Intracellular chloride regulates cell proliferation through the activation of stress‐activated protein kinases in MKN28 human gastric cancer cells

Recently, we reported that reduction of intracellular Cl^? concentration ([Cl^?]_i) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G₁ to S cell‐cycle phase through upregulation of p21, cyclin‐dependent kinase inhibitor, in a p53‐independent manner. However, it is still unknown how intracellular Cl^? regulates p21 expression level. In this study, we demonstrate that mitogen‐activated protein kinases (MAPKs) are involved in the p21 upregulation and cell‐cycle arrest induced by reduction of [Cl^?]_i. Culture of MKN28 cells in a low Cl^? medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G₁/S cell‐cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl^? medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G₁/S cell‐cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl^? medium and rescued MKN28 cells from the low Cl^?‐induced G₁ cell‐cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl^? medium. These results strongly suggest that the intracellular Cl^? affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin‐dependent kinase inhibitor (p21) in a p53‐independent manner in MKN28 cells. J. Cell. Physiol. 223:764–770, 2010. © 2010 Wiley‐Liss, Inc.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21^Cip1 and p27^Kip1 expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G₁ arrest, up-regulation of cell cycle-regulatory proteins p21^Cip1 and p27^Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]     

Mitochondrial membrane potential (DeltaPsi) and Ca<Superscript>2+</Superscript>-induced differentiation in HaCaT keratinocytes

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca²⁺]₀, induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca²⁺]₀ results in acute and sustained increases in intracellular calcium concentration, [Ca²⁺]_i. We find that the Ca²⁺-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca²⁺]_i led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K⁺ channels, which is known to inhibit cell proliferation, and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a blocker of plasma membrane Cl^– channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.     

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl⁻ condition elevated pH_lys and reduced the intra-lysosomal Cl⁻ concentration ([Cl⁻]_lys) via reduction of cytosolic Cl⁻ concentration ([Cl⁻]_c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G₀/G₁ arrest without induction of apoptosis. We also studied effects of direct modification of H⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na⁺/H⁺ exchanger (NHE)] elevated pH_lys and decreased [Cl⁻]_lys associated with inhibition of cell proliferation via induction of G₀/G₁ arrest similar to the culture under a low Cl⁻ condition. However, unlike low Cl⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl⁻]_c compared with low Cl⁻ condition. These observations suggest that the lowered [Cl⁻]_c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl⁻ is a key factor of lysosome acidification and autophagy.     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996     

Optical Imaging of Hippocampal Neurons with a Chloride-Sensitive Dye: Early Effects of In Vitro Ischemia

Abstract: We determined if changes in intraneuronal Cl^? occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14–19 days old) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl^?-sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl^?]_i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26–28°C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia-induced increase in [Cl^?]_i of ～10 mM. Pretreatment with the GABA_A-gated Cl^? channel antagonist picrotoxin (100 µM) blocked the ischemia-induced change in [Cl^?]_i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at ～10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia-induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume-sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl^?]_i secondary to GABA_A receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K⁺ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K⁺-induced increase in [Cl^?]_i. It is possible that an increased [Cl^?]_i, following either an ischemic event or an episode of depolarization, would reduce the Cl^? driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl^?]_i after 20-min reoxygenation.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53^−/− astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53^−/− astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.     

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

Summary Internodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl^– and K⁺ in the excitation of the plasmalemma was studied.[Cl^–] _i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl^–. The lowered [Cl^–] _i was about 0.01mm. Cells with this low [Cl^–] _i generated action potential and showed anN-shapedV–I curve under voltage clamped depolarization like Cl^–-rich cells containing 13 or 29mm Cl^–.E _m at the peak of the action potential was constant at [Cl^–] _i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl^– during excitation is discussed.At [Cl^–] _i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K⁺ concentration to 100mm, NO ₃ ^– , F^–, SO ₄ ^2– , acetate, and propionate inhibited the generation of action potentials like Cl^–, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K⁺ concentration. It decreased as [K⁺] _i (K-methane sulfonate) increased. Increase in [Na⁺] _i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K⁺. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol. 30:249).     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

The ruthenium(II)–arene compound RAPTA-C induces apoptosis in EAC cells through mitochondrial and p53–JNK pathways

An investigation of the molecular mechanism of the anticancer activity demonstrated by the ruthenium(II)–arene compound [Ru(η⁶-p-cymene)Cl₂(pta)] (pta is 1,3,5-triaza-7-phosphaadamantane), termed “RAPTA-C”, in Ehrlich ascites carcinoma (EAC) bearing mice is described. RAPTA-C exhibits effective cell growth inhibition by triggering G₂/M phase arrest and apoptosis in cancer cells. Cell cycle arrest is associated with increased levels of p21 and reduced amounts of cyclin E. RAPTA-C treatment also enhances the levels of p53, and its treatment triggers the mitochondrial apoptotic pathway, as shown by the change in Bax to Bcl-2 ratios, resulting in cytochrome c release and caspase-9 activation. c-Jun NH₂-terminal kinase (JNK) is a critical mediator in RAPTA-C-induced cell growth inhibition. Activation of JNK by RAPTA-C increases significantly during apoptosis. Overall, these results suggest a critical role for JNK and p53 in RAPTA-C-induced G₂/M arrest and apoptosis of EAC-bearing mice. Consequently, RAPTA-C treatment results in a significant inhibition in the progression of cancer in an animal model, which emulates the human disease, and does so with remarkably low general toxicity; hence, RAPTA-C has potential for clinical application.     

p53-dependent change in replication timing of the human genome

The tumor suppressor gene p53 has roles in multiple cell-cycle checkpoints, including the G1/S transition, to prevent replication of cells with DNA damage. p53 is thought to be associated with regulation of replication timing during S-phase in the human genome. In the present study, we used p53-wild-type and p53-null HCT116 colon carcinoma cells to analyze p53-dependent changes in replication timing of the human genome. The percentage of HCT116 p53(−/−) cells in S-phase was higher than that of HCT116 p53(+/+) cells. We compared replication timing of human genes between the two cell lines using 25,000 human cDNA microarray. We identified genes that replicated earlier in HCT116 p53(−/−) cells than in HCT116 p53(+/+) cells. These genes included cell-cycle- and apoptosis-related genes. We propose that p53 plays a role in regulation of replication timing of the human genome through the control of cell-cycle checkpoints.     

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit₂]³⁻) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit₂]³⁻) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit₂]³⁻ was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit₂]³⁻, proteomics results from [YbCit₂]³⁻-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit₂]³⁻-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit₂]³⁻-treated cells. Western blot analysis showed that [YbCit₂]³⁻-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit₂]³⁻-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.     

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53^wt) or being p(HCT-116 p53^−/−), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53^−/− xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53^wt cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53^wt cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75^NTR, p53 and Bax.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G₁/S checkpoint, ATM and p53 signaling pathways in p53-wild-type cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G₁/S checkpoint pathway in p53 wild-type lines and not in p53-mutant cells. These responses are coupled with G₂/G₁ checkpoint effectors p21^CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G₂ cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wild-type and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G₂ arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wild-type and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.Key words: DNA damaging agent, G₂ arrest, microarray, PARP inhibition, p53, topotecan, veliparib (ABT-888)