Renal cell carcinomas (RCC) commonly retain wild-type but functionally inactive p53, which is repressed by an unknown dominant mechanism. To help reveal this mechanism, we screened a diverse chemical library for small molecules capable of restoring p53-dependent transactivation in RCC cells carrying a p53-responsive reporter. Among the compounds isolated were derivatives of 9-aminoacridine (9AA), including the antimalaria drug quinacrine, which strongly induced p53 function in RCC and other types of cancer cells. Induction of p53 by these compounds does not involve genotoxic stress and is mediated by suppression of NF-B activity. In contrast to agents that target I B kinase 2, 9AA and quinacrine can effectively suppress both basal and inducible activities of NF-B, representing inhibitors of a previously undescribed type that convert NF-B from a transactivator into a transrepressor, leading to accumulation of inactive nuclear complexes with unphosphorylated Ser-536 in the p65͞RelA subunit. p53 function in RCC can be restored by ectopic expression of a superrepressor of I B as effectively as by 9AA-derived compounds. These findings suggest that the complete or partial repression of p53 observed in many tumors can be the result of constitutive activation of NF-B. The results demonstrate, in principle, the possibility to kill cancer cells selectively through simultaneous inhibition of NF-B and activation of p53 by a single small molecule and suggest anticancer applications for the well known antimalaria drug quinacrine.anticancer treatment ͉ apoptosis ͉ chemical library ͉ quinacrine T he protein p53 controls genetic stability and reduces the risk of cancer through induction of growth arrest or apoptosis in response to DNA damage or deregulation of proto-oncogenes (1). The efficacy of p53 as a tumor-preventing factor is reflected by the high frequency of p53 loss, in at least 50% of human tumors, due to inactivating mutations (2). Understanding the mechanisms of functional inactivation of wild-type p53 in human tumors, for example, by overexpression of natural antagonists of p53, Mdm2, or the viral protein E6, helps to define prospective targets for treating cancer by restoring p53 function (3).We have recently shown that renal cell carcinomas (RCC), the most frequent and least curable type of kidney cancer, maintain wild-type but functionally inactive p53 (4). The mechanism of p53 repression in RCC is dominant, and therefore ''druggable,'' and different from that of all reported cases of p53 repression in tumors, suggesting the existence of an as-yet-unknown molecular target for restoring p53 function in cancer. As an approach to finding such factor(s), we have isolated a set of compounds that can restore p53 function in RCC and strongly activate p53 in many other types of cancer cells. Among the most effective compounds from this set were derivatives of 9-aminoacridine (9AA), including an old-known antimalaria drug quinacrine (QC). Analysis of the molecular mechanisms of action of 9AA and QC showed that p53 activat...

Prokaryotes of the genus Mycoplasma are the smallest cellular organisms that persist as obligate extracellular parasites. Although mycoplasma infection is known to be associated with chromosomal instability and can promote malignant transformation, the mechanisms underlying these phenomena remain unknown. Since persistence of many cellular parasites requires suppression of apoptosis in host cells, we tested the effect of mycoplasma infection on the activity of the p53 and nuclear factor (NF)-jB pathways, major mechanisms controlling programmed cell death. To monitor the activity of p53 and NF-jB in mycoplasma-infected cells, we used a panel of reporter cell lines expressing the bacterial b-galactosidase gene under the control of p53-or NF-jB-responsive promoters. Cells incubated with media conditioned with different species of mycoplasma showed constitutive activation of NF-jB and reduced activation of p53, common characteristics of the majority of human tumor cells, with M. arginini having the strongest effect among the species tested. Moreover, mycoplasma infection reduced the expression level and inducibility of an endogenous p53-responsive gene, p21 waf1 , and inhibited apoptosis induced by genotoxic stress. Infection with M. arginini made rat and mouse embryo fibroblasts susceptible to transformation with oncogenic H-Ras, whereas mycoplasma-free cells underwent irreversible p53-dependent growth arrest. Mycoplasma infection was as effective as shRNA-mediated knockdown of p53 expression in making rodent fibroblasts permissive to Rasinduced transformation. These observations indicate that mycoplasma infection plays the role of a p53-suppressing oncogene that cooperates with Ras in cell transformation and suggest that the carcinogenic and mutagenic effects of mycoplasma might be due to inhibition of p53 tumor suppressor function by this common human parasite.

Inactivation of p53 function, which frequently occurs in tumors, can significantly modulate tumor cell sensitivity to radiation and chemotherapeutic drugs. However, in addition to acting on malignant cells, anticancer agents act on the cells of tumor stroma, causing activation of a p53 response. The effect of this response on treatment outcome has been the subject of the present study. Tumors with p53-deficient stroma were generated using mouse tumorigenic packaging cells that produce a p53 inhibitory retrovirus, encoding a dominantnegative p53 mutant. Tumors maintaining wild-type p53 in their stroma were formed by cells of similar origin but deficient in retroviral production due to the deletion of the packaging signal in the retroviral vector. Comparison of these tumor models, differing only in p53 status of their stromas, showed that tumors with p53-deficient stroma were significantly more sensitive to experimental chemotherapy and radiotherapy. A similar effect was achieved when anticancer treatment was combined with pharmacologic suppression of p53 by the cyclic form of pifithrin A, a small-molecule inhibitor of p53. Potentiation of the anticancer effect of chemotherapy and radiotherapy by p53 suppression in the tumor stroma is likely to be due to the increased sensitivity of p53-deficient endothelium to genotoxic stress as shown both in cell culture and in experimental tumors. Thus, reversible pharmacologic suppression of p53 may be a viable approach to improving anticancer treatment via an enhanced antiangiogenic effect of chemotherapy and radiotherapy.