradiation. Apoptosis is, in fact, a usual mechanism used by all multicellular organisms
to facilitate normal development, selection of differentiated cells that the organism
needs, and control of tissue size. For example, studies of nematodes (C. elegans), fruit
flies, and mice indicate that apoptotic-mediated mechanisms similar to those
described here are intrinsic and required for normal development. Dysfunction of
these pathway results in developmental abnormalities and disease states (Ruddon,
2007).
In the human, development of the immune system is perhaps the best example
of the role for apoptosis in normal development. In the immune system, apoptosis is a
fundamental process that regulates T- and B-cell proliferation and survival and is used
to eliminate immune cells that would potentially recognize and destroy host tissues
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(‘‘anti-self ’’). Mechanisms involving Apo-1/FAS (CD95)-mediated signaling of the
caspase cascade are employed in lymphocytic cell selection. In the case of T
lymphocytes, pre-T cells are produced in the bone marrow and circulate to the thymus
where they differentiate and rearrange their T-cell receptors (TCRs). Those cells that
fail to rearrange appropriately their TCR genes, and thus cannot respond to self–major
histocompatibility complex (MHC)–peptide complexes, die by ‘‘neglect’’, Figure 2.23.
Those T cells that pass the TCR selection tests mature and leave the thymus to become
the adult peripheral T-cell pool. The mature T-cell pool thus passes through a number
of selection steps to ensure self-MHC restriction and self-tolerance. Apoptosis also is
used to delete mature peripheral T cells that are insufficiently stimulated by positive
growth signals, and this is a mechanism to downregulate, or terminate, an immune
response (Ruddon, 2007).
B lymphocytes undergo selection and maturation in the bone marrow and
germinal centers of the spleen and other secondary lymphoid organs. Those with low
antigen affinity or those autoreactive are eliminated by apoptosis. Those that pass this
test mature into memory B cells and long-lived plasma cells. The ability of lymphoid
progeny cells to avoid apoptosis may lead to lymphatic leukemias or lymphomas. In
addition, cancers develop multiple mechanisms to evade destruction by the immune
system such as a decreased expression of MHC molecules on cancer cell surfaces and
production of immunosuppressive cytokines. Several cell proliferations promoting
events take place in cancer cells as they evolve over time into growth dysregulated,
invasive, metastatic cell types. These events include activation of proliferation-
promoting oncogenes such as ras and myc, overexpression of cell cycle regulatory
factors such as cyclin D, increased telomerase to overcome cell senescence, and
increased angiogenesis to enhance blood supply to tumor tissue (Ruddon, 2007).
The cancer-related alterations in the apoptotic pathway provide a number of
cancer chemotherapeutic targets.
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Figure 2.23 - The role of apoptosis in the development and function of T lymphocytes. Major pro-apoptotic and anti-
apoptotic signals/molecules (from (Zhang, 2005))
2.8 - SUMMARY
At the end of this chapter is possible to conclude that many of the controls that
govern the transition between quiescence and active cell cycling in mammals operate
in G1 phase. Loss of R point control appears to be a common, possibly even universal
step in tumor development, and a number of genetic lesions that can contribute to this
deregulation have been identified.
Loss of survival proteins can also contribute to apoptosis. The antiapoptotic
gene, BCL2, has been shown to be repressed by p53 and, therefore, contributes to
apoptosis by blocking survival signals mediated by BCL2. The choice as to whether a
cell undergoes apoptosis or cell cycle arrest and DNA repair depends on a number of
factors. Some may be independent of p53 such as extracellular survival factors, the
existence of oncogenic alterations and the availability of additional transcription
factors. However, the extent of DNA damage may also contribute to the choice by
affecting the level of activity of p53 induced. Activation of apoptosis has been
associated with higher levels of p53 than those required for cell cycle arrest which may
reflect a lower affinity of cell cycle arrest target gene promoters for p53. In addition,
the type of cell may affect the response to p53. Importantly, it is vital to identify why
transformed cells die in response to p53, whereas normal cells undergo cell cycle
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arrest and DNA repair as this may be of great potential for the development of cancer
therapies (Macdonald, 2005).
This loss of cell cycle check point control by cancer cells may contribute to their
increased susceptibility to anticancer drugs. Normal cells have mechanisms to protect
themselves from exposure to growth-limiting conditions or toxic agents by calling on
these check point control mechanisms. Cancer cells, by contrast, can continue through
these checkpoints into cell cycle phases that make them more susceptible to the
cytotoxic effects of drugs or irradiation (Ruddon, 2007).
Apoptosis occurs in most, if not all, solid cancers. Ischemia, infiltration of
cytotoxic lymphocytes, and release of TNF may all play a role in this and it would be
therapeutically advantageous to tip the balance in favor of apoptosis over mitosis in
tumors, if that could be done.
Clearly, a number of anticancer drugs induce apoptosis in cancer cells but the
problem is that they usually do this in normal proliferating cells as well. Therefore, the
goal should be to manipulate selectively the genes involved in inducing apoptosis in
tumor cells, although understanding how those genes work may go a long way to
achieving this goal.
CHAPTER III
RADIATION EFFECT ON NORMAL AND NEOPLASTIC TISSUES
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3.1 – INTRODUCTION
When cells are exposed to ionizing radiation the standard physical effects
between radiation and the atoms or molecules of the cells occur first and the possible
biological damage to cell functions follows later. The biological effects of radiation
result mainly from damage to the DNA, which is the most critical target within the cell;
however, there are also other sites in the cell that, when damaged, may lead to cell
death (Suntharalingam, 2002).
Many aspects of the response of tissue systems are strongly affected by the
state of the cell in its cycle, for example, the state of oxygenation of the cell. The
supply of metabolic substrates and the removal of metabolic products also play a role
in modifying the response of tissue systems. The most significant aspect of the
radiosensitivity of a tissue or organ system centers on the state of reproductive activity
and this proliferative state varies widely among the tissues of any mammalian species.
At one extreme are the tissues of the central nervous system, some of which rarely, if
ever, undergo division during the organism's adult life, and for which loss of clonogenic
ability is an irrelevant end point. At the other extreme are the blood forming organs,
which are proliferating at a rate approaching that of an exponentially growing, in vitro
culture (Alpen, 1998).
In this chapter, it is provided a detailed description of the effects of radiation
on normal and neoplastic tissues. For this purpose, the chapter starts with the
irradiation carcinogenesis which includes the description of ionizing and UV radiation
effect followed by the description of the types of cell death in mammalian cells. After
this item, it is performed a description of the nature of cell population in tissues and of
the cell population kinetics and radiation damage. Subsequently, the chapter focuses
on the cell kinetics in normal and tumor tissues, on the models for radiobiology
sensitivity of neoplastic tissues and the tumor growth and “cure” models. Finally, it
ends with a description of the radiobiological responses, hypoxia and radiosensitivity
of the tumor cells.
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3.2 - IRRADIATION CARCINOGENESIS
X-rays and ultraviolet (UV) radiation produce damage to DNA inducing DNA
repair processes, some of which are error prone and may lead to mutations. The
development of malignant transformation in cultured cells after irradiation requires
cell proliferation to ‘‘fix’’ the initial damage into a heritable change and then to allow
clonal proliferation and expression of the typical transformed phenotype. Fixation
appears to be complete after the first post irradiation mitotic cycle, thus, a promotion
phase is required for full expression of the initiated malignant alteration. Moreover,
when low doses of chemical carcinogens and X-rays are used together, these two types
of agents act synergistically to produce malignant transformation (Raddon, 2007).
When cells are exposed to UV light in the 240 to 300 nm range, the bases
acquire excited energy states, producing photochemical reactions between DNA bases.
The principal products in DNA at biologically relevant doses of UV light are cyclobutane
dimers formed between two adjacent pyrimidine bases in the DNA chain. Both
thymine–thymine and thymine–cytosine dimmers are formed. That formation of these
dimers is linked to mutagenic events (Raddon, 2007).
3.2.1 - Ionizing Radiation
The history of radiation carcinogenesis goes back a long way. The harmful
effects of X-rays were observed soon after their discovery in 1895 by W. K. Roentgen.
The first observed effects were acute, such as reddening and blistering of the skin
within hours or days after exposure. By 1902, it became apparent that cancer was one
of the possible delayed effects of X-ray exposure. These cancers, which included
leukemia, skin cancers, lymphomas, and brain tumors, were usually seen in radiologists
only after long-term exposure before adequate safety measures were adopted, thus it
was thought that there was a safe threshold for radiation exposure. The hypothesis
that small doses of radiation might also cause cancer was not adopted until the 1950s,
when data from atomic bomb survivors in Japan and certain groups of patients treated
with X-rays for noncancerous conditions, such as enlarged thyroids, were analyzed.
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These and other data led to the concept that the incidence of radiation-induced
cancers might increase as a linear, nonthreshold function of dose (Raddon, 2007).
In radiation carcinogenesis, the damage to DNA, and hence its mutagenic and
carcinogenic effect, is due to the generation of free radicals as the radiation passes
through tissues. The amount of radical formation and ensuing DNA damage depend on
the energy of the radiation. In general, X-rays and gamma rays have a low rate of linear
energy transfer, generate ions sparsely along their tracks, and penetrate deeply into
tissue. This profile contrasts with that of charged particles, such as protons and α
particles, which have a high linear energy transfer, generate many more radical ions
locally, and have low penetration through tissues. The damage to DNA can include
single- and double-strand breaks, point mutations due to misrepair deletions, and
chromosomal translocations, Figure 3.1 (Raddon, 2007).
The molecular genetics events that follow radiation damage to cells include:
1) Induction of early-response genes such as c-jun and Egr-1;
2) Induction of later-response genes such as tumor necrosis factor-α (TNF-α),
fibroblast growth factor (FGF), and platelet-derived growth factor-α (PDGF-α);
3) Activation of interleukin-1 (IL-1) PKC;
4) Activation of oncogenes such as c-myc and K-ras.
Induction of these genes may be involved in the cellular responses to
irradiation and in the longer-range effects that lead to carcinogenesis. At any rate, the
production of clinically detectable cancers in humans after known exposures generally
occurs after long latent periods. Estimates of these latent periods are 7 to 10 years for
leukemia, 10–15 years for bone, 27 years for brain, 20 years for thyroid, 22 years for
breast, 25 years for lung, 26 years for intestinal, and 24 years for skin cancers (Raddon,
2007).
3.2.2 - Ultraviolet Radiation
Ultraviolet radiation–induced lesions, generated by UV-B (280–320 nm
wavelength) or UV-A (320–400 nm wavelength), result from DNA damage, which is
converted to mutations during cellular repair processes. UB-B and UV-A generate
different types of DNA damage and DNA repair mechanisms (Raddon, 2007).
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Figure 3.1 - Direct DNA damage radiation model (from (Price, 2009))
Irradiation with UV-B produces cyclobutane pyrimidine dimers that are
repaired by nucleotide excision repair. If left unrepaired, C T and CC TT base
transitions occur. UV-A induced DNA damage produces mostly oxidative lesions via
photosensitization mechanisms and is repaired by base excision repair (Raddon, 2007).
UV-B and UV-A also produce distinct effects on the immune system and elicit
different transcriptional and inflammatory responses. While the specific mechanisms
by which UV radiation induces basal cell or squamous cell carcinomas or melanoma are
not clear, a number of signal transduction pathways are affected that can either lead
to apoptosis or to increased cell proliferation (Raddon, 2007).
UV irradiation activates receptor tyrosine kinases and other cell surface
receptors. It also enhances phosphorylation by ligand-independent mechanisms via
inhibition of protein tyrosine phosphatase activity. Ligand dependent cell surface
receptor activation can occur as well by activation of autocrine or paracrine release of
growth factors from keratinocytes, melanocytes, or neighboring fibroblasts (Raddon,
2007).
3.3 – CELL DEATH IN MAMMALIAN TISSUES
The clonogenic potential is the essential element for the maintenance of a cell
line, either in vitro or in organized tissues, although there are other important issues in
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the maintenance associated with complex tissue systems. Normal senescence of cells
is one of these important issues and the other is the removal of cells that are in the
wrong place at the wrong time. Examples of this would be the metastatic arrival of
tumor cells transported from a primary tumor elsewhere or the resolution of
inflammatory processes (Alpen, 1998).
It is possible to define at least two different types of cell death that go beyond
the end point of clonogenic potential and involve the actual disappearance of the cell:
necrosis and apoptosis (Alpen, 1998).
Necrosis is characterized by a tendency for cells to swell and ultimately to lyse,
which allows the cell's contents to flow into the extracellular space, this is usually
accompanied by an inflammatory response. In the case of neoplasms, necrosis is most
often seen in rapidly growing tumors, where the tumor mass outgrows its blood supply
and regions of the tumor become undernourished in oxygen and energy sources. In
this case inflammation is not a characteristic of the necrotic process (Alpen, 1998).
