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The adenovirus E1A and E1B proteins, like SV40 large T antigen and the human papillomavirus transforming proteins, specifically interact with cellular proteins encoded by the Rb and p53 tumor suppressor genes. In particular, the adenovirus E1A proteins interact with Rb and the E1B 55 kd protein interacts with p53. It is noteworthy that the adenovirus E1A and E1B proteins— which, like the human papillomavirus E6 and E proteins, are needed in combination to induce transformation—interact separately with Rb and p53. This contrasts with SV40 large T, which can alone induce cell transformation and interacts with both Rb and p53. The interactions of the transforming proteins of SV40, human papillomaviruses, and adenoviruses with the same tumor suppressor gene products clearly indicate that these small DNA tumor viruses induce cell transformation by a common pathway in which inactivating the proteins encoded by the cellular Rb and p tumor suppressor genes plays a central role. Herpesviruses The herpesviruses are among the most complex animal viruses, with genomes ranging from 100 to 200 kb. Several members of the herpesvirus family are of particular interest because they induce tumors in their natural hosts. Such naturally occurring herpesvirus-induced neoplasms in animals include Lucke's carcinoma in frogs and Marek's disease (a T-cell lymphoma) in chickens. Another highly oncogenic member of this family is herpesvirus saimiri, which is nonpathogenic in its natural host (squirrel monkeys) but in- duces virulent T-cell lymphomas in other monkey species. A human herpesvirus, Epstein-Barr virus (EBV), has been identified as a causative agent of African Burkitt's lymphoma, B- cell lymphomas in immunosuppressed individuals (for example, AIDS patients), and nasopharyngeal carcinoma. The epidemiological association of EBV with these lymphomas is further supported by the ability of EBV to transform human lymphocytes in culture. In spite of this important biology, molecular studies have been hindered both by the large size of the EBV genome (-180 kb) and by the lack of cell culture systems for virus propagation. Nonetheless, several EBV genes have been found to be required for transformation of primary human lymphocytes. Moreover, one of these genes (LMP1) is itself capable of inducing transformation of fibroblasts and altering the growth properties of lymphoid cell lines. This gene encodes a plasma membrane protein of currently unknown function. Poxviruses The last family of DNA viruses with transforming activity comprises the poxviruses. Like herpesviruses, the poxviruses are extremely complex animal viruses with genomes of - 200 kb. Unlike all of the other DNA viruses, which replicate in the cell nucleus, the poxviruses replicate in the cy- toplasm of infected cells. Poxviruses do not induce malignant tumors, but they do induce benign neoplasms. Two examples are the Shope fibroma virus, which causes benign fibromas in rabbits, and the Yaba monkey virus, which induces transient superficial tumors on the limbs of rhesus monkeys. The molecular basis of poxvirus transformation is obscure, because of both the large genome size and the lack of in vitro transformation systems
Retroviruses In contrast with the diversity of DNA viruses with oncogenic potential, only one family of RNA viruses has the capacity to induce neoplastic transformation. The unique feature of this family, the retroviruses, is that they replicate in infected cells through a DNA intermediate, called a provirus, which is integrated into chromosomal DNA of the host. The virion RNA consists of two identical subunits of ~9 kb. In infected cells, this RNA is copied into DNA by the viral reverse transcriptase, which is carried in virus particles. The viral DNA is integrated into host DNA to form the DNA provirus. Proviral DNA is replicated along with cellular DNA in dividing cells. In addition, the proviral DNA is transcribed by host cell RNA polymerase to yield progeny viral RNA genomes as well as mRNAs, which are translated into viral proteins. In most cases, productive infection with retroviruses is not cytocidal. Progeny virus particles are released by budding from the plasma membrane without cell lysis. In contrast to most of the DNA tumor viruses, oncogenic retroviruses are consequently able to transform the same cells in which replication proceeds efficiently. Perhaps as a corollary, many retroviruses induce cancer in nature in their normal host species. They are a large virus family found in a wide variety of animals, including reptiles, chickens, mice, cats, monkeys, and human beings. One human retrovirus, human T-lymphotropic virus type I (HTLV I), is the causative agent of adult T cell leukemia and a second, HTLVII, may cause hairy cell leukemia. Although not directly oncogenic, a third human retrovirus, human immunodeficiency virus (HIV), is of major interest as the causative agent of AIDS. The efficiency with which retroviruses induce transformation varies with different viruses. Some viruses of this family are the most potent known carcinogens, both in intact animals and in cell culture. A good example is provided by Rous sarcoma virus, which was the first clearly accepted tumor virus, isolated from a chicken tumor by Peyton Rous in
