119 Harold Varmus TUMOR VIRUSES AND ONCOGENES MMe ular Gets --. , J.M. Bishop, The Molecular Biology of/RNA Tumor Viruses: A Physician's Guide, New England Journal of Medicine 303:673-682, 1980, "Oncogenes", Scientific American 246:80, March 1982;/ Varmus, i? Form and Function of Retroviral ’ I. Further Readings: Proviruses, Science 2164812~820, 1982 Weinberg, A Molecular Basis of Cancer, Sei. Amer., Nov. 19 33;tT. Krontiris, e Emerging Genetics of Human Cancer, NEJM 309: + HOt 409 1983.//T. Hunter, The Proteins of Oncogenes, Sci. Amer. 251:70, Aug. 19 . . Marx, J.C. The Case of the Misplaced Gene. Science 218:983-985, 1982. The Molecular Biology of Tumor Viruses, Part II, DNA Tumor Viruses, 1980 ; Part III, RNA Tumor Viruses, ‘1982, Co Cold Spring Harbor Laboratory. Review of Medical Microbiology, chapter 40. | ° Davis, et al., Microbiology, Chapter 63. Cold Spring Harbor Symposium on Quantitative Biology, Vol. 42, 1979. Bunn et al., Clinical course of retrovirus-associated adult T-cell lymphoma in We ee er items ae meen cenit meena tae Rtas’ sb~redorthrus Seminar and/or instructor for further readings. iI. Introduction JAM, buy poe, bw A The evidenége for a role for viruses in the etiology of most human tumors is sparse: epifemiological and biochemical studies suggest that conventional horizontally- eee ted infectious agents are not important in the majority of human cancers minent exceptions to these statements -- evidence that hepatitis B vird’ is a causative factor in hepatoma, that a human retrovirus may cause a form of T cell leukemia and lymphoma, and that herpes viruses may play some role in a few human cancers -- will be considered in the herpes virus and HPV § hepatitis virus lectures and in the seminar on tumor viruses. /a dicta, 771 5 This lecture is intended primarily to acquaint you with the rapid advances now being made in the understanding of how viruses cause cancers in animals other than man and how tumor virology has introduced us to a class of cellular genes, called proto-oncogenes, that appear to be activated in many human tumors of unknown etiology. Whether or not human cancer is virus-related, animal tumor viruses offer an important opportunity to learn how cells work, how their behavior can be altered by a very small number of viral genes, and how cellular genes related to viral genes might be implicated in human cancer. III. General Considerations A. Transformation Tumor viruses, by definition, have the capacity to produce tumors upon infection of appropriate animals. In practice, most work with tumor viruses is now conducted in cultured animal cells; in the appropriate cells, tumor viruses generally have the capacity to cause an in vitro analogue of tumor production, referred to as "neoplastic transformation" of cells. Transformation of cells by tumor viruses involves a stably inherited change in one or more properties of ~2- cell behavior; most commonly, such change includes altered morphology (transformed cells look more like tumor cells than like normal cells) and freedom from normal growth restraints (transformed cells, in contrast to normal ells, will grow in random array to a very high density, without attachment to a solid surface, and at low serum concentration; they will form a "focus" of piled-up cells, permitting an assay for viral transformation; and they will usually produce tumors when injected into animals). In addition, various biochemical changes (alteration in properties of cell membranes, production of new cellular and viral proteins, reduction of cyclic AMP levels, etc.) may also accompany transformation. As discussed below, a major focus of interest in tumor virology is an understanding of how a single viral gene can produce such a profound effect upon cell behavior. B. DNA and RNA tumor viruses Tumor viruses, like other viruses, are classified according to the nature of their genomes. There are many specific examples of RNA tumor viruses which exhibit a variety of biological effects, but (fortunately for students) the biochemical and structural properties of these viruses all appear very similar. The DNA tumor viruses are more complex, since the major classes of DNA viruses (papova viruses, hepatitis B viruses, adenoviruses, herpesviruses, and pox viruses) all have members which are tumorigenic (see appendix A and Table 1). TABLE 1 DNA TUMOR VIRUSES: GENERAL PROPERTIES Three size classes: small (DNA size 3-5 kb )---papovaviruses (SV40, ‘polyoma, papilloma viruses: other human and