Apoptosis involves shrinkage of the nucleus and cytoplasm, followed by
fragmentation and phagocytosis of these fragments by neighboring cells or
macrophages. The contents of the cell do not usually leak into extracellular space, so
there is no inflammation. Since there is no inflammation accompanying apoptosis, the
process is histologically quite inconspicuous (Alpen, 1998).
Figure 3.2 - Structural changes of cells undergoing necrosis or apoptosis (from (Goodlett, 2001))
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The concept of apoptosis as a mechanism for the control of cell population
numbers and cell senescence has been around for several decades, but the
mechanisms of apoptosis have received extensive research attention only in the
nineties. This interest in apoptosis was engendered by the discovery that tumor
suppressor genes and oncogenes were central control agents for the process. The
principal focus of these studies has been the role of the p53 tumor suppressor gene,
already described in chapter II. The p53 gene is a transcriptional activator that may
include activation of genes that regulate genomic stability, cell cycle progression, and
cellular response to DNA damage. The synthesis of the p53 product is known to be
responsible for the induction of apoptosis in many cell lines in which this gene is
present in unmutated form. The mutational absence of this gene is often accompanied
by the inability of a cell line to initiate apoptosis. For radiation pathology, the
important finding is that even small amounts of DNA damage in G1 cells cause
synthesis of the p53 product and ultimate apoptosis of the cells. It is pertinent for
radiation pathology that cells of the lymphoid system generate high concentrations of
p53 gene product after cell damage. This is particularly true for low doses of ionizing
radiation. Clearly, the generation of the p53 product is not sufficient for the onset of
apoptosis, but it is certainly necessary (Alpen, 1998).
Another significant gene involved in apoptosis is the bcl-2 gene (described in
chapter II). This gene encodes a protein that blocks physiological cell death (apoptosis)
in many mammalian cell types, including neurons, myeloid cells, and lymphocytes. This
gene is able to prevent cell death after the action of many noxious agents (Alpen,
1998).
The role of apoptosis as a mechanism for cell death following ionizing radiation
exposure remains unclear at this time, particularly the relative importance of the
agonistic role of p53 and the antagonistic role of bcl-2. However, it must be important,
as that the detection of small nicks and errors in the DNA of G1 cells is crucial to the
recovery of irradiated tissues and the reduction of genomic misinformation (Alpen,
1998).
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3.4 – NATURE OF CELL POPULATIONS IN TISSUE
One of the earlier systematic overviews of the nature of cell population kinetics
in normal and malignant tissues was that of Gilbert, 1965. Their classification of the
various kinetic systems found in mammalian (and, incidentally, in other organisms)
organs and tissues is shown in Figure 3.3 (Alpen, 1998).
Figure 3.3 - Classification of cell kinetic types in the system of Gilbert, 1965 (from (Alpen, 1998)).
From Figure 3.3, the definitions of each of the systems are the following (the
double arrows in classifications D, E, and F, are meant to signify the mitotic division of
one of the cells of the compartment, giving rise to two daughter cells):
A. Simple transit population. Fully functional cells are added to the
compartment while a population of either aging or randomly destroyed cells disappear
from the pool. There are many examples of functional end cells that are in this
category. Examples are spermatozoa, which are constantly being replaced, as well as
red cells or other end cells of the blood.
B. Decaying population. The cell numbers decrease with time without
replacement. The population of oocytes in the mammalian female is often quoted as
an example, if not the only example. Populations of this classification are rare in
mammalian systems, but not in insects.
C. Closed, static population. There is neither decrease nor increase in cell
numbers during life. It is unlikely that such a population truly exists. The differentiated
neurons of the central nervous system are quoted as an example of a static
population, but there is probably a decline in cell numbers even in this population.
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D. Dividing, transit population. In addition to the transiting cells, division of the
cells within the compartment occurs that leads to a larger number leaving than
entering. It is assumed in this model that the number of cells in the compartment
remains more or less static. The differentiating and proliferating blood cell types (for
example, the proerythroblast of the bone marrow) that follow the stem cell are
examples of this type of population.
E. Stem cell population. A self-sustaining population, that relies on self-
maintenance for its continued existence. All the progeny of this type of cell line
depend upon the continued existence of the stem cell pool. Every self-maintaining,
dividing cell population must have such a precursor pool. Examples are the stem cells
responsible for sustained spermatogenesis or hematopoiesis.
F. Closed, dividing population. Such a population is best represented by
neoplastic growth. No cells enter or leave the compartment in the early stages of
tumor growth. In the long run, neoplastic growth is probably best represented as a
stem cell population, since as the tumor enlarges, there is cell death, suppression of
growth by metabolic and other nutrient shortages, and a highly variable rate of
division. The epithelial cells responsible for cell renewal in the lens of the eye are
another example of this type of population (Alpen, 1998).
3.5 – CELL POPULATION KINETICS AND RADIATION DAMAGE
It should be almost self-evident that the kinetic types represented by D, E, and
F of Figure 3.3 will be most vulnerable to radiation damage. It has been established
that for clonogenic death of the cell the principal target of ionizing radiation is the
genome, and the genome is certainly at its most vulnerable to radiation damage during
G2 and mitosis (M), when replication has been completed. The principal outcome of
disturbances to the dynamic replicative activity of the genome is altered clonogenic
ability. That is indeed the case, and the most critically sensitive of these systems would
be the stem-cell-type tissue (E), which depends for its continuing function on its own
continued clonogenic potential, since there is no precursor compartment to replace
deficiencies (Alpen, 1998).
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The ultimate functional viability of a tissue that is dependent on stem cell
activity will be determined by whether, after radiation exposure, there are adequate
numbers of surviving and still clonogenic stem cells to repopulate the compartment
and finally to produce functionally competent progeny. The most resistant tissues are
those that require neither input of cells from a prior compartment nor division within
the compartment. The closed static model is such a case, and in the case of the central
nervous system, its high degree of radioresistance can be attributed to its lack of need
for cell replication and replacement (Alpen, 1998).
3.5.1 – Growth Fraction and its significance
The concept of growth fraction as a descriptive parameter for the kinetics of
proliferating tissue appears to have been first proposed by Mendelsohn (1962) as the
result of his observations that all cells in a growing tumor are not in the active process
of proliferation as determined by the cellular incorporation of radioactive labels of
DNA synthesis. Lajtha (1963), based on his own studies as well as those of others,
proposed the concept of the G0 phase of the cell cycle, a state of the cell in which the
cell was not engaged in active proliferation, but in which the cell could reenter the
proliferative state. The G0 cell was visualized as a cell that has been removed from the
actively dividing population by regulatory activities rather than as a result of metabolic
deprivation. Subsequently, it became apparent that cells also could be removed from
active division in a reversible manner by deprivation of oxygen, glucose, or other
metabolites (Hlatky et al., 1988). Restoration of the lacking nutrient led to reentry of
the cell into active proliferation (Alpen, 1998).
Figure 3.4 – Cell cycle phases (from (Goldwein, 2006))
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The growth fraction is defined as the fraction of the total cellular population
that is clonogenically competent and is actually in the active process of DNA replication
and cell division. The growth fraction may be estimated by any one of several
techniques, most of which depend on incorporation of a radioactively labeled DNA
precursor into those cells that are actively dividing. One of the simpler methods for
determination of the growth fraction is the exposure of a growing culture of cells, in
vitro or in vivo, to an appropriate radioactive label for the synthesis of DNA. A typical
and frequently used label is 3H-thymidine. The cells are exposed to the radioactive
label in the medium or by injection into the intact animal for at least the full length of a
cell cycle (and usually for half again as long). Under these conditions, all cells that
synthesize DNA, thus indicating their passage through the S period of the cell cycle, are
labeled and can be identified by autoradiography. The percentage of cells that is
labeled constitutes the growth fraction, since every cell in cycle will have passed
through the S period at least once during exposure to the radioactive label (Alpen,
1998).
The radiobiological significance of the growth fraction was unclear until the
appearance of new data in the late 1980s. In 1980, Dethlefsen indicated that the role
of quiescent cells in radiobiological response was not satisfactorily delineated. Recent
studies indicate that cells that are out of cycle are capable of a more significant
amount of repair of potentially lethal damage, simply because there is more time
before the cell is called on to replicate its DNA. It is possible, but by no means proved,
that the concentration of enzymes necessary for repair of DNA damage may be
depleted in the noncycling cell, but, in spite of this, the additional time allows effective
repair to proceed with the lower concentration of repair enzymes (Alpen, 1998).
3.6 – CELL KINETICS IN NORMAL TISSUES AND TUMORS
Both normal and neoplastic tissues have a cellular kinetic pattern that follows
the accepted model of a G1-S-G2-M cycle, and, indeed, the cell cycle parameters are
not very different for tumors as compared to other growing tissues. The total cycle
time and the time devoted to DNA synthesis in the S period are very much alike for
both tissue types. However, there are, significant differences in some of the
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characteristics of the kinetic pattern as the tumor reaches a size where vascularization
is required for continued tumor growth. The orderly vascularization of normal tissues
that originates in embryonic life and that is maintained throughout the existence of
normal, nonpathological function assures that the supply of oxygen and nutrients is
adequate for survival of cells. Most, if not all, tumors, on the other hand, originate as
nonvascularized aggregations of cells and develop a vascular supply sometime after
the origination of tumor growth. The development of vascular supply in a tumor
depends on the activities of angiogenic factors that occur in normal tissues. The newly
developing vascular supply is, at best, chaotic and disorganized (Alpen, 1998).
Some parts of the tumor tissue will be so far from the source of oxygen and
nutrients that cell survival will be impossible, Figure 3.5. Other parts of the tumor will
have nutrient and oxygen supplies that are adequate only for survival of cells without
replication. The lack of oxygen and glucose can lead to a decrease in the growth
fraction, and probably to cell death and necrosis. Several nutrients and metabolic
products, including oxygen, glucose, and lactic acid, play an important role in the
determination of quiescent and proliferating cells in tumors (Alpen, 1998).
One important difference between normal tissues and tumor tissues is the
determinant of the fraction of quiescent cells in the organ or tumor. Because of the
orderly vascular architecture of normal tissue, the movement of cells from the
proliferating to the quiescent compartment is probably not the result of nutrient lack,
but, rather, the result of the activity of normal soluble growth factors and naturally
occurring inhibitors that regulate the growth and development of the tissue (Alpen,
1998).
3.7 – MODELS FOR RADIOBIOLOGICAL SENSITIVITY OF NEOPLASTIC TISSUES
The earliest attempts to assay the sensitivity of organized tissue systems were
directed at establishing the radiosensitivity of tumor tissues. This was partly because
these tissues offered opportunities for analysis that were not available for normal
tissues. The possibility for syngeneic transplantation of the cell lines from host to
recipient animal was the most important characteristic of these in vivo tissue systems.
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Figure 3.5 - Role of hypoxia in tumour angiogenesis (from (Carmeliet, 2000))
After irradiation of the tumor in the host in which it was growing, it was
possible to transplant the tumor cells to an unirradiated recipient animal and to
observe the growth response of the irradiated tumor cells. There was also strong
interest in understanding tumor biology arising from the treatment of cancer by
radiotherapy. It was important to establish the role of oxygen in the sensitivity of
cancer cells, as well as the importance of the fraction of G0 cells and repair or
repopulation in these tissues. The overall goal was practical: to maximize the
effectiveness of radiotherapy for cancer control in patients, while reducing damage to
normal tissues in the radiation field (Alpen, 1998).
3.7.1 – Hewitt Dilution Assay
Probably the first in vivo assay for mammalian tissues was that developed by
Hewitt and Wilson (1959) with a syngeneic mouse tumor system. At that time a
number of tumor cell lines that were grown in the peritoneal cavity of mice had been
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developed. The cells from these ascites tumors could be harvested or allowed to
continue to grow in the peritoneal cavity of the host, which would cause the death of
the animal. It occurred to Hewitt and Wilson that this end point - death of the host
animal could be used to measure the clonogenic potential of the tumor cells after
irradiation. Figure 3.6 shows the essentials of a Hewitt assay for a single dose point at
10 Gy (Alpen, 1998).
Figure 3.6 - Typical data set for a Hewitt dilution assay (from (Alpen, 1998))
Cells harvested from the mouse ascites tumor P388 and unirradiated cells were
collected from the donor and a series of dilutions was prepared from a stock
suspension of the tumor cells. A typical microbiological-type binary dilution was
carried out to produce cell suspensions with low concentrations of cells that will allow
the recipient animal to be injected with cell numbers that are correct for killing about
half of the animals. For the tumor line used, the usual cell dose required to kill half of
the animals is about two to three cells. A small number of animals (5-10) are injected
with the same cell dose and the survival is followed. The same procedure is used for
several additional cell doses. The resulting data on percent survival at each of the cell
doses are plotted as shown in Figure 3.6, and the LD50 (lethal dose for 50% of the
animals) is determined by graphical or analytical means. The procedure is repeated,
but with the cell suspension prepared from animals that were irradiated before cell
collection. Animals are irradiated at several doses and injections proceed as just
described for each dose. The LD50 values can be used to construct a survival curve.
