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BioSci 145A lecture 15 page 1 ©copyright Bruce Blumberg 2000. All rights reserved
BioSci 145A Lecture 15 - Oncogenes and Cancer
• Topics we will cover today
– Introduction to normal and cancer cells
– Characteristics of cells in culture
– Cancerous changes in cells
– Viruses can harbor transforming genes
– DNA from tumor cells can transform normal cultured cells
– Oncogenes and cell growth
– tumor suppressor genes
• Last year’s final exam is posted
• Don’t forget that the next two lectures will be held in the Beckman Laser Institute Library
• No office hours on the following dates (I will be out of town)
– 3/1
– 3/6
• Office hours for 3/8 will be held on 3/9 from 2-3
BioSci 145A lecture 15 page 2 ©copyright Bruce Blumberg 2000. All rights reserved
Introduction to normal and cancer cells
• Most cells in the organism have a finite lifetime
– majority of differentiated cells are postmitotic
• stem cells can divide nearly endlessly
• other cell types that typically divide
– skin
– lining of gut
– hematopoeitic stem cells
– hair follicles
• liver cells can dedifferentiate, re-enter the cell cycle
– cell growth and division are tightly controlled
• most cells that can divide are only capable of a finite number of cell divisions
– so-called Hayflick limit
• cancer cells are a notable exception
• Cancer cells have lost their ability to regulate their own growth or to respond to normal growth regulatory cues or to sense their proper location in the organism
– each of these characteristics of cancer cells contributes to disease progression
– a variety of genetic events are responsible
– generally speaking, different genetic events can be associated with characteristics of the developing tumor.
BioSci 145A lecture 15 page 3 ©copyright Bruce Blumberg 2000. All rights reserved
Introduction to normal and cancer cells (contd)
• Three types of changes occur as a cell becomes tumorigenic
– immortalization - cells retain the ability to divide endlessly
• not necessarily detrimental to organism
• telomerase– transformation - cells
stop responding to normal growth controls
• do not need growth factors and/or
• do not respond to growth inhibitors
• transformed cells typically form tumors in situ
– metastasis - cells gain the ability to move from their normal location and invade other tissues
• very dangerous feature of cancer cells
• aberrant regulation of extracellular matrix proteases
BioSci 145A lecture 15 page 4 ©copyright Bruce Blumberg 2000. All rights reserved
Cells in culture
• growth characteristics of normal and tumor cells differ
– normal cells do not grow well, in vitro, typical cancer cells grow very well
– primary cells are the immediate descendents of cells taken directly from a tissue.
• such cells divide a small number of times and then stop growing - senescence
• subsequently, most cells will die.
– Lewin calls this the crisis stage
– if the cells are kept and fed for a long time, a small number may begin to grow
– cell lines are cells that successfully pass through crisis and gain the ability to divide indefinitely
• many, if not most, overexpress telomerase
• Fundamental rule - Cells (even primary cells) change their phenotype almost immediately when they are placed in culture
– degree of difference depends on the similarity of their microenvironment to their usual environment
• extracellular matrix
• type and density of surrounding cells
– change usually comes after several cell divisions.
• primary cells that stop dividing will maintain more of their phenotype than those that divide
BioSci 145A lecture 15 page 5 ©copyright Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
• Characteristics of cells in culture - most cells grow as a monolayer for the following reasons:
– anchorage dependence - cells require a substrate to grow on
• solid or semi solid medium
– serum dependence - cells require substances in serum to grow
• commonly called growth factors but in reality there are two different types
– mitotic factors - required for cells to grow and divide
» typically peptide growth factors, e.g. FGF, EGF, PDGF, etc
– survival factors - not strictly required for cell division, but required for cells to survive in culture
» typically lipids or other small molecules, e.g. retinol, 14-hydroxy retroretinol
– density-dependent inhibition (contact inhibition) - cells only grow until confluence
• surface is completely covered
• at this time cells go into G0 and exit the cell cycle
– cytoskeletal organization - cells are flat and extended on the surface
BioSci 145A lecture 15 page 6 ©copyright Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
• illustrates– morphological differences
• flat vs rounded up– contact inhibition
• transformed cells pile up on plates and cluster in 3D
– nuclear morphology• note strong staining in transformed cells, a higher
resolution picture would show multinucleate cells and mitotic figures
BioSci 145A lecture 15 page 7 ©copyright Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
• How does one judge the “normalcy” of cultured cells?
– much can be surmised from morphology
– what is the chromosomal constitution of the cells?
• chromosomal duplications, deletions and translocations are common in culture
• cells that have changed from normal, diploid state are aneuploid
– are the cells anchorage dependent?
• most normal cells (except blood cells) are anchorage dependent
• many transformed cells can grow in soft agar
– are the cells serum dependent?
