ARCONEL

Theories were formulated in the 1950s to explain why the incidence of cancer increased with age. Both Nordling (1953) and Armitage & Doll (1954) proposed that this was due to the accumulation of a succession of genetic mutations. Nordling recognized that such mutations were most likely to occur in naturally proliferating cells. Whilst Armitage & Doll suggested that the effect of a carcinogen, was dependent not only on the cell type exposed, but also upon the time of life at which the carcinogen was encountered. Ashley (1969) attempted to refine these multi-stage theories of carcinogenesis, and concluded that two major steps were involved: initiation and promotion. It seemed likely that a number of discrete structural and functional changes were

involved in both initiation and promotion of tumour growth. In 1971 Knudson put forward his two hit hypothesis for the development of inherited malignancies. An epidemiological study of the rare childhood malignancy retinoblastoma, revealed that tumours in both eyes were more frequent in patients with the dominantly inherited form of the disease. Knudson concluded that such individuals had inherited a germline mutation, the "first hit", which predisposed them to develop retinoblastoma. The "second hit", required for tumour development, was acquired somatically, in cells of the developing retina. In patients with no family history of the disease, both the first and second hits would occur in the retina. Statistically this hypothesis was in agreement with the observed frequency of bilateral tumours and early age of onset, in familial retinoblastoma.

Conclusive evidence that cancer has a genetic basis was therefore provided by the occurrence of inherited cancers, the observation of chromosomal damage in the cells of certain cancers and the association between mutagens and

carcinogenesis. As the precise nature of the genetic mechanisms responsible for the development of cancers were studied, it became apparent that two distinct classes of genes were involved. There were oncogenes, whose activation resulted in the promotion of carcinogenesis, and tumour suppressors or anti-oncogenes, whose inactivation allowed tumourigenesis to proceed (Land, et al, 1983a, Nowell, 1988), What also became apparent from experimental evidence, was that more than one genetic change was required for expression of the cancer phenotype, this was in agreement with the early theories put forward.

1.41. Oncogenes.

The existence of genes with the ability to promote tumourigenesis was alluded to from the study of tumourigenic retroviruses, and DNA transfection experiments. The first oncogene to be identified was v-src, isolated from the Rous sarcoma virus (Martin, 1970) . This retrovirus, which can induce sarcomas in vivo and transform fibroblasts in vitro, was found to contain a gene {v-src) encoding a tyrosine kinase (pp60src)(Brugge & Erikson, 1977, Hunter & Sefton, 1980) . Closer examination of the v-src gene, revealed that it was derived from a normal gene of the chicken, which is the host of the Rous sarcoma virus (Stehelin, et ai, 197 6) . This therefore led to the conclusion that a normal cellular gene (proto-oncogene), had the potential to exhibit strong transforming properties if activated, in this case by transduction into a retrovirus. A number of oncogenes, and their cellular homologues have been identified from the study of retroviruses, including Harvey-ras (H-ras), Kirsten-ras

(K-ras) and c-myc (Land, efc ai, 1983a).

C ellular oncogenes were also i d e n t i f i e d from transfection studies, in which DNA from cancer cells was used to transfect normal cells in culture. Foci of transformed cells were then examined, and dominantly acting oncogenes were detected (Shih, et ai, 1979a) . As with the retroviral studies, the genes identified also had cellular homologues. Although these assays were initially performed using DNA from chemically induced cancer cell lines, it soon became apparent that similar results were obtainable from tumours of various cell types and from different species (Shih, et ai, 1981).

It has become increasingly evident that proto-oncogenes and their products constitute a heterogeneous group of genes and p eptides. Products of different proto-oncogenes have been reported in the cytoplasm, associated with the nuclear and plasma membranes and in the nucleus. The biological functions of these gene products are frequently associated with cell cycle regulation, and include protein kinases, GTPases and transcription factors (Land, et al, 1983a, Bishop, 1985). However, it would appear that there is only a limited group of cellular genes with transforming potential, as the same oncogenes have been isolated both from different viruses, and from transfection studies (Eva, et al, 1982, D e r , et al , 1982).

