P53 In Tumor Cells Research Articles

Transient depletion of p53 followed by transduction of c-Myc and K-Ras converts ovarian stem-like cells into tumor-initiating cells

Although the existence of tumor-initiating cells (T-ICs) in several types of human cancer has been documented, the contribution of somatic stem cells to the development of T-ICs has remained unclear. Here, we show that normal mouse ovary contains epithelial cell adhesion molecule (EpCAM)-expressing stem-like cells that possess the ability to differentiate into cytokeratin 8 (CK8)-expressing epithelial progeny cells. Furthermore, RNA interference-mediated transient depletion of the tumor suppressor p53 followed by retrovirus-mediated transfer of c-Myc and K-Ras oncogenes in EpCAM-expressing ovarian stem-like cells resulted in the generation of ovarian T-ICs. The established ovarian T-ICs gave rise to hierarchically organized lethal tumors in vivo and were able to undergo peritoneal metastasis. Finally, subsequent RNA interference-mediated knockdown of p53 in tumor cells triggered the expansion of EpCAM-expressing stem-like tumor cells and induced further tumor growth. These data reveal a role for p53 in the development and expansion of ovarian stem-like tumor cells and subsequent malignant progression.

Restoring p53 active conformation by zinc increases the response of mutant p53 tumor cells to anticancer drugs

Absence of p53 expression or expression of mutant p53 (mtp53) are common in human cancers and are associated with increased cancer resistance to chemo- and radiotherapy. Therefore, significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. We previously reported that, in HIPK2 knockdown background, p53 undergoes misfolding with inhibition of DNA binding and transcriptional activities that correlate with increased chemoresistance, and that zinc rescues wild-type p53 activity. Zinc has a crucial role in the biology of p53, in that p53 binds to DNA through a structurally complex domain stabilized by zinc atom. In this study, we explored the role of zinc in p53 reactivation in mutant p53-expressing cancer cells. We found that zinc re-established chemosensitivity in breast cancer SKBR3 (expressing R175H mutation) and glioblastoma U373MG (expressing R273H mutation) cell lines. Biochemical studies showed that zinc partly induced the transition of mutant p53 protein (reactive to conformation-sensitive PAb240 antibody for mutant conformation) into a functional conformation (reactive to conformation-sensitive PAb1620 antibody for wild-type conformation). Zinc-mediated p53 reactivation also reduced the mtp53/p73 interaction restoring both wtp53 and p73 binding to target gene promoters by ChIP assay with in vivo induction of wtp53 target gene expression, which rendered mutant p53 cells more prone to drug killing in vitro. Finally, zinc administration in U373MG tumor xenografts increased drug-induced tumor regression in vivo, which correlated with increased wild-type p53 protein conformation. These results show that the use of zinc might restore drug sensitivity and inhibit tumor growth by reactivating mutant p53.

Abstract 2973: Differential regulation of p21waf1 protein half-life by DNA damage and nutlin-3 in p53 wild-type tumors and its therapeutic potential