• many abnormal cells are serum independent
• but many “normal” cell lines can be adapted to low or serum-free conditions
– do the cells express normal protein complement?
– do the cells form tumors if injected into animals?
• if not, they are not “transformed”
• cells originating from tumors are typically transformed
– reduced serum dependence
– reduced anchorage independence
– reduced contact inhibition - cells grow in foci
– will cause tumors if injected into animals
• typically use nude mice (lack significant part of immune system). somewhat cheating
BioSci 145A lecture 15 page 8 ©copyright Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells
• benign vs malignant tumors
– benign tumors contain cells that look and function like normal cells
• express normal complement of proteins
• typically remain localized to appropriate tissues
– often surrounded by a fibrous capsule of connective tissues
• can become problematic if:
– their size interferes with normal function of the tissue (e.g. brain tumor)
– they secrete excessive amounts of biologically active substances such as hormones (e.g. pituitary tumor)
– malignant tumors look qualitatively different from normal tissues of origin
• close enough to determine tissue of origin but not identical to normal tissue
• express only a subset of normal proteins
• many grow and divide more rapidly than normal
• can remain encapsulated in situ for a time (e.g. carcinoma in situ)
• later become invasive and metastatic (definition of malignant)
– many tumors produce growth factors that increase the local blood supply
BioSci 145A lecture 15 page 9 ©copyright Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells (contd)
• Induction of tumors
– discovery of oncogenes led to the model that genetic changes could cause cancer
– tumor incidence increases with age -> a series of events is required to cause a tumor
• believed that 6-7 discrete genetic events are required to get a cancer
– agents that increase frequency of cell transformation are called carcinogens
• can be classified according to properties
• tumor initiators cause tumors
– typically cause DNA damage (e.g. benzapyrene-diol-epoxide)
• tumor promoters aid in the growth of transformed cells, typically by inhibiting growth control (e.g. phorbol esters)
BioSci 145A lecture 15 page 10 ©copyright Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells (contd)
– two classes of genes are targets of mutations that cause transformation
• oncogenes encode proteins that can transform cells or cause cancer in animals
– most are dominant gain of function mutations - three basic types
– point mutations that cause constitutively active protein products
– gene amplification that leads to overexpression
– translocations that result in inappropriate expression (Dr. La Morte)
• tumor suppressor genes are recessive, loss-of-function mutations that inactivate cellular genes that regulate growth or cell cycle
– five classes of tumor suppressor genes
– intracellular proteins that regulate or inhibit progression through the cell cycle
– receptors for secreted hormones that should inhibit cell proliferation (e.g. TGF-beta)
– checkpoint control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal
– proteins that promote apoptosis (programmed cell death)
– enzymes that participate in DNA repair
BioSci 145A lecture 15 page 11 ©copyright Bruce Blumberg 2000. All rights reserved
Viruses can harbor transforming genes
• Peyton Rous (1911)
– took chicken fibrosarcomas, ground them up, filtered out all cells, cellular debris and things as small as bacteria
– injected this filtrate into other chickens -> fibrosarcomas
• Rous sarcoma virus remains one of the most virulent tumor viruses ever discovered
– received the Nobel Prize in 1966 (55 years later) when it was finally discovered that a virus was the cause of the cancer
• RSV contains an oncogene v-src that was demonstrated to be required for cancer induction
– RSV is a retrovirus with only 4 genes so this was relatively easy to demonstrate
• Bishop and Varmus (1977)
– showed that normal cells from chickens and other species contained a cellular homolog of v-src.
