In this diagram, activating mutations are represented by solid red boxes, inactivating mutations by hollow red boxes. (A) Oncogenes act in a dominant manner: a gain-of-function mutation in a single copy of the cancer-critical gene can drive a cell toward cancer. (B) Mutations in tumor suppressor genes, on the other hand, generally act in a recessive manner: the function of both alleles of the cancer-critical gene must be lost to drive a cell toward cancer. Although in this diagram the second allele of the tumor suppressor gene is inactivated by mutation, it is often inactivated instead by loss of the second chromosome. Not shown is the fact that mutation of some tumor suppressor genes can have an effect even when only one of the two gene copies is damaged.
Different Searches for Oncogenes Converged on the Same Gene—Ras
In an attempt to answer the above question, other researchers searched directly for oncogenes in the genomes of human cancer cells. They did this by searching for DNA fragments from cancer cells that could provoke uncontrolled proliferation when introduced into noncancerous cell lines. As tester cells for the assay, cell lines derived from mouse fibroblasts were used. These cells had been previously selected for their ability to proliferate indefinitely in culture, and they are thought to already contain alterations that take them part of the way toward malignancy. For this reason, the addition of a single oncogene can sometimes be enough to produce a dramatic effect.
When DNA was extracted from the human tumor cells, broken into fragments, and introduced into the cultured cells, occasional colonies of abnormally proliferating cells began to appear in the culture dish. These cells showed a transformed phenotype, outgrowing the untransformed cells in the culture and piling up in layer upon layer (see Figure 20–11). Each colony was a clone originating from a single cell that had incorporated a DNA fragment that drove cancerous behavior. This fragment, which carried markers of its human origin, could be isolated from the transformed cultured mouse cells. And once isolated and sequenced, it could be recognized: it contained a human version of a gene already known from study of a retrovirus that caused tumors in rats—an oncogene called v-Ras.
The newly discovered oncogene was clearly derived by mutation from a normal human gene, one of a small family of proto-oncogenes called Ras. This discovery in the early 1980s of the same oncogene in human tumor cells and in an animal tumor virus was electrifying. The implication that cancers are caused by mutations in a limited number of cancer-critical genes transformed our understanding of the molecular biology of cancer.
As discussed in Chapter 15, normal Ras proteins are monomeric GTPases that help transmit signals from cell-surface receptors to the cell interior (see Movie 15.7). The Ras oncogenes isolated from human tumors contain point mutations that create a hyperactive Ras protein that cannot shut itself off by hydrolyzing its bound GTP to GDP. Because this makes the protein hyperactive, its effect is dominant—that is, only one of the cell’s two gene copies needs to change to have an effect. One or another of the three human Ras family members is mutated in perhaps 30% of all human cancers. Ras genes are thus among the most important of all cancer-critical genes.
Figure 20–18 summarizes the types of accidents that can convert a proto-oncogene into an oncogene. (1) A small change in DNA sequence such as a point
Figure 20–18 The types of accidents that can convert a proto-oncogene into an oncogene.
extracellular domain binding of growth factor of receptor triggers intracellular signaling mutation or deletion may produce a hyperactive protein when it occurs within a protein-coding sequence, or lead to protein overproduction when it occurs within a regulatory region for that gene. (2) Gene amplification events, such as those that can be caused by errors in DNA replication, may produce extra gene copies; this can lead to overproduction of the protein. (3) A chromosomal rearrangement— involving the breakage and rejoining of the DNA helix—may either change the protein-coding region, resulting in a hyperactive fusion protein, or alter the control regions for a gene so that a normal protein is overproduced.