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Published OnlineFirst December 20, 2013; DOI: 10.1158/1541-7786.MCR-13-0485

Molecular Cancer Cell Death and Survival Research

T-Type Ca2þ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through Activation of p38-MAPK in Colon Cancer Cells

Barbara Dziegielewska1, David L. Brautigan2,3, James M. Larner1,3, and Jaroslaw Dziegielewski1,3

Abstract þ Epithelial tumor cells express T-type Ca2 channels, which are thought to promote cell proliferation. This study þ investigated the cellular response to T-type Ca2 channel inhibition either by small-molecule antagonists or by þ RNAi-mediated knockdown. Selective T-type Ca2 channel antagonists caused growth inhibition and apoptosis more effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116 mutant p53 / cells. These antagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at speciﬁc target þ sequences. The knockdown of a single T-type Ca2 channel subunit (CACNA1G) reduced cell growth and induced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53 / cells. Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdown þ when p53wt was stably expressed. Upon T-type Ca2 channel inhibition, p38-MAPK promoted phosphorylation at Ser392 of p53wt. Cells treated with the inhibitor SB203580 or speciﬁc RNAi targeting p38-MAPKa/b þ (MAPK14/MAPK11) showed resistance to T-type Ca2 channel inhibition. Finally, the decreased sensitivity to channel inhibition was associated with decreased accumulation of p53 and decreased expression of p53 target genes, p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA).

Implications: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumor þ therapies that target T-type Ca2 channels. Mol Cancer Res; 12(3); 348–58. 2013 AACR.

Introduction with activity in vivo in combination with chemo- (9) or þ þ 2 Intracellular Ca2 regulates many cellular processes, radiotherapy (10). Aberrant expression of T-type Ca including cell cycle, proliferation, transcription, exocytosis, channels in cancer cells is thought to promote cell survival, hormone release, cell motility, and apoptosis (1, 2). Voltage proliferation, and motility (3, 11); however, the molecular 2þ 2þ mechanisms for these effects are poorly understood. gated Ca channels facilitate transient Ca influx from þ Signaling through voltage gated Ca2 channels involves the environment into the cytoplasm and appear mostly in þ Ca2 -binding proteins such as calmodulin (CaM) and excitable tissues, but also are unusually expressed in cancer þ þ 2 – cells. Low-voltage activated Ca2 channels, termed T-type activation of its binding partner Ca /calmodulin depen- 2þ dent kinase II (CaMKII), which in turn regulates T-type Ca channels, recently gained attention in cancer therapy, 2þ because their inhibition decreased proliferation of glioblas- Ca channels activity (12, 13). Consequently, proteins toma cells (3, 4), breast adenocarcinoma cells (5, 6), mel- downstream from activated CaMKII, such as mitogen-acti- anoma cells (7), and esophageal carcinoma cells (8). In vated protein 3 kinase 5 (MAP3K5, also known as apoptosis þ – addition, an antagonist selective for T-type Ca2 channels, signal regulating kinase 1 or ASK-1) or prosurvival protein kinase B (PKB also known as AKT) are affected by changes in mibefradil, has been proposed recently as a sensitizing agent þ intracellular Ca2 concentration (14–16). Increased CaM- KII activity regulates gene expression directly through phos- Authors' Affiliations: Departments of 1Radiation Oncology, 2Microbiol- phorylation of transcription factors, such as cyclic-AMP ogy, Immunology, and Cancer Biology, Center for Cell Signaling, University response element binding protein (CREB; reviewed in of Virginia School of Medicine; and 3Cancer Center, University of Virginia, Charlottesville, Virginia ref. 17) or indirectly involving p53 activation (18, 19). For example, CREB transcription factor could be responsible for Note: Supplementary data for this article are available at Molecular Cancer radiation resistance through regulation of DNA repair genes Research Online (http://mcr.aacrjournals.org/). þ (20), whereas intracellular Ca2 and CaMKII could regulate Corresponding Author: Jaroslaw Dziegielewski, Department of Radiation fi fl Oncology, University of Virginia School of Medicine, P.O. Box 800383, ef cient p53 activation upon 5- uorouracil treatment that Charlottesville, VA 22908. Phone: 434-982-0076; Fax: 434-243-9789; involves activated p38-MAPK (19). E-mail: [email protected] Under physiologic conditions the levels of p53 protein doi: 10.1158/1541-7786.MCR-13-0485 and its activity are low, but exposing cells to stress results 2013 American Association for Cancer Research. in p53 induction and activation (21). Activated p53