The transition from pro to-oncogene to oncogene m.ay occur either as a result of aberrant expression of the gene or by expression of a mutated product (Land, et al, 1983a, Bishop, 1985) . Although a large number of genes with an oncogenic potential have now been identified, it would still appear to be the case that activation of a single proto-oncogene is insufficient for tumourigenesis (Land, et al, 1983b, Weinberg, 1989) . Furthermore, it has been revealed that c- myc and K-ras, amongst others, can co-operate with each other to produce a transformed phenotype (Weinberg, 1985, Land, et al, 1986) . Thus, demonstrating that different oncogenes may have distinct and complementary effects on the normal cellular phenotype.

1,42. Tumour Suppressor Genes (Anti-oncogenes).

In contrast to the tumour promoting properties of oncogenes, tumour suppressor genes appear to be involved in restraining tumourigenesis. The existence of such genes was s u g g e s t e d by cell hybrid experiments, c y t o g e n e t i c observations and the occurrence of familial cancers

(Marshall, 1991).

In the cell hybrid experiments, it was observed that fusions between normal cells and certain tumour cell lines, resulted in non-tumourigenic hybrids. Thus, the normal cells were able to suppress the tumour phenotype (Stanbridge, 197 6) . These experiments were extended to fusions between different tumour cell lines. It was found that whilst hybrids between carcinoma cell lines of epithelial origin

remained tumourigenic, complementation occurred in those between lines of differing cellular origin, resulting in a non-tumourigenic hybrid (Weissman & Stanbridge, 1983) . Therefore, it was concluded that tumourigenicity was essentially recessive, or dependent on gene dosage, and that because complementation only occurred between lines of different cell type, a variety of genes existed with the ability to suppress neoplastic expression (Weissman & Stanbridge, 1983) .

Chromosomal instability and non-random loss of specific regions was observed by karyotypic analysis in numerous tumours (Mitelman, 1985). The loss of chromosomal material was in direct agreement with the concept of genes acting as tumour suppressors, in that loss or inactivation of at least one copy of the gene allowed tumourigenesis to proceed. Furthermore, the occurrence of consistent chromosomal alterations in certain cancers, ultimately 1 e 8 to the identification and isolation of a number of tumour suppressor genes by positional cloning. The first tumour suppressor gene to be cloned was that associated with retinoblastoma. Following reports of cytogenetic deletions of chromosome 13ql4 in retinoblastomas (Balaban, at al , 1982), Cavenee, at al (1983) pioneered the use of polymorphic DNA markers in the search for chromosomal deletions in tumour DNA. U s i n g markers known to map into the correct region, allele losses were observed in the DNA of tumour samples, one of the markers used was esterase-D. A chromosomal walk from the esterase-D region ultimately le& to isolation of the retinoblastoma susceptibility gene (Rb) (Lee, at ai, 1987a) , The tumour suppressor genes involved in the familial cancer syndromes Wilms' tumour (WT), neurofibromatosis type 1 (NFl) and familial adenomatous polyposis (FAP) have now all been cloned (Rose, at ai, 1990, Wallace, at ai, 1990, Viskochill, at ai, 1990, Joslyn, at ai, 1991, Kinzler, at ai, 1991b). In addition, the genes deleted in colorectal cancer (DCC) and mutated in colorectal cancer (MCC), have also been cloned (Fearon, at ai, 1990, Kinzler, at ai, 1991a) . Whilst the chromosomal regions associated with Wilms' tumour, FAP, MCC and DCC were identified by cytogenetics (Francke, at ai, 1979, Herrera, at ai, 1986, Reichmann, at ai, 1981), NFl was

localized to 17qll.2 by linkage analysis in affected families (Goldgar, et al, 1989) . However, reports of chromosomal translocations in two NFl patients hastened identification of the NFl gene. The specific chromosomal regions associated with a number of other malignancies have now been identified (Green, 1988), including 3p21 for small cell carcinoma of the lung (SCLC) (Wang-Peng, et a l , 1982) and llql3 for multiple endocrine neoplasia type 1 (MENl) (Larson, et a l , 1988, Thakker, et al, 1989). But the genes responsible have yet to be cloned.