simian isolates) ; hepatitis B viruses (man, woodchuck) medium (DNA size 40 kb )—-adenoviruses (from several animal species, e.g., Chickens, monkeys, man) large (DNA Size 100-150kb) ~herpesviruses (from several animal species, e.g., frogs, chickens, monkeys, man); pox viruses (generally produce benign tumors; found in several speci C. The lysogeny model severa tes) Since tumor viruses are able to produce stable alterations in host cells and since those changes appear to be maintained by the activity of viral genes, it is not surprising to learn that tumor virus DNA has the capacity to integrate covalently into the genome of the host cell; thus tumor viruses appear to be at least partially related to lysogenic bacteriophage (Table 2). As in the case of most bacteriophage, the genomes of many tumor viruses appear to pass through a stage in which they are in the form of double stranded DNA circles; this is even true of RNA tumor viruses (see below). There is no evidence that tumor viruses produce repressors of the sort responsible for the maintenance of lysogeny. However, in many cases, there is limited expression of viral genes in cells transformed by tumor viruses. 121 -2a- TABLE ©. RELEVANCE OF LYSOGENY MODEL TO TUMOR VIROLOGY CIRCULAR LIMITED dsDNA VIRAL EXPRESSION OF CLASS EXAMPLE GENOME PHASE INTEGRATION REPRESSOR __ VIRAL GENES Temperate Lambda phage Linear dsDNA + + + + phage DNA turor SV40 Circular dsDNA + + ~ + virus RNA tumor Rous sarcoma Linear ssRNA + + - Vitus vi In some cases FIGURE 1. ALTERNATIVE CONSEQUENCES OF svho INFECTION IN DIFFERENT CELL TYPES MONKEY CELL (African green monkey) SV40 MOUSE CELL Viral DNA replicated Viral DNA integrated Early and late genes expressed “Early” gene(s) expressed (T antigen) Virus produced Cell lysed No virus produced Cell Transformed 122 -3- D. Permissive and nonpermissive cells DNA tumor viruses, with the exception of some herpes viruses and perhaps hepatitis B viruses, do not normally produce tumors in their natural hosts, and cells from the natural host are generally permissive for virus replication. Ordinarily, DNA tumor viruses only transform cells when some component of the virus or the host cell is defective for replication. Most commonly, transformation by DNA tumor viruses is studied in foreign (or heterologous) host cells which, for generally unknown reasons, are defective in their ability to support viral replication (i.e., are nonpermissive for that virus). For example (Fig. 1) the DNA tumor virus SV4O (simian virus 40) replicates in monkey cells, produces cytopathic effects (e.g., vacuolization in rhesus monkey cells or lysis in African green monkey cells) but it transforms rodent cells (e.g., mouse, rat, hamster) in which it is unable to replicate. r , adtuo™ RNA tumor viruses, by contrast, are frequently implicated in cancers in their natural hosts, and they are capable of transforming cells while replicating within them. However, RNA tumor viruses can also transform heterologous host. cells in which they are unable to replicate. In other words, RNA tumor viruses can transform permissive or non-permissive host cells. IV. Properties of RNA Tumor Viruses ( Rats Yi eriey) Pa tumolveee, have several unusual characteristics which are not considered elsewhere in this course; these characteristics have profound implications for molecular biology and they have strongly influenced the search for human tumor viruses. ‘ A. Structural and genetic features es have been identified in a wide variety of animals (e.g., viper, fish, birds, rodents, ungulates, cats, and several primates including man) but their biochemical and structural features are highly similar: 1. The viruses are enveloped, leaving infected cells by budding through cytoplasmic membranes and entering cells by interacting with host receptors, as deseribed for several virus classes in lectures on replication. 2. The genome is in the form of two identical subunits of 5-10 thousand bases of single stranded RNA. . 3. The virus core contains a virus-coded RNA-directed DNA polymerase ("reverse transcriptase") that converts the RNA genome to double stranded DNA during the virus life cycle. . 