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accumulates as a tetramer in the cell nucleus and acts as a blockers as chemotherapeutic agents that can induce cancer transcription factor, mediating expression of cell cycle-reg- cell death. ulating, senescence-inducing, and proapoptotic genes (22). Previously, mibefradil was shown to decrease proliferation of Materials and Methods esophageal carcinoma cells by increasing transcription of the Cell culture and drug treatment cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 Colon carcinoma HCT116 p53wt and p53-deleted (8). This suggested to us that cellular responses to T-type 2þ mutant were described previously (35) and were maintained Ca channels might be p53 dependent. in McCoy 5A medium (Life Technologies) supplemented In general, p53 is phosphorylated by protein kinases of with 10% FBS (Life Technologies). CaCo2 colon adeno- the phosphatidylinositol 3-kinase-related kinase family carcinoma cell line was purchased from American Type that are activated by DNA damage, such as ataxia telan- Culture Collection and cultured in Minimum Essential giectasia mutated (ATM), ataxia telangiectasia mutated Medium (MEM) supplemented with L-glutamine, nones- and Rad3 related (ATR), and DNA-dependent protein sential amino acids, and 20% FBS (Life Technologies). The kinase (DNAPK). Phosphorylation releases p53 from its cell lines were not authenticated. Mibefradil and TTL1177 interactions with MDM2 ubiquitin ligase, which stabilizes were generously provided by Tau Therapeutics LLC. fi p53, and allows for its speci c DNA binding (23, 24). NNC55–0396 hydrate was from Sigma-Aldrich. The Following DNA damage induced by ionizing radiation or p38-MAPK inhibitor SB203580 was from Santa Cruz UV radiation, at least 17 Ser/Thr residues are phosphor- Biotechnology. All inhibitors were prepared in dimethyl fi ylated within p53. Signi cant redundancies are observed sulfoxide (DMSO; 10 mmol/L stock) and diluted in media as a single residue can be subject to phosphorylation by before the use. multiple protein kinases, for example, Ser15-p53 is report- edly phosphorylated by ATM, ATR, DNAPK, p38- Drug-induced growth inhibition MAPK, and ERK (extracellular signal–regulated kinase) HCT116 cells were seeded in 96-well plates at 1,000 cells kinases (reviewed in ref. 21). Interestingly, some of the per well. Twenty-four hours later, cells were treated in protein kinases that interact and phosphorylate p53 þ triplicates with different concentrations of T-type Ca2 belong to the calmodulin-dependent kinase super family channels inhibitors (mibefradil or TTL1177). After contin- (which includes Chk1, Chk2, and death associated protein uous exposure to drug for 4 days, cells were fixed with kinase 1 and 3; ref. 25), and these may phosphorylate p53 2þ trichloroacetic acid (10%) and stained with sulforhodamine in response to pathologic Ca signaling. 2þ B solution (0.1%; ref. 36). Percentage of growth inhibition Cross-talk is apparent between T-type Ca channel signaling and mitogen-activated protein kinase (MAPK) was determined after background subtraction based on the comparison of the absorbance signal of drug-treated cells pathways with consequences for p53. Mobilization of 2þ 2þ versus the control. Ca through T-type Ca channels (Cav 3.1; CACNA1G) produces a transient decrease in activity of Raf/MEK/ERK (26). Although ERK tends to induce prosurvival pathways, Plasmid DNA and siRNA transfection and growth stress-activated MAPKs, such as p38-MAPK and JNK (c- inhibition jun– – NH2 kinase), are linked to induction of apoptosis (27, CaCo2 cells were transfected with pcDNA3.1 p53wt or 28). In particular, upon UV-induced DNA damage or pcDNA3.1 empty vector plasmids [kind gift from Dr. A. osmotic shock, p38-MAPK phosphorylates p53 at Ser46 Dutta, University of Virginia (UVA), Charlottesville, VA] (29) or Ser33 (30), thus stabilizing p53. Moreover, UV- using lipofectamine LTX and plus reagent (Life Technolo- induced DNA damage activates p38-MAPK to activate gies) according to the manufacturer's instructions. Colonies casein kinase 2 (CK2), which in turn also phosphorylates were selected for 8 weeks in media containing selection p53 at Ser392, increasing p53 transcriptional activity (31– antibiotic geneticin G418 (600 mg/mL; Life Technologies). In RNAi experiments the cells were transfected with 25 33). Thus, p38-MAPK supports increased p53 levels and 0 0 activity. nmol/L of siRNA [scrambled: 5 -gacgaaagaccacucaauu/5 - þ In this study, we evaluated the actions of T-type Ca2 aauugaguggucuuucguc, siCACNA1G-ORF (#3166; 1): 50-uuguagaggacuuuguucc/50-ggaacaaaguccucuacaa, siCAC- channels using two isogenic epithelial colon carcinoma cell 0 0 lines, HCT116 p53 wild-type (p53wt) and the HCT116 NA1G-ORF (#5356; 2): 5 -auuuccuccagcgugaugc/5 -gcau- p53-deleted counterpart (HCT116 p53 / ). In addition, cacgcuggaggaaau; all from Invitrogen/Life Technologies], or we compared CaCo2 cells that do not express detectable p53 with 10 nmol/L of siRNA pool of four different sequences protein due to a nonsense mutation in exon 6 of the p53 gene for each gene (Santa Cruz Biotechnology) against MAPK14 (Glu to Stop codon; ref. 34) with cells rescued for p53 by and MAPK11 (p38-MAPKa and p38-MAPKb, respective- stable transfection with either vector (pcDNA3.1) or p53wt ly), using lipofectamine RNAiMax (Life Technologies) gene (pcDNA3.1 p53wt). We uncovered a relationship according to the manufacturer's instructions. HCT116 or þ 5 between T-type Ca2 channel inhibition and p53-depen- CaCo2 cells were seeded at 10 per well and transiently dent growth inhibition and apoptosis that implicate a p38- transfected 24 hours later with appropriate siRNA con- MAPK kinase signaling circuit. Our results support the structs. HCT116 cells were trypsinized after 72 to 96 hours, þ rationale for future development of T-type Ca2 channel counted using trypan blue exclusion assay and collected by

