Retroviruses

Retroviruses have their genetic information encoded in RNA and replicate through DNA by coding for an enzyme known as reverse transcriptase (p. 17), which makes a double-stranded DNA copy of the viral RNA. This DNA intermediate integrates into the host cell genome, allowing the appropriate proteins to be manufactured, resulting in repackaging of new progeny virions.

Naturally occurring retroviruses have only the three genes necessary to ensure replication: gag, encoding the structural proteins for the core antigens; pol, coding for reverse transcriptase; and env, the gene for the glycoprotein envelope proteins (Figure 14.1). Study of the virus responsible for the transmissible tumor in chickens, the so-called Rous sarcoma virus, identified a fourth gene that results in transformation of cells in culture, a model for malignancy in vivo. This viral gene, which transforms the host cell, is known as an oncogene.

FIGURE 14.1 Model for acquisition of transforming ability in retroviruses. A, Normal retroviral replication. B, The Rous sarcoma virus has integrated near a cellular oncogene. The transforming ability of this virus is due to the acquired homolog of the cellular oncogene, v-src. C, A defective transforming virus carries an oncogene similar to src but is defective in the structural genes, (e.g., Moloney murine sarcoma virus, which carries mos).

Identification of Oncogenes at Chromosomal Translocation Breakpoints

Chromosome aberrations are common in malignant cells, which often show marked variation in chromosome number and structure. Certain chromosomes seemed to be more commonly involved and it was initially thought that these changes were secondary to the transformed state rather than causal. This attitude changed when evidence suggested that chromosomal structural changes, often translocations (p. 44), resulted in rearrangements within or adjacent to proto-oncogenes. It has been found that chromosomal translocations can lead to novel chimeric genes with altered biochemical function or level of proto-oncogene activity. There are numerous examples of both types, of which chronic myeloid leukemia is an example of the former and Burkitt lymphoma an example of the latter.

Chronic myeloid leukemia

In 1960, investigators in Philadelphia were the first to describe an abnormal chromosome in white blood cells from patients with chronic myeloid leukemia (CML). The abnormal chromosome, referred to as the Philadelphia, or Ph¹, chromosome, is an acquired abnormality found in blood or bone marrow cells but not in other tissues from these patients. The Ph¹ is a tiny chromosome that is now known to be a chromosome 22 from which long arm material has been reciprocally translocated to and from the long arm of chromosome 9 (Figure 14.2), i.e., t(9;22)(q34;q11). This chromosomal rearrangement is seen in 90% of those with CML. This translocation has been found to transfer the cellular ABL (Abelson) oncogene from chromosome 9 into a region of chromosome 22 known as the breakpoint cluster, or BCR, region, resulting in a chimeric transcript derived from both the c-ABL (70%) and the BCR genes. This results in a chimeric gene expressing a fusion protein consisting of the BCR protein at the amino end and ABL protein at the carboxy end, which is associated with transforming activity.

FIGURE 14.2 Karyotype from a patient with chronic myeloid leukemia showing the chromosome 22 (arrow) or Philadelphia chromosome, which has material translocated to the long arm of one of the number 9 chromosomes (arrow).

Oncogene Amplification

Proto-oncogenes can also be activated by the production of multiple copies of the gene or what is known as gene amplification, a mechanism known to have survival value when cells encounter environmental stress. For example, when leukemic cells are exposed to the chemotherapeutic agent methotrexate, the cells acquire resistance to the drug by making multiple copies of the gene for dihydrofolate reductase, the target enzyme for methotrexate.

Gene amplification can increase the number of copies of the oncogene per cell up to several hundred times, leading to greater amounts of the corresponding oncoprotein. In mammals the amplified sequence of DNA in tumor cells can be recognized by the presence of small extra chromosomes known as double-minute chromosomes or homogeneously staining regions of the chromosomes. These changes are seen in approximately 10% of tumors and are often present more commonly in the later rather than the early stages of the malignant process.

Amplification of specific proto-oncogenes appears to be a feature of certain tumors and is frequently seen with the MYC family of genes. For example, N-MYC is amplified in approximately 30% of neuroblastomas, but in advanced cases the proportion rises to 50%, where gene amplification can be up to 1000-fold. Human small cell carcinomas of the lung also show amplification of MYC, N-MYC, and L-MYC.