Figure 3.6 shows an example for only one radiation dose on the right panel and for
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unirradiated cells on the left panel, with the calculated surviving fraction. The surviving
fraction is estimated for each of the other doses, and a survival curve of surviving
fraction against dose is plotted in the usual way (Alpen, 1998).
The Hewitt assay has been the tool used for a number of significant studies of
tumor cell sensitivity to radiation. Figure 3.7 is a very good example of such studies.
Andrews and Berry (1962) developed survival curves for three mouse tumors, two
leukemias, and a sarcoma. Some of the data were Berry's own previously unpublished
observations and some were provided by Hewitt. The clonogenic survival curves were
developed for both anoxic and oxic conditions. All three cell lines could be plotted on
the same curve for oxic cells or for anoxic cells as appropriate, and the line produced
was a good fit for the appropriate condition of oxygenation. The oxygen enhancement
ratio (OER) for these cells was about 2.4, which is not far from the 2.8 or so for cell
lines that are irradiated in vitro and analyzed for clonogenic survival in vitro. The Do for
the cells irradiated under oxic conditions was about 150 cGy, and the extrapolation
number was about 3-4 for this set of data (Alpen, 1998).
A significant shortcoming of the dilution assay system is that donor cells that
are grown in ascites fluid are usually irradiated when the cell number in the peritoneal
cavity is very large. Under these conditions, it is not always clear that the cells are fully
oxygenated at the time of irradiation. If that is indeed the case, there is the possibility
of significant anoxic protection of the cells and, subsequently, there is an
overestimation of the resistance of the cells to the irradiation. The data reported in the
Berry study do not seem to be affected by such hypoxia. The Do (oxic) is about 150 cGy,
a number quite consistent with that found for many cell systems in vitro. The OER of
2.4 or so is, again, not very different from the 2.5-2.8 seen for in vitro systems. We
must conclude, at least for the cell lines reported in this study, that adequate
oxygenation probably existed at the time of irradiation (Alpen, 1998).
Another shortcoming of the Hewitt method is that the irradiated tumor cells
must be capable of expressing clonogenic potential while growing in the ascites
medium. For example, most leukemias grow readily in this environment, and usually
require an inoculum of only 1-3 cells to cause the death of 50% of the recipient
animals. For the Berry data just described, the sarcoma cells required an inoculum of
more than 80 cells to kill 50% of the recipients. In many cases no cell growth is seen
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and no assay is possible. To avoid this shortcoming, other assays have been developed
(Alpen, 1998).
Figure 3.7 - The survival curve obtained by Berry (1964) via the Hewitt assay method for two mouse leukemias and a sarcoma (from (Alpen, 1998))
3.7.2– Lung Colony Assay System
A modification to the Hewitt assay was developed by Hill and Bush (1969) to
measure clonogenic survival of cells derived from solid tumors. In principle, the assay
measures the clonogenic survival of tumor cells by determining their ability to form
colonies in the lung of recipient syngeneic mice. The cells from a tumor, irradiated
either in vivo or, after dissection and cell dissociation, in vitro, are injected into a
recipient mouse, and after 18-20 days the animals are killed, the lungs are dissected,
and the number of tumor colonies in the lung is counted. Hill and Bush were able to
demonstrate a linear relationship between cell number injected and the number of
colonies formed in the lung. A very large enhancement of the number of colonies in
the lung was found if, along with the experimentally irradiated cells, a large number of
heavily irradiated, nonclonogenic cells were injected. Typically, such a procedure
produced a 10-50-fold increase in the number of colonies formed from the clonogenic
survivors. Hill and Bush were not able to establish the mechanism of this
enhancement, but it was not due to an immune response on the part of the recipient.
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Very consistent survival curves were obtained, and, for the KHT transplantable
sarcoma, the Do was 134 cGy, with an extrapolation number of about 9.5. Again, these
data were found to be quite consistent with the values found for the same tumor with
the Hewitt assay. Such an agreement not only validates the lung colony assay, it also
demonstrates that there was little protection from radiation damage due to partial
hypoxia for the KHT cells irradiated as solid tumors and tested by the dilution assay
(Alpen, 1998).
A significant limitation of the lung colony assay is that cells must be injected
into syngeneic recipient mice, that is, inbred mouse lines of the same genotype as that
from which the tumor is derived (Alpen, 1998).
3.8 – TUMOR GROWTH AND TUMOR “CURE” MODELS
Since there is a very limited set of models for examining the clonogenic
potential of tumor cells, much of the radiation biology of tumors has been developed
using a set of tools that was developed for general use in tumor biology. Therefore,
some of these tools have been more valuable than others for radiation effect studies
because of the inherent inability to effect precise quantitation.
3.8.1 – Tumor Volume Versus Time
A widely used and relatively powerful tool in tumor radiobiology is the tumor
growth curve after implantation of an inoculum of cells, usually in the flank region of
recipient syngeneic mice or rats. The simplest application of the growth curve for
implanted tumors is the analysis on the increase rate of the tumor volume. For analysis
of the radiation effect we can measure the time for the tumor to reach a preselected
volume. The measurements of tumor volume are at best imprecise. The volume is
usually determined from a caliper measurement of two or more diameters of the
growing tumor and calculation of the volume from the average diameter (Alpen,
1998).
After the tumor has been irradiated, the time course of volume change is as
shown in Figure 3.8. There may be a slowing of growth for a brief time, followed by a
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period of decreasing tumor volume. This decrease is due to lack of replacement of the
normal cell loss from tumors, associated with local necrosis, nutrient lack, or other
causes unrelated to the radiation exposure. It is not due to the interphase death of
cells as the result of irradiation. As the surviving clonogenic cells repopulate the tumor,
regrowth will be observed; the surviving clonogenic cells will ultimately produce
progeny exceeding the cell-loss factor (Alpen, 1998).
Figure 3.8 - Tumor volume versus time (from (Alpen, 1998))
The criterion for measurement of the radiation dependent response is the time
for the cell volume to again reach the value observed at the time of irradiation. This
time is shown in Figure 3.8, and it is measured, as shown, as the time from irradiation
until the tumor volume achieves the value existing at the time irradiation occurred.
This time value is called the growth delay. The important limitation of the growth delay
model for testing the radiobiological response of tumors is that a significant number of
transplantable tumors does not show any decrease in the volume of tumor after
irradiation (Alpen, 1998).
Presumably, this failure to decrease in volume is the result of a small cell-loss
fraction in the growing tumor. When irradiation takes place, clonogenic activity is
reduced until repopulation from competent clonogenic cells occurs. During the period
before regrowth commences as the result of repopulation, the normally small cell-loss
fraction of the tumor does not lead to reduction in tumor volume. In these cases it is
necessary to revert to the simpler measure of tumor volume versus time and the use
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of the time to reach a preset volume. Alternatively, differences in this time for control
and irradiated tumors may be taken as the end point (Alpen, 1998).
3.8.2 – TCD50, Tumor Cure
Another end point that is widely used in tumor biology is the dose required to
"cure" an implanted tumor. For this model, a large number of implanted tumors are
irradiated with graded doses at the same time period after implantation of the tumor
inoculum. The end point is the fraction of animals that has received a given dose in
which the growth of the tumor is controlled. This local control index can be plotted for
each of the doses, and the dose required to control tumor growth in 50% of the
animals is estimated by a variety of statistical techniques. This value is usually called
the 50% tumor cure dose -TCD50 (Alpen, 1998).
3.9 – RADIOBIOLOGICAL RESPONSES OF TUMORS
Using a number of end points, including dilution assay, lung colony assay,
primary cell cultures, and tissue derived in vitro cultures, it has been possible to define
rather clearly the radiobiological responsiveness of various tumor lines, both animal
and human. With only a few important exceptions, the various tumor cell lines in wide
and long term experimental use have been found to have clonogenic survival
characteristics that are generally stable and for which the relevant survival parameters
are not very variable, considering the range of cell types and tissues from which these
transformed and immortal cell lines have been derived (Alpen, 1998).
Rather different findings have been reported for the survival curve parameters
of freshly derived culture systems grown from naturally occurring malignant tumors.
Extensive efforts have been devoted to characterization of the radiosensitivity of cell
lines from human tumors. The best fit to the data for a large number of human cell
lines, both nontransformed fibroblasts and tumors, is the linear-quadratic (LQ) model.
The radiosensitivity of the various cell lines can be divided into three groups with a
very good correlation with the known responsiveness of the tumors to radiotherapy:
lymphomata, known to be highly curable, were the most radiosensitive of the derived
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cell lines, and melanomata revealed to be the most resistant for tumor curability and
the most radioresistant in the survival of the cell lines in culture (Alpen, 1998).
It is important to realize that the immediate responsiveness of a tumor to
radiation, as determined by reduction in the tumor volume, does not necessarily
predict the curability of the tumor with high efficiency. The degree of responsiveness
will be determined by many of the cell kinetic parameters of the tumor system. A high
cell-loss factor and a high growth factor associated with a small fraction of cells out of
cycle and associated with inherent cellular radiosensitivity, will assure a high degree of
responsiveness of the tumor, as measured by volume changes. Curability, on the other
hand, will depend in a complex way on the ability of the few remaining clonogenic cells
to repopulate the tumor after irradiation is over (Alpen, 1998).
3.10 – HYPOXIA AND RADIOSENSITIVITY IN TUMOR CELLS
Under circumstances where severe anoxia can occur in tissues or cellular
preparations, one should expect to see significant protection from the effects of
ionizing radiation. It is expected to find conditions of moderate to severe anoxia in
growing tumors in vivo. For cells grown in suspension, careful attention to culture
conditions usually will prevent the development of such anoxic conditions with
concomitant radioprotection. For the tissue assay systems, such as the Hewitt dilution
assay and others, there is clearly a protective effect of oxygen lack under the correct
conditions. Figure 3.7 shows such radioprotection for cells deliberately made anoxic by
killing the host animal or by allowing the cell number for cells growing in the peritoneal
cavity to reach very high levels. Figure 3.9 demonstrates methods by which the
fraction of hypoxic cells in a mixture with fully oxygenated cells can be detected and
measured quantitatively. The radioresistant "tail" for the dashed line survival curve
shown in Figure 3.9 (10% anoxic cells) is a common observation for cells from tumors
and indicates the presence of a mixed population of cells, part of which have a
radioresistance relative to the remainder of the population. This resistant fraction may
be due to hypoxia and the radioprotection that this state affords (Alpen, 1998).
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Figure 3.9 - Survival curve for the irradiation of a cell suspension containing a fraction of hypoxic cells (from (Alpen, 1998))
The well known work of Thomlinson and Gray (1955) laid the foundations for
our understanding of hypoxia as well as reoxygenation in tumors during growth and
regrowth. Figure 3.10 (from Thomlinson, 1967) illustrates the processes proposed by
this author. The very young tumor is well oxygenated, since it is so small that no cells
are beyond the effective diffusion distance of oxygen from nearby capillaries. As the
tumor continues to grow, portions of the tumor volume may be beyond easy access to
diffusing oxygen. The tumor must depend for its supply of oxygen on the development
of newly formed vessels that arise from the adjacent normal tissue and penetrate the
tumor volume. This neovascularization of the tumor is not as well organized as the
blood supply in normal tissues, and the expanding volume of tumor will contain
regions in which oxygen is inadequate for the maintenance of metabolism, and some
fraction of the cells will be anoxic. Figure 3.10 illustrates that the fraction of anoxic
cells in the growing tumor may rise to several percent and in some tumor types, to as
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much as 10%. According to the model of Thomlinson, when the tumor is irradiated
(position R 1 in the figure) the more radiosensitive, fully oxygenated cells are killed,
and the remaining hypoxic cells are in an environment of dead and dying cells with
lesser demand for metabolic oxygen (Alpen, 1998).
Figure 3.10 - Development of hypoxia and reoxygenation in an irradiated tumor (from (Alpen, 1998))
Shrinking of the tumor volume and lowered oxygen demand allow for
reoxygenation of the hypoxic cells, which is indicated by a rapid fall to near zero for the
anoxic fraction. After this period of reoxygenation, tumor regrowth commences and
the complete cycle is repeated. The significance of the reoxygenation phase in
fractionated radiotherapy of human tumors is undergoing careful reexamination,
partly because treatment modalities designed to optimize the kill of anoxic cells (high
linear energy transfer (LET) radiation, radiation under hyperbaric oxygen conditions,
and so on) have not been particularly successful. According to Figure 3.10, the
optimum time for a second irradiation of a fractionated scheme would be at point H in
the curve, when the population of hypoxic clonogenic cells is at a minimum. Recent
data suggest that the reoxygenation phenomenon actually occurs very soon after
irradiation, and indeed may take place while the irradiation is in progress.
