Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1 Functions Permits Ras-Induced Anchorage-Independent Growth in Telomerase-Immortalized Human Fibroblasts

A state of irreversible growth arrest, commonly referred to as replicative senescence, has been documented in many normal human cells after a period of rapid proliferation in cell culture (20, 68). Since the proliferative period seems to be limited by the number of elapsed cell divisions, rather than chronological time, and indefinite proliferation (referred to as immortalization) depends on the accumulation of genetic lesions, it has been proposed that the senescence response may have evolved as a defense against the development of malignancy (8, 11). Indeed, most tumor cells bear mutations in the p53 and/or Rb pathways, both of which have been implicated in the establishment of replicative senescence (3, 60). A number of significant differences have been documented between human and rodent cells in the regulation of the senescence response. Many rodent cell types either express telomerase or can spontaneously activate telomerase after a relatively limited culture period (46). Rodent cells are also more susceptible to malignant transformation. For example, normal mouse embryo fibroblasts are easily transformed by the combined expression of an activated oncogene, such as Ha-RasG12V (referred to hereafter simply as Ras), and an immortalizing function, such as Myc (29), adenovirus E1a (53), simian virus 40 (SV40) large T antigen (LT) (38), or human papillomavirus E6 or E7 (32, 45). These viral proteins all have the ability to interfere with the normal functions of the cellular p53 and/or retinoblastoma (Rb) proteins. The importance of the p53 and Rb pathways in preventing tumor formation was further confirmed by mouse knockout studies, which showed that mouse embryo fibroblasts derived from p53−/− (22), p19 Arf−/− (24), or Rb/p107/p130−/− (55) animals could be transformed by activated Ras alone. In contrast, both the senescence and transformation mechanisms are more stringently regulated in human cells (11, 56). The great majority of normal human cells do not express human telomerase (hTERT) activity (27), and immortalization is an extremely rare event. Likewise, Myc and Ras fail to transform primary human cells on their own (10, 11, 17, 81). More recent work has shown that Ras actually elicits a senescence-like arrest in both primary human and rodent cells (58). This somewhat unexpected finding can be viewed as yet another defense mechanism against inappropriate oncogenic signaling present in normal cells. In rodent cells, Ras-induced arrest can be eliminated by lesions in either the p53 or Rb pathways (58); however, in human cells, both pathways must be compromised (18, 44, 58, 75). Furthermore, bypassing Ras-induced arrest is not sufficient for full oncogenic transformation of human cells (18, 39, 44). Transformation of human foreskin fibroblasts, mammary epithelial cells, or keratinocytes has been shown to require the additional expression of SV40 small antigen (ST) (12, 18), which interferes with the function of protein phosphatase 2A (PP2A) (43, 80). We have previously used gene targeting to knock out the p21 (6) and p53 (7) genes in normal, nonimmortalized human fibroblasts and used the resulting cell lines to study both replicative and induced senescence states. We presented data indicating that p53, p21, and Rb act sequentially and constitute the major pathway for establishing growth arrest in response to telomere attrition (75). p21 appears to be the major effector downstream of p53 responsible for both the establishment of replicative senescence (6) and p14ARF-induced premature senescence (75). In the studies reported here, we have expanded this genetic system by constructing additional isogenic cell lines to investigate the roles that p53, p21, and p16 play in premature senescence and transformation induced by oncogenic Ras. These experiments were stimulated by our desire to deduce clear-cut genetic functions for the steps required to convert a normal human cell into a malignant one. Most of the prior work in this area was predicated on the expression of viral transforming proteins, which are known to target multiple cellular proteins. We found that abolition of p21 and p16 functions was sufficient to bypass Ras-induced growth arrest and elicit limited anchorage-independent growth in response to Ras transformation. Loss of p53 gave the same results as loss of p21, confirming that p21 is a major p53 effector. Somewhat surprisingly, loss of p53 and p16 function did not mimic the introduction of LT in transformation elicited by the combination of Ras and ST, indicating that LT may target additional cellular function(s). In the presence of LT and ST, Myc cooperated to a limited extent with Ras to enhance anchorage-independent growth but did not further enhance in vivo tumorigenicity. Finally, in the absence of Ras, Myc cooperated with ST to elicit aggressive anchorage-independent growth, but the resulting cells did not form tumors in nude mice.