Amplification of ERB-B2, MYC, and cyclin D1 is a feature in 20% of breast carcinomas, where it has been suggested that it correlates with a number of well-established prognostic factors such as lymph node status, estrogen and progestogen receptor status, tumor size, and histological grade.

Detection of Oncogenes by DNA Transfection Studies

The ability of DNA from a human bladder carcinoma cell line to transform a well established mouse fibroblast cell line called NIH3T3, as demonstrated by the loss of contact inhibition of the cells in culture, or what is known as DNA transfection, led to the discovery of the human sequence homologous to the ras gene of the Harvey murine sarcoma virus. The human RAS gene family consists of three closely related members, H-RAS, K-RAS, and N-RAS. The RAS proteins are closely homologous to their viral counterparts and differ from one another only near the carboxy termini. Oncogenicity of the ras proto-oncogenes has been shown to arise by acquisition of point mutations in the nucleotide sequence. In approximately 50% of colorectal cancers and 95% of pancreatic cancers, as well as in a proportion of thyroid and lung cancers, a mutation in a ras gene can be demonstrated. The RAS gene family has been shown to be the key pathway (RAS-MAPK) in neurofibromatosis type 1 (p. 298) and the Noonan/cardio-facio-cutaneous/Costello syndromes, all of which demonstrate some increased risk of tumor formation.

DNA transfection studies have also led to identification of other oncogenes that have not been demonstrated through retroviral studies. These include MET (hereditary papillary renal cell carcinoma), TRK, MAS, and RET (multiple endocrine neoplasia type 2, see Tables 14.5, 14.9).

Growth Factors

The transition of a cell from G₀ to the start of the cell cycle (p. 39) is governed by substances called growth factors. Growth factors stimulate cells to grow by binding to growth factor receptors. The best known oncogene that acts as a growth factor is the v-SIS oncogene, which encodes part of the biologically active platelet-derived growth factor B subunit. When v-SIS oncoprotein is added to the NIH 3T3 cultures, the cells are transformed, behaving like neoplastic cells; that is, their growth rate increases and they lose contact inhibition. In vivo they form tumors when injected into nude mice. Oncogene products showing homology to fibroblast growth factors include HST and INT-2, which are amplified in stomach cancers and in malignant melanomas, respectively.

Growth Factor Receptors

Many oncogenes encode proteins that form growth factor receptors, with tyrosine kinase activity possessing tyrosine kinase domains that allow cells to bypass the normal control mechanisms. More than 40 different tyrosine kinases have been identified and can be divided into two main types: those that span the cell membrane (growth factor receptor tyrosine kinases) and those located in the cytoplasm (non-receptor tyrosine kinases). Examples of tyrosine kinases include ERB-B, which encodes the epidermal growth factor receptor, and the related ERB-B2 oncogene. Mutations, rearrangements, and amplification of the ERB-B2 oncogene result in ligand-independent activation, which has been associated with cancer of the stomach, pancreas, and ovary. Mutations in KIT occur in the hereditary gastrointestinal stromal tumor syndrome. These oncogenes are not activated by translocation (as in Burkitt lymphoma) but rather by point mutations. When germline or inherited, the mutations are not lethal, nor are they sufficient by themselves to cause carcinogenesis. In the case of MET (located on chromosome 7), the papillary renal cell carcinoma tumors are trisomic for chromosome 7 and two of the three copies of MET are mutant. A ratio of one mutant to one wild-type copy of MET is not sufficient for carcinogenesis, but a 2:1 ratio is.

Intracellular Signal Transduction Factors

Two different types of intracellular signal transduction factor have been identified, proteins with GTPase activity and cytoplasmic serine threonine kinases.

DNA-Binding Nuclear Proteins

The FOS, JUN, and ERB-A oncogenes encode proteins that are specific transcription factors that regulate gene expression by activating or suppressing nearby DNA sequences. The function of MYC and related genes remains uncertain but appears to be related to alterations in control of the cell cycle. The MYC and MYB oncoproteins stimulate cells to progress from the G₁ into the S phase of the cell cycle (p. 39). Their overproduction prevents cells from entering a prolonged resting phase, resulting in persistent cellular proliferation.