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3.11 – SUMMARY
Human tumors strongly differ in radiosensitivity and radiocurability and this is
thought to stem from differences in capacity for repair of sub-lethal damage.
Radiosensitivity varies along the cell cycle, S being the most resistant phase and G2 and
M the most sensitive. Therefore, cells surviving an exposure are preferentially in a
stage of low sensitivity (G1), i.e. synchronized in a resistant cell cycle phase. They
progress thereafter together into S and then to the more sensitive G2 and M phases. A
new irradiation exposure at this time will have a larger biological effect (more cell kill).
However, while this synchronization effect has explained some experimental results,
redistribution has never been shown to play a measurable role in the clinic of
radiotherapy (Mazeron, 2005).
Cells surviving an irradiation keep proliferating, increasing the number of
clonogenic cells, i.e. the number that must eventually be sterilized to eradicate cancer.
An inappropriate development of intratumoral vasculature leads to a large proportion
of poorly oxygenated cells and the proportion of hypoxic cells increases with the tumor
size (Mazeron, 2005).
Acutely hypoxic cells are far more radioresistant than well oxygenated cells.
Hypoxic cells usually survive irradiation, but they progressively (re)oxygenate due to
the better supply of oxygen available after well oxygenated cells have died. This
restores radiosensitivity in the tumor by several mechanisms, but re-oxygenation
occurring at long intervals is probably due to tumor shrinkage leading to a reduction of
the intercapillar distance (Mazeron, 2005).
CHAPTER IV
CELL CULTURE AND FLOW CYTOMETRY
CHAPTER IV – CELL CULTURE AND FLOW CYTOMETRY
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4.1 – INTRODUCTION
Cell culture is an invaluable tool for researchers in numerous fields. It facilitates
the analysis of biological properties and processes that are not readily accessible at the
level of the intact organism. Successful maintenance of cells in culture, whether
primary or immortalized, requires knowledge and practice of a few essential
techniques (Helgason, 2005).
The use of cells in analytical chemistry, engineering, and biology requires a
dedicated space for cell culture and maintenance. The proper handling of cells and
tissues requires a level of diligence and constant education, to mitigate health and
safety risks. Cell culture requires a system of mutual separation of sample and scientist
to avoid contamination of either. Each time a culture flask and the dish is opened is, in
essence, an opportunity for a single bacterium or fungal cell to ruin an experiment.
Likewise, every time cell cultures or tissues are handled, there is a risk to the scientist.
It is therefore needed to understand the protective countermeasures required to
handle cells properly (Pappas, 2010).
This chapter presents the importance of the laboratory conditions in the
manipulation and maintenance of cell culture. Subsequently, it is explained the
cytogenetic analysis of cell line and I performed a description of the methods to induce
cell cycle checkpoints. In the end of the chapter, it is presented a description of the
methods for synchronizing mammalian cells and the analysis of the mammalian cell
cycle by flow cytometry.
4.2 - CELL-CULTURE LABORATORY
Setting up a laboratory (or space within an existing lab) for cell culture is not a
daunting task, but requires some planning and strict adherence to regulations. Most
universities, research institutes, and hospitals have a safety committee (some
committees specialize in biosafety) that is in place in part to help a research establish a
cell lab. While the government guidelines typically set the standard for safety rules, the
research institution may have additional guidelines to follow. Therefore, the safety
committee is therefore indispensable in the planning and setting up of a cell lab, as
well as in the subsequent (and often frequent) safety inspections. The main issues
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when setting up and maintaining a culture lab are safety, sterility, and contamination.
All three of these issues are linked by the common safe practices and proper use of
equipment, and all three require that individuals working in the lab are properly
educated (Pappas, 2010).
Working in the lab requires universal precautions, assuming that all cell cultures
and related materials may contain hazardous pathogens. This assumption maintains a
more vigilant attitude, and reduces the risk of accidental exposure to a real pathogen.
Moreover, the possibility that cultures can be cross-contaminated requires additional –
albeit similar – precautions. In short, careful procedures will result in productive
research in a safe environment for cells and individuals. For those new to cells and cell
culture, this chapter will not only serve as an introduction to the tools required for a
cell lab, but will also detail some of the practical aspects to setting up a culture facility.
For those with cell culture experience, the discussion of analytical equipment should
prove useful (Pappas, 2010).
4.3 – MAINTAINING CULTURES
The proper maintenance of cells includes homeostasis during culture, cell
storage and the correct preparation of cells for analysis. The latter case is of the most
importance, as often analysis and homeostasis are incongruent. Buffers must be
changed, different media used, and the cells, at times, are exposed to drastically
diverdse conditions for analysis. In some cases, the change in conditions can affect the
outcome of the experiment negatively. In other instances, the conditions suitable for
cell analysis are fatal to the cell (e.g., electron microscopy). There are many works
available on the culture of almost every cell type imaginable (Pappas, 2010).
When culturing primary or immortal cells for analysis, sterility and cross-
contamination must also be monitored at all times. A few bacteria in a sample can
wreak havoc in a short time, rendering any analytical data useless. The cross-
contamination of cultures is at best a nightmare, as extensive genetic testing is
required to purify cell populations and yield accurate data. Considering the cost of
cells, reagents, instrumentation, and lab upkeep, at least as much thought should be
placed on the maintenance of cell cultures for appropriate analysis. The type of
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environment the cell encounters can directly affect the outcome of an analytical
experiment: cell-growth conditions, analysis buffers and reagents can affect the cell
phenotype, cell signaling, and a host of other parameters. By careful maintenance of
primary and immortal cells, accurate and reproducible cell analyses can be conducted
(Pappas, 2010).
4.3.1 – Medium
More than any other reagent in a cell-analysis laboratory, a steady supply of
culture medium – and the choice of correct medium type – is essential for cell analysis.
There are, in general, two classes of medium one can consider for cell analysis. First,
medium that is used to maintain a culture in between experiments, and second,
medium used in the analysis itself. Often these two can be one in the same, although
in some cases a modified medium or supplemented buffer is needed during the
analysis or processing phase (Pappas, 2010).
There are many types of medium available and the supplements that can be
added to them expand the palette of options even further. Table 4.1 lists some
medium types that are common to cellular analysis, by cell type. The table is not
inclusive, but serves to highlight the differences in medium types, and that some
medium formulations are applicable to many cell lines. In most cases, the medium in
Table 4.1 is used during the culture (maintenance) phase, and a different buffer or
medium may be used during the analysis itself (Pappas, 2010).
Medium can be classified as basic or complete, depending on whether or not
serum is included, respectively. Basic medium has many of the components required
for cell metabolism. Basic media, such as DMEM and RPMI 1640 (see Table 4.1),
contain salts (partly from buffer action), amino acids, vitamins (such as biotin, folic
acid, B-12, etc.), and molecules involved in energy production (glucose, pyruvate).
Basic medium also often contains other buffers (such as HEPES) and a colorimetric
acid–base indicator, such as phenol red. The latter serves as a quick visual inspection
of the “age” of the medium in culture. As cells consume nutrients and produce waste,
the culture medium acidifies, resulting in a shift in color for the pH indicator. The
formulations of most culture media are available and should be examined for potential
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interference in the analysis. For example, staining using Annexin-V-based apoptosis
probes requires relatively high Ca2+ concentrations and at the same time, the presence
of phenol red in the medium will interfere with fluorescence measurements of
fluorescein, green fluorescent protein (GFP), and other fluorophores with similar
emission spectra. Fluorescence from phenol red itself makes sensitive fluorescence
measurements nearly impossible (Pappas, 2010).
Table 1 – Medium types common to cell analysis (from (Pappas, 2010))
Medium Serum Additives Cell lines
RPMI 1640 10% FBS Antibacterial-Antifungal
Jurkat, HuT 78, RPMI
8226, CCRF-CEM, U937,
HL-60
Dulbecco`s modified
Eagle Medium (DMEM) 10% FBS
Antibacterial-Antifungal,
L-Glutamine
NIH 3T3, RBL-1, HT-29,
HeLa
Clavcomb`s Medium 10% FBS
Antibacterial-Antifungal,
Norepunephrine, L-
Glutamine
HL-1
Cell Mab 0-10% FBS Varies
Designed for
monoclonal antibody
production
Leibovitz`s L-15 Hemolymph Bag neuronal cells
Eagle`s Minimum
Essential Medium 0-10% FBS L-Glutamine
F-12 0-10% FBS L-Glutamine Designed for primary
cells
Iscove`s Modified
DMEM 0-10% FBS L-Glutamine HuT 78 T Cells
FBS = Fetal Bovine Serum
Medium is, in essence, a man-made attempt to mimic the life support found in
vivo. It is, therefore, lacking in many essential compounds for cell growth. Many cell
lines can function in basic medium without additional materials, but for the most
routine culture and analysis, serum must be added to form the complete medium
(Pappas, 2010).
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Serum is typically derived from animal sources, the most common being fetal
bovine serum (FBS). FBS and other sera contain growth factors such as epidermal
growth factor (EGF), some interleukins, and transferrin. Furthermore, present are
adhesion-promoting proteins and peptides, for example, fibronectin and laminin and
other components including insulin and various minerals. FBS and other animal-based
sera are by far the most common supplements used for culture maintenance (Pappas,
2010).
Being derived from animal sources, serum is inherently difficult to use from a
quality-control perspective and since it is derived from different animal types this can
affect experiment outcome. For example, the use of FBS instead of native rat serum
was shown to affect the outcome of rat leukocyte immunological response. In addition
to species variability, serum varies from lot to lot, as well as by country of origin, so if
cell products are to be analyzed over long time periods (months of experimentation) it
is best to purchase a large quantity of serum from one particular lot. Given the high
cost of medium, this may not always be practical since serum cost increases as the
level of quality control improves. The more consistent and well characterized the
medium, the higher the cost (Pappas, 2010).
Another negative aspect of dealing with serum is that the serum, or animal of
origin, is subject to contamination, just like any other primary derived material. Certain
viruses, bacteria, and mycoplasma have been shown to be transmitted via serum.
There are several replacement sera that can be substituted for FBS. For example, the
FetalClone series and Bovine Growth Serum, both from HyClone, are non-fetal animal
sera supplemented with various growth factors, minerals, and other compounds. Since
they are not derived from fetal animals, there is less variability between lots (especially
for the added compounds). None of the alternative sera offers much relief as far as
cost is concerned, but the increase in quality control is a major improvement (Pappas,
2010).
Some cells readily grow in serum-free medium; most, however, must be
acclimated to a serum-free environment. This requirement is especially true if the cell
line in question is already being cultured in serum-enriched medium (typically 10%
v/v). It is possible to reduce serum content in medium; in some cases, it is advisable to
do so, because reducing the amount of serum added can reduce costs, as serum is the
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most expensive component of the complete medium. Reducing serum also lowers the
total protein content of the medium, facilitating collection of cell products, and
minimizing sources of contamination. For cells growing in serum-enriched medium, a
method of systematically reducing medium can be implemented (Pappas, 2010).
One must first consider the growth of cells in culture, before discussion of how
to achieve serum reduction can initiate, Figure 4.1. Cell growth in culture – whether
the cells are adherent or suspended – is characterized by several stages. The lag phase,
during which minimal or no cell division occurs, is a brief period after inoculation. The
lag phase occurs as cells adjust to a new cell-culture environment, and as adherent
cells begin the process of reattaching to the culture substrate. The lag phase is
followed by the log or exponential phase. This is the major phase of cell division. The
doubling time, an indicator of cell growth, is determined during this period (Pappas,
2010).
Figure 4.1 - Cell growth in culture (from (Pappas, 2010))
The time for the cell population to double, Figure 4.1, can be determined at any
point during the log phase, although it is most accurate at the center of that phase.
After the log phase, the culture reaches the stationary phase (Pappas, 2010).
High cell density, contact inhibition, and consumption of nutrients signal a
slowing of the cell cycle, and the cell concentration remains constant. Cell crowding,
depletion of nutrients and accumulation of waste eventually causes a sharp drop in cell
concentration, called the death phase. This latter phase can be confirmed by
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microscopy, where the presence of a large number of dead cells, cell debris, and
acidified medium (if an indicator is present) can be observed (Pappas, 2010).
The glucose content of basic medium varies and is sometimes supplemented
with additional glucose. The high glucose content of many medium types is intended
to stimulate growth of the culture. However, some cell lines change phenotypic
properties in high or low glucose. When culturing for conditions close to those
encountered in vivo, the glucose concentration should be adjusted to reflecting the
physiological value as much as possible. Like serum reduction, the impact of changes in
glucose concentration can be monitored using the culture doubling time (Pappas,
2010).