4, Newly synthesized viral DNA forms a closed circle and is then integrated into host chromosomes by a precise mechanism tht generates a provirus structurally similar to many transposable elements (see previous lecture). The genes of the provir expressed by host cell machinery. Note that permissive cells arttireistentiy infected aad often transformed, ysed, by retroviruses. an a usbeh nk 5. Retroviruses require Panes ones for PePiication, but many viruses are defective for replication (due to dele f one or more of these three genes) A PbS (OLS Ge 123 ~ 4e and must be complemented by a helper virus that supplies the missing functions in co-infected cells (much as described for defective transducing phages in previous lectures). Many of these replication defective viruses carry a gene that mediates the oncogenic effects of the virus (a viral oncogene, derived from a normal cellular gene; see below). But retroviruses without oncogenes can also produce a variety of tumors and other kinds of pathology (e.g., anemia, osteopetrosis, etc.). 6. The most commonly studied RNA tumor viruses are: avian sarcoma viruses (e.g., Rous sarcoma virus) avian leukosis virus mouse leukemia virus mouse sarcoma virus mouse mammary tumor virus feline leukemia and sarcoma Views simian sarcoma virus a Oe human T cell re viruses AWSs Vinee 7. Retroviruses are able to infect germ line cells and retroviral . proviruses are thus endogenous to the chromosomes of most if not all species, including man. Some properties of endogenous retroviruses are listed below: -- Genetically transmitted in the form of proviruses ~- May be induced chemically (see Section VI.C below) -- Often xenotropic (grow well in foreign hosts, poorly in species from which they are isolated) ~~ Occasionally oncogenic (e.g., murine leukemia virus and murine mammary tumor virus) but more often not pathogenic -- Probably present in all vertebrates, including man V. How do Viruses Transform Cells? The major attraction of tumor viruses as laboratory tools is their capacity to alter cell behavior with very few genes. The dependence of transformation upon the continued synthesis of the products of these "viral oncogenes" has been shown by the use of temperature sensitive mutants with lesions in these genes (Table 3). The genome of the most intensely studied DNA virus, SV-40, is divided into an "early" region (expressed prior to replication of viral DNA) and a "late" region. The "late" region consists of three overlapping genes for coat proteins, and it is not expressed in transformed cells (which do not produce virus, see above). The "early" region also @ppears=te- contain® overlapping genes, encoding at least two proteins; genetic studies with viral mutants indicate that one or more of these "early" proteins are necessary for transformation of non-permissive cells and for replication of viral DNA in permissive cells. The best studied RNA tumor virus, Rous sarcoma virus of chickens, has four genes, only one of which (the viral one gene, called sre in Rous sarcoma virus) mediates transformation. This gene, in contrast to the early region of SV4O0 DNA, is not required for virus replication; in addition, the sre gene product transforms permissive as well as non-permissive cells. The product, a 124 -5- phosphoprotein of 60,000 daltons, has the intriguing capacity to phosphorylate tyrosine residues in certain proteins and it is found mainly in plasma membranes. Furthermore, this gene has been derived from a normal cellular gene, Called c-sre (see below Section VII). TABLE 3 EXAMPLES OF VIRAL ONCOGENES Property Simian Virus 40 Rous Sarcoma Virus Name Early region src gene (A gene) Size of protein(s) ca. 85,000 and ca. 60,000 17,000 Required for viral Yes No replication Required for trans- Yes Yes formation Functions of product DNA binding (stimu- Protein kinase (phos- lates DNA synthesis) phorylates tyrosine ATPase residues) (others) Location of product Primarily nuclear Plasma membrane (mainly) ae homologue No Yes (see Section VIT) " In addition to sre, there are over 15 distinguishable retroviral oncogenes, each of which has been derived from normal cellular genes, called c-onc's or proto-oncogenes. Several of these other oncogenes also encode protein kinases specific for tyrosine and found at the plasma membrane, but most produce proteins that clearly have other kinds of biochemical properties (Table 4). In several cases, determination of the nucleotide sequences of the genes show that they are closely related, suggesting that retroviral oncogenes are derived from a few families of related cellular genes. Such genes are thought to function under normal circumstances as regulators of growth and development. In one case (sis) the gene has been shown to be derived from a host gene for a peptide hormone called platelet-derived growth factor (PDGF); in another case (erb B), the gene encodes the receptor for the epidermal growth factor (EGF). The importance of these proto-oncogenes in human cancer is discussed further below (Section VII). aa 125 -5a- TABLE 4. A SAMPLING OF RETROVIRAL ONCOGENES : ANCESTRIES AND PROTEIN PRODUCTS — << SRC TYR KINASE FPS/FES ROS ABL RAF/MIL PM op $1 -R_ ns CYTOPLASM EGF-R————- ErB-B MOS J PDGF e——__-_—_ srs STP B hee | INDING _ 1CTPASE HA RAS/BAS MYB | DNA BINDING — myc NUCLEUS FOS SKI Amino AcIpD HOMOLOGY Ml 126 - 6 - VI. Mechanisms of virus rescue It is of inherent interest to know whether viral genes can be recovered from transformed cells which fail to produce virus (e.g., permissive cells transformed by defective viruses, or nonpermissive cells transformed by non- defective viruses). In addition, development of such techniques may be impor- tant in searches for human tumor viruses. Three types of approaches are gen- erally used. A. Cell fusion(Fig. 3). A transformed non-permissive cell is fused with an uninfected permissive cell to form a heterokaryon (cell with two or more different nuclei) which provides the factors necessary for virus replication. This method can be used with both RNA and DNA viruses. RESCUE OF TUMOR VIRUS FROM NON-PERMISSIVE HOST f BLA TS il’ NON-PESMISSIVE FIBROBLAST TRANSFORMED CELL WITH INFECTED BY TUMOR VIRUS INTEGRATED VIRAL GENOAMF, NO VIRUS PRODUCTION Figure 3 HETEROKARYGON PRODUCING FUSION OF TRANSFORMED TUMOR VIRUS NON-PERMISSIVE CELL WITH NORMAL, PERMISSIVE CELL {paramyxo virus used as facing agent) B. Helper virus. A permissive cell transformed by a virus defective in 127 -7T- replication function(s) is superinfected by a "helper" virus with normal replicative powers. The helper virus provides the factors required for replication of the defective transforming virus, and both are produced by the cell. This phenomenon has been observed with both RNA and DNA viruses. C. Chemical induction. 1) Treatment of certain non-permissive cells transformed by DNA tumor viruses with a variety of chemical agents (some of which induce lysogenic phage in bacteria) results in production of virus, presumably by facilitating excision of the integrated DNA genome. 2) Treatment of normal cells with similar inducing agents most commonly halogenated pyrimidines, such as iododeoxyuridine (IUdR) or bromodeoxyuridine (BUdR) often results in the production of endogenous RNA tumor viruses (see above Section IV). The mechanism of the induction is unknown. Only endogenous RNA viruses have been observed; their properties are considered more completely below. Production of retroviruses from exogenously-infected cells may also be augmented by such inducing agents (see Tumor Virus Seminar). VII. Cellular proto-oncogenes "Proto-oncogenes" in the genomes of normal cells were first discovered by molecular hybridization experiments performed with the viral gene (v-sre) shown to be responsible for the oncogenic properties of Rous sarcoma virus (see part V, above). With each of over 15 different retroviral oncogenes the same extraordinary result has been obtained: a very similar gene is present in the normal genome of the animal species from which the virus was isolated. Moreover, these genes are not part of some endogenous retroviral provirus, but instead constitute genes recognized as cellular in origin by their architecture (introns and exons), their mode of expression, and their conservation throughout evolution. (All are present throughout vertebrates, including man, and some are even found in insects, worms, and yeast). Though the functions of most proto- oncogenes are not known, they generally produce proteins very similar to the protein products of viral oncogenes in small amounts, and it is presumed that their high degree of conservation bespeaks some fundamental role in growth or development (see Table 4). The appearance of these genes in the genomes of oncogenic RNA viruses implies that the genes were "transduced" by retroviruses that lacked oncogenes during their passage through host animals. The fact that each member of this group of genes can mediate oncogenic events (transformation of cultured celis and tumor induction in animals) when it is part of a viral genome suggests that each might have oncogenic potential in its natural setting, within the genomes of normal cells, e.g., if altered by mutations that affect the level of expression or the nature of the protein product (Fig. 4). Several lines of experimentation support this