1. 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 –Important motifs in transcription factors helix-turn-helix –homeobox genes helix-loop-helix –myogenic genes bZIP proteins –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 Next two lectures are by Dr. LaMorte in BLI No lecture on 3/12 Review session on 3/14 Final on Tuesday 3/19

2. BioSci 145A lecture 15 page 2 © copyright Bruce Blumberg 2000. All rights reserved Homeobox genes helix-turn-helix motif is widely used in transcription factors –phage repressors –2 helices come to lie at almost right angles to each other –the recognition helix fits in the major groove of DNA while other helices make minor groove contacts –target sequence discrimination is by only a few residues in the recognition helix

3. BioSci 145A lecture 15 page 3 © copyright Bruce Blumberg 2000. All rights reserved Homeobox genes (contd) Homeobox is a highly-conserved, 180 bp DNA sequence –only DNA has a homeobox homeodomain is the protein product of the homeobox and. –proteins encoded by homeobox genes are homeodomain proteins very large family of genes –first discovered in Drosophila homeotic selector genes, Antennapedia and Ultrabithorax –low stringency hybridization showed that there were many such genes in the Drosophila genome –in a “secret” experiment, Bill McGinnis (Walter Gehring’s lab) and Andrés Carrasco (Eddy De Robertis’s lab) decided to test whether such sequences occurred in vertebrates low stringency Southern blot was performed, the first “zoo blot” several genes were identified, Cell papers published and feelings were hurt Drosophila (Hom-C) and vertebrate Hox genes control the identity of body segments during development. Loss of function mutations cause changes in segment identity –incredibly, corresponding vertebrate genes can completely rescue fly mutations –Transcription factors are developmental switches

4. BioSci 145A lecture 15 page 4 © copyright Bruce Blumberg 2000. All rights reserved Homeobox genes (contd) this residue is very important for determining specificity

5. BioSci 145A lecture 15 page 5 © copyright Bruce Blumberg 2000. All rights reserved Homeobox genes (contd) We will talk more about homeobox genes in the last two lectures –for now, it is sufficient to note that homeobox genes are critical for normal development –more than 400 different types already known –homeodomain proteins can act as transcriptional activators or repressors many people spent years trying to demonstrate activation of reporter genes by homeodomain proteins with little success it later turned out that the ones that were being tested were repressors mutations in homeobox genes cause developmental defects in humans –mutations in emx2 homeobox gene (related to Drosophila empty spiracles) causes schizencephaly cortical malformation that manifests developmental delay, blindness, seizures, and other neurological disabilities –mutations in MSX-2 lead to Boston-type craniosynostosis cranial bones fuse inappropriately

6. BioSci 145A lecture 15 page 6 © copyright Bruce Blumberg 2000. All rights reserved Helix-loop-helix proteins HLH proteins are a large group of dimeric proteins –signature motifs are two stretches of amphipathic  - helices flanking a central loop (linker) of variable size protein protein interaction is mediated via hydrophobic interactions regions of strong sequence conservation within the helices among related proteins –not all have the ability to bind DNA, these are typically negative regulators –those that can bind DNA tend to have a basic region adjacent to the HLH motif these are called bHLH proteins dimerization regulates function –two basic regions are required for DNA binding –two groups of bHLH proteins exist Class A are ubiquitously expressed (eg E12/E47) Class B are tissue-specific (MyoD, myogenin) –a common strategy among tissue-specific proteins is to heterodimerize with ubiquitous partners –homodimers are not very stable and do not bind DNA with high affinity –heterodimers between bHLH and HLH proteins are typically nonfunctional, an important regulatory mechanism

7. BioSci 145A lecture 15 page 7 © copyright Bruce Blumberg 2000. All rights reserved Helix-loop-helix proteins (contd)

8. BioSci 145A lecture 15 page 8 © copyright Bruce Blumberg 2000. All rights reserved Helix-loop-helix proteins (contd) bHLH proteins and muscle development –MyoD was the first discovered. Identified in an expression screen as a single protein that could transform cultured fibroblasts (3T3) into muscle (myotubes) –MyoD acts first to kick ID off of E12 and/or E47 and initiates the muscle program. Later bHLH genes such as myogenin and myf5 are also important –this family of genes illustrates the general principle that combinatorial associations of transcription factors can yield complexes with different functions DNA binding transcriptional regulation

9. BioSci 145A lecture 15 page 9 © copyright Bruce Blumberg 2000. All rights reserved Leucine zipper (b-ZIP) proteins Leucine zipper is a protein:protein interaction domain characterized by coiled-coil  -helical structure –coiled-coil is a common structural motif in proteins (e.g. myosin) –coiled-coil is formed from two helices wound around each other and typified by large hydrophobic amino acids (leu, ile) repeated every 7 residues –the helices are usually amphipathic –leucine zipper flanked by a basic region is common in transcription factors, so-called b-ZIP motif.

10. BioSci 145A lecture 15 page 10 © copyright Bruce Blumberg 2000. All rights reserved Leucine zipper (b-ZIP) proteins (contd) bZIP is a common motif in viral transcriptional activators and some enhancer binding proteins –eg C/EBP (CAAT box enhancer binding protein) like bHLH proteins, bZIP proteins are regulated by heterodimerization. –dimers have distinct functions –not all proteins can homodimerize eg c-jun can homodimerize to bind DNA c-fos can not c-jun and c-fos can heterodimerize to produce the transcription factor AP-1 –the jun/fos heterodimer binds DNA ~10x better than the jun homodimer although both prefer the same DNA target sequence target sequences for all dimeric proteins have two half-sites. –dyad symmetry AGGTCACACTGACCT AGGTCAAAGGAGGTCA –this is a signature feature and should always cause you to suspect a dimeric transcription factor

11. BioSci 145A lecture 15 page 11 © 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 –different genetic events can be associated with characteristics of the developing tumor.

12. BioSci 145A lecture 15 page 12 © 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

13. BioSci 145A lecture 15 page 13 © 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

14. BioSci 145A lecture 15 page 14 © 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, NGF, 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

15. BioSci 145A lecture 15 page 15 © 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

16. BioSci 145A lecture 15 page 16 © 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

17. BioSci 145A lecture 15 page 17 © copyright Bruce Blumberg 2000. All rights reserved Cancerous changes in cells benign vs malignant tumors –Tumor = swelling caused by new tissue growth –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 Inhibiting angiogenesis is promising treatment

18. BioSci 145A lecture 15 page 18 © 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)

19. BioSci 145A lecture 15 page 19 © 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

20. BioSci 145A lecture 15 page 20 © 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 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, at age 86) when it was finally discovered that a virus was the cause of the cancer –http://www.nobel.se/medicine/laureates/1966/rous- bio.html –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 in 1989) 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

21. BioSci 145A lecture 15 page 21 © 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

22. BioSci 145A lecture 15 page 22 © 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

23. BioSci 145A lecture 15 page 23 © 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

24. BioSci 145A lecture 15 page 24 © 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

25. BioSci 145A lecture 15 page 25 © 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 proto-oncogenes 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

26. BioSci 145A lecture 15 page 26 © 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

27. BioSci 145A lecture 15 page 27 © 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

28. BioSci 145A lecture 15 page 28 © 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

29. BioSci 145A lecture 15 page 29 © 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

30. BioSci 145A lecture 15 page 30 © 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