When formulating complete medium, care must be taken to preserve sterility
of the final mixture. If all components are sterile to begin with, then aseptic handling in
the biosafety cabinet will prevent contamination of the complete medium. If any of
the reagents are not sterile at the onset, then filtration can be employed to remove
contaminating organisms.
4.3.2 – The use of medium in analysis and alternatives
Medium is primarily used to maintain cultures and samples before analysis. The
medium can also be used during the analysis; in other instances, components of the
medium may produce artifacts or otherwise interfere. The presence of several
components of medium can interfere with fluorescence measurements. Phenol red,
one of the most common pH indicators added to medium, has a broad absorption
band that interferes with most green fluorescence. Phenol red is also weakly
fluorescent, creating an additional problem for green-emitting fluorophores. If the cell
homeostasis is not required, then any buffer devoid of phenol red will work for
fluorescence. On the other hand, if the cells are to be kept alive for long periods, then
phenol-red-free medium is available from most medium manufacturers. In addition to
the weakly fluorescent properties of phenol red, other compounds present at
relatively high concentrations can interfere with fluorescence detection. Riboflavin is
also weakly fluorescent, but the relatively large volume of the medium contributes to
an unacceptable background signal. Proteins such as albumin, one of the major
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components of serum, also contribute strongly to autofluorescence of medium. The
exact medium used for culture depends on the cell type, the culture conditions, and
the desired end result. For analysis, a similar selection process must be undertaken.
The final medium or buffer used for analysis must be of low background, minimal
interference, and – when possible – capable of sustaining cell viability and function for
the experiment duration (Pappas, 2010).
4.4 – CYTOGENETIC ANALYSIS OF CELL LINES
4.4.1 - The Utility of Cytogenetic Characterization
Countless cell lines have been established—more than 1000 from human
hematopoietic tumors alone —and the novelty and utility of each new example should
be proven prior to publication. For several reasons, karyotypic analysis has become a
core element for characterizing cell lines, mainly because of the unique key
cytogenetics provides for classifying cancer cells. Recurrent chromosome changes
provide a portal to underlying mutations at the DNA level in cancer, and cell lines are
rich territory for mining them. Cancer changes might reflect developmentally
programmed patterns of gene expression and responsiveness within diverse cell
lineages. Dysregulation of certain genes facilitates evasion of existing antineoplastic
controls, including those mediated by cell cycle checkpoints or apoptosis. The
tendency of cells to produce neoplastic mutations via chromosomal mechanisms,
principally translocations, duplications, and deletions, renders these changes
microscopically visible, facilitating cancer diagnosis by chromosome analysis. Arguably,
of all neoplastic changes, those affecting chromosomal structures combine the
greatest informational content with the least likelihood of reversal. This is particularly
true of the primary cytogenetic changes that play key roles in neoplastic
transformation and upon the presence of which the neoplastic phenotype and cell
proliferation ultimately depend. Nevertheless, the usefulness of karyotype analysis for
the characterization of cell lines lies principally among those derived from tumors with
stronger associations with specific chromosome rearrangements (i.e., hematopoietic,
mesenchymal, and neuronal, rather than epithelial tumors) (Helgason, 2005).
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Cytogenetic methods facilitate observations performed at the single-cell level,
thus allowing detection of intercellular differences. Accordingly, a second virtue of
cytogenetic data lies in the detection of distinct subclones and the monitoring of
stability therein. Except for doublings in their modal chromosome number from 2n to
4n “tetraploidization,” cell lines appear to be rather more stable than is commonly
supposed. Indeed, chromosomal rearrangement in cells of the immune system could
reach peak intensity in vivo during the various phases of lymphocyte development in
vivo. A further application of cytogenetic data is to minimize the risk of using false or
misidentified cell lines. At least 18% of new human tumor cell lines have been cross-
contaminated by older, mainly “classic,” cell lines, which tend to be widely circulated.
This problem, first publicized over 30 years ago but neglected of late, poses an
insidious threat to research using cell lines (Helgason, 2005).
In the event of cross-contamination with cells of other species, cytogenetic
analysis provides a ready means of detection. Although modal chromosome numbers
were formerly used to identify cell lines, their virtue as descriptors has declined along
with the remorseless increase in the numbers of different cell lines in circulation. Thus,
species identification necessarily rests on the ability to distinguish the chromosome
banding patterns of diverse species. Fortunately, cells of the most prolific mammalian
species represented in cell lines (primate, rodent, simian, as well as those of domestic
animals) are distinguishable by experienced operators (Helgason, 2005).
4.5 – METHODS TO INDUCE CELL CYCLE CHECKPOINTS
The way cells respond to radiation or chemical exposure that damages
deoxyribonucleic acid (DNA) is important because induced lesions left unrepaired, or
those that are misrepaired, can lead to mutation, cancer, or lethality. Prokaryotic and
eukaryotic cells have evolved mechanisms that repair damaged DNA directly, such as
nucleotide excision repair, base excision repair, homology-based recombinational
repair, or nonhomologous end joining, which promote survival and reduce potential
deleterious effects. However, at least eukaryotic cells also have cell cycle checkpoints
capable of sensing DNA damage or blocks in DNA replication, signaling the cell cycle
machinery, and causing transient delays in progression at specific phases of the cell
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cycle. These delays are thought to provide cells with extra time for mending DNA
lesions before entry into critical phases of the cell cycle, such as S or M, events that
could be lethal with damaged DNA (Lieberman, 2004).
The precise mechanisms by which checkpoints function is under intensive
investigation and details of the molecular events involved are being pursued
vigorously. This owes not only to the complexity and the intellectually and technically
challenging aspects of the process but also to the relevance of these pathways to the
stabilization of the genome and carcinogenesis. Nevertheless, it is clear that
checkpoint mechanisms are very sensitive and can be induced by the presence of
relatively small amounts of DNA damage. For example, in the yeast Saccharomyces
cerevisiae, as little as a single double-strand break in DNA can cause a delay in cell
cycle progression. One important aspect of studying cell cycle checkpoint mechanisms
is an understanding of how to induce the process (Lieberman, 2004).
The application of radiations, such as gamma rays and ultraviolet (UV) light, are
capable of causing DNA damage, and thus leading to the induction of cell cycle
checkpoints. Certain chemicals or the use of temperature- sensitive mutants to disrupt
DNA replication, are also used routinely to induce checkpoints. Gamma rays cause
primarily single- and double-strand breaks in DNA but can infrequently induce
nitrogenous base damage as well. In contrast, UV light (i.e., 254 nm) causes a
preponderance of bulky lesions, such as pyrimidine dimers, although single-base
damage and strand breaks are a smaller part of the array of lesions that can be
produced. Regulation of cell cycle checkpoints induced by ionizing radiation versus UV
light is mediated by overlapping but not identical genetic elements (Lieberman, 2004).
4.6 – METHODS FOR SYNCHRONIZING MAMMALIAN CELLS
When studying cell cycle checkpoints, it is often very useful to have large
numbers of cells that are synchronized in various stages of the cell cycle. A variety of
methods have been developed to obtain synchronous (or partially synchronous) cells,
all of which have some drawbacks. Many cell types that attach to plastic culture dishes
round up in mitosis and can then be dislodged by agitation. This mitotic shake-off
method is useful for cells synchronized in metaphase, which on plating into culture
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dishes move into G1 phase in a synchronous manner. A drawback to the mitotic shake-
off method is that only a low percentage (2–4%) of cells are in mitosis at any given
time, so the yield is very small. Also, cells rapidly become asynchronous as they
progress through G1 phase, so the synchronization in S phase and especially G2 phase is
not very good. The first limitation can be overcome by plating multiple T150 flasks with
cells, using roller bottles, or blocking cells in mitosis by inhibitors such as Colcemid or
nocodazole (Lieberman, 2004).
Mitotic cells that are collected can be held on ice for an hour or so while
multiple collections are done to obtain larger numbers of cells. To obtain more highly
synchronous populations of cells in S phase, the mitotic shake-off procedure can be
combined with the use of deoxyribonucleic acid (DNA) synthesis inhibitors, such as
hydroxyurea (HU) or aphidicolin (APH), to block cells at the G1/S border (but probably
past the G1 checkpoint). APH inhibits DNA polymerase α, whereas HU inhibits the
enzyme ribonucleotide reductase, though it may operate by other mechanisms also.
On release from the block, cells move in a highly synchronized fashion through S phase
and into G2 phase. In terms of the number of synchronized cells, this method has the
same limitation as discussed above, because the starting cell population derives from
the mitotic shake-off procedure. In addition, the block of cells with drugs can cause
unbalanced cell growth, so one cannot necessarily conclude that all biochemical
processes are also synchronized (Lieberman, 2004).
Large numbers of synchronous cells can be obtained using centrifugal
elutriation, Figure 4.2. This method requires the use of a special rotor in a large floor
centrifuge and separates cells into the cell cycle based on cell size. Cells may be
obtained in early or late G1 phase, or primarily in S phase. However, the cell
populations are not highly synchronous in S phase but instead have significant
populations of G1- and G2-phase cells included. Nevertheless, it is possible to
synchronize very large numbers of cells using this method, and biochemical processes
are not perturbed (Lieberman, 2004).
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Figure 4.2 - Centrifugal elutriation (from (Wahl, 2001))
Another method that results in highly synchronous populations is based on
labeling cells with a viable dye for DNA (Hoechst 33342). Cells stained with this dye can
then be sorted by cell cycle phase. Sorted G1 cells will be distributed throughout G1,
cells in S phase can be sorted into a small window in S phase and thus will be highly
synchronized, but only a small number of cells can be obtained. G2 phase cells will be
contaminated with late S phase cells. Furthermore, some cell types do not stain well
with Hoechst 33342, so sufficiently good DNA histograms cannot be obtained Hoechst
33342 (Lieberman, 2004).
4.7 – ANALYSIS OF THE MAMMALIAN CELL CYCLE BY FLOW CYTOMETRY
One of the most common uses of flow cytometry is to analyze the cell cycle of
mammalian cells. Flow cytometry can measure the deoxyribonucleic acid (DNA)
content of individual cells at a rate of several thousand cells per second and thus
conveniently reveals the distribution of cells through the cell cycle (Lieberman, 2004).
The DNA-content distribution of a typical exponentially growing cell population
is composed of two peaks (cells in G1/G0 and G2/M phases) and a valley of cells in S
phase, Figure 4.3. G2/M-phase cells have twice the amount of DNA as G1/G0-phase
cells, and S-phase cells contain varying amounts of DNA between that found in G1 and
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G2 cells. Most flow-cytometric methods of cell cycle analysis cannot distinguish
between G1 and G0 cells or G2 and M cells, so they are grouped together as G1/G0 and
G2/M. However, there are flow cytometric methods that can distinguish four or even
all five cell cycle subpopulations: G0, G1, S, G2, and M. Furthermore, each
subpopulation can be quantified. Obviously, flow cytometry with these unique
features is irreplaceable for monitoring the cell cycle status and its regulation
(Lieberman, 2004).
Figure 4.3 - A typical cell cycle distribution of DNA content (from (Cooper,2004))
Cell cycle checkpoint genes are key elements in cell cycle regulation.
Checkpoint gene mutation can lead to defects in one or more cell cycle checkpoint
controls, which can then result in cell death or cancer. Many of the cell cycle
checkpoint genes are tumor suppressors, such as p53, ataxia-telangiectasia mutant
(ATM), ataxia-telangiectasia and Rad3 (ATR), and BRCA1 (Lieberman, 2004).
In mammalian cells, the cell cycle checkpoint controls that can be analyzed by
flow cytometry are G1 arrest, suppression of DNA replication, and ATM dependent as
well as independent G2 arrest. Exposure to a genotoxic agent can activate some or all
the checkpoints (Lieberman, 2004).
4.8 – CONCLUSION
Effective in vitro maintenance and growth of animal cells requires culture
conditions similar to those found in vivo with respect to temperature, oxygen and
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carbon dioxide concentrations, pH, osmolality, and nutrients. Within normal tissue in
vivo, animal cells receive nutrients through blood circulation. For growth in vitro,
animal cells require an equivalent supply of a complex combination of nutrients. For
this reason, the first attempts in animal cell culture were based on the use of biological
fluids such as plasma, lymph and serum, as well as on extracts from embryonic-derived
tissue (Castilho, 2008).
Medium composition is one of the most important factors in the culture of
animal cells. Its function is to provide appropriate pH and osmolality for cell survival
and multiplication, as well as to supply all chemical substances required by the cells
that they are unable to synthesize themselves. Some of these substances can be
provided by a culture medium consisting of low molecular weight compounds, known
as basal media. However, most basal media fail to promote successful cell growth by
themselves and require supplementation with more complex and chemically
undefined additives such as blood serum (Castilho, 2008).