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centrifugation for total RNA isolation. For CaCo2, cells Chromatin immunoprecipitation assay were reseeded after 24 hours at 500 cells per well into new Chromatin immunoprecipitation (ChIP) assay was per- dishes and allowed to form colonies for 14 days before formed according to the published protocol (39). Briefly, staining with Coomassie Brilliant Blue R-250. cells were seeded in 100-mm dishes at 2.5 106 2days before treatment with mibefradil at 10 mmol/L, or expo- Caspase-3/7 activity assay sure to ionizing radiation (6 Gy). After 9 hours drug Cells seeded at 3 105 per well in 6-well plates were treatment or 6 hours after irradiation, macromolecules transfected with 25 nmol/L of specific siRNA [scrambled were cross-linked with 1% formaldehyde for 10 minutes. control or siCACNA1G (1)]. Twenty-four hours later, cells Cells were lysed in buffer containing 1% SDS, protease were trypsinized, counted, and reseeded at 6,000 cells per inhibitors (Pierce; Thermo Fisher Scientific) and 100 mg/ well into an opaque 96-well plate. Caspase-3/7 activity was mL of sonicated herring sperm genomic DNA. Detergent- measured 48 hours posttransfection using the Apo-ONE insoluble fractions were collected by centrifugation at caspase activity assay kit according to the manufacturer's 14,000 rpm for 10 minutes at 4C, resuspended in instructions (Promega). Caspase-3/7 activity was measured 0.25% SDS, 0.25 mmol/L NaCl, protease inhibitors in triplicates and represented as a fold-increase of fluores- (Pierce), and 100 mg/mL herring sperm genomic DNA, cence calculated by comparing siRNA targeted cells with and sonicated using Branson Sonifier 400 W Cell Dis- cells treated with scrambled siRNA. ruptor. Extracts were diluted with three volumes of 350 mmol/L NaCl, 1% NP40 plus protease inhibitors, and Reverse transcriptase quantitative PCR specific antibodies (mouse monoclonal anti-p53 or mouse Total RNA was isolated using the RNeasy Kit (Qiagen) monoclonal IgG; EMD Millipore) were added for over- m and 1 g was used for cDNA synthesis using the iScript night incubation. Protein–DNA complexes were collected cDNA Synthesis Kit (Bio-Rad Laboratories) according to with protein G sepharose beads (Sigma-Aldrich) and the instructions. Each quantitative PCR (qPCR) reaction washed four times with 350 mmol/L NaCl, 1% NP40, was done in triplicate and included 50 ng of cDNA as a 50 mg/mL of herring sperm genomic DNA. Complexes fi template, speci c primers (Supplementary Table S1; were dissociated from beads, cross-links were reversed with ref. 37) and SsoFast EvaGreen master mix (Bio-Rad heat (65C/4 h), and DNA was phenol–chloroform fi Laboratories). Conditions for ampli cation were as fol- extracted and precipitated with 0.7 volume of 100% lows: initial denaturation 98C for 30 seconds, followed isopropanol. Mibefradil or ionizing radiation induced by 40 cycles of denaturation for 5 seconds at 98 C, and fi enrichment of p53 binding to speci c sites was deter- annealing with extension for 5 seconds at 62 C. Relative mined on the basis of the qPCR reaction normalized to the fi 2þ gene expression of speci c genes of T-type Ca channels input and to the signal of untreated HCT116 p53wt ðDDC Þ wasnormalizedonthebasisoftheglyceraldehyde-3- control according to the formula: 2 t .Specificpri- phosphate dehydrogenase (GAPDH) expression level. mers were designed using Primer3Plus software (40) and Normalized gene expression was calculated by the formula either included or were in a close proximity to a specific 2ðDDCtÞ C by subtracting the t value of GAPDH and then binding site for p53 according to previously published C the t value of untreated control. data (Supplement Table S2; ref. 41). Western blotting Statistical analyses HCT116 cells were seeded at 2.5 105 in 35-mm plates All values are expressed as the means of at least three 48 hours before mibefradil treatment for 9 or 24 hours. After independent experiments SEM. Results were compared treatment cells were trypsinized, counted, and resuspended using one-factor ANOVA analysis. A P value of less than at 1 107 cells/mL in a modified RIPA buffer (20 mmol/L 0.05 indicated statistically significant differences. Tris pH 7.6, 150 mmol/L NaCl, 5% glycerol, 1 mmol/L EDTA, 1% deoxycholate plus protease inhibitors, and Results þ phosphatase inhibitors; Sigma-Aldrich). Equivalent of Inhibition of T-type Ca2 channels induces p53- 30,000 cells per lane were resolved by electrophoresis in dependent apoptosis in HCT116 cells SDS–PAGE, transferred to the nitrocellulose membrane We tested mibefradil and the structurally unrelated T-type þ (0.2 mm; Bio-Rad Laboratories) and probed with specific Ca2 channel blocker TTL1177 in HCT116 p53wt and antibodies anti-p53 (1:200; Santa Cruz Biotechnology), p53 / cells, a well-established model system to compare anti-CACNA1G (1:500; anti-Cav 3.1; Abcam), anti-CAC- p53-dependent cellular responses (35). Cells were treated NA1H (1:500; anti-Cav 3.2; clone N55/10; UC Davis/NIH with increasing concentrations of mibefradil or TTL1177, NeuroMab Facility), anti-phospho Ser392 p53 and anti- or vehicle as control, for 96 hours and assayed for prolifer- phospho Ser15 p53 (1:1,000; Cell Signaling Technology), ation as described in Materials and Methods. Both mibe- anti-p21 and anti-PUMA (1:1,000; Cell Signaling Tech- fradil and TTL1177 decreased growth of HCT116 cells nology), and anti-b-actin (1:10,000; Sigma-Aldrich). The relative to controls in a dose-dependent manner; however, proteins of interest were visualized using a two-color Li- the p53wt cells showed greater sensitivity than HCT116 COR Odyssey Imager (LI-COR) and quantified using p53 / cell line (3.3- and 3.1-fold difference in sensitivity to ImageJ software (38). mibefradil and TTL; Fig. 1A and C, respectively).

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þ Figure 1. T-type Ca2 channel antagonists inhibit growth and induce p53-dependent apoptosis in HCT116 cells. HCT116 cells were treated with increasing mibefradil (Mib; A) or TTL1177 (C) concentrations. Drug-induced growth inhibition was measured after 96 hours treatment using sulforhodamine B (SRB) staining assay. Plots present data from at least three independent experiments SEM. To measure apoptosis, HCT116 cells were treated with Mib (B) or TTL1177 (D) and after 24 hours, apoptosis induction was assessed using ﬂuorescence caspase-3/7 activity assay as described in Materials and Methods. Plots present data from at least three independent experiments SEM. Statistical signiﬁcance: , P < 0.05; , P < 0.001.

Preferential inhibition of HCT116 p53wt over p53 / cells To rule out the possibility that the observed effects of also was observed with NNC55–0396, an analog of mibe- mibefradil and TTL1177 on HCT116 cells were due to off- þ þ fradil with greater selectivity for T-type Ca2 channels (data target actions rather than inhibition of T-type Ca2 chan- not shown; ref. 42). Notably, the reduction in cell prolif- nels, we used siRNA-mediated knockdown of CACNA1G. eration in response to either mibefradil or TTL1177 was Cells were transiently transfected with two different siRNAs accompanied by a significant increase in activation of cas- targeting CACNA1G, si(1) and si(2). The efficiency of pase-3/7 (Fig. 1B and D). Treatment of p53wt cells for 24 knockdown was approximately 75% to 80% as measured hours with 10 mmol/L mibefradil resulted in a 9 1.8–fold by RT-qPCR (Fig. 2B). Reduction of CACNA1G expres- increase in caspase-3/7 activity as compared with the vehicle- sion significantly decreased growth of HCT116 p53wt cells treated control, whereas TTL1177 gave a 2.9 0.1–fold (Fig. 2C). Interestingly, depletion of CACNA1G also increase. Under the same conditions neither mibefradil nor reduced the proliferation of HCT116 p53 / cells, although TTL1177 induced caspase activity in p53 / HCT116 not as extensively as in p53wt cells, suggesting that decreased þ cells. Thus, reduced proliferation and induction of apoptosis expression of T-type Ca2 channels can affect proliferation þ in response to blockade of T-type Ca2 channels by different independent of p53 status (Fig. 2C). Inhibition of cell agents seems to depend upon p53. proliferation induced by specific siRNA was accompanied by a significant increase of caspase-3/7 activity in HCT116 þ Knockdown of CACNA1G T-type Ca2 channel in p53wt cells (2.4 0.15–fold increase over vehicle-treated HCT116 cells results in a p53-dependent reduction of cells; Fig. 2D). cell proliferation and induction of apoptosis þ We measured the relative expression of T-type Ca2 Expression of p53wt sensitizes CaCo2 cells to a þ channels subunits CACNA1G (Cav3.1) and CACNA1H knockdown of T-type Ca2 channels (Cav3.2) in HCT116 p53wt and p53 / cells using reverse We used CaCo2, another colon carcinoma cell line þ transcriptase quantitative PCR (RT-qPCR; Fig. 2A). previously reported to express T-type Ca2 channels HCT116 p53wt and p53 / cells express mRNA for both (43). CaCo2 are deficient in functional p53 due to a CACNA1G and CACNA1H subunits at comparatively nonsense mutation in exon 6 of the p53 gene (Glu to Stop similar levels. Consistent with similar mRNA levels, using codon; ref. 34); thus, for a pairwise comparison, we specific antibodies, we observed the same levels of CAC- established cell lines stably transfected with the p53wt þ NA1G (Cav3.1) and CACNA1H (Cav3.2) proteins by gene. The expression of mRNA for T-type Ca2 channels immunoblotting extracts of HCT116 p53wt and p53 / subunits CACNA1G and CACNA1H (Fig. 3A) and p53 cells (Fig. 2A, inset). protein level (Fig. 3A, inset) were confirmed in the

