Ceramide triggers caspase activation during gamma-radiation-induced apoptosis of human glioma cells lacking functional p53

We have previously shown that treatment of human glioma U87-MG cells expressing wild-type p53 with a DNA topoisomerase II inhibitor, etoposide resulted in ceramide-dependent apoptotic cell death. However, U87-W E6 cells lacking functional p53 due to the expression of human papilloma virus type 16 (HPV-16) E6 oncoprotein were resistant to etoposide. In order to gain insight into the roles of p53 and ceramide in gamma-radiation-induced glioma cell death, we used U87-W E6 and vector-infected U87-LXSN cells. U87-LXSN glioma cells expressing wild-type p53 were relatively resistant to gamma-radiation. U87-W E6 cells, which lost functional p53, became susceptible to radiation-induced apoptosis. Activation of caspase-3, and formation of ceramide by acid sphingomyelinase, but not by neutral sphingomyelinase, were associated with p53-independent apoptosis. Radiation-induced caspase activation and apoptotic death in U87-W E6 cells were modified by the agents which affected ceramide metabolism. SR33557, an inhibitor of acid sphingomyelinase, suppressed radiation-induced caspase activation and then apoptotic cell death. In contrast, N-oleoylethanolamine (OE) and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibit ceramidase and UDP-glucose:ceramide glucosyltransferase-1, respectively, and then augment ceramide formation, enhanced radiation-induced caspase activation. These results indicate that glioma cells with functional p53 were relatively resistant to gamma-radiation, and that ceramide may play an important role in caspase activation during gamma-radiation-induced apoptosis of glioma cells lacking functional p53.     

Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells

Ceramide has recently been regarded as a potential mediator of apoptosis. In the present study, the effects of Bcl-2 and Bax on the ceramide-mediated apoptotic pathways were examined in glioma cells overexpressing Bcl-2 or Bax. Etoposide, cisplatin and tumor necrosis factor-alpha induced apoptosis of C6 rat glioma cells which was associated with ceramide formation due to activation of neutral sphingomyelinase, followed by release of mitochondrial cytochrome c into the cytosol and activation of caspases-9 and -3. The growth of C6 cells stably overexpressing either Bcl-2 or Bax was almost equal to that of the vector-transfected cells. Bax overexpression enhanced etoposide-induced apoptosis through acceleration of cytochrome c release and caspases activation. However, Bax had no effect on ceramide formation. Similar findings were obtained in C6 cells and U87-MG human glioblastoma cells which were transiently overexpressed with Bax. In contrast, Bcl-2 overexpression resulted in a retardation of the apoptotic process via prevention of cytochrome c release and caspases activation, and ceramide formation was also blocked when Bcl-2 was highly overexpressed in glioma cells. In addition, transient overexpression of Bcl-xL also exerted inhibitory effects on ceramide formation and apoptotic cell death induced by etoposide. These results indicate that Bax promotes apoptosis regardless of ceramide formation and that Bcl-2 or Bcl-xL prevents ceramide formation by repressing neutral sphingomyelinase as well as ceramide-induced cytochrome c release. Oncogene (2000) 19, 3508 - 3520     

Overcoming resistance to gamma-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels

Recent strategies to sensitize radioresistant tumours are based on combining gamma-irradiation with inducers of apoptosis. We report that the combination of three inhibitors of sphingolipid metabolism, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl(DL-PDMP)+imipramine +/- D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP), with 10-Gy irradiation triggers both mitotic and apoptotic killing in radioresistant SQ20B squamous carcinoma cells. In these cells, apoptosis is defective due to a lack of ceramide generation upstream, which cannot be explained by sphingomyelinase (neutral and acidic) deficiency or rapid derivation to the sphingolipid pathway. We present evidence of a functional transduction death pathway when ceramide generation is restored, which involves the mitochondrial-mediated pathway coupled to alterations in redox status and to executive caspases activation. The poly-drug treatment restored apoptosis to levels similar to those observed in radiosensitive SCC61 squamous carcinoma cells. Simultaneous exposure to gamma-irradiation and poly-drug treatment acted synergistically in SQ20B cells to produce a marked increase in both mitochondrial dysfunction and caspase cleavage, which led to a 7.8-fold increase in apoptosis within 48 h, relative to irradiated cells. Moreover, the results suggest that the ceramide released by irradiation or poly-drug treatment converges upon common cellular targets. Modulation of endogenous ceramide levels by inhibitors of sphingolipid metabolism may represent a new cellular target for the sensitization of radioresistant tumours to gamma-ray therapy.     

