Involvement of p53 in insulin-like growth factor binding protein-3 regulation in the breast cancer cell response to DNA damage

Chemotherapy drugs that induce apoptosis by causing DNA double-strand breaks, upregulate the tumor suppressor p53. This study investigated the regulation of the growth-regulatory protein insulin-like growth factor binding protein-3 (IGFBP-3), a p53 target, by DNA-damaging agents in breast cancer cells. IGFBP-3 was upregulated 1.4- to 13-fold in response to doxorubicin and etoposide in MCF-10A, Hs578T, MCF-7 and T47D cells, which express low to moderate basal levels of IGFBP-3. In contrast, IGFBP-3 was strongly downregulated by these agents in cells with high basal levels of IGFBP-3 (MDA-MB-231, MDA-MB-436 and MDA-MB-468). In MDA-MB-468 cells containing the R273H p53 mutation, reported to display gain-of-function properties, chemotherapy-induced suppression of IGFBP-3 was not reversed by the p53 reactivating drug, PRIMA-1, or by p53 silencing, suggesting that the decrease in IGFBP-3 following DNA damage is not a mutant p53 gain-of-function response. SiRNA-mediated downregulation of endogenous IGFBP-3 modestly attenuated doxorubicin-induced apoptosis in MDA-MB-468 and Hs578T cells. IGFBP-3 downregulation in some breast cancer cell lines in response to DNA-damaging chemotherapy may have clinical implications because suppression of IGFBP-3 may modulate the apoptotic response. These observations provide further evidence that endogenous IGFBP-3 plays a role in breast cancer cell responsiveness to DNA damaging therapy.     

The cell death machinery governed by the p53 tumor suppressor in response to DNA damage

The cellular response to genotoxic stress that damages DNA includes cell cycle arrest, activation of DNA repair, and in the event of irreparable damage, induction of apoptosis. However, the signals that determine cell fate, that is, survival or apoptosis, are largely unclear. The tumor suppressor p53 has been implicated in many important cellular processes, including regulation of apoptotic cell death. When cells encounter genotoxic stress, certain sensors for DNA lesions eventually stabilize and activate p53. Subsequently, p53 exerts its tumor suppressor function by transactivating numerous target genes. Active p53 is subjected to a complex and diverse array of covalent post‐translational modifications, which selectively influence the expression of p53 target genes. In this regard, the molecular basis for how p53 induces apoptosis has been extensively studied; however, the relative contribution of each downstream effecter is still to be explored. Moreover, little is known about precise mechanisms by which modified p53 is capable of apoptosis induction. A thorough understanding for the whole picture of p53 modification in apoptosis will be extremely valuable in the development of highly effective and specific therapies for caner patients. This review is focused on the current views regarding the regulation of cell fate by p53 in the apoptotic response to DNA damage. (Cancer Sci 2010; 101: 831–835)     

Programmed cell death 6, a novel p53‐responsive gene,targets to the nucleus in the apoptotic response to DNA damage

The cellular response to genotoxic stress is multifaceted in nature. Following DNA damage, the tumor suppressor gene p53 activates and plays critical roles in cell cycle arrest, activation of DNA repair and in the event of irreparable damage, induction of apoptosis. The breakdown of apoptosis causes the accumulation of mutant cells. The elucidation of the mechanism for the p53‐dependent apoptosis will be crucial in applying the strategy for cancer patients. However, the mechanism of p53‐dependent apoptosis remains largely unclear. Here, we carried out ChIP followed by massively parallel DNA sequencing assay (ChIP‐seq) to uncover mechanisms of apoptosis. Using ChIP‐seq, we identified PDCD6 as a novel p53‐responsive gene. We determined putative p53‐binding sites that are important for p53 regulation in response to DNA damage in the promoter region of PDCD6. Knockdown of PDCD6 suppressed p53‐dependent apoptosis. We also observed that cytochrome c release and the cleavage of PARP by caspase‐3 were suppressed by depletion of PDCD6. We further observed that PDCD6 localizes in the nucleus in response to DNA damage. We identified the nuclear localization signal of PDCD6 and, importantly, the nuclear accumulation of PDCD6 significantly induced apoptosis after genotoxic stress. Therefore, we conclude that a novel p53‐responsive gene PDCD6 is accumulated in the nucleus and induces apoptosis in response to DNA damage.     

