BASE, FRAMESHIFT, AMPLIFICATION, AND INSERTIONAL OR SUBSTITUTIONAL MUTATIONS IN SPECIFIC GENES IN SOMATIC CELLS AS FACTORS IN NEOPLASTIC DEVELOPMENT
Somatic Mutations in Proto- and Cellular Oncogenes
With the advent, during the last two decades, of a clearer understanding of primary target genes for the action of carcinogenic agents, the search for mutations within specific genes has ad- vanced dramatically. Prior to the development of modern methods of DNA sequencing and the use of the polymerase chain reaction (PCR), analysis of specific mutations in oncogenes was determined by indirect methods followed by specific sequencing. This procedure was first exem- plified by demonstrating that the Ha-ras-1 proto-oncogene bearing a single base substitution in the 12th codon resulted in the substitution of valine for glycine in the mutated gene product. Initial evidence for this alteration came from studies of transfection of DNA from the human bladder carcinoma cell line, T24, transfected into mouse 3T3 cells, which resulted in their mor- phological transformation (Chapter 9; Tabin et al., 1982). The transfection assay became a method for the rapid screening of “activated” proto-oncogenes in neoplasms. When such activa- tion was discovered, analysis of mutations in candidate proto- and cellular oncogenes could be determined by more detailed nucleic acid sequence methods.
It has now become apparent that the Ha-ras-1 proto-oncogene is only one of a family of genes coding for G proteins whose function is involved in the mediation of signal transduction pathways (Chapter 7). Several other ras proto- and cellular oncogenes have been studied (Tables 6.3 and 6.4), but the most extensive investigations into the structure and effects of mutations on the protein function have been carried out on the Ha-ras-1 proto-oncogene and its product. Fig- ure 6.1 shows a ribbon drawing of the Ha-ras-1 (p21) proto-oncogene product showing the loops (λ1-10) and helices (shown as multilined bands). The positions of important amino acid residues are labeled for orientation, as noted in the figure. Most of the known mutations in the ras gene family occur in codons 12, 13, and 61 (Bos, 1989). While the details of how amino acid substitutions at these positions affect the function of the p21 molecule have not been completely clarified (Bokoch and Der, 1993), such mutations cause a loss of the ability of the p21 protein to become inactivated, thus resulting in a constant stimulation of the signal transduction path- way(s) resulting in cell growth, cell function, or cell differentiation on a constitutive basis (cf. Kiaris and Spandidos, 1995). Through such a mechanism, mutations at codons 12, 13, or 61 would thus confer a proliferative advantage to the cell bearing such mutations, allowing its se- lective overgrowth and overfunction in a general population of its normal peers.
Since the ras proto- and cellular oncogene family has been most extensively studied, most investigations have observed specific mutations in one member of this family. Table 6.3 lists examples of human and animal neoplasms in which 50% or more of the tumors exhibited a mu- tated oncogene. This is not meant to imply that other lesions exhibited no evidence of such mu- tations. Rather, one finds that in the majority of human neoplasms as well as many animal
tumors similar mutations may be found. In humans an average of 5% to 10% of all neoplasms exhibit mutation(s) in at least one oncogene studied (Nishimura and Sekiya, 1987). However, several human neoplasms including some sarcomas (Castresana et al., 1993), adrenal neoplasms (Moul et al., 1993), and neuroendocrine neoplasms of the lung (Wagner et al., 1993) are not associated specifically with mutations in ras oncogenes. While spontaneous hepatomas arising in the B6C3F1 strain exhibited a high incidence (65%) of mutations in the c-Ha-ras gene, other mouse strains having a lower incidence of spontaneous liver tumors, including the C57BL/6J and BALB/c strains, possess few if any such mutations in hepatomas arising spontaneously (Buchmann et al., 1991). Interestingly, Sugio et al. (1994) indicated that mutations in the c-Ki- ras gene occurred relatively late in the development of lung cancer, since early lesions did not exhibit these mutagenic changes. c-Ki-ras mutations may be found in normal colonic mucosa in the human (Ronai et al., 1994) as well as in very early lesions prior to the appearance of frank cancer (Smith et al., 1994).
Proto- and cellular oncogenes other than those of the ras family may also be found in a variety of human neoplasms, as exemplified in Table 6.4. While many of the changes noted in the table were reported only in single cases, the variety of mutation type and loci involved exem- plify the extent of such changes in the human. A number of examples of amplification of proto- oncogenes have been described. Similarly, mutations in proto-and cellular oncogenes other than the ras gene family have also been reported in lower animals including c-myb (Shen-Ong and
Figure 6.1 Schematic representation of the three-dimensional structure of the ras protein. In the figure, loops and several important amino acid residues are labeled, including glycine-12 and glutamine-61. (After Pai et al., 1989, with permission of the authors and publisher.)
Wolff, 1987), the neu activated in rat neoplasms of the central nervous system (Perantoni et al.,1994), and the Spi-1 cellular oncogene in murine erythroleukemia (Moreau-Gachelin, 1994).