Cell-Cycle Factors

Cancer cells can increase in number by increased growth and division, or accumulate through decreased cell death. In vivo, most cells are in a non-dividing state. Progress through the cell cycle (p. 39) is regulated at two points: one in G₁ when a cell becomes committed to DNA synthesis in the S phase, and another in G₂ for cell division in the M (mitosis) phase, through factors known as cyclin-dependent kinases. Abnormalities in regulation of the cell cycle through growth factors, growth factor receptors, GTPases or nuclear proteins, or loss of inhibitory factors lead to activation of the cyclin-dependent kinases, such as cyclin D1, resulting in cellular transformation with uncontrolled cell division. Alternatively, loss of the factors that lead to normal programmed cell death, a process known as apoptosis (p. 85), can result in the accumulation of cells through prolonged cell survival as a mechanism of development of some tumors. Activation of the bcl-2 oncogene through chromosomal rearrangements is associated with inhibition of apoptosis, leading to certain types of lymphoma.

Signal Transduction and Phakomatoses

Phakomatosis derives from the Greek phakos, meaning ‘lentil’ (in this context ‘lentil-shaped object’), and originally referred to three diseases that included scattered benign lesions: neurofibromatosis, tuberous sclerosis, and von Hippel-Lindau disease. To this list has now been added nevoid basal cell carcinoma (Gorlin) syndrome, Cowden disease, familial adenomatous polyposis, Peutz-Jegher syndrome, and juvenile polyposis. The genes for all of these conditions are now known and are normally active within intracellular signal transduction, and their protein products are tumor suppressors.

‘Two-Hit’ Hypothesis

In 1971, Knudson carried out an epidemiological study of a large number of cases of both types of Rb and advanced a ‘two-hit’ hypothesis to explain the occurrence of this rare tumor in patients with and without a positive family history. He proposed that affected individuals with a positive family history had inherited one non-functional gene that was present in all cells of the individual, known as a germline mutation, with the second gene at the same locus becoming inactivated somatically in a developing retinal cell (Figure 14.5). The occurrence of a second mutation was likely given the large number of retinal cells, explaining the autosomal dominant pattern of inheritance. This would also explain the observation that in hereditary Rb the tumors were often bilateral and multifocal. In contrast, in the non-heritable or sporadic form, two inactivating somatic mutations would need to occur independently in the same retinoblast cell (see Figure 14.5), which was much less likely to occur, explaining the fact that tumors in these patients were often unilateral and unifocal, and usually occurred at a later age than in the hereditary form. Hence, although the hereditary form of Rb follows an autosomal dominant pattern of inheritance, at the molecular level it is recessive because a tumor occurs only after the loss of both alleles.

FIGURE 14.5 Retinoblastoma and Knudson’s ‘two-hit’ hypothesis. All cells in the hereditary form (A) have one mutated copy of the gene, RB1 (i.e., the mutation is in the germline). In the non-hereditary form (B) a mutation in RB1 arises as a post-zygotic (somatic) event some time early in development. The retinoblastoma tumor occurs only when both RB1 genes are mutated—i.e., after a(nother) somatic event, which is more likely to be earlier in life in the hereditary form compared with the non-hereditary form; it is also more likely to give rise to bilateral and multifocal tumors.

It was also recognized, however, that approximately 5% of children presenting with Rb had other physical abnormalities along with developmental concerns. Detailed cytogenetic analysis of blood samples from these children revealed some of them to have an interstitial deletion involving the long arm of one of their number 13 chromosome pair. Comparison of the regions deleted revealed a common ‘smallest region of overlap’ involving the sub-band 13q14 (Figure 14.6). The detection of a specific chromosomal region involved in the etiology of these cases of Rb suggested that it could also be the locus involved in the autosomal dominant familial form of Rb. Family studies using a polymorphic enzyme, esterase D, which had previously been mapped to that region, rapidly confirmed linkage of the hereditary form of Rb to that locus.

FIGURE 14.6 Two homologs of chromosome 13 from a patient with retinoblastoma showing an interstitial deletion of 13q14 in the right-hand homolog, as indicated.

Loss of Heterozygosity

Analyses of the DNA sequences in this region of chromosome 13 in the peripheral blood and in Rb tumor material from children who had inherited the gene for Rb showed them to have loss of an allele at the Rb locus in the tumor material, known as loss of heterozygosity (LOH), or sometimes as loss of constitutional heterozygosity. An example of this is shown in Figure 14.7, A