Recessive Oncogenic Mutations, Tumor Suppressors
Transformation studies like those used to find the DNA change associ-ated with a bladder cancer have shown that only about 10% of tumors can be associated with dominant mutations. Similarly, fusion of trans-formed tumor cells and normal cells shows that in most cases, the genotype of the normal cells can suppress the oncogene being expressed in the transformed cells. If the fusion cells possess an unstable karyotype and lose chromosomes, then the descendants of these fused cells can again express the transformed phenotype. Thus, oncogenesis can derive from two different pathways. The first is dominant mutations that generate transformed cells independent of the other cellular genes, and the second is a product of two events. One of these is a mutation that would produce uncontrolled cell growth were it not for the presence of another cellular regulator, and the second is the loss of the regulator.
The simplest example of the two-step pathway is a mutation in a growth factor such that it no longer prevents uncontrolled growth. Ordinarily the mutation to inability to repress growth is not revealed because the mutation is recessive and the nonmutated copy of the gene product from the other copy of the chromosome suppresses growth. If, however, the nonmutated copy is lost by recombination or mutated, then the growth inhibition ceases. Retinoblastoma is a form of cancer that develops in young children. Most cases of retinoblastoma are familial, and the pattern of inheritance indicates that the gene involved is not on a sex chromosome. The patterns of retinoblastoma develop-ment suggest that in addition to inheriting a defective gene, a second event must occur in the stricken individuals. We now know that this second event frequently is recombination of the defect from one chro-mosome onto the homologous chromosome in a way that leaves both chromosomes defective for the critical gene. Gene conversion as dis-cussed earlier could do this.
Through good luck and hard work the gene whose defect gives rise to retinoblastoma was cloned. The first step in the process came from the finding that an appreciable fraction of retinoblastoma cell lines possess a gross defect or deletion in part of chromosome 13. This suggests that the gene in question lies on chromosome 13. With this as a starting point it was possible to clone the gene. The original idea was that perhaps a clone could be found containing some of the DNA that is deleted from chromosome 13 in some of the retinoblastoma lines. By chromosome walking, it might then be possible to clone the desired retinoblastoma gene.
A library of about 1,000 clones of segments of chromosome 13 was available. These were then screened against a large panel of retinoblas - toma lines to see if any of the clones contained DNA absent from the transformed cell lines. One clone indeed contained DNA absent from two of the retinoblastoma lines. Best of all, it was complementary to an RNA that is synthesized in normal cells, but not in retinoblastoma cells. This suggests that the protein could, in fact, be the gene for the suppres-sor of retinoblastoma.
Further study of the retinoblastoma protein required sensitive assays for its presence. Two approaches were used to make antibodies against the supposed retinoblastoma suppressor protein. Part of the cloned and sequenced gene was fused to the trpE gene and a fusion protein was synthesized in E. coli, purified by making use of the trp tag, and used to immunize animals for antibody synthesis (Fig. 23.11). The second approach was to synthesize chemically peptides corresponding to sev-eral regions of the presumed protein and use these to immunize ani-mals.
With antibodies against the protein, the synthesis and properties of the retinoblastoma protein could be studied. It is indeed synthesized in normal cells, but frequently is missing in retinoblastoma cells. The protein is phosphorylated, and has a molecular weight of about 105 kD. This is the molecular weight of one of the cellular proteins to which the adenovirus E1a protein, the papillomavirus E7 protein, and the SV40 T antigen bind. Since the E1a-associated protein is phosphorylated like the Rb protein, and since phosphorylation of proteins is relatively rare, it seemed possible that the retinoblastoma suppressor protein and the E1a-associated protein were one and the same. Indeed, they were. Both antibody specificity and proteolysis patterns confirm the identity. Thus it appears that the normal role of the retinoblastoma suppressor gene is to counteract the positive activity of some other cell growth inducing pathway. Hence one of the steps in transformation by adenovirus, papillomavirus, and SV40 is to inactivate suppressor proteins rather than to synthesize a dominant growth-inducing gene in the cells.
In as many as 50% of human tumors, the p53 protein is found to have been altered. Thus, this protein appears to play a key role in regulating cell growth. What is the nature of the regulation expressed by the p53 protein? Is it required at all times for proper cell growth or regulation of differentiation? A conceptually simple way to answer these questions is to attempt to make a homozygous mouse deficient in p53 protein. Such mice turn out to develop normally, but they are highly likely to develop cancers before they are as old as six months. The tumors are found in many different tissues. Further study has shown that p53 prevents the onset of DNA replication in a cell possessing damaged DNA or a broken chromosome. By blocking DNA synthesis, and allowing time for DNA repair, the spontaneous mutation frequency in cells is greatly reduced.
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