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þ Figure 2. RNAi-mediated downregulation of a T-type Ca2 channel subunit gene, CACNA1G, results in p53-dependent decrease in cell proliferation and þ apoptosis induction. A, RT-qPCR reaction assessing relative expression level of different T-type Ca2 subunits, CACNA1G and CACNA1H, present in HCT116 cells. The plot represents data from three independent experiments SEM. Inset represents Western blot analysis of CACNA1G and CACNA1H proteins expressed in HCT116 cells. HCT116 were transiently transfected with scrambled (scrmbl, 25 nmol/L) or two different siRNA targeting a single T-type þ Ca2 channel subunit CACNA1G [siCACNA1G (1) and siCACNA1G (2) 25 nmol/L]. After 96 hours total RNA was isolated and used for RT-qPCR reaction. B, RT-qPCR confirms specific downregulation of CACNA1G gene expression with si(1) and si(2). The plot represents data from at least two independent experiments SEM. C, growth inhibition induced by siCACNA1G (1) or siCACNA1G (2) was determined by counting cells 96 hours after transfection using trypan blue exclusion assay. The plot represents data from four independent experiments SEM. D, apoptosis assay in HCT116 cells transfected with 25 nmol/L of siCACNA1G (1). Cells were transfected 48 hours prior measuring caspase-3/7 activity. The plot presents data from three independent experiments SEM. Statistical significance: , P < 0.05; , P < 0.01.

þ selected clones. Next, the parental CaCo2 and those Inhibition of T-type Ca2 channels increases p53 expressing p53wt were transiently transfected with two binding at the promoters and regulatory sequences of different speciﬁc siRNA to knockdown CACNA1G target genes expression. The extent of CACNA1G knockdown was In response to mibefradil treatment, p53 protein accu- demonstrated with RT-qPCR (Fig. 3B). Expression of mulated in HCT116 cells and its phosphorylation at p53 sensitized CaCo2 cells to knockdown of CACNA1G Ser392 increased in a mibefradil dose-dependent manner gene expression (Fig. 3C), with cell survival reduced for si (Fig. 4A). In agreement with previous reports (8), mibe- (1) to 33% 4.3% (CaCo2p53wt-11) versus 74% fradil also induced expression of the CDK inhibitor 6.1% for control cells and for si(2) cell survival at 64% CDKN1A (p21Cip1/Waf1), and the proapoptotic protein 1.4% (CaCo2p53wt-11) versus 93% 6.3% for controls. BBC3 (PUMA; Fig. 4A). The expression of p53-depen- Importantly, the decrease in survival of CaCo2p53wt-11 dent genes induced by mibefradil was conﬁrmed at the cells was associated with increased expression of p53- mRNA level (Fig. 4B). Total RNA from either DMSO- dependent genes, such as CDKN1A (p21Cip1/Waf1) and (0.1%) or mibefradil- (10 mmol/L) treated cells was BBC3 (PUMA; Fig. 3D), indicating that p53 transcrip- isolated and analyzed for expression of p53-responsive tional activity was induced in response to knockdown of genes using RT-qPCR. As a positive control of p53 CACNA1G. function, irradiated cells (6 Gy/6 hours after irradiation)

