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Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.
Holland-Frei Cancer Medicine. 6th edition.
Show detailsThe first oncogenes were discovered through the study of retroviruses, RNA tumor viruses whose genomes are reverse-transcribed into DNA in infected animal cells.9 During the course of infection, retroviral DNA is inserted into the chromosomes of host cells. The integrated retroviral DNA, called the provirus, replicates along with the cellular DNA of the host.10 Transcription of the DNA provirus leads to the production of viral progeny that bud through the host cell membrane to infect other cells. Two categories of retroviruses are classified by their time course of tumor formation in experimental animals. Acutely transforming retroviruses can rapidly cause tumors within days after injection. These retroviruses can also transform cell cultures to the neoplastic phenotype. Chronic or weakly oncogenic retroviruses can cause tissue-specific tumors in susceptible strains of experimental animals after a latency period of many months. Although weakly oncogenic retroviruses can replicate in vitro, these viruses do not transform cells in culture.
Retroviral oncogenes are altered versions of host cellular protooncogenes that have been incorporated into the retroviral genome by recombination with host DNA, a process known as retroviral transduction.11 This surprising discovery was made through study of the Rous sarcoma virus (RSV) (Figure 6-1). RSV is an acutely transforming retrovirus first isolated from a chicken sarcoma over 80 years ago by Peyton Rous.12 Studies of RSV mutants in the early 1970s revealed that the transforming gene of RSV was not required for viral replication.13–15 Molecular hybridization studies then showed that the RSV transforming gene (designated v-src) was homologous to a host cellular gene (c-src) that was widely conserved in eukaryotic species.16 Studies of many other acutely transforming retroviruses from fowl, rodent, feline, and nonhuman primate species have led to the discovery of dozens of different retroviral oncogenes (see below and Table 6-1). In every case, these retroviral oncogenes are derived from normal cellular genes captured from the genome of the host. Viral oncogenes are responsible for the rapid tumor formation and efficient in vitro transformation activity characteristic of acutely transforming retroviruses.
Figure 6-1
Retroviral transduction. A ribonucleic acid (RNA) tumor virus infects a human cell carrying an activated src gene (red star). After the process of recombination between retroviral genome and host deoxyribonucleic acid (DNA), the oncogene c-src is incorporated (more...)
Table 6-1
Oncogenes.
In contrast to acutely transforming retroviruses, weakly oncogenic retroviruses do not carry viral oncogenes. These retroviruses, which include mouse mammary tumor virus (MMTV) and various animal leukemia viruses, induce tumors by a process called insertional mutagenesis (Figure 6-2).8 This process results from integration of the DNA provirus into the host genome in infected cells. In rare cells, the provirus inserts near a protooncogene. Expression of the protooncogene is then abnormally driven by the transcriptional regulatory elements contained within the long terminal repeats of the provirus.17,18 In these cases, proviral integration represents a mutagenic event that activates a protooncogene. Activation of the protooncogene then results in transformation of the cell, which can grow clonally into a tumor. The long latent period of tumor formation of weakly oncogenic retroviruses is therefore due to the rarity of the provirus insertional event that leads to tumor development from a single transformed cell. Insertional mutagenesis by weakly oncogenic retroviruses, first demonstrated in bursal lymphomas of chickens, frequently involves the same oncogenes (such as myc, myb, and erb B) that are carried by acutely transforming retroviruses.19–21 In many cases, however, insertional mutagenesis has been used as a tool to identify new oncogenes, including int-1, int-2, pim-1, and lck.22
Figure 6-2
Insertional mutagenesis. A, The process is independent of genes carried by the retrovirus. Retrovirus, for example, mouse mammary tumor virus (MMTV), infects a human cell. The proviral deoxyribonucleic acid (DNA) is integrated into the host genome in (more...)
