THE REPLICATION OF THE TWO HOTSPOTS OF BREAKAGE

LOCATED WITHIN THE HUMAN COMMON FRAGILE SITE FRA11D

OCCURS IN MID TO LATE S PHASE: A PRELIMINARY STUDY

By

Omar El Mawas

A thesis submitted to the Department of Biology in partial fulfillment of the requirements for

the degree of Master of Science in Biology

Faculty of Sciences

University of Balamand

December 2015

Copyright © 2015, Omar El Mawas

All Rights Reserved

ii

University of Balamand

Faculty of Sciences

This is to certify that I have examined this copy of a Master’s thesis by

Omar El Mawas

and have found that it is complete and satisfactory in all the respects,

and that any and all revisions required by the final

examining jury have been made.

JURY MEMBERS:

Approved: ____________________

Full name, Ph.D.

President of the jury

Approved: ____________________

Full name, Ph.D.

Jury Member

Approved: ____________________

Full name, Ph.D.

Supervisor

Date of thesis defense: December 16, 2015

iii

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. .…… for his continuous

support, for his patience, motivation, enthusiasm, and immense knowledge. His guidance

helped me in all the time of research and writing of this thesis.

I also would like to thank my family for supporting me spiritually throughout my life.

iv

ABSTRACT

Cancer is a genetic disease characterized by the transformation of normal cells into

malignant cells through uncontrollable divisions. Genomic instability, a key feature of

genomic regions called fragile sites (FS), has been shown to be a hallmark of cancer. FS are

specific DNA loci that show propensity to form gaps and breaks on metaphase chromosomes

following DNA replication stress…. Calyculin A, which triggers premature chromosome

condensation at any phase of the cell cycle, also induces CFS. The causes of their fragility are

not yet fully deciphered; several causes are so far described such as replication fork stalling,

paucity in initiation events, and late or slow replication. This latency might be due to CFS

forming secondary structures, which upset the progression of the replication fork. In our

work, we propose to investigate the replication timing at the CFS FRA11D which is located

within the chromosomal band 11p14.2. …

v

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

ABSTRACT iv

TABLE OF CONTENTS v

LIST OF ABBREVIATIONS viii

LIST OF TABLES x

LIST OF FIGURES xi

CHAPTER 1: INTRODUCTION 1

1.1 The Cell and Cell Cycle 1

1.1.1 Cell Discovery 1

1.1.2 Cell Cycle: Description and Regulation 2

1.2 Cancer 6

1.2.1 Cancer Hallmarks 7

1.2.1.1 Limitless replication potential 7

1.2.1.2 Sustaining proliferative potential 8

1.2.1.3 Insensitivity to antigrowth signals 8

1.2.2 Proto-Oncogenes and Tumor Suppressor Genes 12

1.2.3 Cancer Risk Factors 13

1.2.3.1 Genetic predisposition 13

1.2.4 Cancer Sign and Symptoms 16

1.2.4.1 Unexpected weight loss 16

1.3.6.2.5 Inability to recover stalled forks 39

1.4 Calyculin A 40

1.5 Fluorescent in Situ Hybridization 42

CHAPTER 2: MATERIALS AND METHODS 49

2.1 Lymphocyte Culture and CFS Induction 49

2.2 Metaphase Spreading 49

CHAPTER 3: RESULTS 54

3.1 Lymphocyte Culture: Induction of Premature Chromosome Condensation and

Metaphase Spreading54

3.2 Bacterial Culture 57

vi

CHAPTER 4: DISCUSSION 95

LIST OF REFERENCES 100

APPENDIX A: Gene Maps 105

vii

LIST OF ABBREVIATIONS

AML Acute Myelogenous Leukemia

APC Anaphase-Promoting Complex

APH Aphidicolin

AT Ataxia Telangiectasia

ATM Ataxia Telangiectasia Mutated

ATR Ataxia Telangiectasia and Rad3-Related Protein

BAC Bacterial Artificial Chromosome

BRCA1 Breast Cancer 1

BrdU Bromodeoxyuridine

CDK Cyclin-Dependent Kinase

CFS Common Fragile Sites

DM Double Minutes

dsDNA Double Stranded DNA

FHIT Fragile Histidine Triad

FISH Fluorescent In Situ Hybridization

FMR1 Fragile Mental Retardation 1

FMR2 Fragile Mental Retardation 2

FS Fragile Sites

viii

LIST OF TABLES

Table 1.1 CACGs and Molecularly Mapped CFS Involved in Cancer. 31

Table 2.1 Standard PCR Protocol. 50

Table 3.1 Forward and Reverse Primers Used for the Amplification of our Target

Sequences.