Abstract DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. We have used the topoisomerase I inhibitor SN38 to induce DNA damage in three p53 wild-type cell lines: the immortalized MCF10A, and tumorigenic HCT116 and U2OS. Both phospho-Chk1 and p53 were elevated to a similar extent in all three cell lines. However, the HCT116 and U2OS cells exhibited far less accumulation of p21waf1 protein. The p21waf1 mRNA was induced to a similar extent, and the protein half-life was similar (∼ 30 min) in all three cell lines. These results suggest that the two tumor cell lines have a reduced rate of translation of the p21waf1 mRNA. To obtain further insight into the regulation of p21waf1, we incubated the three cell lines with the non-genotoxic drug nutlin-3, which disrupts p53:MDM2 binding thereby inducing a p53 response. Nutlin-3 induced similar levels of p53 in the three cell lines, and these levels were comparable to those seen with SN38. While nutlin-3 had little additional impact on p21waf1 induction in MCF10A cells and the half-life remained at ∼ 30 min, there was a much stronger induction in HCT116 and U2OS cells which was attributed to a much longer protein half-life (∼ 80 to 90 min). Incubation of p53 mutant cells with Chk1 inhibitors such as SCH900776 can overcome SN38-induced cell cycle arrest and drive cells through a lethal mitosis. p53 wild-type cells can be protected from this lethal mitosis through induction of p21waf1. However, the defective p21waf1 induction in HCT116 and U2OS cells is insufficient to protect them from Chk1 inhibition. In contrast, incubation with nutlin-3 induced sufficient p21waf1 that the cells arrested in either G1 or G2. This arrest protected the cells from subsequent incubation with the S phase-specific DNA damaging agents SN38 and hydroxyurea, either alone, or when combined with checkpoint inhibition. This protection was not observed in MCF10A or HCT116 cells deleted for p21waf1, or in MDA-MB-231 cells that have mutant p53. The results demonstrate that some p53 wild-type tumors have defects in p21waf1 expression, and may retain sensitivity to a combination of DNA damage plus checkpoint inhibition. Furthermore, the results suggest nutlin-3 could be used to protect normal cells from these combinations such that therapy can be targeted selectively to p53-defective tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2011-2973

Abstract 4470: Kevetrin™, a novel small molecule, activates p53, enhances expression of p21, induces cell cycle arrest and apoptosis in a human cancer cell line

Abstract We have previously shown that Kevetrin, a small molecule currently under development, has potent antitumor activity in human multi-drug resistant carcinoma xenograft models while being well tolerated. To investigate the mechanism of action for its potent antitumor activity, we assessed Kevetrin's effect on apoptosis, cell cycle progression, including underlying molecular mechanisms. Here we report that Kevetrin strongly induced cell cycle arrest and apoptosis in a human lung adenocarcinoma cell line (A549) using the TUNEL assay and FACS analysis; similar results were observed using a human breast carcinoma cell line (MDA-MB-231). Treatment of A549 cells with 400 µM of Kevetrin for 48 hours resulted in G2/M phase cell cycle arrest that was associated with a marked decline in levels of G2/M regulatory proteins, including CDK1 and cdc25B, and increased expression of Wee1. Further, Kevetrin-mediated growth inhibition of A549 correlated with apoptosis induction that was characterized by cleavage of procaspase-3 and poly (ADP-ribose) polymerase (PARP). The p53 tumor suppressor is a well characterized transcription factor controlling cell growth and apoptosis during times of cellular stress. The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. In cancer development, the resistance of cells to apoptosis is one of the crucial steps. The reactivation of p53 in tumor cells should trigger massive apoptosis and eliminate the tumor. Interestingly, Kevetrin treatment caused enhanced activated p53 levels in A549 cells. Activation of p53 can lead to cell cycle arrest and apoptosis. Western blotting revealed a concentration dependent increase in phosphorylation of p53 at serine 15. The phosphorylation of p53 at serine 15 leads to reduced interaction between p53 and its negative regulator, the oncoprotein MDM2. We also found that Kevetrin increased expression of p53 target genes such as p21 (Waf1). The tumor suppressor protein p21 (Waf1) acts as inhibitor of cell cycle progression. Finally, we also found that p53 induced apoptosis by inducing the expression of PUMA. Our study shows that Kevetrin activates p53 functions in tumor cells. Thus Kevetrin is a strong candidate as an anticancer drug that targets p53. Based on our studies we plan to initiate a Phase I clinical trial in 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2011-4470

Abstract 3242: New chemical tools for disrupting the MDM2/p53 protein-protein interaction: Identification, synthesis and biological evaluation of a novel class of MDM2/p53 inhibitors