view: (1) In vitro manipulation. It has been shown that a few (though not all) cellular proto-oncogenes, e.g., c-ras and c-mos, directly isolated from normal cell genomes, can transform cells and make tumors, if programmed to be expressed efficiently when reintroduced into normal cells. (2) Retroviral insertion mutations activate proto-oncogenes. Some of the 128 -~ 8. ad retroviruses that lack their own oncogenes (e.g., the chicken or mouse leukosis viruses or mouse mammary tumor virus) can nevertheless induce tumors of various types. Tumor production appears to be dependent upon the stimulation of efficient expression of a "cellular oncogene" by proviral DNA inserted nearby during infection. This mechanism has been clearly implicated in the case of B cell lymphomas in chickens, since proviral DNA is almost always found adjacent to a known proto-oncogene (first identified by its virtual identity to the viral oncogene in an acute leukemia virus of birds); in these tumors, the proto- oncogene, c-myc, is expressed about 50 times more efficiently than in normal B celjs. , , (3) Point mutations of cellular ras genes make them oncogenic. DNA isolated from some tumors of animals and man, regardless of whether a causative agent (e.g., chemical or virus) is known, can transform the behavior of cultured mouse fibroblasts from a normal to malignant phenotype. The most likely interpretation of these results is that a mutation has occurred during the development of the original tumor, rendering a normal cellular proto-oncogene into a “transforming gene". Remarkably, several of these "transforming genes", including some isolated from human tumors of the lung, colon, and bladder, have proven to be members of the group of proto-oncogenes identified by their homology with viral oncogenes (Table 6). In several cases, the mutant "transforming" genes, all members of the ras gene family thus far (Table 4), have been cloned in bacteria and subjected to nucleotide sequencing. The eaere show that single base substitutions, different in each case but confined eal io posistions, codons 12 (and 61 in the ras genes, are responsible for the altered properties of the c Llular genes. TABLE 6. Cellular Genes Implicated in Tumorigenesis By the 3T3 Cell Transformation Assay . Gene Tumor Alteration c-Ha-ras Bladder CA Single base substitition Lung CA Mammary CA e-Ki-ras Lung CA, Colon CA, Single base substitution others N-ras Neuroblastoma Single base substitution Myeloid leukemia oo ‘ . - Rhabdomyosarcoma © , eo. - (4) Rearrangements of proto-oncogenes affects their expression and may render them oncogenic. In a number of human and murine tumors, the cellular homologues of retroviral oncogenes have been found to be grossly altered, either by (a) chromosomal translocations or (b) gene amplification (some examples are provided in Table 7.) (a) The translocations frequently involve the same chromosomal sites in many tumors of the same type; most attention has been given to translocations in (human) Burkitt's lymphoma that join c-myc on chromosome 8, band q24, to the immunoglobulin heavy chain locus on chromosome 14, band q32, and translocations bern ath 19 that join the same loci (gn chromosomes 15 and 12) in mouse plasmacytomas. The translocations appear to/alter the level of expression of c-myc in at least some cases, but the effect. the translocations upon the neoplastic process has yet to be defined. y (b) Amplification has been shown to occur in several types of tumors, affecting several oncogenes (Table 7). The amplified unit is large and can generally be seen during karyotyping as a homogeneously staining region within a chromosome or as multiple, small chromosomes lacking a centromere (double minute chromosomes). The increase in gene dosage is generally matched by an increment in oncogene expression. Amplification sometimes correlates with the staging of human cancers (e.g., N-myc in neuroblastomas) and may be useful in diagnosis. TABLE 7. CELLULAR GENES IMPLICATED IN NON-VIRAL HUMAN AND MURINE TUMORS BY REARRANGEMENTS Gene Tumor Alteration c-myc Burkitt's lymphoma Translocations Plasmacytoma e~-abl Myeloid leukemia Translocation (Philadelphia chromosome) c-myc Myeloid leukemia Amplification Apudoma Small cell lung carcinoma c-Ki-ras Adrenocortical Ca Amplification Geboni-o—CA__. N-myc Neuroblastomas Amplification Rats pcs Ca Su ce2l More than one of these var (bos that affect cellular oncogenes are sometimes found within the same tumor cell. This is consistent