p53 functional loss by mutation and p53 antagonizing proteins during tumor development

Abstract: p53 is the most common target for genetic alterations in human cancers. Inactivation of wild type p53 function by point mutation, deletion or complexing with antagonizing proteins such as MDM2 and DNA tumor viral oncoproteins, is frequently found in human and rodent tumors. p53-induced cell cycle arrest and apoptosis in response to DNA damage, hypoxia and oncogene activation are probably fundamental mechanisms for prevention of tumor development. The possible involvement of MDM2 and p21WAF1 gene alterations in the development of nasopharyngeal carcinoma (NPC) was investigated in a series of 46 tumors by Southern blotting. All 46 tumors showed normal size p21WAF1 hybridizing bands, indicating that gross structural alteration of this gene is infrequent in NPC. Likewise, most tumors showed a normal size MDM2 hybridizing band at normal intensity. However, one tumor had a 10 to 12-fold more intense normal size MDM2 fragment, indicative of MDM2 gene amplification. MDM2 overexpression due to gene amplification may thus represent an infrequent p53 -inactivating mechanism in NPC. Unlike SV40 LT, polyoma virus (Py) LT protein complexes only with RB, not with p53. Therefore, we tested whether p53 mutation is required for the development of polyoma virus-induced tumors. Eight of fifteen polyoma virus-induced sarcomas showed p53 mutation or deletion. Another six sarcomas expressed low levels of wt p53. One tumor expressed high levels of wt p53 protein and overexpressed MDM2 mRNA. Mitomycin C treatment indicated that p53-mediated transcriptional activation was unaltered in these tumors. These results demonstrate that p53 mutation is frequently but not invariably involved in polyoma virusinduced tumorigenesis. We also addressed whether polyoma virus can block p53 function indirectly. Polyoma virus mT or st alone, or together, were transfected into J3D mouse T lymphoma cells carrying temperature-sensitive p53 (tsp53). Induction of wt p53 by temperature shift to 32OC triggers both GI cell cycle arrest and apoptosis in parental J3D-ts p53 cells. In contrast, J3D-ts p53 cells co-expressing mT and st showed only a weak GI cell cycle arrest response at 32OC. Moreover, FACS analysis revealed that nearly half of the mT-expressing cells, 30% of the st-expressing cells, and a majority of the cells co-expressing mT and st were resistant to p53-induced apoptosis. The P13 kinase inhibitor wortmannin partially abrogated the protective effect of mT but not st on p53-induced apoptosis, indicating that mT prevents p53-induced apoptosis through the P13 kinase signal transduction pathway. Our results thus establish a mechanism for polyoma virus-mediated inhibition of p53 function and reinforce the need of small DNA tumor viruses to block p53-dependent apoptosis. Established ascites tumor cells grow under highly crowded, virtually anoxic conditions. We wished to determine whether the conversion of relatively well-vascularized solid mouse tumors into freely growing ascitic cell variants favors cell with inactivated p53. DNA sequence analysis showed that all solid tumors carried exclusively wild type p53, whereas most of the ascites tumors harbored mutant p53. The naturally occurring alternatively spliced p53 (p53as) mRNA was detected in all solid tumors, but not in five of the eight ascites tumors. Our findings indicate that conversion of solid into ascites tumors favors the selection of cell variants with mutated p53 and of cells that lack the alternatively spliced form of p53. p53 inactivation and increased telomerase activity are both associated with tumor development. Therefore, we asked if p53 was able to regulate telomerase. We found that activation of either exogenous ts p53 in BL41 Burkitt lymphoma cells or endogenous wt p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of the telomerase catalytic subunit (hTERT) mRNA expression, independently of the induction of the p53 target gene p2l. Activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Thus, p53 appears to downregulate hTERT by interfering with Sp1-mediated transactivation. Inhibition of hTERT/telomerase expression may represent a novel mechanism of p53-dependent tumor suppression.