Abstract MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting its degradation. In particular, MDM2 is overexpressed in some of human tumors, and with MDMX contributes directly to loss of p53 function during the development of nearly 50% of human cancers. Due to p53 inactivation, MDM2 in many tumors confers tumor survival; therefore it is an important molecular target for anticancer therapy. Several studies showed that reactivation of wild type p53 in tumor cells can be obtained by disrupting the MDM2/p53 interaction with peptidic, peptidomimetic, and small molecule p53-mimetics. Specific successful examples include the Nutlins and spirooxindole analogs (MI-219 and MI-63). Amongst the peptidic and peptidomimetic inhibitors examined to date, none is nearly as effective as Nutlins and MI-219 in tumor killing in vitro. Hence, new inhibitors against MDM2 and/or MDMX are needed: as cell permeable chemical probes of the p53 pathway in cancer biology, and as templates for structure-based rational design of p53 activators for future therapeutic use. As part of our drug discovery program to identify antagonists of the p53/MDM2 and p53/MDMx protein-protein interactions, a high-throughput in-silico screen of a 3.2 millions virtual library of compounds (from Schrödinger, Inc.). A physical restraint was applied during the screen, in order to mimic binding to the hydrophobic cleft of MDM2 normally occupied by three p53 side chains (F19, W23, and L26) that are critical for MDM2/p53 binding. The top highest ranked 160 compounds were then assessed for their ability to block p53 interaction with MDM2 and MDMx in an ELISA assay. This resulted in the identification of E12/DP3-117, a small molecule disruptor of the p53/MDM2 protein-protein interaction with an IC50 value of 47 ± 14 μM. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and on compounds showing improved activity. Structure activity relationship studies around the hits will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. Binding of E12/DP3-117 to MDM2 is currently being assessed via co-crystallization and other biophysical techniques. We will describe the use of the crystal structure of p53-like mutant peptides in complex with the N-terminal domains of Mdm2, as the basis for rational design of more potent MDM2 small-molecule/peptide hybrid inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2011-3242

OTX1 expression in breast cancer is regulated by p53

The p53 transcription factor has a critical role in cell stress response and in tumor suppression. Wild-type p53 protein is a growth modulator and its inactivation is a critical event in malignant transformation. It has been recently demonstrated that wild-type p53 has developmental and differentiation functions. Indeed an over-expression of p53 in tumor cells induces asymmetrical division avoiding self-renewal of cancer stem cells (CSCs) and instead promoting their differentiation. In this study, 28 human breast carcinomas have been analyzed for expression of wild-type p53 and of a pool of non-clustered homeobox genes. We demonstrated that orthodenticle homolog 1 gene (OTX1) is transcribed in breast cancer. We established that the p53 protein directly induces OTX1 expression by acting on its promoter. OTX1 has been described as a critical molecule for axon refinement in the developing cerebral cortex of mice, and its activity in breast cancer suggests a synergistic function with p53 in CSC differentiation. Wild-type p53 may regulate cellular differentiation by an alternative pathway controlling OTX1 signaling only in breast cancer cells and not in physiological conditions.

Understanding wild-type and mutant p53 activities in human cancer: new landmarks on the way to targeted therapies

Three decades of p53 research have led to many advances in understanding the basic biology of normal and cancer cells. Nonetheless, the detailed functions of p53 in normal cells, and even more so in cancer cells, remain obscure. A major breakthrough is the realization that mutant p53 has a life of its own: it contributes to cancer not only through loss of activity, but also through gain of specific 'mutant functions'. This new focus on mutant p53 is the rationale behind the meeting series dedicated to advances on mutant p53 biology. This review provides an overview of results presented at the Fourth International Workshop on Mutant p53, held in Akko, Israel in March 2009. New roles and functions of p53 relevant for tumor suppressions were presented, including the regulation of microRNAs networks, the modulation of cell-stroma interactions and the induction of senescence. A main focus of the meeting was the rapidly growing body of knowledge on autonomous properties of mutant p53 and on their oncogenic 'gain of function' impact. Importantly, the meeting highlighted that, 30 years after p53 discovery, research on mutant p53 is entering the clinical and translational era. Two major steps forward in this respect are a better understanding of the active mechanism of small drugs targeting mutant p53 in tumor cells and an improved definition of the prognostic and predictive value of mutant p53 in human cancer.