– This c-src (cellular src) was the first proto-oncogene
– fundamental discovery that revolutionized the field (and got them a Nobel prize) was that cancer may be induced by the action of normal, or nearly normal cellular genes that were incorporated into transducing viruses
– turns out that c-src is a protein tyrosine kinase that is constitutively active when mutated
BioSci 145A lecture 15 page 12 ©copyright Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
• many acutely transforming retroviruses exist
– affect a variety of species
– impact many cellular signaling pathways
• fundamental mechanism is transduction of cellular gene and later mutation due to inaccurate viral reverse transcriptases
BioSci 145A lecture 15 page 13 ©copyright Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
• oncogenes may be involved in many types of cancers
– same c-onc (cellular oncogene) may be represented as v-onc (viral oncogenes) in a variety of cancers
• sis in both simian and feline sarcoma viruses
– viruses may contain related v-onc genes
• Harvey and Kirsten sarcoma viruses contain v-ras genes derived from two different members of the c-ras family
• evidence exists directly linking oncogenes from acutely transforming retroviruses with cancer
– first obtained from RSV using temperature sensitive mutations in v-src that allowed the phenotype to be reverted and regained
• identification of dominant oncogenes from acutely transforming retroviruses led to the model that single gene changes could cause cancers
– major opponent to this idea was Peter Duesberg who later became somewhat infamous for his criticism of the involvement of HIV in AIDS
• in this case, Duesberg was correct
– although the data linking acutely transforming retroviruses with cancer are strong, this mechanism is considered to be a relatively minor cause of cancer in humans
BioSci 145A lecture 15 page 14 ©copyright Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
• most acutely transforming retroviruses require normal retroviruses to get packaged into infective particles
• growth-promoting genes transduced by retroviruses confer a selective advantage because they increase the proliferation of infected tissues
– retroviruses cannot replicate unless cell is proliferating
• viruses can be transferred laterally from one organism to another, carrying the cancer potential along
• viruses can also be transferred to offspring
BioSci 145A lecture 15 page 15 ©copyright Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
• Not all viruses are acutely carcinogenic
– slow-acting retroviruses
• cause cancers by integrating near cellular protooncogenes and activating them inappropriately
• act slowly because integration into cellular protooncogenes is a rare event and other mutations may be required
– various DNA viruses
• oncogenic potential resides in a single function or group of related functions that are activated early in viral lytic cycle
• many oncogenes act by inactivating tumor suppressor genes
– polyoma T antigens
– papilloma virus E6,7 antigens and cervical cancer
– adenovirus E1A,B
BioSci 145A lecture 15 page 16 ©copyright Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
• Models for differences in properties between c-onc and v-onc
– quantitative model
• viral genes are functionally indistinguishable from normal cellular genes
• oncogenesis comes from
– overexpression
– expression in inappropriate cell types
– failure to turn expression off
– qualitative model
• c-onc genes are not intrinsically oncogenic
• mutations can convert into oncogenes
– that acquire new properties
– or lose old properties
– as usual, both models are correct
• mos, sis and myc genes can confer oncogenesis without significant mutation
• ras and src are changed by point mutations into dominant transforming oncogenes
BioSci 145A lecture 15 page 17 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor cell DNA can transform cultured cells
• DNA from any of a variety of tumors can be transfected into cultured cells (typically NIH 3T3 cells)
– a small number take up DNA and form foci of transformed cells
– DNA is extracted from these foci and re-transfected into fresh cells to enrich for the specific human sequence
– genomic library is prepared and human clones selected by hybridizing with repetitive DNA (Alu)
– oncogene responsible is isolated and characterized
BioSci 145A lecture 15 page 18 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor cell DNA can transform cultured cells (contd)
• Using such methods, a variety of human oncongenes were identified
– two important properties were identified in oncogenes isolated in this way
• many have closely related sequences in the DNA of normal cells
– this argues that the transformation was caused by mutation of a normal cellular gene (proto-oncogene)
– could be a point mutation or reorganization of genomic DNA
• many have counterparts in the oncogenes carried by acutely transforming retroviruses
– e.g.mutations were found in human bladder cancer DNA that corresponded those in the Ha-ras gene from harvey sarcoma virus.
– oncogenes found in this manner frequently do not cause tumors when introduced into normal cells
• NIH-3T3 cells already have a mutation in a tumor suppressor gene that, in combination with the introduced oncogene, could lead to transformation
• It is important to note that DNA with transforming activity can only be isolated from tumorigenic cells
– not present in normal DNA
– in general, this is not such a great way to identify oncogenes
BioSci 145A lecture 15 page 19 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth
• Seven classes of proteins control cell growth
– Collectively, these genes comprise the known set of genes involved in tumor formation
BioSci 145A lecture 15 page 20 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• Dominant transforming oncogenes are frequently created from proteins involved in regulating cell growth
– Growth factors
– Growth factor receptors
– Intracellular transducers of above
– Transcription factors that mediate the terminal effects of extracellular signaling
BioSci 145A lecture 15 page 21 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• Growth factors - proteins secreted by one cell that act on another cell (eg sis, wnt, int)
– oncoprotein growth factors can only transform cells that harbor the specific receptor