The demonstration of activated protooncogenes in human tumors was first shown by the DNA-mediated transformation technique.23,24 This technique, also called gene transfer or transfection assay, verifies the ability of donor DNA from a tumor to transform a recipient strain of rodent cells called NIH 3T3, an immortalized mouse cell line (Figure 6-3).25,26 This sensitive assay, which can detect the presence of single-copy oncogenes in a tumor sample, also enables the isolation of the transforming oncogene by molecular cloning techniques. After serial growth of the transformed NIH 3T3 cells, the human tumor oncogene can be cloned by its association with human repetitive DNA sequences. The first human oncogene isolated by the gene transfer technique was derived from a bladder carcinoma.27,28 Overall, approximately 20% of individual human tumors have been shown to induce transformation of NIH 3T3 cells in gene-transfer assays. The value of transfection assay was recently reinforced by the laboratory of Robert Weinberg, which showed that the ectopic expression of the telomerase catalytic subunit (hTERT), in combination with the simian virus 40 large T product and a mutated oncogenic H-ras protein, resulted in the direct tumorigenic conversion of normal human epithelial and fibroblast cells.29 Many of the oncogenes identified by gene-transfer studies are identical or closely related to those oncogenes transduced by retroviruses. Most prominent among these are members of the ras family that have been repeatedly isolated from various human tumors by gene transfer.30,31 A number of new oncogenes (such as neu, met, and trk) have also been identified by the gene-transfer technique.32,33 In many cases, however, oncogenes identified by gene transfer were shown to be activated by rearrangement during the experimental procedure and are not activated in the human tumors that served as the source of the donor DNA, as in the case of ret that was subsequently found genuinely rearranged and activated in papillary thyroid carcinomas.34–36
Figure 6-3
Transfection assay. Deoxyribonucleic acid (DNA) from a tumor (eg, bladder carcinoma) was used to transform a rodent immortalized cell line (NIH 3T3). After serial cycles, DNA from transformed cells was extracted and then inserted into λ vector, (more...)
Chromosomal translocations have served as guideposts for the discovery of many new oncogenes.37,38 Consistently recurring karyotypic abnormalities are found in many hematologic and solid tumors. These abnormalities include chromosomal rearrangements as well as the gain or loss of whole chromosomes or chromosome segments. The first consistent karyotypic abnormality identified in a human neoplasm was a characteristic small chromosome in the cells of patients with chronic myelogenous leukemia.39 Later identified as a derivative of chromosome 22, this abnormality was designated the Philadelphia chromosome, after its city of discovery. The application of chromosome banding techniques in the early 1970s enabled the precise cytogenetic characterization of many chromosomal translocations in human leukemia, lymphoma, and solid tumors.40 The subsequent development of molecular cloning techniques then enabled the identification of protooncogenes at or near chromosomal breakpoints in various neoplasms. Some of these protooncogenes, such as myc and abl, had been previously identified as retroviral oncogenes. In general, however, the cloning of chromosomal breakpoints has served as a rich source of discovery of new oncogenes involved in human cancer.
Oncogenes, protooncogenes, and their functions
Protooncogenes encode proteins that are involved in the control of cell growth. Alteration of the structure and/or expression of protooncogenes can activate them to become oncogenes capable of inducing in susceptible cells the neoplastic phenotype. Oncogenes can be classified into five groups based on the functional and biochemical properties of protein products of their normal counterparts (proto-oncogenes). These groups are (1) growth factors, (2) growth factor receptors, (3) signal transducers, (4) transcription factors, and (5) others, including programmed cell death regulators. Table 6-1 lists examples of oncogenes according to their functional categories.
Growth Factors
Growth factors are secreted polypeptides that function as extracellular signals to stimulate the proliferation of target cells.41,42 Appropriate target cells must possess a specific receptor in order to respond to a specific type of growth factor. A well-characterized example is platelet-derived growth factor (PDGF), an approximately 30 kDa protein consisting of two polypeptide chains.43 PDGF is released from platelets during the process of blood coagulation. PDGF stimulates the proliferation of fibroblasts, a cell growth process that plays an important role in wound healing. Other well-characterized examples of growth factors include nerve growth factor, epidermal growth factor, and fibroblast growth factor.