Targeting wild-type and mutant p53 with small molecule CP-31398 blocks the growth of rhabdomyosarcoma by inducing reactive oxygen species-dependent apoptosis.

Rhabdomyosarcoma (RMS) is a common soft-tissue sarcoma of childhood in need of more effective therapeutic options. The expression of p53 in RMS is heterogeneous such that some tumors are wild-type whereas others are p53 mutant. The small molecule CP-31398 modulates both the wild-type and the mutant p53 proteins. Here, we show that CP-31398 blocks the growth of RMS cells that have either wild-type or mutant p53 status. In wild-type A204 cells, CP-31398 increased the expression of p53 and its downstream transcriptional targets, p21 and mdm2; enhanced the expression of apoptosis-related proteins; and reduced proliferation biomarkers. Flow profiling of CP-31398-treated cells indicated an enhancement in sub-G(0) and G(1) populations. CP-31398 inhibited proliferation in a manner associated with co-induction of SOX9 and p21. Apoptosis induced by CP-31398 occurred with translocation of p53 to mitochondria, leading to altered mitochondrial membrane potential, cytochrome c release, and reactive oxygen species release. In vivo, CP-31398 decreased the growth of tumor xenografts composed of wild-type or mutant p53 tumor cells, increasing tumor-free host survival. Our findings indicate that the ability of CP-31398 to modulate wild-type and mutant p53 results in the inhibition of RMS growth and invasiveness.

Abstract SY18-02: Targeting transcriptional regulators for simultaneous modulation of p53 and NF-κB in cancer treatment

Abstract There are three advantages of phenotype-based versus traditional target-based drug discovery strategy. It (i) avoids placing premature focus on a target that may not be the optimal one, (ii) permits the biological system to drive the investigator towards the optimal target through the selection process and (iii) eliminates a number of pharmacological optimization steps by filtering out compounds with unacceptable toxicity or intracellular stability. We used cell phenotype-based readout to screen for chemicals capable of activation of dormant p53 in tumor cells. As a result, we isolated two classes of compounds that simultaneously inhibit NF-kappaB through previously unknown mechanism and activate p53 without causing any genotoxicity. These compounds appeared to have strong anticancer activity in all tested human tumor xenografts grown in mice and were named Curaxins. The mechanisms of activity of Curaxins involves targeting SSRP1, a component of “facilitate chromatin transcription” (FACT) complex, which is required for transcription from NF-kappaB-inducible promoters. In the presence of Curaxins, SSRP1 is trapped in chromatin thereby blocking NF-kappaB-mediated gene expression what results in CK2-mediated activation of p53 through its phosphorylation at a single C-terminal serine residue. Besides NF-kappaB, Curaxins suppress initiation of transcription driven by Heat Shock Factor-1 (HSF1), thereby blocking activation of cell response to proteotoxic stresses (heat shock response). Constitutive activation of NF-kappaB and HSF1 is frequently observed in tumors making them “addicted” to these pro-survival pathways. The mechanism of antitumor activity of Curaxins suggests their rational combinations with other cancer treatment approaches. For example, inhibition of HSF1 explains high antitumor efficacy of these compounds in tumor-bearing mice treated by Curaxins in combination with proteotoxic stress (local hyperthermia or proteotoxic drugs) Simultaneous targeting of three signal transduction pathways commonly deregulated in cancer (suppression of NF-kappaB and HSF1 and activation of p53) and lack of genotoxicity opening the opportunity for using Curaxins not only for safe chemotherapy but also for chemoprevention. In fact, efficacy of Curaxins as chemopreventive agents given in drinking water was demonstrated in the mouse model of spontaneous breast cancer (transgenic mice carrying MMTV promoter-driven expression of her2/neu). In summary, Curaxins are triple-targeted non-genotoxic anticancer drug candidates acting through the inhibition of SSRP1 function via non-genotoxic DNA binding. The Curaxin story indicates principle advantages of phenotype-driven “non-targeted” drug discovery strategy, which enables finding new anticancer targets and approaches that could not be predicted based on the preexisting knowledge. Citation Format: Katerina Gurova, Andrei V. Gudkov. Targeting transcriptional regulators for simultaneous modulation of p53 and NF-κB in cancer treatment [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY18-02