Aberrant p53 alters DNA damage checkpoints in response to cisplatin: downregulation of CDK expression and activity

The p53 tumor suppressor protein is a critical mediator of cell cycle arrest and apoptosis in response to genotoxic stress. Abrogation of p53 function is a major feature of tumor development and may result in a compromised DNA-damage response. In our study, we examined the effect of expressing a human p53 cDNA, encoding a histidine to leucine amino acid substitution at codon 179 (H179L), on the ability of wild-type p53-containing NIH3T3 cells to respond to treatment with the chemotherapeutic cisplatin. After 72 hr of cisplatin treatment control cells underwent apoptosis preceded by a combination of S- and G(2) arrest, as judged by flow cytometry of propidium iodide-stained cells, and TUNEL and caspase-3 assays. This correlated with increased expression of the pro-apoptotic protein Bax. In contrast, cells stably expressing H179L-p53 arrested in S-phase following cisplatin treatment, which correlated with a marked decrease in the expression of cdc2, cyclin B1 and cyclin A, and a decrease in CDK2 and cyclin A-associated kinase activity. Interestingly, H179L p53 expressing cells underwent apoptosis earlier than control cells, indicating that this aberrant p53 may enhance cisplatin chemosensitivity. These data suggest that dominant-negative p53 can influence the expression and activity of CDK complexes, thereby modifying cell behavior following cisplatin-induced genotoxicity.     

p51A (TAp63gamma), a p53 homolog, accumulates in response to DNA damage for cell regulation

p51A, or TAp63gamma, a translation product of gene p51, or p63, was identified as a homolog of p53 in its primary structure and transactivating function. p53 plays a decision-making role in inducing either cell cycle arrest or apoptosis in response to DNA damage, and thereby preserves genome integrity of living cells. To compare the biological activities between p51A and p53, cell lines with low-level, constitutive expression of each protein were obtained by cDNA transfection of mouse erythroleukemic cells. Production of p51A with an apparent molecular mass of 57-kilodalton (kD) accompanied induction of p21waf1 and appearance of hemoglobin-producing cells. After DNA-damaging treatment either with ultraviolet light (UV) irradiation or with actinomycin D, the p51A protein accumulated in time courses corresponding to those of wild-type p53, and caused an increase in the hemoglobin-positive cell count. In contrast, p53-accumulated cells underwent apoptosis without exhibiting the feature of erythroid differentiation. The mode of p21waf1 and Bax-alpha upregulations varied between p51A- and p53-expressing cells and between the types of DNA damage. These results suggest the possibility that p51A induces differentiation under genotoxic circumstances. There may be cellular factors that control p51A protein stability and transactivating ability.     

Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells

Background  

The importance of various inflammatory cytokines in maintaining tumor cell growth and viability is well established. Increased expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) has previously been associated with various types of adenocarcinoma.     

Vascular endothelial growth factor, p53, Rb, Bcl-2 expression and response to chemotherapy in advanced non-small cell lung cancer

Vascular endothelial growth factor (VEGF) increases microvascular permeability and stimulates endothelial cell growth. p53 Overexpression has been associated with resistance to cisplatin-based chemotherapy in patients (pts) with NSCLC. The aim of this study was to evaluate the predictive role of VEGF for chemotherapy response, its relationship with p53, Rb, Bcl-2 and hemoglobin levels and its impact on overall survival in pts with advanced NSCLC. Bronchial or fine-needle biopsy specimens from 85 pts with NSCLC obtained before chemotherapy were analyzed using an immunohistochemical method for VEGF, p53, Rb and Bcl-2. There were 73 males and 12 females with a median age of 62.6 years. The majority of pts (48%) had squamous cell histology. Ten pts had stage IIIA, 25 stage IIIB and 50 stage IV. Thirty six (43%) pts had positive immunostaining for VEGF, 37 (44%) had positive p53, 53 (62%) had negative Rb and 4 (5%) had positive Bcl-2. VEGF was negatively correlated with Rb (r(s) = 0.26; P = 0.015), positively with Bcl-2 (r(s) = 0.22; P = 0.42), whereas no statistically significant correlation with p53, age, stage and histological type was found. In a logistic regression model, adjusting for treatment, VEGF expression was not associated with chemotherapy response (odds ratio (OR) = 1.01; P = 0.085 ), unlike p53 positivity and Rb negativity ( OR = 4.0, P = 0.005; OR = 2.6, P = 0.016, respectively). A statistically significant higher VEGF expression was detected in the subgroups defined, using as cut-off value Hb median level (13.3g/dl) (chi-square = 5.00; ; one d.f.; P = 0.025). At a median follow-up time of 8.4 years, 2-year survival was 21%. After adjustment for stage and chemotherapy treatment, VEGF expression was not associated with a better overall survival (OR = 1.06; P = 0.80), unlike Bcl-2 positivity showed a statistically significant effect (OR = 0.28; p = 0.02). Our results suggest that VEGF is weakly correlated with regulators of apoptosis and has not been shown to be an independent predictive factor for resistance to cisplatin-based chemotherapy and prognostic for survival.     