The link between growth factors and retroviral oncogenes was revealed by study of the sis oncogene of simian sarcoma virus, a retrovirus first isolated from a monkey fibrosarcoma. Sequence analysis showed that sis encodes the beta chain of PDGF.44 This discovery established the principle that inappropriately expressed growth factors could function as oncogenes. Experiments demonstrated that the constitutive expression of the sis gene product (PDGF-β) was sufficient to cause neoplastic transformation of fibroblasts but not of cells that lacked the receptor for PDGF.45 Thus, transformation by sis requires interaction of the sis gene product with the PDGF receptor. The mechanism by which a growth factor affects the same cell that produces it is called autocrine stimulation .46 The constitutive expression of the sis gene product appears to cause neoplastic transformation by the mechanism of autocrine stimulation, resulting in self-sustained aberrant cell proliferation. This model, derived from experimental animal systems, has been recently demonstrated in a human tumor. Dermatofibrosarcoma protuberans (DP) is an infiltrative skin tumor that was demonstrated to present specific cytogenetic features: reciprocal translocation and supernumerary ring chromosomes, involving chromosomes 17 and 22.47,48 Molecular cloning of the breakpoints revealed a fusion between the collagen type Ia1 (COL1A1) gene and PDGF-β gene. The fusion gene resulted in a deletion of PDGF-β exon 1 and a constitutive release of this growth factor.49 Subsequent experiments of gene transfer of DPs genomic DNA into NIH 3T3 cells directly demonstrated the occurrence of an autocrine mechanism by the human rearranged PDGF-b gene involving the activation of the endogenous PDGF receptor.50,51 Another example of a growth factor that can function as an oncogene is int-2, a member of the fibroblast growth factor family. Int-2 is sometimes activated in mouse mammary carcinomas by MMTV insertional mutagenesis.52
Growth Factor Receptors
Some viral oncogenes are altered versions of normal growth factor receptors that possess intrinsic tyrosine kinase activity.53 Receptor tyrosine kinases, as these growth factor receptors are collectively known, have a characteristic protein structure consisting of three principal domains: (1) the extracellular ligand-binding domain, (2) the transmembrane domain, and (3) the intracellular tyrosine kinase catalytic domain (see Figure 6-2). Growth factor receptors are molecular machines that transmit information in a unidirectional fashion across the cell membrane. The binding of a growth factor to the extracellular ligandbinding domain of the receptor results in the activation of the intracellular tyrosine kinase catalytic domain. The recruitment and phosphorylation of specific cytoplasmic proteins by the activated receptor then trigger a series of biochemical events generally leading to cell division.
Because of the role of growth factor receptors in the regulation of normal cell growth, it is not surprising that these receptors constitute an important class of protooncogenes. Examples include erb B, erb B-2, fms, kit, met, ret, ros, and trk. Mutation or abnormal expression of growth factor receptors can convert them into oncogenes.54 For example, deletion of the ligand-binding domain of erb B (the epidermal growth factor receptor) is thought to result in constitutive activation of the receptor in the absence of ligand binding.55 Point mutation in the tyrosine kinase domain or of the extracellular domain and deletion of intracellular regulatory domains can also result in the constitutive activation of receptor tyrosine kinases. Increased expression through gene amplification and abnormal expression in the wrong cell type are additional mechanisms through which growth factor receptors may be involved in neoplasia. The identification and study of altered growth factor receptors in experimental models of neoplasia have contributed much to our understanding of the normal regulation of cell proliferation.
Signal Transducers
Mitogenic signals are transmitted from growth factor receptors on the cell surface to the cell nucleus through a series of complex interlocking pathways collectively referred to as the signal transduction cascade.56 This relay of information is accomplished in part by the stepwise phosphorylation of interacting proteins in the cytosol. Signal transduction also involves guanine nucleotide-binding proteins and second messengers such as the adenylate cyclase system.57 The first retroviral oncogene discovered, src, was subsequently shown to be involved in signal transduction.