Some cultivation processes are based on operational strategies that allow cells
to remain viable, but in a nonproliferative state, so as to prolong the productive phase
and to increase the productivity of the process. By these strategies cell proliferation
may be controlled by adding chemical additives that arrest the cell cycle, usually in the
G1 phase, increasing specific productivity. However, concomitantly undesirable effects
such as cytotoxicity may be observed, which result in a decrease in cell viability and in
the impossibility of maintaining the culture in a nonproliferative state for long periods
of time. Deprivation of specific nutrients and growth factors can also stop cell
proliferation, but in this case cell viability decreases and programmed cell death –
apoptosis – is activated. Currently, much research on the biochemical control of cell
cultures based on preventing the cell death mechanisms, to avoid cell death instead of
inhibiting cell growth, is being carried out with the aim of prolonging the productive
period of a cell culture process (Castilho, 2008).
Any process, industrial or laboratory-based, presents a series of important
variables that represent its state. In the case of cell culture, there are the variables
related to the environment to which the cells are exposed, such as temperature, pH,
dissolved oxygen, nutrients in the culture medium, and metabolite concentrations, as
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well as those related to the cell itself, such as concentration, average size, or the
profile of intracellular enzyme activities (Castilho, 2008).
CHAPTER V
MATERIALS AND METHODS
CHAPTER V – MATERIALS AND METHODS
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5.1 – INTRODUCTION
The main goal of this chapter is to perform a description of the materials and
methods of the following papers: “Lack of p53 function promotes radiation-induced
mitotic catastrophe in mouse embryonic fibroblast cells” of Fiorenza Ianzini,
Alessandro Bertoldo, Elizabeth A Kosmacek, Stacia L Philips and Michael A Mackey,
Cancer Cell International 2006 6:11, and “Nuclear accumulation and activation of p53
in embryonic stem cells after DNA damage” of Valeriya Solozobova, ALexandre
Rolletschek and Christine Blattner, BMC Cell Biology 2009 10:46.
The previous chapters enable the acquisition of theoretic knowledge regarding
the methods employed in the execution of the laboratorial work performed in the
mentioned papers.
The images resulting from the mentioned papers will be analyzed and
processed, as will be demonstrate in the next chapter.
5.2 – MATERIALS AND METHODS OF THE PAPER “LACK OF P53 FUNCTION PROMOTES RADIATION-
INDUCED MITOTIC CATASTROPHE IN MOUSE EMBRYONIC FIBROBLAST CELLS”
5.2.1 - CELL CULTURE
Mouse embryonic fibroblast (MEF) cells were grown in monolayer in Dulbecco's
modified eagle medium (DMEM) (GIBCO) containing 10% heat-inactivated fetal bovine
serum (Hyclone), non-essential aminoacids (GIBCO) and antibiotics (100 U/ml penicillin
and 100 μg/ml streptomycin) (GIBCO). Under these growth conditions, cells grew with
a doubling time of about 14 using an Olympus AX-70 microscope equipped with a
mercury lamp and ultraviolet filters. Only intact cells containing three or more
fragmented nuclei were scored for these experiments.
5.2.2 - Light microscopy
The same microscopic slides prepared for the cytology end point (described
above) were used to depict cell morphology changes post-irradiation. Images were
acquires using an epifluorescence Olympus BX51 microscope at a magnification of 10×.
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5.2.3 - Bivariate BrdUrd-PI (bromodeoxiuridine-propidium iodide) flow cytometry
Analysis of cell cycle distribution during the post-irradiation interval was
determined using 10 μM BrdUrd pulse labeling techniques, followed by bivariate
analysis using anti-BrdUrd-PI staining, to monitor cells in G1, S, and G2 phases. Flow
cytometric analysis was performed using a FacSTAR, with excitation of fluorochromes
by an argon laser emitting at 488 nm with 300 mW power; red fluorescence (PI) was
detected using a 640 nm low-pass filter, and green fluorescence (FITC) using a 525 nm
band-pass filter.
5.2.4 - Bivariate cyclin B1-PI flow cytometry
Estimates of the relative intracellular levels of cyclin B1 were made using anti-
cyclin B1-PI analysis. Briefly, cells fixed in 95% ethanol were incubated (1 hour, room
temperature) with an anti-cyclin B1 monoclonal antibody, rinsed and incubated as
before with FITC-conjugated goat-anti-mouse IgG, treated with RNAse (1 mg/ml, 30
min, room temperature) after which 0.5 ml of 70 μg/ml of PI was added. The details
for flow cytometric data acquisition were the same as for the anti-BrdUrd-PI analysis
above. In all samples analyzed, cyclin fluorescence was detected only in cells with early
S DNA content.
5.2.5 - Western blotting
Western Blotting was performed to determine cyclin B1 protein expression in
the mutant p53 MEF 10 cells and in the wild-type p53 MEF 12 cells. Thirty μg total
proteins from whole cell extracts were boiled for 10 min in Laemmli sample buffer and
separated using 10–12% 1D SDS-PAGE. The separated proteins were transferred to
Immobilon-P membranes using a semi-dry blotting apparatus and probed with an anti-
cyclin B1 monoclonal antibody (anti-mouse), at a dilution of 1:500. Beta actin was
detected using goat anti-actin polyclonal antibody. Peroxidase-conjugated AffiniPure
goat anti-mouse IgG Fcy fragment specific and donkey anti-goat IgG HRP were used as
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secondary antibodies at a dilution of 1:6000 and 1:2000, respectively. Detection was
performed using Western Lightning Chemiluminescence Reagent.
5.3 – MATERIALS OF THE PAPER “NUCLEAR ACCUMULATION AND ACTIVATION OF P53 IN EMBRYONIC
STEM CELLS AFTER DNA DAMAGE”
5.3.1 - Cell lines and their treatments
R1 and D3 ES cells were cultured in GlutaMAX™-I medium supplemented with
15% fetal bovine serum, 0.1 mM β-mercaptoethanol, 40 μg/ml gentamycin and 1000
units/ml LIF in culture dishes that had been coated with 0.1% gelatine. Mouse
embryonal fibroblasts that had been irradiated with 6.3 Gray served as feeder cells.
p53-/- ES cells were cultured in GlutaMAX™-I medium supplemented with 15% fetal
bovine serum, 0.1 mM β-mercaptoethanol, 1000 units/ml LIF and 300 μg/ml G418 in
culture dishes that had been coated with 0.1% gelatine.
SNL cells irradiated with 6.3 Gray served as feeder cells. CGR8 cells were grown
in Glasgow Minimum Essential Medium supplemented with 10% fetal bovine serum,
40 μg/ml gentamycin, 100 units/ml LIF, 0.05 mM β-mercaptoethanol, 2 mM L-
glutamine and 1 mM non-essential aminoacids without feeder cells. Culture dishes
were coated with 0.2% gelatine.
All ES cell lines were sub-cultured each day. 3T3 cells were grown in Dulbecco's
Modified Eagle Medium supplemented with 10% donor bovine serum and 1%
penicillin/streptomycin. All cells were cultured at 37°C and 6% CO2 in a humidified
atmosphere.
Cells were irradiated with a 60Co γ-ray source at a dose rate of 1 Gray per
minute in cell culture medium. For UV irradiation, the culture medium was removed
and saved. The cells were washed with PBS and irradiated with 30 J/m2. After
irradiation, the original culture medium was added back to the cells. Transfections
were performed using the mouse ES cell Nucleofector Kit according to the
manufacturer's recommendations.
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5.3.2 - Immunofluorescence staining
ES cells were grown on feeder cells that had been grown on cover slips. After
washing twice with ice cold PBS, cells were fixed with ice-cold acetone/methanol (1:1)
for 8 min on ice. Cover slips were washed 3 times with PBS and cells were
permeabilized with 0.5% Triton-X-100 in PBS for 10 min. Cover slips were washed 3
times with PBS and incubated for 30 min in blocking buffer (1% bovine serum albumin;
1% goat serum in PBS). After blocking, cells were incubated overnight with an antibody
directed against p53 (Pab421) that had been diluted 1:200 in blocking buffer. Cover
slips were washed 3 times with PBS and incubated for 30 min at room temperature in
the dark with an antibody directed against mouse IgG coupled to Alexa-Fluor-488 and
Draq5 both diluted 1:1000 in blocking buffer. Cover slips were washed 3 times and
mounted with Hydromount on microscope slides.
5.3.3 - RT-PCR
Total RNA was prepared from cells using the RNeasy kit according to the
manufacturer's recommendation and treated with DNase I to remove residual genomic
DNA. RNA was transcribed into cDNA using random primers and RevertAidtm H
MinusM-MuLV reverse transcriptase. Real-time PCR was performed in duplicates with
a SYBR Green PCR mixture. The cDNA was denatured for 15 min at 95°C followed by 40
cycles of 95°C for 15 s and 50°C for 1 min using the 7000 ABI sequence detection
system and gene specific primers. The signals were normalized to the signals for the
housekeeping gene 34B4. Sequences of primers are available on request.
5.3.4 - Beta-Galactosidase staining
Cells were washed with PBS and fixed with 3.5% paraformaldehyde in PBS for
10 min on ice. Cells were washed 3 times with PBS, incubated with X-gal (0.25 mg/ml)
and solubilised in reaction buffer (5 mM potassium ferricyanide, 5 mM potassium
ferrocyanide, 2 mM magnesium chloride in PBS) for 16 h at 37°C.
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5.3.5 - MTT-assay
106 D3 cells or p53-/- cells were plated on 0.1% gelatinecoated 6 cm-plates.
After attachment, cells were irradiated with 2 Gray or left unirradiated for control.
Each day, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Bromide) was
added to two plates at a final concentration of 2.5 mg/ml and incubated for 4 h. The
reaction was stopped by removal of the medium and solubilization of the MTT-
precipitate in 2 ml isopropanol. Absorbances were read at 595 nm.
5.3.6 - Colony assay
200 ES cells were plated in 3.5 cm dishes coated with 0.2% gelatine. Twenty-
four hours after plating α-pifithrin and μ-pifithrin were added to a final concentration
of 10 μM. Two hours after drug addition, cells were irradiated with 0.5 Gy, 1 Gy, 2 Gy
and 4 Gy. After additional two hours, the culture medium was replaced with fresh
medium without inhibitors and the cells were incubated for two weeks with a daily
change of culture medium. The cells were washed with PBS, fixed with methanol,
stained with 1% crystal violet in PBS and counted. For colony assays in the absence of
an inhibitor, cells were irradiated at four hours after plating and incubated for two
weeks with a daily change of culture medium.
5.3.7 - Apoptosis Assay by Annexin V staining
1 × 106 D3 or p53-/- ES cells were washed with ice-cold PBS and resuspended in
400 ml Ca-containing buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM CaCl2). 5 μl of
annexin V-FITC and 1 μg propidium iodide were added for 10 min and the cells were
immediately analyzed by flow cytometry.
CHAPTER VI
IMAGE PROCESSING AND ANALYSIS
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6.1 – INTRODUCTION
Digital image processing is an area characterized by the need for extensive
experimental work to establish the viability of proposed solutions to a given problem.
An important characteristic underlying the design of an image processing system is the
significant level of testing and experimentation that normally is required before
arriving at an acceptable solution. This characteristic implies that the ability to
formulate approaches and quickly prototype candidate solutions generally plays a
major role in reducing the cost and time required to arrive at a viable system
implementation (González, 2004).
MATLAB is a high-performance language for technical computing. It integrates
computation, visualization, and programming in an easy-to-use environment where
problems and solutions are expressed in familiar mathematical notation. Typical uses
include the following:
Math and computation;
Algorithm development;
Data acquisition;
Modeling, simulation and prototyping;
Data analysis, exploration and visualization;
Scientific and engineering graphics;
Application development, including graphical user interface building.
MATLAB is an interactive system whose basic data element is an array that
does not require dimensioning. This allows formulating solutions to many technical
computing problems, especially that involving matrix representation, in a fraction of
the time it would take to write a program in a scalar non-interactive language such as C
or Fortran (González, 2004).
The name MATLAB stands for matrix laboratory and was written originally to
provide easy access to matrix software developed by the LINPACK (Linear System
Package) and EISPACK (Eigen System Package) projects (González, 2004).
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The Image Processing Toolbox is a collection of MATLAB functions (called M-
functions or M-files) has extended the capability of the MATLAB environment for the
solution of digital image processing problems (González, 2004).
6.2 – IMAGE PROCESSING AND ANALYSIS
In this work, it is performed the image processing and analysis of the images
withdrawn from the following papers:
“Nuclear accumulation and activation of p53 in embryonic stem
cells after DNA damage”, of Valeriya Solozobova, ALexandre Rolletschek
and Christine Blattner, BMC Cell Biology 2009 10:46;
“Lack of p53 function promotes radiation-induced mitotic
catastrophe in mouse embryonic fibroblast cells”, of Fiorenza Ianzini,
Alessandro Bertoldo, Elizabeth A Kosmacek, Stacia L Philips and Michael A
Mackey, Cancer Cell International 2006 6:11.
