Modulation of the vitamin D3 response by cancer-associated mutant p53.

The p53 gene is mutated in many human tumors. Cells of such tumors often contain abundant mutant p53 (mutp53) protein, which may contribute actively to tumor progression via a gain-of-function mechanism. We applied ChIP-on-chip analysis and identified the vitamin D receptor (VDR) response element as overrepresented in promoter sequences bound by mutp53. We report that mutp53 can interact functionally and physically with VDR. Mutp53 is recruited to VDR-regulated genes and modulates their expression, augmenting the transactivation of some genes and relieving the repression of others. Furthermore, mutp53 increases the nuclear accumulation of VDR. Importantly, mutp53 converts vitamin D into an antiapoptotic agent. Thus, p53 status can determine the biological impact of vitamin D on tumor cells.

A cell chip for sequential imaging of individual non-adherent live cells reveals transients and oscillations.

Advances in molecular cell biology, medical research, and drug development are driving a growing need for technologies that enable imaging the dynamics of molecular and physiological processes simultaneously in numerous non-adherent living cells. Here we describe a platform technology and software--the CKChip system--that enables continuous, fluorescence-based imaging of thousands of individual living cells, each held at a given position ("address") on the chip. The system allows for sequential monitoring, manipulation and kinetic analyses of the effects of drugs, biological response modifiers and gene expression in both adherent and non-adherent cells held on the chip. Here we present four specific applications that demonstrate the utility of the system including monitoring kinetics of reactive oxygen species generation, assessing the intracellular enzymatic activity, measuring calcium flux and the dynamics of target cell killing induced by conjugated cytotoxic T-lymphocytes. We found large variations among individual cells in the overall amplitude of their response to stimuli, as well as in kinetic parameters such as time of onset, initial rate and decay of the response, and frequency and amplitude of oscillations. These variations probably reflect the heterogeneity of even cloned cell populations that would have gone undetected in bulk cell measurements. We demonstrate the utility of the system in providing kinetic parameters of complex cellular processes such as Ca++ influx, transients and oscillations in numerous individual cells. The CKChip opens up new opportunities in cell-based research, in particular for acquiring fluorescence-based, kinetic data from multiple, individual non-adherent cells.

Mutant p53 attenuates the SMAD-dependent transforming growth factor beta1 (TGF-beta1) signaling pathway by repressing the expression of TGF-beta receptor type II.

Both transforming growth factor beta (TGF-beta) and p53 have been shown to control normal cell growth. Acquired mutations either in the TGF-beta signaling pathway or in the p53 protein were shown to induce malignant transformation. Recently, cross talk between wild-type p53 and the TGF-beta pathway was observed. The notion that mutant p53 interferes with the wild-type p53-induced pathway and acts by a "gain-of-function" mechanism prompted us to investigate the effect of mutant p53 on the TGF-beta-induced pathway. In this study, we show that cells expressing mutant p53 lost their sensitivity to TGF-beta1, as observed by less cell migration and a reduction in wound healing. We found that mutant p53 attenuates TGF-beta1 signaling. This was exhibited by a reduction in SMAD2/3 phosphorylation and an inhibition of both the formation of SMAD2/SMAD4 complexes and the translocation of SMAD4 to the cell nucleus. Furthermore, we found that mutant p53 attenuates the TGF-beta1-induced transcription activity of SMAD2/3 proteins. In searching for the mechanism that underlies this attenuation, we found that mutant p53 reduces the expression of TGF-beta receptor type II. These data provide important insights into the molecular mechanisms that underlie mutant p53 "gain of function" pertaining to the TGF-beta signaling pathway.

Mutant p53 enhances nuclear factor kappaB activation by tumor necrosis factor alpha in cancer cells.

Mutations in the p53 tumor suppressor are very frequent in human cancer. Often, such mutations lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting gain of function. We now report that cancer-associated mutant p53 can augment the induction of nuclear factor kappaB (NFkappaB) transcriptional activity in response to the cytokine tumor necrosis factor alpha (TNFalpha). Conversely, down-regulation of endogenous mutant p53 sensitizes cancer cells to the apoptotic effects of TNFalpha. Analysis of human head and neck tumors and lung tumors reveals a close correlation between the presence of abundant mutant p53 proteins and the constitutive activation of NFkappaB. Together, these findings suggest that p53 mutations may promote cancer progression by augmenting NFkappaB activation in the context of chronic inflammation.

Inactivation of myocardin and p16 during malignant transformation contributes to a differentiation defect.

Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFbeta required for TGFbeta-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16ink4a/Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.

Transactivation of the EGR1 gene contributes to mutant p53 gain of function.

Tumor-associated mutants of the p53 tumor suppressor protein exert biological activities compatible with an oncogenic gain of function. To explore the underlying molecular mechanism, we performed microarray analysis, comparing p53-null cells to mutant p53-expressing cells. One of the genes up-regulated in the presence of mutant p53 was EGR1, a transcription factor implicated in growth control, apoptosis, and cancer. EGR1 induction by various types of stress is markedly augmented in cells expressing mutant p53. Moreover, chromatin immunoprecipitation analysis indicates that mutant p53 is physically associated with the EGR1 promoter. Functional assays indicate that induction of EGR1 by mutant p53 contributes to enhanced transformed properties and resistance to apoptosis. We propose that EGR1 is a significant contributor to mutant p53 gain of function.

Mutant p53 gain of function: repression of CD95(Fas/APO-1) gene expression by tumor-associated p53 mutants.

Tumor-associated mutant forms of p53 can exert an antiapoptotic gain of function activity, which probably confers a selective advantage upon tumor cells harboring such mutations. We report that mutant p53 suppresses the expression of the CD95 (Fas/APO-1) gene, encoding a death receptor implicated in a variety of apoptotic responses. Moderate (40-50%) downregulation of CD95 mRNA and surface protein expression by mutant p53 correlates with partial protection against CD95-dependent cell death. Excess mutant p53 represses the transcriptional activity of the CD95 promoter, with the extent of repression varying among different tumor-associated p53 mutants. Furthermore, mutant p53 protein binds the CD95 promoter in vitro, in a region distinct from the one implicated in tight interactions of the CD95 gene with wild-type p53. Hence, the CD95 promoter is likely to be a direct target for downregulation by mutant p53. This activity of mutant p53 may contribute to its gain of function effects in oncogenesis.