Abstract 3197: Differential senescent and apoptotic responses to HDM2 antagonism in p53 wild-type human solid cancer cells

Abstract The p53 protein is a primary regulator of the cellular response to DNA damage and is a critical tumor suppressor. Restoration of p53 function in soft tissue sarcomas and hepatocellular carcinomas in mice was reported to cause tumor regressions associated with cellular senescence. These findings led us to investigate the ability of HDM2 antagonism to induce cellular senescence as well as apoptosis in a broad panel of wild-type p53 cell lines derived from human solid tumors. Here we report that the small molecule HDM2 antagonist Nutlin-3a activates p53 and induces cellular senescence in 10 out of 12 human tumor cell lines tested. Only two of these tumor cell lines also demonstrated an apoptotic response to Nutlin-3a. The kinetics of senescence induction by Nutlin-3a was slower than that of the apoptotic response, suggesting that apoptosis may be the first-line response, while senescence may be triggered as a back-up program when the apoptotic response fails to execute. A distinct expression profile of p53 target genes associated with apoptosis, cell cycle and senescence was observed and corresponded with the distinct phenotypic responses in this panel of cell lines. Depletion of p53 protein expression using shRNA blocked cellular senescence induced by HDM2 antagonism demonstrating the critical role for p53 in this process. The pronounced effects of Nutlin-3a on senescence in multiple wild-type p53 tumor cell lines underscores the unique anti-tumor mechanisms of HDM2 antagonism. This approach may provide a novel therapeutic strategy for future cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3197.

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

In vitro and In vivo Radiation Sensitization of Human Tumor Cells by a Novel Checkpoint Kinase Inhibitor, AZD7762

Inhibition of checkpoint kinase 1 has been shown to enhance the cytotoxicity of DNA-damaging targeted chemotherapy through cell cycle checkpoint abrogation and impaired DNA damage repair. A novel checkpoint kinase 1/2 inhibitor, AZD7762, was evaluated for potential enhancement of radiosensitivity for human tumor cells in vitro and in vivo xenografts. Survival of both p53 wild-type and mutant human cell lines was evaluated by clonogenic assay. Dose modification factors (DMF) were determined from survival curves (ratio of radiation doses for control versus drug treated at 10% survival). Flow cytometry, Western blot, and radiation-induced tumor regrowth delay assays were conducted. AZD7762 treatment enhanced the radiosensitivity of p53-mutated tumor cell lines (DMFs ranging from 1.6-1.7) to a greater extent than for p53 wild-type tumor lines (DMFs ranging from 1.1-1.2). AZD7762 treatment alone exhibited little cytotoxicity to any of the cell lines and did not enhance the radiosensitivity of normal human fibroblasts (1522). AZD7762 treatment abrogated radiation-induced G(2) delay, inhibited radiation damage repair (assessed by gamma-H2AX), and suppressed radiation-induced cyclin B expression. HT29 xenografts exposed to five daily radiation fractions and to two daily AZD7762 doses exhibited significant radiation enhancement compared with radiation alone. AZD7762 effectively enhanced the radiosensitivity of mutated p53 tumor cell lines and HT29 xenografts and was without untoward toxicity when administered alone or in combination with radiation. The results of this study support combining AZD7762 with radiation in clinical trials.

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.