59

Table 3.2 Classification of FISH Signals at the Level of All Clones According to

Mitotic Stages and Signal Shape.

66

ix

LIST OF FIGURES

Figure 1.1 The Stages of the Cell Cycle. 3

Figure 1.2 APC Targeting Securing and Mitotic Cyclin for Degradation

Allowing for Cell Cycle Progression.

4

Figure 1.3 The Cell Goes Through Several Stages Before It Reaches Metastasis

and Becomes Malignant.

7

Figure 1.4 Cancer Cells Detach From the Primary Tumor, Squeeze through

Blood Vessels and Form Secondary Tumors.

11

Figure 1.5 Acquired Capabilities in Parallel Pathways, Regardless of their

Chronological Order, Eventually Lead to Colorectal Cancer.

12

Figure 1.6 Chromosome Ideogram Depicting the Locations of Frequently (Red)

and Less Frequently (Blue) Expressed CFS.

25

Figure 1.7 Repeat Copy Number Increase as Suggested by Okazaki Fragment-

Mediated Model.

32

Figure 1.8 Broad Scheme Depicting Possible Sources of Replication Stress at

CFS.

34

1

CHAPTER 1

INTRODUCTION

1.1 The Cell and Cell Cycle

1.1.1 Cell Discovery

The mid-17th century witnessed one of the possibly greatest scientific breakthroughs,

the discovery of the cell. Thanks to the advances in microscopy, English physicist Robert

Hooke was able to examine thin slices of cork in 1665. He noticed that such tissues are

formed by tiny pores, he termed cells from the Latin word Cella meaning 'a small room'. …

Further studies into the cell led Walther Flemming to the discovery that cells undergo cellular

division or Karyomitosis (meaning threadlike metamorphosis of the nucleus) and his work

was published in 1882. Cell division is required both in developmental and adult stages to

maintain homeostasis. In the adult stage, it replaces old, warn out cells with new functional

ones (Mazzarello, 1999).

1.1.2 Cell Cycle: Description

The order of events required through which a cell can pass from one cell division to the

next is termed cell cycle. Today, we know that the cell cycle consists of two successive

stages: interphase, a highly regulated long preparatory stage, and mitosis, a unidirectional and

tightly regulated process by which one parent cell divides to give rise to two identical

daughter cells. In most mammalian cells, Mitosis, lasting one hour, includes prophase,

metaphase, anaphase and telophase. While interphase, lasting around twenty-three hours,

includes G1, S and G2 phases. G1 is a preparatory growth stage for DNA replication and

division, S is the stage where replication takes place, and G2 is the stage where the cell

prepares for mitosis (Vermeulen, Van Bockstaele, & Berneman, 2003).

2

1.1.3 Cell Cycle Regulation: Cyclin – Cyclin Dependent Kinases

Most cells in an adult organism are not dividing. They remain inactive in a quiescent

stage called G0. Yet still each second twenty five million cells are undergoing cellular

division in an adult human. Such an enormous number demands an accurate regulation. As

such, specific cyclin proteins and cyclin-dependent kinases (CDKs) coordinately control the

progression of the cell from one stage to the next. CDKs are serine/threonine kinases.

Unbound CDKs are present in an inactive conformation (Collins & Garrett, 2005). The

association of cyclin proteins to CDKs triggers conformational changes in CDKs allowing

them to become catalytically active. It is noteworthy that the levels of CDKs in the cell

remain moderately constant throughout the cell cycle. Apart from cyclin D levels, which

increases in the beginning of G1 and remains constant throughout, the levels of cyclin

fluctuate while the cell progresses through its cycle. Cyclins are classified according to the

stage where they are mostly elevated. The level of cyclin in the cell is determined by the rate

of synthesis, transcriptional regulation of the cyclin gene, and protein degradation by

proteasome (Pecorino, 2012). The following Figure 1.1 highlights the different stages of the

cell cycle.