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þ Figure 3. Expression of p53wt gene sensitizes CaCo2 cells to downregulation of T-type Ca2 channels. p53-mutant CaCo2 cells were stably transfected with þ p53wt gene (CaCo2p53wt-11) or with pcDNA3.1 vector (CaCo2Vec). A, evaluation of mRNA expression levels of the T-type Ca2 channels in CaCo2 cells. RT- qPCR reaction was performed using total RNA isolated from cells as described in Materials and Methods. The plot represents data from at least three independent experiments SEM. In the inset, Western blot analysis of p53 protein level in CaCo2 cells. B, RT-qPCR reaction confirms RNAi-mediated downregulation of CACNA1G gene expression in CaCo2 cells with specific siRNA [siCACNA1G (1) and siCACNA1G (2)]. The plot represents data from at least two independent experiments SEM. C, transient transfection of CaCo2 cells with siRNA targeting CACNA1G decreases cell survival of cells with restored p53wt gene. CaCo2 cells were transfected with siCACNA1G (1) or siCACNA1G (2) and 24 hours later reseeded for colonies formation. The plot represents data from at least three independent experiments SEM. D, RNAi-mediated downregulation of CACNA1G subunit increases expression of p53-dependent genes in CaCo2p53wt-11 cells. Total RNA was used for RT-qPCR reaction using specific primers for p53-dependent gene expression listed in Supplementary Table S1. The plot represents averaged data obtained from transfections with si(1) and si(2) targeting CACNA1G gene expression that was independently repeated SEM. Statistical significance: , P < 0.05; , P < 0.01. were used, and demonstrated similar levels of enrichment from either nontreated control, ionizing radiation (6 Gy/6 of gene expression as cells treated with mibefradil (data not hours after irradiation), or mibefradil-treated cells. Both shown). Increased expression of p53-dependent genes ionizing radiation and mibefradil increased association of CDKN1A (p21Cip1/Waf1) and BBC3 (PUMA) have p53 at specific p53BS (expressed as fold of enrichment). In been observed before (8), but we also uncovered addi- agreement with our previous observation, mibefradil treat- tional p53-induced genes that were elevated by inhibition ment increased p53 binding to CDKN1A, GADD45A, and þ of T-type Ca2 channels, such as growth arrest and DNA BBC3 genes (Fig. 4C), corresponding well with increased damage–inducible gene GADD45A (Fig. 4B). As a neg- gene expression (Fig. 4A and B). Surprisingly, not all selected ative control of p53-dependent gene expression, we eval- proapoptotic p53 target genes (e.g., FAS, TP53AIP1, or uated mibefradil-treated HCT116 p53 / cells (Fig. 4A BAX) showed an increase in p53 binding upon mibefradil and B). Lack of p53-dependent gene expression in treatment (Fig. 4C and data not shown). HCT116 p53 / cells was consistent with their resistance to mibefradil-induced apoptosis (Fig. 1B). p38-MAPK is required for signaling during T-type þ To explore in more detail the action of p53 in mibefradil- Ca2 channels inhibition induced gene expression, cells treated with mibefradil (10 We found that mibefradil treatment of HCT116 cells led mmol/L) for 9 hours were subjected to ChIP. The specific to an increase in Ser392 phosphorylation of p53 (Fig. 4A). sequences within selected genes containing p53-binding Activated p38-MAPK associates with CK2 to enhance sites (p53BS), or in close proximity to p53BS, were detected phosphorylation of p53 at Ser392 (31–33). Therefore, we using qPCR in immunoprecipitated p53-DNA complexes investigated whether p38-MAPK was involved in the

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þ Figure 4. T-type Ca2 channel inhibitor, Mib, increases p53 phosphorylation on Ser392, expression of p53-dependent genes and p53 binding at the promoters and regulatory sequences within genomic DNA. A, Western blot analysis of the whole-cell extracts isolated from HCT116 cells treated with increasing concentrations of Mib. Blot is representative of at least three independent experiments. B, gene expression of p53-dependent genes: Fas, CDKN1A, BBC3, and GADD45A upon Mib treatment in HCT116 cells. RT-qPCR was performed using total RNA isolated from Mib-treated cells (10 mmol/L/9 h) and speciﬁc primers used for gene expression as listed in Supplementary Table S1. Graph represents data from at least three independent experiments SEM. C, association of p53 with its binding sites within a genome induced by Mib treatment as measured by ChIP assay. Cells were treated with Mib (10 mmol/L) for 9 hours prior isolation of protein–DNA cross-links. As a positive control, HCT116 cells were irradiated with 6 Gy and collected 6 hours after irradiation. qPCR reaction was done in triplicates using primers listed in Supplementary Table S2. Data were normalized to the input and to the nondrug-treated nonirradiated HCT116 p53wt control. The plot represents data from at least two independent experiments SEM.

þ response to T-type Ca2 channel inhibition. HCT116 cells tion was clearly evident at both the mRNA (Fig. 6A) and were preincubated with either a pharmacologic inhibitor of protein level (Fig. 6B). Taken together, our results point to p38-MAPK, SB203580, or specific siRNA to knockdown p38-MAPK activation of p53-dependent transcription in þ the expression of both MAPK14 and MAPK11 (p38- cells upon inhibition of T-type Ca2 channels. MAPKa/b), and then treated with mibefradil. Inhibition or knockdown of p38-MAPK significantly relieved inhibi- Discussion þ tion of HCT116 cell proliferation by mibefradil (Fig. 5A and T-type Ca2 channels are expressed in epithelial cancer B). These results provide evidence for the involvement of cells, such as prostate, breast, and colon carcinoma (5, p38-MAPK in the cellular response to mibefradil. In support 6, 37, 43), but not in the corresponding normal tissues, of these data, either SB203580 or knockdown of p38- making them a potentially selective target for anticancer þ MAPK prevented accumulation of p53 and phosphorylation therapy. Pharmacologic inhibition of T-type Ca2 channels of Ser392 in response to mibefradil (Fig. 5C for SB203580 was previously shown to inhibit cell growth in several and Fig. 5D for si-p38-MAPKa/b). We observed higher different cancer cell lines (4–6). Although p53 was impli- levels of p53 Ser15 phosphorylation in HCT116 cells treated cated in the response (8), the signaling pathway(s) connect- þ with mibefradil (Fig. 5D); however, these increases were not ing T-type Ca2 channels to proliferation and cell death have statistically significant (P ¼ 0.671, N ¼ 3). Our data indicate not been defined. that p38-MAPK is required for signaling to p53 in response Results from this study show that both p38-MAPK and þ to inhibition of T-type Ca2 channels. p53 are required for immediate response (growth inhibition Finally, HCT116 cells were subjected to combined treat- and induction of apoptosis) following inhibition of T-type þ ment with SB203580 and mibefradil and assayed for expres- Ca2 channels in cancer cells. The evidence suggests that sion of CDKN1A (p21Cip1/Waf1) and BBC3 (PUMA). p38-MAPK promotes phosphorylation of Ser392 in the p53 Combination treatment significantly reduced the expression protein to increase p53 levels that in turn activates tran- of CDKN1A (p21Cip1/Waf1) and BBC3 (PUMA) genes scription of genes leading to cell-cycle arrest and caspase- compared with treatment with mibefradil alone. The reduc- mediated apoptosis (Fig. 7). This signaling pathway