Immunohistochemical analysis of P53and bcl-2 in benign and malignant salivary glands tumors

Salivary glands tumors are relatively uncommon neoplasm with widely variable histopathologic and biologic characteristics. Alteration in some proto-oncogenes and tumor suppressor genes may lead to the development and progression of these tumors. The purpose of this study was to analyze the immunohistochemical expression of P(53) and bcl-2 in some salivary gland tumors in relation to tumor size, histologic grade, and extent of invasion. The sample consisted of 22 formalin-fixed paraffin embedded blocks of benign and malignant salivary glands tumors. P(53) and bcl-2 immunoreactivity was semi-quantitatively evaluated in at least 1000 cells examined under the microscope at 40x magnification and recorded as percentage of P(53) or bcl-2 positive tumor cells over the total number of cells examined in the same area. Percentage scores were subsequently categorized using the 5% cut-off point for positive staining. Pleomorphic adenoma (PA) showed negative expression of P(53) and bcl-2 in 70% of cases, whereas all malignant salivary glands tumors were positive for P(53) and bcl-2. P(53) positive/bcl-2 positive immunostaining reaction was week in small size PA and was totally absent in larger lesions. However, all malignant tumors expressed P(53), with the highest record in low-grade adenocarcinoma (76%) and the lowest score was observed in both low-grade carcinoma in PA and adenocystic carcinoma. bcl-2 immunostaining was also assessed, the highest recorded score was in high-grade adeno cystic carcinoma (90%) and the lowest in both low-grade carcinoma ex-PA and low-grade cystic mucoepidermoid carcinoma (3%). P(53)/bcl-2 immunostaining reactivity could be helpful in demonstrating salivary glands tumor behavior in terms of progression and extent of invasion.

Specific activation of the p53 pathway by low dose actinomycin D: A new route to p53 based cyclotherapy

The activation of p53 has been proposed as a novel anti-cancer treatment in two distinct contexts. In the first activation of p53 in tumor cells can promote apoptosis and senescence and enhance the anti-tumor activity of cytotoxic chemotherapeutic drugs. In the second application activation of p53 in normal tissues can cause a reversible cell cycle arrest that can be used to protect normal cells from the action of anti-mitotics. In this cyclotherapy role p53 mutant tumor cells are not arrested and remain sensitive to anti-mitotics. The advent of specific p53 activating molecules such as nutlin-3 has encouraged both approaches. We have sought for a clinically approved drug that can mimic nutlin-3. We show here that low doses of actinomycin D mimic nutlin-3 in the highly specific activation of p53 dependant transcription, in the induction of a reversible protective growth arrest in normal cells and in the enhancement of the activity of chemotherapeutic drug induced killing of p53 positive human tumor cells. While high doses of actinomycin D reveal its more non-specific activities, low doses of the drug will allow exploration of the value of p53 activation in preclinical and clinical models before nutlin-3 like drugs are approved.

Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells

Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

Tumor-specific induction of apoptosis by a p53-reactivating compound

The tumor suppressor function of p53 is disabled in the majority of tumors, either by a point mutation of the p53 gene, or via MDM2-dependent proteasomal degradation. We have screened a chemical library using a cell-based assay and identified a low molecular weight compound named MITA which induced wild-type p53-dependent cell death in a variety of different types of human tumor cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fibrosarcoma-derived cells. MITA inhibited p53–MDM2 interaction in vitro and in cells, which in turn prevented MDM2-mediated ubiquitination of p53 and resulted in a prolonged half-life and accumulation of p53 in tumor cells. Notably, p53 induction by MITA resulted in upregulated expression of p53 target genes MDM2, Bax, Gadd45 and PUMA, on protein and mRNA level. Importantly, neither p53 nor these target genes were induced in normal human fibroblasts (HDFs), which correlated with the absence of growth suppression in fibroblasts after treatment with MITA. However, upon activation of oncogenes in fibroblasts an induction and activation of p53 was observed, suggesting that activation of p53 by MITA occurs predominantly in tumor cells.