- Inoculation of chickens with this virus results in the formation of large sarcomas within one to two weeks. Likewise, Rous sarcoma virus transforms chicken embryo fibroblasts in culture with high efficiency, such that nearly every infected cell is converted into the transformed phenotype. The oncogenic retroviruses have proved to be extraordinarily informative objects of study for the molecular biologist interested in cancer. A few such retroviruses, including the HTLVs, induce neoplasia as a result of the activity of viral gene products that also function directly in virus replication. These gene products (for example, the HTLV I Tax protein) apparently serve as transcriptional activators of both the viral genome and some genes of the host cell. The induction of neoplastic transformation by most retroviruses, however, is not mediated by genes that also function in virus replication. As discussed in the next chapter, the retroviral oncogenes are instead a unique set of genes that function specifically to induce transformation without playing a direct role in virus replication. Most importantly, as will be appreciated throughout this text, the identification of retroviral oncogenes has served to elucidate multiple different aspects of carcinogenesis and cellular oncogene activation.
attention on attempting to elucidate the molecular basis for this difference in virus pathogenicity. These studies led to the identification of retroviral oncogenes. The src Gene of RSV The difference in transforming activity of RSV and ALV carried a critical implication. These two viruses were closely related and shared a common mode of replication in the same host cells, chicken embryo fibroblasts. Yet, only infection with RSV resulted in cell transformation. It thus appeared possible that RSV contained genetic information that was responsible for transformation and that was lacking in ALV. Identification of this information in the genome of RSV was the first step in the study of retroviral oncogenes. Analysis of the sizes of genomic RNAs of RSV and ALV strains revealed an important difference. In 1970, Peter Duesberg and Peter Vogt found that the genomic RNA of RSV was ~10 kb, whereas that of ALV was smaller, ~8.5 kb. The suggestion was that the extra 1.5 kb of RSV RNA contained the information responsible for its unique
biological activity: induction of transformation. However, substantiating this hypothesis required the isolation of RSV mutants that were defective in transforming ability. Two types of RSV mutants have contributed to our understanding of transformation: (1) deletion mutants that are totally defective in transforming activity and (2) point mutants that are temperature sensitive for transformation. Transformation-defective deletion mutants were first isolated by Peter Vogt in 1971. Vogt infected microwell cultures of chick embryo fibroblasts with a limiting dilution of a clonal RSV stock such that each microwell culture received on average only a single transforming virus particle. As expected from the statistics of random sampling distributions, approximately two thirds of the microwell cultures developed foci of transformed cells. The remaining cultures could have received no virus and been uninfected. Alternatively, some of these non-transformed cultures could have been infected with a non-transforming mutant virus of the type that Vogt hoped to isolate. To distinguish these possibilities, Vogt tested all of the nontransformed cultures for the presence of replicating nontransforming virus. He was able to isolate such transformation-defective viruses at high frequency, thus demonstrating that RSV could lose its transforming ability by mutation. These transformation-defective RSV mutants had also lost the ability to induce sarcomas in infected birds; so the effect of the mutation was not limited to tissue culture. Because the transformation-defective mutants were capable of normal replication, an important conclusion of these experiments was that wild-type RSV contained genetic information that was specifically required for transformation but not for virus replication.
