Download - 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

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


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.


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


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


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



• 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



• 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


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


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)


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


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


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



• 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



• 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



• 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



• 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


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


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


Oncogenes and cell growth

• Seven classes of proteins control cell growth

– Collectively, these genes comprise the known set of genes involved in tumor formation


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



• 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



• 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



• 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



• 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



• modulation of transcription factor activity is important for oncogenesis

– can’t cause cancer without altering gene expression!



• 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


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


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



• 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



• 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)



• 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


Cancer - putting it all together

Download - 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

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