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Autophagy is a process by which cells re-cycle organelles and macromolecular proteins during cellular stress. Defects in the regulation of autophagy have been associated with various human pathologies including heart failure. In the heart tumor suppressor p53 protein is known to promote apoptotic and autophagic cell death. We found p53 over-expression increased endogenous protein level of the hypoxia-inducible Bcl-2 death gene Bnip3 which leads to loss of mitochondrial membrane potential (ΔΨm). This was accompanied by autophagic flux and cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3-/- mouse embryonic fibroblasts prevented mitochondrial targeting of p53 and autophagic cell death. These data provide the first evidence for the dual regulation of autophagic cell death of cardiac myocytes by p53 that is mutually dependent on Bnip3 activation. Hence, our findings may explain how autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

The tumour suppressor p53 has evolved a MDM2-dependent feedback loop that has a dual role as either a stimulator of p53 protein translation through mRNA binding or a stimulator of p53 protein degradation through the ubiquitin-proteasome system. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic drug-binding pocket and we have evaluated whether the lid of MDM2 is a physiological regulator of this dual function of MDM2. Deletion of this flexible pseudosubstrate motif inhibits MDM2 indicating that this peptide stretch can function as a positive regulatory motif. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2S17D) stabilizes the binding of MDM2 towards p53. Molecular modeling orientates the pseudo-substrate motif over a charged surface patch on the MDM2 surface at Arg97/Lys98 and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phosphomimetic mutation. Transient or inducible low level expression of MDM2WT can promote an increase in p53 protein steady-state levels whilst the expression of MDM2S17D in cells results in p53 protein de-stabilization. Phospho-specific antibodies to the MDM2 lid demonstrate two physiological conditions that alter lid phosphorylation: (i) lid hypo-phosphorylation occurs after DNA damage where p53 protein is stabilized and (ii) lid hyper-phosphorylation occurs at high cell density under conditions where p53 protein is de-stabilized. Expression of MDM2S17D in cells also de-stabilizes hyperubiquitinated mutant p53 under conditions where MDM2WT has no effect on mutant p53 protein degradation. The lid functions as a flexible regulatory motif whose phosphorylation switches MDM2 from a synthesis mode to a degradation mode with implications for defining the physiological signals that control the MDM2-p53 feedback regulatory loop.

Cancer is a group of diseases that are the second leading cause of human mortality in the United States. Discovering new therapies is vital to conquer cancer. Thymoquinone (TQ) is found in the plant Nigella sativa. TQ was found to be cytotoxic to the human ovarian cancer cell lines PA-1, CAOV-3 and SKOV-3, which have varying p53 status. PA-1 cells were the most sensitive, indicating that TQ was effective against cells having wild-type (WT) p53. Western blots indicated an increase in p53 in cell lines having WT p53. TQ when given concurrently with cisplatin resulted in antagonism for PA-1, A172 and H460 cell lines. Sequential exposure to TQ followed by cisplatin resulted in synergy or additive effects in these cell lines. Sequential exposure to cisplatin followed by TQ resulted in additive or moderate antagonism in these cell lines. Concurrent exposure to TQ and paclitaxel showed synergy in PA-1 and H460 cells. Sequential exposure to TQ followed by paclitaxel resulted in synergism or antagonism in A172, PA-1, and H460 cells. Paclitaxel followed by TQ resulted in antagonism or synergism in these cells. These results demonstrate that TQ has a potential as an antineoplastic agent and may affect p53 levels.

Autophagy is a process by which cells re-cycle organelles and macromolecular proteins during cellular stress. Defects in the regulation of autophagy have been associated with various human pathologies including heart failure. In the heart tumor suppressor p53 protein is known to promote apoptotic and autophagic cell death. We found p53 over-expression increased endogenous protein level of the hypoxia-inducible Bcl-2 death gene Bnip3 which leads to loss of mitochondrial membrane potential (ΔΨm). This was accompanied by autophagic flux and cell death. Conversely, loss of function of Bnip3 in cardiac myocytes or Bnip3-/- mouse embryonic fibroblasts prevented mitochondrial targeting of p53 and autophagic cell death. These data provide the first evidence for the dual regulation of autophagic cell death of cardiac myocytes by p53 that is mutually dependent on Bnip3 activation. Hence, our findings may explain how autophagy and cell death are dually regulated during cardiac stress conditions where p53 is activated.

Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

Konstanz, Univ., Diss., 2007.

Zugl.: Berlin, Techn. Univ., Diss., 2007

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.