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Figure 5. p38-MAPK regulates antiproliferative action of p53 upon treatment of cells with Mib. A, HCT116 p53wt cells were treated with specific inhibitor of p38-MAPK (SB203580, 10 mmol/L) for 1 hour and subsequently with Mib (10 mmol/L) or (B) transfected with specific siRNA to decrease expression of p38-MAPKa/b (10 nmol/L) and then treated with Mib (10 mmol/L) for 24 hours. Next day cells were trypsinized, counted using trypan blue exclusion assay and number of live cells plotted as a percentage of control. Plots present data from at least three independent experiments SEM. C, Western blot analysis of whole- cell lysates isolated from HCT116 p53wt cells treated with Mib or in combination with SB203580. The plot presents quantification of p53 and P-Ser392 p53 signal relative to actin and nondrug-treated control from at least two independent experiments SEM. D, Western blot analysis of whole-cell lysates isolated from HCT116 p53wt cells transfected with sip38-MAPKa/b and treated with Mib (10 mmol/L/24 h). The plot represents quantification of p53 and P-Ser392 p53 signal relative to actin and nondrug-treated control from at least three independent experiments SEM. Statistical significance: , P < 0.05; ns, P > 0.05. presumably is kept in a relatively inactive state by the activity regulation (Fig 3C). As demonstrated by ChIP and gene þ of T-type Ca2 channels. The HCT116 and CaCo2 epi- expression analyses, p53 binds to specific promoter/enhancer thelial cancer cells studied here both express two subunits of sequences and thereby facilitates transcription of genes such þ T-type Ca2 channels, CACNA1G (Cav3.1) and CAC- as CDKN1A and BBC3. The increase in p53 transcriptional NA1H (Cav3.2). Either pharmacologic inhibition of the activities correlates with its phosphorylation at Ser392. channels or RNAi-mediated knockdown of the Cav3.1 Pharmacologic inhibition or knockdown of MAPK14/11 subunit was sufficient for activation of the signaling path- (p38-MAPKa/b) significantly decreased both Ser392 phos- way. Interestingly and in agreement with our other pub- phorylation and protein levels of p53, corresponding to lished results (4), knockdown of CACNA1H (Cav3.2) had reduced expression of CDKN1A and BBC3 genes. significantly smaller effects compared with the knockdown Phosphorylation of p53 at C-terminal residue Ser392 is of CACNA1G, suggesting differences between the func- believed to be important for tetramerization, stability, and þ tional role of these two subunits of T-type Ca2 channels transcriptional activities of p53 (44–46). Mutation of (data not shown). On the basis of our results, we propose that Ser392 to Ala, but not to phosphomimetic Asp, increased þ CACNA1G (Cav3.1) subunit of T-type Ca2 channels chemoresistance of some cancer cells and increased colony promotes survival of epithelial cancer cells by suppressing formation in soft agar, an indication of aggressive growth and a proapoptotic signaling pathway (Fig. 7), whereas CAC- greater oncogenic properties of p53 (46). In agreement, p53 NA1H (Cav3.2) may be necessary to allow for cell-cycle Ser392 seems to be not phosphorylated in mutant p53 that is transitions. Disruption of this signaling pathway at the expressed in breast cancer (47), and most likely the loss of channel, or p38-MAPK or p53 prevents apoptosis in phosphorylation mark at this residue is responsible for þ response to inhibition or knockdown of T-type Ca2 maintenance of p53 oncogenic properties (reviewed in; channels. ref. 47). Thus, phosphorylation of p53 at Ser392 may be This cell death pathway shows strong dependence on p53 important not only to stimulate p53-dependent transcrip- function. Expression of p53 in CaCo2 cells converted them tion, but also could potentially decrease oncogenic proper- þ from resistant to sensitive to T-type Ca2 channel down- ties of mutant p53.

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Figure 6. p38-MAPK activity increases expression of p53- dependent genes upon Mib treatment. A, normalized gene expression of CDKN1A and BBC3 in HCT116 p53wt cells treated with SB203580 (10 mmol/L), Mib (10 mmol/L) alone or combination of both for 9 hours. Graph represents data from two independent experiments SEM. B, protein analysis of whole-cell extracts isolated from HCT116 p53wt cells treated with SB203580, Mib (10 mmol/L) or with combination of both for 9 hours. Membranes were stripped and reprobed with anti- BBC3 (PUMA) antibody. Western blot analysis represents data from two independent experiments. Statistical signiﬁcance: , P < 0.05.

þ p38-MAPK is involved in cellular response to UV- in response to inhibition of T-type Ca2 channels. These induced DNA damage and DNA replication inhibition, phosphorylation events activate p53 to execute growth leading to p53 phosphorylation at Ser15, 37, and 46 inhibition and apoptosis programs. It has been shown that (29), and has been suggested to indirectly (through activa- mouse embryonic ﬁbroblasts (MEFs) derived from tion of CK2) phosphorylate p53 at Ser392 (31–33). p38- MAPK14 knockout mice (p38-MAPKa / ) are more resis- MAPK was also reported to phosphorylate p53 at Ser33 tant to apoptosis induced by several stimuli (49), which during osmotic shock (30). However, based on the recent correlated well with decreased expression of p53-regulated report in the literature (48), we cannot exclude other kinases proapoptotic genes. from being involved in phosphorylation of p53 Ser392 Our data not only suggest involvement of p38-MAPK in p53-dependent growth inhibition and apoptosis induc- þ tion, but also implicate that T-type Ca2 channels fuel prosurvival processes in cells, especially cancer cells. This occurs most likely through cross-talk with MAPK, given þ that the inhibition of T-type Ca2 channels results in activation of stress-activated p38-MAPK and at the same time downregulation of ERK1/2 kinase (data not shown). These effects could be due to the changes in activity of þ Ca2 -dependent protein phosphatases, such as calcineurin or PP2A (AB70C) that were previously reported to regulate the MAPK pathway (50–52). A recent study from our group suggests that mibefradil inhibits Akt and mTORC2 signaling (4), indicating additional cross-talk þ of T-type Ca2 channels with intracellular prosurvival signaling pathways. It remains to be elucidated how p38- 2þ 2þ Figure 7. The model of p53 activation upon T-type Ca channel MAPK is activated in response to T-type Ca channel þ inhibition. inhibition or knockdown, and what Ca2 -dependent