The objective of that image processing and analyze was to improve, through
the image interpretation, the results achieved in the referred papers relating to the
cellular response to irradiation regarding the p53 gene.
For these propose, the images were enhanced and segmented through the
following procedure:
1. Read the input image;
2. Execute contrast adjustment to better understand the image
data, namely for images with low-contrast as it is the case. This is a fairly
low-contrast image, so I thought it might help;
3. Remove noise from the image and then overlay the perimeter on
the original image;
4. To separate the cells properly, one possible approach is the
marker-based watershed segmentation. With this method it is possible to
connect a partial group of connected pixels inside each object to be
segmented. The extended maxima operator is used to identify groups of
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pixels that are significantly higher than their immediate surroundings and
then the image is overlaid again;
5. Remove noise from the result and then overlay it;
6. Complement the image so that the peaks become valleys.
7. Modify the image so that the background pixels and the
extended maxima pixels are forced to be the only local minima in the
image.
8. Trace the object boundary to better visualization of the
individual/group of cells.
These steps correspond to the following sequence of MATLAB commands, in
this case, of one of the images from the first paper mentioned earlier:
Read and show the image:
g1=imread('p53_IA.png'); g=rgb2gray(g1);
imshow (g)
Contrast adjustment, and here there is the representation of
both the histogram and the equalized histogram to a better
understanding of what is done:
g_eq = adapthisteq(g);
subplot (1,2,1), imhist (g), title('Histogram') subplot (1,2,2), imhist (g_eq), title ('Equalized Histogram')
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o Cleaning the image and overlay the perimeter on the original image:
bw2=imfill(bw,'holes');
bw3=imopen(bw2,ones(5,5)); bw4=bwareaopen(bw3,40); bw4_perim=bwperim(bw4);
overlay1=imoverlay(g_eq,bw4_perim, [.3 1 .3]); imshow (overlay1)
o Extended-maxima transform and carrying out some morphological
operations followed by cleaning and image overlay:
mask_em=imextendedmax(g_eq,30);
mask_em=imclose(mask_em,ones(5,5)); mask_em=imfill(mask_em,'holes'); mask_em=bwareaopen(mask_em,40);
overlay2=imoverlay(g_eq,bw4_perim | mask_em, [.3 1 .3]); imshow (overlay2)
0
200
400
600
800
1000
1200
1400
1600
Histogram
0 50 100 150 200 250
0
200
400
600
800
1000
1200
Equalized Histogram
0 50 100 150 200 250
0
200
400
600
800
1000
1200
1400
1600
Histogram
0 50 100 150 200 250
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o Complement the image and impose minima to the image:
g_eq_c=imcomplement(g_eq);
g_mod=imimposemin(g_eq_c,~bw4 | mask_em);
o Trace the object boundary:
dim = size (g_mod)
col = round(dim(2)/2)-90; row = min(find(g_mod(:,col)))
boundary = bwtraceboundary(g_mod,[row, col],'N'); imshow(g_mod)
hold on; plot(boundary(:,2),boundary(:,1),'b','LineWidth',2);
g_mod_filled = imfill(g_mod,'holes'); boundaries = bwboundaries(g_mod_filled);
for k=1:13 b = boundaries{k};
plot(b(:,2),b(:,1),'b','LineWidth',2); end
This procedure was executed for every image from both articles mentioned
before, and the result is:
1. “Nuclear accumulation and activation of p53 in embryonic stem cells after DNA
damage”:
a. Light microscopy photos of the mutant p53 cell line MEF 10(1) following
10 Gy γ-irradiation.
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i. Morphology of MEF 10(1) cells at 32 hours post-irradiation:
ii. Morphology of sham irradiated MEF 10(1) cells, at time zero:
b. Light microscopy photos of wild-type p53 cell line MEF 12(1) following
10 Gy γ-irradiation:
i. Morphology of MEF 12(1) cells at 40 hours post-irradiation:
ii. Morphology of sham irradiated MEF 12(1) cells, at time zero:
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2. “Lack of p53 function promotes radiation-induced mitotic catastrophe in
mouse embryonic fibroblast cells” of Fiorenza Ianzini, Alessandro Bertoldo,
Elizabeth A Kosmacek, Stacia L Philips and Michael A Mackey, Cancer Cell
International 2006 6:11:
a. Cytoplasmic localisation of p53 in proliferating ES cells. R1, D3 and CGR8
mouse embryonal stem cells were plated on cover slips (R1 and D3 in
the presence of feeders, CGR8 in the absence of feeders):
i. P53 in R1, D3 and CGR8:
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ii. Cells were incubated in Draq5 (R1, D3 and CGR8):
b. p53 accumulates in the nucleus of irradiated ES cells:
i. R1 (p53 and Draq5) and D3 (p53 and Draq5) stem cells, grown on
feeders, were irradiated two days after plating with 7.5 Gray IR
(0, 1 and 2h first row, 4,8 and 24h second row):
1. R1 – p53
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2. R1 – Draq5
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3. D3 – p53
4. D3 – Draq5
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ii. R1 stem cells, grown on feeders, were irradiated with 30 J/m2
UVC light (0, 1 and 2h first row, 4,8 and 24h second row):
1. R1 – p53
2. R1 – Draq5
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6.3 - COMPARISON BETWEEN ORIGINAL AND PROCESSED IMAGES
In the paper “Nuclear accumulation and activation of p53 in embryonic stem
cells after DNA damage”, nuclear fragmentation is persistent during the time course of
the experiment and reaches values of about 80% at 48 hours post-irradiation.
Morphological changes in irradiated p53 mutant MEF 10(1) cells were detected. The
presence of fragmented nuclei and changes in cell shape are evident for the irradiated
population. Irradiated MEF 12(1) cells also confirm that the morphological features of
the irradiated cell population do not greatly differ from the control population, except
that a greater number of large cells is observed, probably reflecting the persistent G2
arrest.
In order to highlight these results, the light microscopy photos obtained with
this study were processed using MATLAB. The comparison between the images is the
following:
i. Morphology of MEF 10(1) cells at 32 hours post-irradiation:
ii. Morphology of sham irradiated MEF 10(1) cells, at time zero:
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iii. Morphology of MEF 12(1) cells at 40 hours post-irradiation:
iv. Morphology of sham irradiated MEF 12(1) cells, at time zero:
The processed image allows a better visualization of points with higher
intensity in the cells as well as improved individualization of the cells/group of cells.
With these results the conclusion of the articles are highlighted since the
morphological changes due to irradiation are evident with the contoured cells, as well
as nuclear fragmentation in the MEF 10(1) cells at 32 hours post-irradiation (more
diffuse intracellular content).
In the paper “Lack of p53 function promotes radiation-induced mitotic
catastrophe in mouse embryonic fibroblast cells”, they tried to clarify the activity of
p53 in stem cells, determining the localization of the p53 protein in mouse ES cells,
investigating three different mouse ES cell lines, R1, D3 and CGR8 and determined p53
localization by immuno-fluorescence staining. R1 and D3 cells were cultured on feeder
cells while CGR8 stem cells do not require feeders for maintaining an undifferentiated
phenotype. They found the majority of p53 localized in the cytoplasm. However, these
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results do not exclude the possibility that a small fraction of p53 exists in the nucleus.
As a transcription factor, p53 usually needs to be nuclear to be active.
One particularly important function of p53 is DNA damage signaling. Here, to
suppress tumorigenesis, p53 halts the cell cycle and induces apoptosis in primary cells
and in tumor cell lines. Since stem cells provide the pool of proliferative
pluri/toti/omni-potent cells within organisms, they are more likely to propagate DNA
lesions and mutations to daughter cells compared to differentiated cells.
Since p53 is primarily a transcription factor, nuclear localization of p53 should
be essential for its transcriptional activity and p53 accumulates in the nucleus of ES
cells after DNA damage.
In unstressed ES cells p53 was localized mainly to the cytoplasm. However, at
one hour after irradiation, p53 accumulated in the nucleus of irradiated ES cells. At
four hours after irradiation, p53 was still present in the nucleus of some cells, while in
others it had mostly disappeared. At eight hours after irradiation p53 had essentially
disappeared from the nucleus of all cells. Surprisingly, at 24 hours after IR, p53 was
again localized in the nucleus of most cells. Nevertheless, a minority of cells still
showed cytoplasmic localization of p53.
After UV-irradiation, p53 also accumulated in the nucleus of ES cells. In contrast
to IR-irradiated cells, p53 remained in the nucleus of UV-irradiated cells. At 24 hours,
most of the ES cells had died while the few remaining ones showed a very intensive
nuclear staining for p53.
Once again, to highlight these results, the light microscopy photos obtained
with this study were processed using MATLAB and here is the comparison between the
images:
i. Cytoplasmic localization of p53 in proliferating ES cells, R1, D3 and CGR8 mouse
embryonal stem cells:
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- R1 – p53
- D3 – p53
-
-
-
- CGR8 – p53
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– R1 – Draq5
-
-
-
- D3 - Draq5
- CGR8 - Draq5
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- R1 - p53 - 0h
-
-
-
- R1 - p53 - 1h
-
-
- R1 - p53 - 2h
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- R1 - p53 - 4h
-
-
-
- R1 - p53 - 8h
-
-
-
- R1 - p53 - 24h
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- R1 – Draq5 - 0h
-
-
-
- R1 – Draq5 - 1h
-
-
- R1 – Draq5 - 2h
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- R1 – Draq5 - 4h
-
-
-
- R1 – Draq5 - 8h
-
-
-
- R1 – Draq5 - 24h
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- D3 - p53 – 0h
-
-
-
- D3 - p53 – 1h
-
-
-
-
- D3 - p53 – 2h
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- D3 - p53 – 4h
-
-
-
- D3 - p53 – 8h
-
-
-
- D3 - p53 – 24h
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- D3 - Draq5 – 0h
-
-
-
- D3 - Draq5 – 1h
-
-
-
- D3 - Draq5 – 2h
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- D3 - Draq5 – 4h
-
-
-
- D3 - Draq5 – 8h
-
-
-
- D3 - Draq5 – 24h
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- R1 – p53 – UV - 0h
-
-
-
- R1 - p53 – UV - 1h
-
-
-
- R1 - p53 – UV - 2h
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- R1 - p53 – UV - 4h
-
-
-
- R1 - p53 – UV -8h
-
-
-
- R1 - p53 – UV - 24h
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- R1 – Draq5 – UV - 0h
-
-
-
- R1 – Draq5 – UV - 1h
-
-
-
- R1 – Draq5 – UV - 2h
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- R1 – Draq5 - UV - 4h
- R1 – Draq5 – UV - 8h
- R1 – Draq5 – UV - 24h
Once again, the processed images enable a superior visualization of points with
higher intensity in the nucleus (shown with Draq5) and in the cytoplasm. The results
described in the paper were better understood with the processed images.
CHAPTER VI – IMAGE PROCESSING AND ANALYSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 129
6.4 – SUMMARY
It is possible that p53 functions to repress radiation-induced mitotic
catastrophe (MC) through its activity as a modulator of the G2 checkpoint mechanisms,
and the lack of p53 promotes MC as a mechanism for removing damaged mouse
embryonic fibroblast cells from populations (Ianzini, 2006).
The experimental results obtained through image processing and analysis
demonstrates the morphological changes in irradiated p53 mutant MEF 10 (1) cell line.
The presence of fragmented nuclei and changes in cell shape are evident for irradiated
population and are hallmarks of MC. In the irradiated MEF 12 (1) cells there are a
greater number of larger cells probably due to the persistent G2 arrest, suggesting the
important role of p53 in the induction of MC following irradiation.
P53 is localized in the cytoplasm of embryonic stem (ES) cells and activates
transcription of a reporter gene in resting ES cells and of endogenous target genes in
response to DNA damage. The activity of p53 in resting stem cells shows that the p53
protein is also in ES cells in a latent state and can be activated when its activity is
required. However, after DNA damage, p53 did not activate all, but activates at least
some of its target genes in ES cells and the transcription of these genes was facilitated
by the presence of active p53 in the nucleus of irradiated cells (Solozobova, 2009).
After IR, p53 accumulated in two waves in the nucleus of ES cells. The first
nuclear accumulation occurred at one to two hours after irradiation and correlated
with an increase in the amount of the p53 protein. During the second wave of nuclear
accumulation of p53 at twenty-four hours after irradiation, there is not an increase in
p53 abundance suggesting that for the second wave of nuclear accumulation p53 was
translocated from the cytoplasm into the nucleus (Solozobova, 2009).