Many protooncogenes are members of signal transduction pathways.58,59 These consist of two main groups: nonreceptor protein kinases and guanosine triphosphate (GTP)-binding proteins. The nonreceptor protein kinases are subclassified into tyrosine kinases (eg, abl, lck, and src) and serine/threonine kinases (eg, raf-1, mos, and pim-1). GTP-binding proteins with intrinsic GTPase activity are subdivided into monomeric and heterotrimeric groups.60 Monomeric GTP-binding proteins are members of the important ras family of protooncogenes that includes H-ras, K-ras, and N-ras.61 Heterotrimeric GTP-binding proteins (G proteins) implicated as protooncogenes currently include gsp and gip. Signal transducers are often converted to oncogenes by mutations that lead to their unregulated activity, which in turn leads to uncontrolled cellular proliferation.62
Transcription Factors
Transcription factors are nuclear proteins that regulate the expression of target genes or gene families.63 Transcriptional regulation is mediated by protein binding to specific DNA sequences or DNA structural motifs, usually located upstream of the target gene. Transcription factors often belong to multigene families that share common DNA-binding domains such as zinc fingers. The mechanism of action of transcription factors also involves binding to other proteins, sometimes in heterodimeric complexes with specific partners. Transcription factors are the final link in the signal transduction pathway that converts extracellular signals into modulated changes in gene expression.
Many protooncogenes are transcription factors that were discovered through their retroviral homologs.64 Examples include erb A, ets, fos, jun, myb, and c-myc. Together, fos and jun form the AP-1 transcription factor, which positively regulates a number of target genes whose expression leads to cell division.65,66 Erb A is the receptor for the T3 thyroid hormone, triiodothyronine.67 Protooncogenes that function as transcription factors are often activated by chromosomal translocations in hematologic and solid neoplasms.68 In certain types of sarcomas, chromosomal translocations cause the formation of fusion proteins involving the association of EWS gene with various partners and resulting in an aberrant tumor-associated transcriptional activity. Interestingly, a role of the adenovirus E1A gene in promoting the formation of fusion transcript fli1/ews in normal human fibroblasts was recently reported.69 An important example of a protooncogene with a transcriptional activity in human hematologic tumors is the c-myc gene, which helps to control the expression of genes leading to cell proliferation.70 As will be discussed later in this chapter, the cmyc gene is frequently activated by chromosomal translocations in human leukemia and lymphoma.
Programmed Cell Death Regulation
Normal tissues exhibit a regulated balance between cell proliferation and cell death. Programmed cell death is an important component in the processes of normal embryogenesis and organ development. A distinctive type of programmed cell death, called apoptosis, has been described for mature tissues.71 This process is characterized morphologically by blebbing of the plasma membrane, volume contraction, condensation of the cell nucleus, and cleavage of genomic DNA by endogenous nucleases into nucleosome-sized fragments. Apoptosis can be triggered in mature cells by external stimuli such as steroids and radiation exposure. Studies of cancer cells have shown that both uncontrolled cell proliferation and failure to undergo programmed cell death can contribute to neoplasia and insensitivity to anticancer treatments.
The only protooncogene thus far shown to regulate programmed cell death is bcl-2. Bcl-2 was discovered by the study of chromosomal translocations in human lymphoma.72,73 Experimental studies show that bcl-2 activation inhibits programmed cell death in lymphoid cell populations.74 The dominant mode of action of activated bcl-2 classifies it as an oncogene. The bcl-2 gene encodes a protein localized to the inner mitochondrial membrane, endoplasmic reticulum, and nuclear membrane. The mechanism of action of the bcl-2 protein has not been fully elucidated, but studies indicate that it functions in part as an antioxidant that inhibits lipid peroxidation of cell membranes.75 The normal function of bcl-2 requires interaction with other proteins, such as bax, also thought to be involved in the regulation of programmed cell death (Figure 6-4). It is unlikely that bcl-2 is the only apoptosis gene involved in neoplasia although additional protooncogenes await identification.
Figure 6-4
Effect of bcl-2 activity on the control of the cell life. In the presence of BAX only, the cell goes to apoptosis; bcl-2 regulates the cycle of the cell by the interaction with BAX. When bcl-2 is overexpressed, the cell cycle is deregulated and the apoptosis (more...)
- Discovery and identification of oncogenes - Holland-Frei Cancer MedicineDiscovery and identification of oncogenes - Holland-Frei Cancer Medicine
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