with the generally accepted notion that full fledged cancer cells arise as a result of multiple events. A major goal of contemporary cancer research is the explicit ese several steps in the genesis of a cancer. The general implication of these several findings is that tumor viruses (in particular, retroviruses) may have led investigators to recognize a class of normal cellular genes ("cellular oncogenes") that may be involved in cancers initiated by various infectious, chemical, physical, or genetic mechanisms. Therefore, the normal functions of these genes, the kinds of changes they suffer during tumorigenesis, and the properties that make them oncogenic under certain circumstances are all matters of extraordinary interest to oncologists. As viewed in Table 8, these genes can be thought to have undergone a variety of "activating" events: transduction to become a viral oncogene, point mutation to produce a gene that transforms cultured cells; or insertion mutation, amplification, or translocation that affect gene expression. Lastly, in vitro, 130 ~-~ 10 - some of these genes can be made oncogenic by addition of strong regulatory Signals. ,, VRANSDUCTION ——-—® RETROVIRAL ONCOGENE INSERTION.» ACTIVATED GENE MUTATION. “NORMAL —> AMPLIFIED GENE — CELLULAR REARRANGEMENTS ONCOGENES ————} TRANSLOCATED GENE POINT ______s TRANSFORMING GENE MUTATION | IN VITRO yy OVEREXPRESSED GENE MANIPULATION : Appendix A. Additional Historical Information About DNA Tumor Viruses Papova viruses ---viruses with small, circular (double-stranded) DNA genomes ca. 5-5 kilobase pairs); "papova" derived from common members of this group: Papilloma viruses (from several natural hosts); Polyoma virus of mice; and Vacuolating viruses (causes vacuole formation upén infection of natural hosts) OF which simian virus 40 (SV 40) is most famous example. wn «| Papilloma viruses ---found in several species (ineluding ma in which they replicate and cause warts (papillomata); grow poorly in cell culture and therefore not well studied. \e payee & UAVE® al th fetes virus --~common in wild mice in which the virus occasionally produces a variety of types of tumor (hence "poly" "oma," or many tumors); in cell culture, undergoes lytic, replicative cycle in its natural host (mouse) and transforms certain heterologous, nonpermissive hosts (e.g., hamster). SV 4O -~~-discovered in rhesus monkey cells during development of polio vaccine; causes tumors in and transforms cells from certain heterologous, nonpermissive hosts (e.g., mouse and other rodents); undergoes replicative cycle in monkey cells. Hepatitis B-type viruses ---discovered first in man (see hepatitis lectures), later in woodchucks, ground squirrels, and ducks; associated with hepatocellular earcinomas in man and woodchucks; inability to grow virus in tissue culture has slowed study of its replication and oncogenicity. -11- ; , 131 Adenoviruses ---viruses with medium-sized, linear (double-stranded) DNA genomes (ea. 35-40 kilobase pairs); found in many species, including man; the several human types show low, medium, or high oncogenicity when used to infect newborn rodents (especially hamsters) or rodent cells in culture (these are nonpermissive, heterologous hosts); replicate in cells from natural host (man) and. cause mild, acute GI and respiratory illness (see lectures on respiratory infections). Herpes viruses ---enveloped viruses with large, linear (double-stranded) DNA genome (ca. 150 kilobase pairs); associated with a variety of diseases, including tumors, in the several hosts in which these viruses have been found (e.g., chicken, frog, subhuman primates, and man); evidence for oncogenicity in man is not conclusive; virus replicates in cells from natural host, and, after irradiation to damage replication genes, it can transform certain heterologous host cells in culture (see herpes virus lecture). Epstein-Barr virus (EBV) --~-a herpes-like virus originally found in cells from African patient with Burkitt's lymphoma; now commonly seen in human lymphocytes from normal as well as diseased persons; it is the causative agent of infectious mononucleosis and has been indirectly implicated in causation of African Burkitt's lymphoma and nasopharyngeal carcinoma (see herpes virus lecture). LT, 7 132 -12- B mms THE DOMINANT PARADIGM NORMAL CELLS >> -—-—-—_> CANCER CELLS proTo-oncocenes —MUTATION(S) ONCOGENE(S) BASE CHANGES REARRANGEMENTS | NORMAL REGULATORS INAPPROPRIATE AMOUNTS OF GROWTH | OR ALTERED KINDS OF REGULATORY PROTEINS