The experimental results obtained through image processing and analysis
emphasize the changes in intracellular content due to irradiation of cells, and
emphasize the cytoplasmic localization of p53 in proliferating ES cells and the evidence
that p53 accumulates in the nucleus of irradiated ES cells.
Defining the mechanisms underlying the role of p53 in MC might lead to
strategies to improve clinical radiation response for those human tumors with defects
in p53, or p53-related pathways, and that avoid radiation induced apoptosis. If
CHAPTER VI – IMAGE PROCESSING AND ANALYSIS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 130
radiation-induced MC is the predominant mode of cell death in p53-deficient cells,
clinical interventions designed to enhance its production might not affect surrounding
normal tissue, and thus lead to a therapeutic gain (Ianzini, 2006).
CHAPTER VII
CONCLUSIONS AND FUTURE WORKS
CHAPTER VII – CONCLUSIONS AND FUTURE WORKS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 133
7.1 – FINAL CONCLUSIONS
Clearly that wild-type (WT) p53 not only has anti-proliferative and anti-
transforming activity but also possesses the ability to induce programmed cell death
(apoptosis) after exposure of cells to DNA-damaging agents, such as γ-irradiation or
anticancer drugs. The concept that p53 is a growth regulatory protein fits with its short
half-life, its nuclear location and transcription factor activity, and its increased
synthesis in DNA damaged cells. WT p53 regulates the transcription of a number of cell
replication associated genes (Ruddon, 2007).
Growth arrest induced by WT p53 blocks cells prior to or near the restriction
point in late G1 phase and produces a decrease in the mRNA levels for genes involved
in DNA replication and cell proliferation, such as histone H3, proliferating cell nuclear
antigen (PCNA), DNA polymerase α, and b-myb. To carry out these gene regulatory
events, WT p53 has to assume a certain conformational structure, apparently
modulated by its phosphorylation state, and oligomerize so that it can bind to DNA.
Mutant p53 cannot achieve the appropriate conformation and can block WT p53
function by forming oligomers with it. Even though a lot is known about the biological
actions of p53, e.g., the ability to induce G1 arrest, to induce apoptosis following DNA
damage, to inhibit tumor cell growth, and to preserve genetic stability, the way in
which it does all this is not totally clear (Ruddon, 2007).
The p53 network can be activated by at least three mechanisms. The first is
DNA strand breaks triggered by ionizing radiation or other DNA-damaging agents. This
mechanism is dependent on activation of the ATM (ataxia telangiectasia-mutated)
protein, Chk2, or other kinases. Interestingly, mice that are deficient in p53 function
and in the ability to repair DNA double-strand breaks because of a failure in non-
homologous end-joining (NHEJ) repair, develop highly aggressive pro-B cell
lymphomas. The second mechanism is overexpression or aberrant expression of
growth factor signals such as those turned on by oncogene proteins Ras or Myc.
Finally, cellular stress is induced by chemotherapeutic drugs, ultraviolet light, or
protein kinase inhibitors (Ruddon, 2007).
The role of p53 in maintaining genetic stability appears to involve induction of
genes that stimulate nucleotide excision repair, chromosomal recombination,
CHAPTER VII – CONCLUSIONS AND FUTURE WORKS
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 134
chromosome segregation and induction of the gene for ribonucleotide reductase. p53
also stimulates the expression of genes that inhibit angiogenesis (Ruddon, 2007).
The main scientific reports in which this work is based on are concerning the
role of p53 in the cellular behavior. The right and normal performance of these check
point mechanisms are very important to keep the equilibrium in the cellular turnover.
To be able to perform the adequate study of cells it is important to always have
in mind the proper behavior in laboratory, to ensure that there is no contamination in
the medium for culture cells (chapter IV).
To understand this kind of study, it is imperative to have knowledge regarding
the radiation effects in normal and neoplastic tissues (chapter III). It´s also important
to understand the mechanisms of cell cycle regulation and apoptosis (chapter II).
7.2 – FUTURE WORKS
The future prospect of this thesis is to continue the study with cells, performing
the analysis and image processing of breast cancer cells submitted to brachytherapy.
Hence, the study of morphological changes that occur in the irradiated cells, as well as
the modifications in the cellular environment to obtain the maximum information of
the electron microscopy images of these cells, will be done. Additionally,
computational algorithms will be developed to help that study in an automate and
robust manner.
REFERENCES
REFERENCES
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 137
(ALPEN, 1998) - EDUARD L. ALPEN
RADIATION BIOPHYSICS, SECOND EDITION
ACADEMIC PRESS, 1998
(BESSON, 2004) - ARNAUD BESSON
REGULATION OF THE CYTOSKELETON: AN ONCOGENIC FUNCTION FOR CDK INHIBITORS?
NATURE REVIEWS CANCER 4, 948-955, 2004
(CARMELIET,2000) - PETER CARMELIET
ANGIOGENESIS IN CANCER AND OTHER DISEASES
NATURE 407, 249-257, 2000
(CASTILLO, 2008) - LEDA CASTILLO
ANIMAL CELL TECHNOLOGY: FROM BIOPHARMACEUTICALS TO GENE THERAPY
T&F, 2008
(CHARLES, 2004) – ROBERTS W. CHARLES
THE SWI/SNF COMPLEX – CHROMATIN AND CANCER
NATURE REVIEWS CANCER 4, 133-142, 2004
(COOPER, 2004) – GEOFFREY M. COOPER
THE CELL: A MOLECULAR APPROACH, THIRD EDITION
BOSTON UNIVERSITY, 2004
(DASH, 2003) - PHIL DASH
APOPTOSIS, BASIC MEDICAL SCIENCES
ST.GEORGE’S, UNIVERSITY OF LONDON, 2003
(DETHLEFSEN, 1980) - L. A. DETHELEFSEN
IN QUEST OF THE QUAINT QUIESCENT CELL
RADIATION BIOLOGY IN CANCER RESEARCH, PP. 415-435. RAVEN PRESS, NEW YORK, 1980
REFERENCES
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 138
(FREDERIK, 2002) - IGNEY H. FREDERIK
DEATH AND ANTI-DEATH: TUMOR RESISTANCE TO APOPTOSIS
NATURE REVIEWS CANCER 2, 277-288, APRIL 2002
(FROUIN, 2003) - ISABELLE FROUIN
DNA REPLICATION: A COMPLEX MATTER
EMBO REPORTS 4, 7, 666–670, 2003
(GIL, 2006) - JESÚS GIL
REGULATION OF THE INK4B-ARF-INK4A TUMOR SUPPRESSOR LOCUS: ALL FOR ONE OR ONE FOR ALL
NATURE REVIEWS MOLECULAR CELL BIOLOGY 7, 667-677, 2006
(GILBERT AND LAJTHA, 1965) - C. W. GILBERT AND L. G. LAJTHA
THE IMPORTANCE OF CELL POPULATION KINETICS IN DETERMINING THE RESPONSE TO IRRADIATION OF
NORMAL AND MALIGNANT TISSUE
IN CELLULAR RADIATION BIOLOGY, PP. 474-497, WILLIAMS AND WILKINS, BALTIMORE, 1965
(GOLDWEIN, 2006) – JOEL W. GOLDWEIN
CHEMOTHERAPY: THE BASICS
ABRAMSON CANCER CENTER OF THE UNIVERSITY OF PENNSYLVANIA, 2006
(GONZÁLEZ, 2004) - RAFAEL GONZÁLEZ
DIGITAL IMAGE PROCESSING USING MATLAB, FIRST EDITION
2004
(GOODLETT, 2001) - CHARLES R. GOODLETT
MECHANISMS OF ALCOHOL-INDUCED DAMAGE TO THE DEVELOPING NERVOUS SYSTEM
ALCOHOL RESEARCH & HEALTH 25(3):175–184, 2001
(GRIFFITHS, 1999) - ANTHONY GRIFFITHS
MODERN GENETIC ANALYSIS
NEW YORK, 1999
REFERENCES
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 139
(HELGASON, 2005) - CHERYL D. HELGASON
BASIC CELL CULTURE PROTOCOLS, THIRD EDITION
HUMANA PRESS, 2005
(HILL, 1969) - R. P. HILL
A LUNG COLONY ASSAY TO DETERMINE THE RADIOSENSITIVITY OF THE CELLS OF A SOLID TUMOR
INT. J. RADIATION BIOL. 15, 435-444, 1969
(HLATKY, 1988) - L. HLATKY
JOINT OXYGEN GLUCOSE DEPRIVATION AS THE CAUSE OF NECROSIS IN A TUMOR ANALOG
J. CELL PHYSIOL. 134, 167-178, 1988
(IANZINI, 2006) – FIORENZA IANZINI, ALESSANDRO BERTOLDO, ELIZABETH KOSMACEK, STACIA PHILIPS,
MICHAEL MACKEY
“LACK OF P53 FUNCTION PROMOTES RADIATION-INDUCED MITOTIC CATASTROPHE IN MOUSE
EMBRYONIC FIBROBLAST CELLS”
CANCER CELL INTERNATIONAL, 6:11, 2006
(KASTAN, 2004) - MICHAEL B. KASTAN
CELL-CYCLE CHECKPOINTS AND CANCER
NATURE 432, 316-323, 2004
(LAJTHA,1963) - L. G. LAJTHA
ON THE CONCEPTS OF THE CELL CYCLE
CELL COMP. PHYSIOL. 62, 143-145, 1963
(MACDONALD, 2005) - F. MACDONALD
MOLECULAR BIOLOGY OF CANCER, SECOND EDITION
ADVANCED TEXT, 2005
(MENDELSOHN, 1962) - M. L. MENDELSOHN
REFERENCES
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 140
AUTORADIOGRAPHIC ANALYSIS OF CELL PROLIFERATION IN SPONTANEOUS BREAST CANCER OF C3H
MOUSE
J. NAT. CANCER INST. 28, 1015-1029, 1962
(NOVÁK, 2010) – BÉLA NOVÁK
DYNAMICS OF MOLECULAR REGULATORY NETWORKS
DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF OXFORD, 2010
(PAPPAS, 2010) - DIMITRI PAPPAS
PRACTICAL CELL ANALYSIS
WILEY, 2010
(PRISE, 2009) - KEVIN M. PRICE
RADIATION-INDUCED BYSTANDER SIGNALLING IN CANCER THERAPY
NAT. REV. CANCER; 9(5): 351-360, 2009
(RUDDON, 2007) - RAYMOND RUDDON
CANCER BIOLOGY, FOURTH EDITION
OXFORD UNIVERSITY PRESS, 2007
(RUSS, 1998) - JOHN RUSS
THE IMAGE PROCESSING HANDBOOK, THIRD EDITION
CRC, 1998
(SEELEY, 2004) - ROD R. SEELEY
ANATOMY AND PHYSIOLOGY, SIXTH EDITION
MCGROW-HILL, 2004
(SHAPIRO, 1994) - GEOFFREY SHAPIRO
ANTICANCER DRUG TARGETS: CELL CYCLE AND CHECKPOINT CONTROL
J CLIN INVEST. VOLUME 104, ISSUE 12, 1994
REFERENCES
EVALUATION OF THE EFFECT OF P53 IN CELLULAR RESPONSE FROM ELECTRON MICROSCOPY IMAGES 141
(SHAPIRO, 2001) - LINDA SHAPIRO
COMPUTER VISION
PP 279-325, NEW JERSEY, PRENTICE-HALL, 2001
(SOLOZOBOVA, 2006) - VALERIYA SOLOZOBOVA, ALEXANDRE ROLLETSCHE, CHRISTINE BLATTNER
“NUCLEAR ACCUMULATION AND ACTIVATION OF P53 IN EMBRYONIC STEM CELLS AFTER DNA DAMAGE”
BMC CELL BIOLOGY, 10:46, 2009
(SUQING, 2005) - XIE SUQING
REGULATION OF CELL CYCLE CHECKPOINTS BY POLO-LIKE KINASES
ONCOGENE 24, 277–286, 2005
(SUNTHARALINGAM,2002) - N. SUNTHARALINGAM
N. BASIC RADIOBIOLOGY
CHAPTER 14, 2002
(THOMLINSON, 1955) - R. H. THOMLINSON
THE HISTOLOGICAL STRUCTURE OF SOME HUMAN LUNG CANCERS AND THE POSSIBLE IMPLICATIONS FOR
RADIOTHERAPY
BRITISH J. CANCER 9, 539-549, 1955
(WAHL, 2001) - ALAN WAHL
CENTRIFUGAL ELUTRIATION TO OBTAIN SYNCHRONOUS POPULATIONS OF CELLS
CURRENT PROTOCOLS IN CELL BIOLOGY, 2001
(ZHANG, 2005) - NU ZHANG, HEATHER HARTIG AND IVAN DZHAGALOV
THE ROLE OF APOPTOSIS IN THE DEVELOPMENT AND FUNCTION OF T LYMPHOCYTES
CELL RESEARCH 15, 749–769, 2005