The question of whether this information was required for maintenance or initiation of transformation could then be resolved by temperature-shift experiments with infected cells. If a non-transformed culture at 41° was shifted to 35°, the cells rapidly became morphologically transformed. Conversely, if a transformed culture at 35° was shifted to 41°, the cells reverted to normal morphology. The temperature-sensitive mutation therefore defined a gene that was required for continuous maintenance of the transformed phenotype in infected cells. A large number of RSV mutants that are temperature sensitive for maintenance of transformation have since been isolated. The standard genetic approaches of complementation and recombination were initially employed to ask whether RSV contained one or more than one transforming gene. Coinfection of cells with different combinations of temperature-sensitive transformation mutants did not result in cell transformation. The mutants therefore did not complement each other, indicating that they all affected a single gene. Moreover, coinfection with a temperature-sensitive mutant and a transformation-defective deletion mutant resulted in neither complementation nor recombination. Both types of mutants therefore defined a single gene of RSV that was required for transformation of fibroblasts in culture and for induction of sarcomas in vivo. Because RSV induces sarcomas, its transforming gene, which was the first oncogene to be identified, was called src. The RSV And ALV Genomes The genomic organization of retroviruses has been analyzed by a variety of molecular biological approaches, including cloning and sequencing. The non-transforming and weakly transforming retroviruses share a common genome structure, of which ALV is a representative example (Fig. 3.4). Typical simple retroviral genomes contain three coding segments, each of which gives rise to multiple proteins as a consequence of posttranslational cleavage. The gag gene encodes core proteins, which are the major structural proteins of the virus particles. The pol gene encodes virion reverse transcriptase and integrase, enzymes that function in synthesis and integration of proviral DNA. The env gene encodes envelope glycoproteins, which interact with receptors on the surface of susceptible host cells. The viral genome also contains a number of cis-acting sequences that play important roles at several stages of the virus life cycle. These sequences include signals for packaging RNA into virus particles, sites for initiation of DNA synthesis, specific sequences that recombine with host DNA during viral DNA integration, and signals for transcription and processing of viral RNA. All of these viruses share a similar basic strategy of expression of their oncogenes: they are transcribed from the proviral 5' LTR as part of the retroviral genome. However, the viruses of this type vary as to the details by which this is accomplished. Some oncogenes (including ras, fos, and erbB) are, like src, expressed as independent translation products that do not contain sequences encoded by viral replicative genes (Fig. 3.6A). In contrast, many of the other oncogenes are expressed as fusion proteins that contain amino acid sequences encoded by viral replicative genes in addition to a transformation-specific sequence. The most common genome organization results in expression of the viral oncogene as a fusion with viral gag sequences (Fig. 3.6B). In these viruses, most of gag is deleted and replaced with oncogene sequences (such as abl, myc, erbA, jun, raf, and several others). The oncogene coding sequences are inframe with the remaining 5' gag sequences;
so the gene is transcribed and translated to yield an oncogene protein with amino acid sequences encoded by gag at its amino terminus. Variants of this basic fusion protein theme include the synthesis of recombinant proteins with a short stretch of amino acid sequences encoded by env instead of gag at the amino terminus of the oncogene product. Finally, some acutely transforming retroviruses contain two distinct oncogenes (such as erbA + erbB, myb + ets, and myc + raf), both of which contribute to the induction of neoplasms.
The Origin of Retroviral Oncogenes
The oncogenes of acutely transforming retroviruses are a distinct group of genes that are responsible for viral pathogenesis but not required for viral replication. The presence of such genes in a viral genome seems paradoxical because they do not function directly in virus growth. Where did the oncogenes originate and how did they become incorporated into retroviruses? Isolation of Acutely Transforming Retroviruses The first clue to the origin of oncogenes derives from the way in which the acutely transforming viruses were isolated. The early isolates of these viruses-for example, RSV- were obtained from tumors of animals that were chronically infected with non-transforming retroviruses (for example, ALV). Such animals occasionally developed tumors from which new acutely transforming viruses could be isolated and then maintained by either animal or tissue culture passage. More recent and informative examples are provided by the laboratory isolation of several murine acutely transforming viruses-for example, Harvey sarcoma virus and Abelson leukemia virus (Fig. 4.1). These viruses were isolated following inoculation of rats or mice with weakly transforming murine retroviruses that, like ALV, did not contain oncogenes. A small fraction of such animals developed tumors from which new, more highly oncogenic viruses could be isolated. These new acutely transforming viruses contained oncogenes that were responsible for their increased oncogenic potential: they now induced neoplasms rapidly in mice or rats and efficiently transformed cells in culture. The common element to the isolation of acutely transforming viruses, illustrated by these examples, is the rare isolation of a new virus from a tumor that developed in an animal infected by a non-transforming retrovirus. The acutely transforming viruses isolated from such tumors contained new oncogenes that were not present in the viruses that initially induced the neoplasms. Normal Cell Homologs of Retroviral Oncogenes The scenario of acutely transforming virus isolation suggested the hypothesis that the retroviral oncogenes were derived from host cells and incorporated into the genomes of