The tumour suppressor gene whose involvement has been reported in the largest number of different human malignancies is p53 (Levine, et al, 1991) , Although p53 was first identified through its ability to form an oligometric complex with the large T-antigen of simian virus 40 (SV40) (Lane & Crawford, 1979) . And despite reports of co­ operation between the ras oncogene and p53 in neoplastic transformation (Parada, et al 1984, Eliyahu, et al, 1984) , subsequent studies have shown p53 to be a genuine tumour suppressor gene (Finlay, et al, 1988). The p53 gene encodes a 393 amino acid nuclear phosphoprotein (Lane & Crawford, 1979, Lamb & Crawford, 1986) , which appears to bind to specific DNA sequences and act as a transcriptional activator, presumably on genes involved in negative regulation of cell growth (Raycroft, et a l , 1990, Kern, et a l , 1992) . This normal function is disrupted in many human malignancies, both by mutations within conserved regions of

the p53 gene, and by deletions encompassing the gene (Nigro, et a l , 1989, Hollstein, et al , 1991) . In common with the tumour suppressor genes identified through their association with particular inherited malignancies, p53 germline mutations have been detected in the cancer-prone condition, Li-Fraumeni syndrome, and in other non-Li-Fraumeni cancer families (Malkin, et a l , 1990, Srivastava, et a l , 1990, Prosser, et al, 1992).

Although the biological properties of the tumour suppressor genes so far identified are generally diverse, the product of the retinoblastoma susceptibility gene (pRB) shares several features with p53 . The pRB peptide is also a nuclear phosphoprotein, with DNA binding properties, and it

forms complexes with oncoproteins of DNA tumour viruses (Lee, et al, 1987b, Whyte, et al, 1988, Dyson, et al , 1989) . In addition, deletions and mutations of the Rb gene have been reported in many cancers, other than those clinically associated with retinoblastoma, notably breast cancer (T'Ang, et a l , 1988, Lee, et al, 1988) and small cell carcinoma of the lung (Harbour, et al, 1988) . It seems likely that the W i l m s ' tumour gene is also involved in transcriptional regulation, as it encodes a zinc finger protein (Call, et a l , 1990) . The products of the NFl, MCC and APC genes are all cytoplasmic. The NFl protein probably acts as a GTPase activating protein, in signal transduction (Martin, et al, 1990), whilst MCC and APC may well be involved in maintaining the normal architecture of the cell, as they both include domains capable of forming coiled coil proteins (Bourne, 1991, Groden, et al, 1991, Kinzler, et al , 1991b) . Finally, it would appear that the product of the DCC gene may be involved in cell-cell interaction, as it shares homology with various cell surface glycoproteins (Fearon, et al, 1990).

Confirmation that candidate genes are truly tumour suppressors, may be provided by transfection assays or from the creation of transgenic animals. The normal Rb and p53 genes have been used in both types of assay. Soon after the Rb gene was cloned, constructs of the normal Rb gene were made and transfected into retinoblastoma and osteosarcoma cell lines. The neoplastic phenotype of the cells was suppressed in both cases (Huang, et a l , 1988) . Similar results were obtained when a normal p53 gene was introduced into a colorectal cancer cell line (SW837) , from which one normal p53 gene had been deleted and the remaining allele was mutated (Baker, et al, 1990b). Transgenic mice homozygous for a null p53 gene, were found to be developmentally normal, but prone to a variety of spontaneous neoplasms (Donehower, et al, 1992) . Homozygosity for null Rb mutations, resulted in non-viable embryos, apparently due to the failure of certain neuronal and haematopoietic cells to undergo terminal differentiation (Lee, et a l , 1992, Jacks, et a l , 1992) . Interestingly, although some heterozygotes for the mutated Rb gene, were susceptible to pituitary tumours, retinoblastomas

were not detected in any of the animals examined (Jacks, et al, 1992, Lee, et al , 1992).