• Growth factor receptors - transmembrane proteins that are activated by binding to extracellular ligand (protein)
– very frequently protein tyrosine kinases
– oncogenicity usually results from constitutive (ligand-independent) activation
• Intracellular transducers - several classes
– protein tyrosine kinases, e.g. src
– G-protein signal transduction pathways - primary effectors of activated growth factors (e.g. ras)
– protein serine/threonine kinases (e.g. mos, raf)
• Transcription factors - these regulate gene expression directly
– myc - HLH protein
– fos, jun - b-ZIP proteins
– erbA - nuclear receptor
• common feature among these is that each type of protein can trigger general changes in cell phenotypes by:
– initiating changes that lead to cell growth
– respond to signals that cause cell growth
– altering gene expression directly
BioSci 145A lecture 15 page 22 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• One example signaling pathway - MAPK (Bardwell lab)
growth factor
receptor tyrosine kinase
ras
kinase cascase (serine/threonine)
transcription factors
• since the signal passes from one component to the next, inappropriate activation of one element in the cascade canl lead to widespread changes in gene expression
– these pathways are not strictly linear but branch and interact with many other signaling pathways
• can cause wider effects
• may require mutations in parallel pathways to get oncogenesis
• central importance of this pathway is illustrated by the number of components that can be mutated into oncogenes
– aberrant activation of mitogenic pathways can contribute to oncogenicity
BioSci 145A lecture 15 page 23 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• Growth factor receptors are ligand modulated dimers
– EGF receptor (v-erbB) is the prototype member
• EGF binding stimulates dimerization and activates tyrosine kinase cascade
• one oncogenic variant can dimerize in the absence of ligand and signals constitutively
• another lacks an internal regulatory domain resulting in constitutive signaling
– activated kinase domain autophosphorylates and can then interact with src family proteins
BioSci 145A lecture 15 page 24 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• transforming activity of src-family kinases is related to kinase activity
– autophosphorylation controls activity
• Y416 -> active
• Y527 -> weak, normally suppresses phosphorylation of Y416
– some oncoproteins activate src by interfering with phosphorylation of Y527
BioSci 145A lecture 15 page 25 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• modulation of transcription factor activity is important for oncogenesis
– can’t cause cancer without altering gene expression!
BioSci 145A lecture 15 page 26 ©copyright Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• transcription factors and cancer
– several prominent families of oncogenes are transcription factors - rel, jun, fos, erbA, myc, myb
– actions may be quantitative or qualitative
• effects may be to increase activity of the oncoprotein
– increased expression could upregulate target genes and influence growth, e.g. AP-1
• alternatively, the mutations could make the oncoprotein a dominant negative inhibitor of other cellular transcription factors (e.g. v-erbA)
– many members are “immediate early” genes
• transcription is immediately upregulated without the requirement for new protein synthesis when cells are treated with mitogens
– likely to be involved with initiating or promoting growth
• increased activity would be expected to increase oncogenesis and it does with some but not others
BioSci 145A lecture 15 page 27 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes
• oncogenesis is not typically dominant.
• A growing number of “tumor suppressor” genes have been identified that confer a genetic predisposition to cancers
– several types of genes are involved
• apoptosis proteins (eg p53)
• cell-cycle control proteins (RB)
• DNA-repair proteins (p53)
– classic examples are RB (retinoblastoma) and p53
– loss of tumor suppressor genes is implicated in several infrequent cancers of childhood
• retinoblastoma
• Wilm’s tumor
BioSci 145A lecture 15 page 28 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
• RB is a nuclear phosphoprotein that influences the cell cycle
– unphosphorylated RB prevents cell proliferation by binding to E2F and blocking G1/S transition
– phosphorylation of RB inhibits binding to E2F and releases block
– some oncogenes (e.g. SV40 T-antigen, E1A) function by sequestering RB and removing block to cell growth
– similar effects by loss of both alleles in human disease
BioSci 145A lecture 15 page 29 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
• A variety of other cell-cycle control proteins are tumor suppressor genes
– p16, p21 and D cyclins
– shown by identification of inactivating mutations in a variety of human tumors
• in quiescent cells
– RB is not phosphorylated
– D cyclin levels are low or absent
– p16, p21 and p27 prevent activity of cdk-cyclin complexes
• cdc2, cdk2 and cdk4,6 interact with cyclins and promote cell cycle
• this is blocked by tumor suppressor genes
BioSci 145A lecture 15 page 30 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
• P53 suppresses cell growth or triggers apoptosis
– more than 50% of human tumors have lost p53 protein or harbor mutations in the gene
– a variety of mutations are possible
• recessive mutations cause loss of p53 function allowing unrestrained growth (eg. ko mice)
• others are dominant negative p53 mutants that interfere with normal p53 subunits in cells and allow unrestrained growth (eg rare cancers)
BioSci 145A lecture 15 page 31 ©copyright Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
• p53 has dual functions
– cells normally have low levels of p53
– DNA damage induces large increase in p53 levels
– increased p53 leads to growth arrest until DNA is repaired if cells are in G1
– cells in S-phase or later are triggered to become apoptotic
• p53 is a transcription factor that typically activates
– one target is p21 -> cell cycle arrest
– another is GADD45 - a DNA repair protein
– role in inducing apoptosis is unknown at present
• apoptosis is an important pathway in preventing tumor formation - blocking it is a common strategy
BioSci 145A lecture 15 page 32 ©copyright Bruce Blumberg 2000. All rights reserved
Cancer - putting it all together