With the availability of src-specific probe, it became possible to test directly for the presence of endogenous oncogene-related sequences in the genomes of normal animals. The results obtained by Varmus, Bishop, and their collaborators were striking. The src- specific probe hybridized extensively to DNAs of normal chickens, demonstrating that chickens regularly inherited DNA sequences that were closely related to the src oncogene. Not only were src-homologous sequences found to be part of the chicken genome, but related sequences were also detected in other vertebrate species, including human. These experiments thus established that normal cells contained genetic information closely related to a viral oncogene, providing the first definitive experimental support for the now well- substantiated hypothesis that the acutely transforming viruses originated by the incorporation of cellular genes into viral genomes. Moreover, these results provided the first clear indication that cells normally contained genes that might have the potential for inducing neoplastic transformation. Analogous experiments with probes for the oncogenes of other acutely transforming viruses yielded results comparable to those obtained for src. In all cases, the retroviral oncogenes were homologous to DNA sequences that were normally found in the species from which the acutely transforming virus was isolated. Furthermore, as with src, the cellular homologs of retroviral oncogenes were conserved throughout vertebrates (and in some cases in lower organisms such as Drosophila and even yeast). It thus appeared that each of the retroviral oncogenes had originated from sequences that were part of the genetic complement of normal cells. The normal cellular sequences from which the viral oncogenes were derived are now called proto-oncogenes in order to distinguish them from the oncogenes in the viruses. As we shall see throughout subsequent chapters, this distinction is critical to understanding the biology of both oncogene and proto-oncogene function. One of the central concepts that has developed is that the proto-oncogenes are normal cell genes that function in a number of aspects of cell growth and differentiation. The oncogenes are abnormally expressed or mutated forms of their normal cell progenitors. As a consequence of such alterations
(including point mutations, deletions, recombination, or amplification), the oncogenes have acquired a new biological activity-the ability to induce cell transformation. Structure and Expression of Proto-oncogenes The discovery of proto-oncogenes as normal cell DNA sequences that were homologous to viral oncogenes immediately raised a number of questions concerning their structure and possible functions. Were the proto-oncogenes functional genetic elements that were expressed in normal cells? If so, what was their activity in normal cell physiology? Finally, did they contribute to the pathogenesis of cancer? Understanding of the structure of proto-oncogenes in detail has come from their isolation and characterization as molecular clones. A typical example continues to be provided by the src gene of RSV. The src proto-oncogene was isolated from genomic libraries of chicken DNA by hybridization with viral src probe. The cellular sequences homologous to src constitute a single locus in the chicken genome that is not linked to sequences related to endogenous nontransforming retroviruses. In addition, the cellular sequences homologous to src are interrupted by several blocks of nonhomologous sequences (Fig. 4.3). These nonhomologous sequences are introns in the proto-oncogene (like those present in almost all cell genes) that have been removed from the viral oncogene as a result of RNA splicing. The structure of src and other proto-oncogenes, as well as their conservation in evolution, is typical of functional genes of normal cells. Expression of proto-oncogenes has been investigated by analysis of both mRNA and protein in a variety of different types of cells. For example, such studies have identified cellular src mRNA in both normal cells and chemically induced tumors. A cellular Src protein, closely related in structure to the product of the viral src gene, also has been found in normal cells of both avian and mammalian species. Both the cellular and viral Src proteins are related in biochemical function: as discussed in detail in chapter 13, both the cellular src proto-oncogene (c-src) and the viral src oncogene (v-src) encode plasma membrane-associated proteins with protein-tyrosine kinase activity. Analogous investigations have found that the other proto-oncogenes are likewise expressed in various types of normal cells as well as in some non-virus-induced tumors. The normal functions of