The disruption of the protein complex mutantp53/p73 increases selectively the response of tumor cells to anticancer drugs

Many in vitro and in vivo evidence have shown that the status of p53 is a key determinant in the response of tumor cells to anticancer treatment. Here we provide evidence that peptide-mediated targeting of the protein complex mutantp53/p73 enhances the response of mutant p53 tumor cells to commonly used anticancer drugs. Indeed, we show that the disruption of the protein complex mutantp53/p73 and the consequent restoration of p73 transcriptional effects, through the activity of short interfering peptides, render mutant p53 cells more prone to the killing of adriamycin and cisplatin. Of note, the activity of the short interfering peptides is mutant p53 specific and causes no effects on wt-p53 and p53 null cells. Our findings highlight the protein complex mutantp53/p73 as a molecular target, whose successful overriding through the selective activity of small interfering peptides, might contribute to the optimization of mutant p53 tumor treatments.

Proteomics analysis of A375 human malignant melanoma cells in response to arbutin treatment

Although the toxicogenomics of A375 human malignant melanoma cells treated with arbutin have been elucidated using DNA microarray, the proteomics of the cellular response to this compound are still poorly understood. In this study, we performed proteomic analyses to investigate the anticancer effect of arbutin on the protein expression profile in A375 cells. After treatment with arbutin (8 μg/ml) for 24, 48 and 72 h, the proteomic profiles of control and arbutin-treated A375 cells were compared, and 26 differentially expressed proteins (7 upregulated and 19 downregulated proteins) were identified by MALDI-Q-TOF MS and MS/MS. Among these proteins, 13 isoforms of six identical proteins were observed. Bioinformatic tools were used to search for protein function and to predict protein interactions. The interaction network of 14 differentially expressed proteins was found to be correlated with the downstream regulation of p53 tumor suppressor and cell apoptosis. In addition, three upregulated proteins (14-3-3G, VDAC-1 and p53) and five downregulated proteins (ENPL, ENOA, IMDH2, PRDX1 and VIME) in arbutin-treated A375 cells were validated by RT-PCR analysis. These proteins were found to play important roles in the suppression of cancer development.

Suppression of Familial Adenomatous Polyposis by CP-31398, a TP53 Modulator, in APCmin/+ Mice

p53 mutations occur in a large number of human malignancies. Mutant p53 is unable to affect downstream genes necessary for DNA repair, cell cycle regulation, and apoptosis. The styrylquinazoline CP-31398 can rescue destabilized mutant p53 expression and promote activity of wild-type p53. The present study examines chemopreventive effects of CP-31398 on intestinal adenoma development in an animal model of familial adenomatous polyposis. Effects were examined at both early and late stages of adenoma formation. Effects of CP-31398 on early-stage adenomas were determined by feeding 7-week-old female C57BL/6J-APC(min) (heterozygous) and wild-type C57BL/6J mice with American Institute of Nutrition-76A diets containing 0, 100, or 200 ppm of CP-31398 for 75 days. To examine activity toward late-stage adenomas, CP-31398 administration was delayed until 15 weeks of age and continued for 50 days. During early-stage intervention, dietary CP-31398 suppressed development of intestinal tumors by 36% (P < 0.001) and 75% (P < 0.0001), at low and high dose, respectively. During late-stage intervention, CP-31398 also significantly suppressed intestinal polyp formation, albeit to a lesser extent than observed with early intervention. Adenomas in treated mice showed increased apoptotic cell death and decreased proliferation in conjunction with increased expression of p53, p21(WAF1/CIP), cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. These observations show for the first time that the p53-modulating agent CP-31398 possesses significant chemopreventive activity in vivo against intestinal neoplastic lesions in genetically predisposed APC(min/+) mice. Chemopreventive activity of other agents that restore tumor suppressor functions of mutant p53 in tumor cells is currently under investigation.