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p53 and p38-MAPK Response to T-Type Ca2þ Channels Inhibition

factors transduce signals to either upstream MAP3K5 Authors' Contributions (ASK1) kinase (14) or directly to p38-MAPK. Conception and design: B. Dziegielewska, J.M. Larner, J. Dziegielewski fi fi Development of methodology: B. Dziegielewska, J.M. Larner Our ndings have signi cant implications for mibefradil- Acquisition of data (provided animals, acquired and managed patients, provided based cancer therapy, since we observe activation of p38- facilities, etc.): B. Dziegielewska Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- MAPK, inhibition of cell growth and an increase in apoptosis tational analysis): B. Dziegielewska, D.L. Brautigan, J.M. Larner, J. Dziegielewski upon treatment of cancer cells with the drug. The data Writing, review, and/or revision of the manuscript: B. Dziegielewska, þ support the notion for use of T-type Ca2 channels D.L. Brautigan, J.M. Larner, J. Dziegielewski inhibitors as potentially effective agents to sensitize cells Study supervision: J. Dziegielewski to DNA damage in novel therapeutic approaches, as tested Acknowledgments in interlaced therapy (9, 53). However, combination The authors thank Tau Therapeutics LLC (Charlottesville, VA) for providing us treatment of mibefradil with any inhibitor(s) of p38- with mibefradil and TTL1177, Dr. Anindya Dutta (University of Virginia, Charlot- fl tesville, VA) for providing us with pcDNA3.1 p53wt plasmid, Dr. Amol Hosing for MAPK that might be used to counteract proin ammatory technical assistance, and Dr. Erin Griner for help with editing the article. responses in cancer (reviewed in ref. 54) should be carefully considered. Grant support This work was supported in part by the UVA Department of Radiation Oncology Dr. George Amornio pilot grant (to B. Dziegielewska) and UVA P30 CA44579 and Disclosure of Potential Conflicts of Interest pilot funding from the James and Rebecca Craig fund (to J. Dziegielewski). Jaroslaw Dziegielewski has received a commercial research grant from Tau Therapeutics LLC. No potential conflicts of interest were disclosed by the other Received September 11, 2013; revised November 19, 2013; accepted December 11, authors. 2013; published OnlineFirst December 20, 2013.

References 1. Clapham DE. Calcium signaling. Cell 2007;131:1047–58. 14. Takeda K, Matsuzawa A, Nishitoh H, Tobiume K, Kishida S, Ninomiya- 2. Monteith GR, Davis FM, Roberts-Thomson SJ. Calcium channels and Tsuji J, et al. Involvement of ASK1 in Ca2þ-induced p38 MAP kinase pumps in cancer: changes and consequences. J Biol Chem 2012; activation. EMBO Rep 2004;5:161–6. 287:31666–73. 15. Liu G, Zhao J, Chang Z, Guo G. CaMKII activates ASK1 to induce 3. Zhang Y, Zhang J, Jiang D, Zhang D, Qian Z, Liu C, et al. Inhibition of T- apoptosis of spinal astrocytes under oxygen-glucose deprivation. Cell type Ca(2þ) channels by endostatin attenuates human glioblastoma Mol Neurobiol 2013;33:543–9. cell proliferation and migration. Br J Pharmacol 2012;166:1247–60. 16. Yano S, Tokumitsu H, Soderling TR. Calcium promotes cell survival 4. Valerie NCK, Dziegielewska B, Hosing AS, Augustin E, Gray LS, through CaM-K kinase activation of the protein-kinase-B pathway. Brautigan DL, et al. Inhibition of T-type calcium channels disrupts Akt Nature 1998;396:584–7. signaling and promotes apoptosis in glioblastoma cells. Biochem 17. Johannessen M, Delghandi MP, Moens U. What turns CREB on? Cell Pharmacol 2013;85:888–97. Signal 2004;16:1211–27. 5. Bertolesi GE, Shi C, Elbaum L, Jollimore C, Rozenberg G, Barnes S, 18. Liu Z-M, Chen GG, Vlantis AC, Tse GM, Shum CKY, van Hasselt et al. The Ca(2þ) channel antagonists mibefradil and pimozide inhibit CA. Calcium-mediated activation of PI3K and p53 leads to apo- cell growth via different cytotoxic mechanisms. Mol Pharmacol ptosis in thyroid carcinoma cells. Cell Mol Life Sci CMLS 2007; 2002;62:210–9. 64:1428–36. 6. Taylor JT, Huang L, Pottle JE, Liu K, Yang Y, Zeng X, et al. Selective 19. Can G, Akpinar B, Baran Y, Zhivotovsky B, Olsson M. 5-Fluorouracil blockade of T-type Ca2þ channels suppresses human breast cancer signaling through a calcium-calmodulin-dependent pathway is cell proliferation. Cancer Lett 2008;267:116–24. required for p53 activation and apoptosis in colon carcinoma cells. 7. Das A, Pushparaj C, Herreros J, Nager M, Vilella R, Portero M, et al. T- Oncogene 2013;32:4529–38. type calcium channel blockers inhibit autophagy and promote apo- 20. Amorino GP, Mikkelsen RB, Valerie K, Schmidt-Ullrich RK. Dominant- ptosis of malignant melanoma cells. Pigment Cell Melanoma Res negative cAMP-responsive element-binding protein inhibits prolifer- 2013;26:874–85. ating cell nuclear antigen and DNA repair, leading to increased cellular 8. Lu F, Chen H, Zhou C, Liu S, Guo M, Chen P, et al. T-type Ca2þ channel radiosensitivity. J Biol Chem 2003;278:29394–9. expression in human esophageal carcinomas: a functional role in 21. Bode AM, Dong Z. Post-translational modiﬁcation of p53 in tumori- proliferation. Cell Calcium 2008;43:49–58. genesis. Nat Rev Cancer 2004;4:793–805. 9. Keir ST, Friedman HS, Reardon DA, Bigner DD, Gray LA. Mibefradil, a 22. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol novel therapy for glioblastoma multiforme: cell cycle synchronization 2007;8:275–83. and interlaced therapy in a murine model. J Neurooncol 2013;111: 23. Hupp TR, Lane DP. Allosteric activation of latent p53 tetramers. Curr 97–102. Biol CB 1994;4:865–75. 10. Sheehan JP, Xu Z, Popp B, Kowalski L, Schlesinger D. Inhibition of 24. Hupp TR, Meek DW, Midgley CA, Lane DP. Regulation of the speciﬁc glioblastoma and enhancement of survival via the use of mibefradil in DNA binding function of p53. Cell 1992;71:875–86. conjunction with radiosurgery. J Neurosurg 2013;118:830–7. 25. Craig AL, Chrystal JA, Fraser JA, Sphyris N, Lin Y, Harrison BJ, et al. 11. Taylor JT, Zeng X-B, Pottle JE, Lee K, Wang AR, Yi SG, et al. Calcium The MDM2 ubiquitination signal in the DNA-binding domain of p53 signaling and T-type calcium channels in cancer cell cycling. World J forms a docking site for calcium calmodulin kinase superfamily mem- Gastroenterol WJG 2008;14:4984–91. bers. Mol Cell Biol 2007;27:3542–55. 12. Welsby PJ, Wang H, Wolfe JT, Colbran RJ, Johnson ML, Barrett PQ. A 26. Choi J, Park J-H, Kwon OY, Kim S, Chung JH, Lim DS, et al. T-type mechanism for the direct regulation of T-type calcium channels by calcium channel trigger p21ras signaling pathway to ERK in Cav3.1- Ca2þ/calmodulin-dependent kinase II. J Neurosci Off J Soc Neurosci expressed HEK293 cells. Brain Res 2005;1054:22–9. 2003;23:10116–21. 27. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing 13. Yao J, Davies LA, Howard JD, Adney SK, Welsby PJ, Howell N, et al. effects of ERK and JNK-p38 MAP kinases on apoptosis. Science Molecular basis for the modulation of native T-type Ca2þ channels in 1995;270:1326–31. vivo by Ca2þ/calmodulin-dependent protein kinase II. J Clin Invest 28. Wada T, Penninger JM. Mitogen-activated protein kinases in apopto- 2006;116:2403–12. sis regulation. Oncogene 2004;23:2838–49.