proto-oncogenes in cell proliferation, differentiation, and development will be extensively discussed in later chapters. In any case, the proto- oncogenes appear to constitute a group of functional genes of normal cells that, when incorporated into the genome of retroviruses as oncogenes, have the capacity to induce neoplastic transformation. Capture of Proto-oncogenes by Retroviruses The life cycle of retroviruses, particularly their stable and non-cytocidal association with their host cells, is a major factor in the ability of these viruses to acquire unique genes from the host through recombination with the viral genome. Recombination is known to occur with very high frequencies when two different retroviruses coinfect the same host cell. The first step in recombination between two viral genomes is the packaging of two different viral RNAs into the same virus particle (Fig. 4.4). This occurs frequently because the virus particles normally contain two copies of the genomic RNA molecules. Stable recombination between the two different viral RNA genomes then occurs when such a
The generation of acutely transforming viruses by such a mechanism would clearly be a rare event, requiring several low probability steps. This is consistent with the rarity of isolation of acutely transforming viruses. It is important to note that any cell gene could be recombined into a viral genome by these mechanisms. However, most such recombinants would lack distinctive new biological properties and consequently would not be detected. In contrast, incorporation of a proto-oncogene is easily detected when the progeny virus has the ability to induce rapid tumor formation and cell transformation. Viral Oncogenes are Altered Versions of Proto-oncogenes How do the proto-oncogenes of normal cells relate in structure and function to the oncogenes of retroviruses? Whereas the viral oncogenes are potent inducers of neoplastic transformation, their progenitors, the proto-oncogenes, are normal components of cellular genomes. Several differences between the viral oncogenes and their proto-oncogene homologs account for these differences in biological activity. The first obvious consequence of incorporation of a proto-oncogene into a retroviral genome is that the resulting oncogene is now expressed as part of the virus. Consequently, the mechanisms that regulated normal expression of the proto-oncogene are abrogated.
For example, the oncogene is transcribed under the control of a strong promoter and enhancer in the viral LTR, whereas transcription of the corresponding proto-oncogene is regulated by its normal cellular transcriptional control sequences. Because the viral LTRs are adapted for high-level transcription of the viral genome, most viral oncogenes are transcribed at much higher levels and can be expressed in different tissues compared with their normal cell counterparts. The viral oncogenes lack introns, which are present in nearly all of the proto- oncogenes. Consequently, any transcriptional regulatory sequences present in introns of the proto-oncogene are lost in the viral oncogenes. In addition, most of the viral oncogenes are truncated versions of their proto-oncogene homologs, which lost either one or both of their normal ends when they were incorporated into viral genomes. Loss of the normal 5' and 3' untranslated regions of the cellular transcripts, and their replacement by viral sequences, can affect both stability and translational efficiency of the viral mRNAs compared with the cellular mRNAs. In addition to these differences in regulation of expression, most of the proteins encoded by oncogenes differ significantly in structure from the corresponding proto- oncogene proteins. For example, as noted in chapter 3, a common genomic organization of acutely transforming viruses results in expression of the oncogene as a fusion protein with viral gag sequences at its amino terminus. In many of these instances, amino acids at the normal amino terminus of the proto- oncogene protein have been deleted. These truncated oncogene proteins are thus very different in structure, and potentially in function, from their proto-oncogene precursors. In many cases, sequences encoding amino acids at the carboxy terminus of the proto- oncogene have also been deleted from the oncogene. Finally, most of the oncogenes have accumulated multiple point mutations relative to their proto-oncogene homologs. Some of these mutations may be neutral. However, because the acutely transforming viruses have been selected for transforming activity during multiple passages both in animals and cell culture, it is to be expected that some of these mutations result in amino acid substitutions that increase transforming potency of the oncogene products. The specific roles of different changes in the altered biological and biochemical activities of oncogenes versus proto-oncogenes will be the subject of later chapters. However, comparison of the RSV src oncogene and proto-oncogene serves as an example to illustrate some of these alterations and their functional significance. Replacement of cellular regulatory sequences with the RSV LTR leads to levels of src mRNA and protein from tenfold to 100-fold higher in RSV-infected chicken embryo fibroblasts than in normal cells. For some proto-oncogenes-for example, mos - increased expression of the normal proto-oncogene protein is sufficient to induce cell transformation. However, although efficient expression of viral src is required for its potent transforming activity, elevated expression of the src proto-oncogene is not by itself sufficient to induce cell transformation. One structural alteration responsible for the transforming activity of viral src is deletion of the nineteen carboxy-terminal amino acids of the proto-oncogene protein. This region of the src proto-oncogene encodes a negative regulatory domain that modulates the protein's tyrosine kinase activity: its deletion from viral src thus releases the oncogene protein from normal controls and enhances its catalytic activity. In addition, RSV src differs from the src proto-oncogene by several point mutations resulting in amino acid
