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It was initially thought that activation of a single oncogene such as Ras would result in tumorigenesis; however, this conclusion was based on studies in which immortalized cells were used as the target cells in transformation assays (1). Thus it was significant when in 1983 Newbold and Overell reported that an activated Ras gene failed to transform normal fibroblasts unless the fibroblasts were first "immortalized by carcinogens" (2). On the basis of these and similar findings, Newbold and Overell and several other groups insightfully hypothesized that Ras gene activation was only one of a number of mutations necessary for the "progression to malignancy" (2, 3). This was borne out when it was found that two different oncogenes that failed to transform normal fibroblasts on their own could cooperate to transform the fibroblasts when expressed together (4, 5). These findings provided the first in vitro evidence that tumorigenesis is a multistep process requiring the activation of several oncogenes.
We now also understand that the process of tumorigenesis not only involves mutations that activate oncogenes; it also requires several mutations that disrupt the activities of proteins that function to suppress tumorigenesis (6). Among these are the cyclin-dependent kinase (cdk) inhibitor pl6^sup INK4a^ and the paradigm tumor-suppressor protein p53. Indeed, the p16^sup INK4a^ and p53 genes are among the most frequently mutated genes in human cancers. When activated by inappropriate proliferative signals or damage to the cell's genome, the proteins expressed by these genes halt cell-cycle progression. Thus, these proteins prevent the proliferation of cells that had developed the potential to undergo transformation. p16^sup INK4a^ arrests cells by inhibiting cyclin D/cdk activity. Cyclin D/cdk activity is necessary to maintain the tumor-suppressor retinoblastoma protein (pRb) in its inactive form, so inhibition of cyclin D/cdk results in arrest mediated by active pRb. The activation of p53 results in increased expression of p21 ^sup WAFl^, a cdk inhibitor that induces cell-cycle arrest. Hence, if p16INK4a or p53 are inactivated, important constraints on inappropriate proliferation are lost.
In the context of our current understanding of the function of tumor-suppressor proteins, previous findings regarding a seemingly paradoxical effect of Ras can be rationalized. Expression of oncogenic forms of Ras will transform most immortalized cell lines;...