Figure 1.1: The Stages of the Cell Cycle (Pecorino, 2012).

3

Growth factors such as c-MYC and c-fos induce the cell to leave G0 and re-enter the

cell cycle by inducing the expression of cyclin D and its partner CDK4/6. The interaction of

cyclin D with CDKs 4/6 leads the cell through G1…. Cyclin E interacts with CDK2 and

promotes the hyperphosphorylation of Rb by means of a positive-feedback loop. As a result,

a conformational change takes place in the pocket domain of Rb causing the release of the

E2F. E2F target genes (cyclin A, thymidylate synthase, etc.) become fully expressed and the

cell can proceed through the S phase (Haering, Lowe, Hochwagen, & Nasmyth, 2002).

…Other CFS that have been shown to be associated with cancer include FRA7G which

is located at the band 7q31.2 and has been shown to be frequently expressed in prostate,

breast, and ovarian cancer (Huang, et al., 1998). Central deletions in FRA6E, where the

candidate tumor suppressor gene Parkin lies, have been shown to exist in ovarian and lung

cancers. The following Table 1.1 shows molecularly mapped CFS and Cancer associated CFS

Genes (CACGs).

Table 1.1: CACGs and Molecularly Mapped CFS Involved in Cancer.

4

1.3.6 Mechanism of Instability at FS

1.3.6.1 Mechanism of instability at RFS

The replication slippage model proposed by Sutherland et al. suggests that the Okazaki

fragment plays an important role during the mechanism of expansion of microsatellite CCG

or AT-rich minisatellites. In one case where the number of copies of repeats is less than 80, it

suggests that slippage of Okazaki fragment might still occur during polymerization though

the 5’ end of the Okazaki fragment might be firmly anchored by a unique sequence of DNA

at one side….

5

LIST OF REFERENCES

Arabshahi, L., Brown, N., Khan, N., & Wright, G. (1988). Inhibition of DNA polymerase

alpha by aphidicolin derivatives. Nucleic Acids Research, 16(11), 5107-5113.

Baynton, K., Otterlei, M., Bjoras, M., von Kobbe, C., Bohr, V. A., & Seeberg, E. (2003).

WRN interacts physically and functionally with the recombination mediator protein

RAD52. Journal of Biological Chemistry, 278(38), 36476-36486. doi:

10.1074/jbc.M303885200

Berchuck, A., Heron, K. A., Carney, M. E., Lancaster, J. M., Fraser, E. G., Vinson, V. L., &

Frank, T. S. (1998). Frequency of germline and somatic BRCA1 mutations in ovarian

cancer. Clinical Cancer Research, 4(10), 2433-2437.

Bianchini, F., Elmstahl, S., Martinez-Garcia, C., van Kappel, A. L., Douki, T., Cadet, J., ...

Kaaks, R. (2000). Oxidative DNA damage in human lymphocytes: Correlations with

plasma levels of alpha-tocopherol and carotenoids. Carcinogenesis, 21(2), 321-324.

Cheng, C. H., & Kuchta, R. D. (1993). DNA polymerase epsilon: Aphidicolin inhibition and

the relationship between polymerase and exonuclease activity. Biochemistry, 32(33),

8568-8574.

Cliby, W. A., Roberts, C. J., Cimprich, K. A., Stringer, C. M., Lamb, J. R., Schreiber, S. L.,

& Friend, S. H. (1998). Overexpression of a kinase-inactive ATR protein causes

sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO

Journal, 17(1), 159-169. doi: 10.1093/emboj/17.1.159

6

APPENDIX A: Gene Maps

Gene maps of the chloroplast transformation vectors. The egfp sequence, coding for

enhanced green fluorescent protein, with the respective psbA promoters and rrnB T1

terminator were cloned into MSC of pBluescript SK+ cloning vector.

Figure A.1: Transformation Vector for P. sativum, Final Size 4 165 bp

Figure A.2: Transformation Vector for V. litorea, Final Size 4 876 bp