TRANSFEKCIJA – ONKOGENI
Identification of Cellular Oncogenes by Gene Transfer The identification of retroviral oncogenes clearly established the fact that specific genes could induce cell transformation. Moreover, the cellular origin of retroviral oncogenes identified the proto-oncogenes as a group of normal cell genes with potential transforming activity. These considerations raised the question whether nonvirus-induced tumors, including human neoplasms, could be caused by alterations in normal cell genes. Such a hypothesis was also consistent with several aspects of the biology of neoplastic disease, particularly the stable inheritance of the transformed phenotype and the mutagenic activity of many carcinogens. Evaluation of this hypothesis depended on identifying cellular genes capable of inducing transformation and specifically altered in tumor cells. Transformation by Cellular DNA The transforming activity of retroviral oncogenes could be readily assessed because the viruses provided a vehicle for introduction of these genes into normal cells. Analogous direct study of the potential transforming activity of cellular genes is dependent on the ability to assay the biological activity of cellular DNA. Transfer of biologically active DNA in bacteria was first demonstrated in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty in classical experiments that established that DNA was the genetic material. Successful transfer of biologically active eucaryotic cell DNA was achieved by Miroslav Hill and Jana Hillova in 1971. These investigators demonstrated that the DNA of RSV-transformed cells could be transferred to recipient cultures of chicken embryo fibroblasts: the biological activity of the RSV provirus was indicated both by transformation of the recipient cells and by production of infectious RSV. These experiments appeared remarkable in that the activity of a single RSV genome could be detected in the presence of nearly a millionfold excess of unrelated cellular DNA. However, technical refinements in this gene transfer assay have allowed the detection and study of a number of single copy genes of either viral or cellular origin, as long as their biological activity is detectable by virtue of inducing an altered phenotype in appropriate recipient cells. For example, subgenomic proviral fragment containing only the src was able to induce cell transformation. As long as src was associated with transcriptional regulatory sequences that allowed its efficient expression, none of the viral replicative genes was required. The ability to detect viral oncogenes by gene transfer set the stage for identifying biologically active cellular oncogenes. Could cellular genes with the potential of inducing neoplastic transformation be detected in the same gene transfer assays as were used for viral oncogenes? The initial approaches to this question were reported in late 1979 and early 1980. Robert Weinberg and colleagues assayed the transforming potential of DNAs isolated from fifteen lines of chemically transformed rodent cells. They found that DNAs from five of these donor cell lines induced transformation of recipient NIH 3T3 cells (an immortalized but non-transformed mouse cell line). The. Thus, some chemically efficiency of transformation induced by the positive donor DNAs was similar to that induced by DNA of murine sarcoma virus-transformed cells transformed cells contained active cellular oncogenes with biological transforming properties analogous to those of retroviruses.
Experiments in our laboratory (Cooper, J) indicated that normal cells also contained genes with a latent potential for inducing transformation. A low level of transformation was detected following transfer of normal chicken and mouse donor DNAs to recipient NIH 3T3 cell cultures. The efficiency of transformation induced by normal cell DNA was about 100-fold lower than that induced by an active viral oncogene. However, when colonies of cells that had been transformed by normal DNA were grown up and used as DNA donors in a second round of gene transfer, their DNAs were found to induce transformation with high efficiencies, comparable to the transforming efficiency of retroviral oncogenes. These results suggested that some normal cell genes could be activated to function as oncogenes as a consequence of DNA rearrangements that occurred during the process of gene transfer. The implication of these combined results was threefold: (1) some chemically transformed rodent cell lines contained potent transforming oncogenes that had apparently been activated at some stage of the carcinogen-induced transformation process; (2) normal cells contained proto-oncogenes that could be activated experimentally; (3) cellular, in addition to viral, oncogenes could be detected by gene transfer. These experiments thus provided the first approach to studying alterations of specific cellular genes in tumors that had the potential of being causal contributors to the development of neoplasms.