www.aacrjournals.org Mol Cancer Res; 12(3) March 2014 357

Dziegielewska et al.

29. Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, 42. Huang L, Keyser BM, Tagmose TM, Hansen JB, Taylor JT, Zhuang H, Appella E, et al. Phosphorylation of human p53 by p38 kinase coordi- et al. NNC 55–0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N- nates N-terminal phosphorylation and apoptosis in response to UV methylamino)ethyl)-6-ﬂuoro-1,2,3,4-tetrahydro-1-isopropyl-2-naph- radiation. EMBO J 1999;18:6845–54. tyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitor 30. Kishi H, Nakagawa K, Matsumoto M, Suga M, Ando M, Taya Y, et al. of T-type calcium channels. J Pharmacol Exp Ther 2004;309: Osmotic shock induces G1 arrest through p53 phosphorylation at 193–9. Ser33 by activated p38MAPK without phosphorylation at Ser15 and 43. Toyota M, Ho C, Ohe-Toyota M, Baylin SB, Issa JP. Inactivation of Ser20. J Biol Chem 2001;276:39115–22. CACNA1G, a T-type calcium channel gene, by aberrant methylation of 31. Sayed M, Kim SO, Salh BS, Issinger OG, Pelech SL. Stress- its 50 CpG island in human tumors. Cancer Res 1999;59:4535–41. induced activation of protein kinase CK2 by direct interaction with 44. Milne DM, Palmer RH, Meek DW. Mutation of the casein kinase II p38 mitogen-activated protein kinase. J Biol Chem 2000;275: phosphorylation site abolishes the anti-proliferative activity of p53. 16569–73. Nucleic Acids Res 1992;20:5565–70. 32. Keller DM, Lu H. p53 serine 392 phosphorylation increases after UV 45. Sakaguchi K, Sakamoto H, Xie D, Erickson JW, Lewis MS, Anderson through induction of the assembly of the CK2.hSPT16.SSRP1 com- CW, et al. Effect of phosphorylation on tetramerization of the tumor plex. J Biol Chem 2002;277:50206–13. suppressor protein p53. J Protein Chem 1997;16:553–6. 33. Keller DM, Zeng X, Wang Y, Zhang QH, Kapoor M, Shu H, et al. A DNA 46. Yap DBS, Hsieh J-K, Zhong S, Heath V, Gusterson B, Crook T, et al. damage-induced p53 serine 392 kinase complex contains CK2, Ser392 phosphorylation regulates the oncogenic function of mutant hSpt16, and SSRP1. Mol Cell 2001;7:283–92. p53. Cancer Res 2004;64:4749–54. 34. Liu Y, Bodmer WF. Analysis of P53 mutations and their expression 47. Walerych D, Napoli M, Collavin L, Del Sal G. The rebel angel: mutant in 56 colorectal cancer cell lines. Proc Natl Acad Sci U S A 2006; p53 as the driving oncogene in breast cancer. Carcinogenesis 103:976–81. 2012;33:2007–17. 35. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, et al. 48. Cox ML, Meek DW. Phosphorylation of serine 392 in p53 is a common Requirement for p53 and p21 to sustain G2 arrest after DNA damage. and integral event during p53 induction by diverse stimuli. Cell Signal Science 1998;282:1497–501. 2010;22:564–71. 36. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cyto- 49. Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, Ambrosino C, toxicity screening. Nat Protoc 2006;1:1112–6. et al. P38 alpha mitogen-activated protein kinase sensitizes cells to 37. Mariot P, Vanoverberghe K, Lalevee N, Rossier MF, Prevarskaya N. apoptosis induced by different stimuli. Mol Biol Cell 2004;15:922–33. Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during 50. Lim HW, New L, Han J, Molkentin JD. Calcineurin enhances MAPK neuroendocrine differentiation of human prostate cancer cells. J Biol phosphatase-1 expression and p38 MAPK inactivation in cardiac Chem 2002;277:10824–33. myocytes. J Biol Chem 2001;276:15913–9. 38. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 51. Wlodarchak N, Guo F, Satyshur KA, Jiang L, Jeffrey PD, Sun T, et al. years of image analysis. Nat Methods 2012;9:671–5. Structure of the Ca2þ-dependent PP2A heterotrimer and insights into 39. Kaeser MD, Iggo RD. Chromatin immunoprecipitation analysis fails to Cdc6 dephosphorylation. Cell Res 2013;23:931–46. support the latency model for regulation of p53 DNA binding activity in 52. Junttila MR, Li S-P, Westermarck J. Phosphatase-mediated crosstalk vivo. Proc Natl Acad Sci U S A 2002;99:95–100. between MAPK signaling pathways in the regulation of cell survival. 40. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA. FASEB J 2008;22:954–65. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 53. Pottle J, Sun C, Gray L, Li M. Exploiting MCF-7 cells' calcium depen- 2007;35:W71–74. dence with Interlaced Therapy. J Cancer Ther 2013;04:32–40. 41. Riley T, Sontag E, Chen P, Levine A. Transcriptional control of human 54. Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK p53-regulated genes. Nat Rev Mol Cell Biol 2008;9:402–12. pathways in cancer development. Nat Rev Cancer 2009;9:537–49.

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T-Type Ca2+ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through Activation of p38-MAPK in Colon Cancer Cells

Barbara Dziegielewska, David L. Brautigan, James M. Larner, et al.

Mol Cancer Res 2014;12:348-358. Published OnlineFirst December 20, 2013.

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