Myosin VI is a mediator of the p53-dependent cell survival pathway

Myosin VI is an unconventional motor protein, and its mutation is responsible for the familiar conditions sensorineural deafness and hypertrophic cardiomyopathy. Myosin VI is found to play a key role in the protein trafficking and homeostasis of the Golgi complex. However, very little is known about how myosin VI is regulated and whether myosin VI has a function in the DNA damage response. Here, we found that myosin VI is regulated by DNA damage in a p53-dependent manner and possesses a novel function in the p53-dependent prosurvival pathway. Specifically, we show that myosin VI is induced by p53 and DNA damage in a p53-dependent manner. We found that p53 directly binds to, and activates, the promoter of the myosin VI gene. We also show that the intracellular localization of myosin VI is substantially altered by p53 and DNA damage in a p53-dependent manner such that the pool of myosin VI in endocytic vesicles, membrane ruffles, and cytosol migrates to the Golgi complex, perinuclear membrane, and nucleus. Furthermore, we show that knockdown of myosin VI attenuates activation of p53 and impairs Golgi complex integrity, which makes myosin VI-deficient cells susceptible to apoptosis upon DNA damage. Taken together, we found a novel function for p53 in the maintenance of Golgi complex integrity and for myosin VI in the p53-dependent prosurvival pathway.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines

Melanoma cell lines and normal human melanocytes (NHM) were assayed for p53-dependent G1 checkpoint response to ionizing radiation (IR)-induced DNA damage. Sixty-six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to IR with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2, and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of 4-yr distant metastasis-free survival. Thus, p53 function, radio-sensitivity, and metastatic spread may be estimated in melanomas from a signature of gene expression.     

Monocyte chemoattractant protein-1 induces endothelial cell apoptosis in vitro through a p53-dependent mitochondrial pathway

The cystine-cystine (CC) chemokine monocyte chemoattractant protein-1 (MCP-1) has been established playing a pathogenic role in the development of atherosclerosis due to its chemotactic ability of leading monocytes to locate to subendothelia. Recent studies have revealed more MCP-1 functions other than chemotaxis. Here we reported that various concentrations (0.1-100 ng/ml) of MCP-1 induced human umbilical vein endothelial cell (HUVEC) strain CRL-1730 apoptosis, caspase-9 activation, and a couple of mitochondrial alterations. Moreover, MCP-1 upregulated p53 expression of HUVECs and the p53-specific inhibitor pifithrin-α (PFTα) rescued the MCP-1-induced apoptosis of HUVECs. Furthermore, PKC (protein kinase C) activation or inhibition might also affect HUVECs apoptosis induced by MCP-1. These findings together demonstrate that MCP-1 exerts direct proapoptotic effects on HUVECs in vitro via a p53-dependent mitochondrial pathway.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

Depletion of endogenous nitric oxide enhances cisplatin-induced apoptosis in a p53-dependent manner in melanoma cell lines

The expression of inducible nitric-oxide synthase in melanoma tumor cells was recently shown to correlate strongly with poor patient survival after combination biochemotherapy (p<0.001). Furthermore, evidence suggests that nitric oxide, a reaction product of nitric oxide synthase, exhibits antiapoptotic activity in melanoma cells. We therefore hypothesized that nitric oxide antagonizes chemotherapy-induced apoptosis. Whether nitric oxide is capable of regulating cell growth and apoptotic responses to cisplatin treatment in melanoma cell lines was evaluated. We demonstrate herein that depletion of endogenously produced nitric oxide can inhibit melanoma proliferation and promote apoptosis. Moreover, our data indicate that the depletion of nitric oxide leads to changes in cell cycle regulation and enhances cisplatin-induced apoptosis in melanoma cells. Strikingly, we observed that the depletion of nitric oxide inhibits cisplatin-induced wild type p53 accumulation and p21(Waf1/Cip1/Sdi1) expression in melanoma cells. When cisplatin-induced p53 binding to the p21(Waf1/Cip1/Sdi1) promoter was examined, it was found that nitric oxide depletion significantly reduced the presence of p53-DNA complexes after cisplatin treatment. Furthermore, dominant negative inhibition of p53 activity enhanced cisplatin-induced apoptosis. Together, these data strongly suggest that endogenously produced nitric oxide is required for cisplatin-induced p53 activation and p21(Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to cisplatin.     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

Bcl-2 controls caspase activation following a p53-dependent cyclin D1-induced death signal

MCF-7 and ZR-75 breast cancer cells infected with an adenovirus constitutively expressing high levels of cyclin D1 demonstrated widespread mitochondrial translocation of Bax and cytochrome c release that was approximately doubled after the addition of all-trans retinoic acid (RA) or Bcl-2 antisense oligonucleotide. By comparison, the percentage of cells in Lac Z virus-infected cultures containing translocated Bax and cytoplasmic cytochrome c was markedly less even after RA treatment. Despite this, RA-treated Lac Z and untreated cyclin D1 virus-infected cultures contained similarly low proportions of cells with active caspase or cells that were permeable to propidium iodide. Bax activation was p53-dependent and accompanied by arrest in G(2) phase. Although constitutive Bcl-2 expression prevented Bax activation, it did not alter cyclin D1-induced cell cycle arrest, illustrating the independence of these events. Both RA and antisense Bcl-2 oligonucleotide decreased Bcl-2 protein levels and markedly increased caspase activity and apoptosis in cyclin D1-infected cells. Thus amplified cyclin D1 expression initiates an apoptotic signal inhibited by different levels of cellular Bcl-2 at two points. Whereas high cellular levels of Bcl-2 prevent mitochondrial Bax translocation, lower levels can prevent apoptosis by inhibition of caspase activation.     

p53-independent checkpoint controls in a plant cell model

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpoint-adapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.     

Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor invitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.     

Emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC(50) value of 42.5, 46.6, and 53.1 microM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G(1) accumulation. Besides, HepG2/C3A cells were found to be arrested in G(2)/M phase after the cells were treated with 60 microM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (419.3 pg/ml), p21 (437.4 units/ml), Fas (6.6 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G(2)/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).     

Cleavage of phospholipase D1 by caspase promotes apoptosis via modulation of the p53-dependent cell death pathway

The enzymatic activity of phospholipase D (PLD) is known to be essential for cell survival and protection from apoptosis. However, the mechanisms regulating PLD activity during apoptosis remain unknown. Here we report that cleavage of PLD1 by caspases facilitates p53-mediated apoptosis. Cleavage of PLD1 into an N-terminal fragment (NF-PLD1) and a C-terminal fragment at the amino-acid sequence, DDVD(545), led to a reduction in PLD1 activity. However, a caspase-resistant mutant form of PLD1 retained significant levels of enzymatic activity and apoptotic function as compared to wild-type PLD1. Exogenous NF-PLD1 expression induced apoptosis through a dominant-negative effect on the activity of endogenous PLD1. During apoptosis, a small fraction of PLD1 is cleaved by caspases in a p53-independent manner and NF-PLD1 amplifies apoptotic signaling through inhibition of the remaining PLD1 activity. As PLD1 suppresses the ATM-Chk2-p53 pathway, elimination of PLD1 activity through NF-PLD1 or si-RNA against PLD1 increases apoptosis in a p53-dependent manner. Taken together, our results reveal that cleavage of PLD1 by caspases promotes apoptosis via modulation of the p53-dependent cell death pathway.     

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.