PML enhances the regulation of p53 by CK1 in response to DNA damage

In response to stress, p53 is accumulated and activated to induce appropriate growth inhibitory responses. This requires the release of p53 from the constraints of its negative regulators Mdm2 and Mdm4. A key event in this dissociation is the phosphorylation of p53 at threonine residue (Thr18) within the Mdm2/4-binding domain. Casein kinase 1 (CK1) plays a major role in this phosphorylation. The promyelocytic leukemia protein (PML) regulates certain modifications of p53 in response to DNA damage. Here, we investigated the role of PML in the regulation of Thr18 phosphorylation. We found that PML enhances Thr18 phosphorylation of endogenous p53 in response to stress. On DNA damage, CK1 accumulates in the cell, with a proportion concentrated in the nucleus together with p53 and PML. Furthermore, CK1 interacts with endogenous p53 and PML, and this interaction is enhanced by genotoxic stress. Inhibition of CK1 impairs the protection of p53 by PML from Mdm2-mediated degradation. Our findings support a role for PML in the regulation of p53 by CK1. We propose that following DNA damage, PML facilitates Thr18 phosphorylation by recruiting p53 and CK1 into PML nuclear bodies, thereby protecting p53 from inhibition by Mdm2, leading to p53 activation.     

PBK/TOPK promotes tumour cell proliferation through p38 MAPK activity and regulation of the DNA damage response

The contribution of the insulin-like growth-factor-I receptor (IGF-IR) to tumour progression is well documented. To identify new mediators of IGF-IR function in cancer, we recently isolated genes differentially expressed in cells overexpressing the IGF-IR. Among these was the serine/threonine kinase PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase), previously associated with highly proliferative cells and tissues. Here, we show that PBK is expressed at high levels in tumour cell lines compared with non-transformed cells. IGF-I could induce PBK expression only in transformed cells, whereas epidermal growth factor could induce PBK in non-transformed MCF-10A breast epithelial cells. Suppression of PBK expression using small interfering RNA did not prevent progression through the cell cycle, but caused decreased proliferation over time in culture, and reduced clonogenic growth in soft agarose. PBK knockdown impaired p38 activation after long-term stimulation with different growth factors and reduced DU145 cells motility. Suppressed PBK expression also resulted in an impaired response to DNA damage that was evident by the decreased generation of gamma-H2AX, increased DNA damage and decreased cell survival. Taken together, the data indicate that PBK is necessary for appropriate activation and function of the p38 pathway by growth factors. Thus, enhanced expression of PBK may facilitate tumour growth by mediating p38 activation and by helping cells to overcome DNA damage.     

Arsenite inhibits p53 phosphorylation, DNA binding activity, and p53 target gene p21 expression in mouse epidermal JB6 cells

Epidemiologic investigations demonstrated that arsenite exposure increases the risk of various human cancers, including skin, lung, bladder, and kidney cancers. However, oral administration of arsenite alone has failed to induce tumors in animal models, suggesting that arsenic may act to enhance mutagenicity induced by other carcinogens. Arsenite may function as a co-carcinogen, acting by inhibiting repair of carcinogen-induced DNA damage mediated by p53 and p21, a p53 target gene. To elucidate the interaction between arsenite and p53 tumor suppressor protein, we studied the effect of arsenite on ultraviolet B (UVB)-induced p53 phosphorylation, p53 DNA binding activity, and p53-induced target gene transactivation in the JB6 Cl41 mouse epidermal skin cell model. Our results indicated that arsenite suppressed UVB-induced p53 phosphorylation and p53 DNA binding activity. Arsenite also inhibited casein kinase 2 (CK2) activity and decreased p53-regulated p21 protein expression. These data suggest that the direct inhibition of p53 functional activation is one of the mechanisms through which arsenite interferes with p53 function, and thus may be a significant mechanism for the co-carcinogenic effects of arsenite.