The tumorigenic potential and cell growth characteristics of p53-deficient cells are equivalent in the presence or absence of Mdm2

Abstract

The Mdm2 oncoprotein forms a complex with the p53 tumor suppressor protein and inhibits p53-mediated regulation of heterologous gene expression. Recently, Mdm2 has been found to bind several other proteins that function to regulate cell cycle progression, including the E2F-1͞DP1 transcription factor complex and the retinoblastoma tumor-suppressor protein. To determine whether Mdm2 plays a role in cell cycle control or tumorigenesis that is distinct from its ability to modulate p53 function, we have examined and compared both the in vitro growth characteristics of p53-deficient and Mdm2͞p53-deficient fibroblasts, and the rate and spectrum of tumor formation in p53-deficient and Mdm2͞p53-deficient mice. We find no difference between p53-deficient fibroblasts and Mdm2͞p53-deficient fibroblasts either in their rate of proliferation in culture or in their survival frequency when treated with various genotoxic agents. Cell cycle studies indicate no difference in the ability of the two cell populations to enter S phase when treated with DNA-damaging agents or nucleotide antimetabolites, and p53-deficient fibroblasts and Mdm2͞p53-deficient fibroblasts exhibit the same rate of spontaneous immortalization following long-term passage in culture. Finally, p53-deficient mice and Mdm2͞p53-deficient mice display the same incidence and spectrum of spontaneous tumor formation in vivo. These results demonstrate that deletion of Mdm2 has no additional effect on cell proliferation, cell cycle control, or tumorigenesis when p53 is absent.

Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras

“…It should be noted that the mutation present in the INK4a/ARF locus, deletion of exons 2 and 3, eliminates the function of p16 INK4a (Serrano et al, 1996), but its e ects on the function of p19 ARF have not been unambiguously determined (see Palmero et al, 1998). As previously reported, wild-type MEFs formed colonies very ine ciently compared to p53 7/7 MEFs, INK4a/ ARF D2,3 MEFs, or the immortal cell line of murine ®broblasts NIH3T3 (Harvey et al, 1993;Jones et al, 1996;Serrano et al, 1996) (see Table 1). Interestingly, the colony-formation e ciency of p21 7/7 MEFs was very similar to that of wt MEFs (see Table 1), suggesting that p21 7/7 cells have not acquired the same proliferative advantages as p53 7/7 or INK4a/ ARF D2,3 MEFs.…”

“…We interpret that in the particular case of p21 7/7 culture number 21 immortalization occurred at a very early passage, thus occluding the senescent phase. As expected, none of the primary p53 7/7 or INK4a/ ARF D2,3 MEFs manifested senescence (see Figure 1) (Harvey et al, 1993;Tsukada et al, 1993;Jones et al, 1996;Serrano et al, 1996). These results indicate that absence of p21 does not result in full immortalization of murine ®broblasts.…”

“…Consequently, it is possible to estimate the impact of a speci®c mutation on the proliferative potential by comparing colony-formation e ciencies (see for example Harvey et al, 1993;Jones et al, 1996;Serrano et al, 1996). We have measured the colonyforming e ciencies of primary mouse embryo fibroblasts (MEFs) derived from wild-type mice or from mice carrying null mutations at the p21, p53, or INK4a/ARF loci, respectively (Table 1).…”

“…This extended proliferative life ends in a senescence-like arrest (Rogan et al, 1995;Medclaf et al, 1996;Gallimore et al, 1997) or in an apoptotic crisis (Bond et al, 1995). In the case of murine ®broblasts, p53 must play also an important role in senescence because cells derived from p53-de®cient mice do not enter senescence (Harvey et al, 1993;Tsukada et al, 1993;Jones et al, 1996), and because spontaneous immortalization of murine ®broblasts is frequently associated to mutation of p53 (Harvey and Levine, 1991;Rittling and Denhardt, 1992;Kamijo et al, 1997). The activation of p53 at senescence might be mediated by the tumor suppressor p19 ARF , which is encoded by the same genetic locus as p16 INK4a (reviewed in Haber, 1997) and is upregulated during senescence (Kamijo et al, 1997).…”

p53-mediated accumulation of hypophosphorylated pRb after the G1 restriction point fails to halt cell cycle progression

“…For example, the Mdm2 protein was ®rst reported to bind and inactivate p53 (Momand et al, 1992). Mdm2 was later shown to interact with pRb and pRb-regulated E2F complexes as well (Martin et al, 1995;Xiao et al, 1995), although recent studies suggest that deletion of Mdm2 has no additional e ect on cell proliferation, cell cycle control or tumorigenesis when p53 is absent (Jones et al, 1996;McMasters et al, 1996). p53 and pRb participate in several cell cycle checkpoint responses that contribute to maintaining genetic stability.…”