Wild-type p53 protein has many properties consistent with its being the product of a tumour suppressor gene. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation as well as in mediating growth arrest by growth-inhibitory cytokines. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.

No abstract

A method is described for the cloning of normal mouse "mast" cells in tissue culture in a soft agar medium. The colonies contain cells in different stages of differentiation. It was shown that a colony can be initiated by a single colony forming unit, and that colonies are formed as a result of cell multiplication.Cell suspensions from adult spleen gave about 3 colonies per lo5 cells seeded. A re-cloning of these colonies gave about 3 colonies per lo3 cells seeded.The frequency of colonies from SWR mice was higher with adult spleen than with adult thymus. No such colonies were obtained with adult lymph node cells.The formation of colonies was shown to require the presence of an embryo cell feeder layer. Since the feeder layers were seeded underneath the agar, the results indicate that the substance(s) required for the growth and differentiation of "mast" cells can pass through agar.

Wild-type p53 is a tumor-suppressor gene that encodes a shortlived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy. W ild-type p53 is a tumor-suppressor gene that is mutated in more than 50% of human cancers (reviewed in refs. 1 and 2). The tumor-suppressing activity of wild-type p53 is a result of its ability to induce growth arrest (1, 2) or apoptosis (3-6). Wild-type p53 is a short-lived protein (7), and its cellular level is controlled by the rate of its degradation in the proteasomes. This degradation is mainly regulated by association with the E3 ubiquitin ligase protein Mdm-2 that ubiquitinates p53 and targets it to the proteasomes (8, 9). After ␥-irradiation or other types of stress, p53 and Mdm-2 undergo posttranslational modifications that diminish their association, leading to diminished p53 degradation (reviewed in ref. 2). Mdm-2 expression is induced by wild-type p53, thus creating a negative feedback loop that maintains p53 at low levels (10, 11). Unlike wild-type p53 in normal cells, mutant p53 accumulates in cancer cells because of its inability to induce expression of Mdm-2 (12) and its formation of ternary complexes with Mdm-2 and heat shock protein 90 (hsp90), which prevents mutant p53 degradation (13). After treatment with hsp90 inhibitors, these complexes are disrupted (13), resulting in degradation of the mutant p53 protein (13-16). However, hsp90 inhibitors such as geldanamycin and radicicol were reported not to cause a similar degradation of wild-type p53 in some cancer cells (14-16).We have shown previously that dicoumarol, an inhibitor of NAD(P)H: quinone oxidoreductase 1 (NQO1), caused degradation of wild-type p53 in various cell types and suppressed its abili...

The tumor suppressor gene wild-type p53 encodes a labile protein that accumulates in cells after different stress signals and can cause either growth arrest or apoptosis. One of the p53 target genes, p53-inducible gene 3 (PIG3), encodes a protein with significant homology to oxidoreductases, enzymes involved in cellular responses to oxidative stress and irradiation. This fact raised the possibility that cellular oxidation-reduction events controlled by such enzymes also may regulate the level of p53. Here we show that NADH quinone oxidoreductase 1 (NQO1) regulates p53 stability. The NQO1 inhibitor dicoumarol caused a reduction in the level of both endogenous and ␥-irradiation-induced p53 in HCT116 human colon carcinoma cells. This reduction was prevented by the proteasome inhibitors MG132 and lactacystin, suggesting enhanced p53 degradation in the presence of dicoumarol. Dicoumarol-induced degradation of p53 also was prevented in the presence of simian virus 40 large T antigen, which is known to bind and to stabilize p53. Cells overexpressing NQO1 were resistant to dicoumarol, and this finding indicates the direct involvement of NQO1 in p53 stabilization. NQO1 inhibition induced p53 degradation and blocked wild-type p53-mediated apoptosis in ␥-irradiated normal thymocytes and in M1 myeloid leukemic cells that overexpress wild-type p53. Dicoumarol also reduced the level of p53 in its mutant form in M1 cells. The results indicate that NQO1 plays an important role in regulating p53 functions by inhibiting its degradation.