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.     

Novel quinuclidinone derivative 8a induced apoptosis in human MCF-7 breast cancer cell lines

Novel quinuclidinone derivatives that cause cytotoxicity in human non-small lung carcinoma epithelial cells null for p53 (H1299) have been previously reported. The current study investigates the effect of these derivatives on cytotoxicity of human MCF-7 cells and normal breast epithelial cells (MCF-12a). This study shows that quinuclidinone derivatives 8a and 8b induce growth inhibition mainly through apoptosis of breast cancer cells (MCF-7) with less cytotoxic effect in normal breast epithelial cells (MCF-12a) for derivative 8a while 8b induced similar cytotoxicity for both breast cancer cells and normal breast epithelial cells. Derivative 8a was chosen for further investigation. 8a induced G(1) phase arrest, presumably sensitizing the breast cancer cells to apoptosis by increasing expression level of p21 and cyclin E. Moreover, 8a increased expression level of ERK1, p53 and BAX, and it reduced expression level of AKT and BCL-2. By investigating the sphingomyelinase apoptosis pathway, it was observed that 8a significantly increased sphingomyelinase activity and increased formation of ceramide as well as increased expression levels of JNK phoshorylation, caspase-8 and caspase-9. Based on previous results it is proposed that quinuclidinone derivative 8a provokes apoptosis in human breast cancer cells (MCF-7) via the sphingomyelinase pathway.     

p53 and redox state in etoposide-induced acute myeloblastic leukemia cell death

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.     

Ceramide triggers p53-dependent apoptosis in genetically defined fibrosarcoma tumour cells.

p53 mutations are among the most common genetic alterations in human cancer and are frequently described in intrinsic or acquired radio- and chemotherapy resistance. Radiation-induced cell kill is not only mediated by DNA damage but also by the activation of signal transduction cascades generated at the plasma membrane like the sphingomyelin pathway. We used genetically defined wild-type p53 or p53-deficient mouse fibrosarcoma cells to investigate the p53-dependence of tumour response upon activation of the sphingomyelin pathway. Treatment of the tumour cells with neutral sphingomyelinase drastically reduced the amount of wild-type p53 fibrosarcoma cell proliferation over 72 h in a clear dose-response (0.2-1.0 U ml(-1) nSMase). Sphingomyelinase had no effect on cell proliferation in tumour cells lacking p53. Similarly, cell proliferation was abolished by C2-ceramide (5-20 microM) only in wild-type p53 cells. FACS-analysis revealed that C2-ceramide induced massive p53-dependent apoptosis (40-50% after 12-24 h) and cell cycle analysis showed a transient G1 arrest in p53-deficient tumour cells 12-24 h after C2-ceramide exposure. These results suggest that ceramide-induced apoptosis in tumour cells can be dependent on the status of p53 and imply that p53 is also important for stress-induced apoptotic signal transduction cascades generated at the plasma membrane.     

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.     

ATM and LKB1 dependent activation of AMPK sensitizes cancer cells to etoposide-induced apoptosis

The present study aims to determine the effect of AMPK on etoposide-induced apoptosis of cancer cells. Our results revealed that etoposide induced AMPK activation in prostate C4-2 cancer cells, an event that was attenuated by ATM siRNA. In A549 cells that lack LKB1, AMPK was unable to be activated by etoposide, which was restored by introduction of LKB1. Likewise, silencing LKB1 in C4-2 cells impaired AMPK activation. Finally, etoposide displayed a potent pro-apoptotic effect in cancer cells with functional LKB1 and AMPK. Thus, our results establish a linear relationship of ATM, LKB1 and AMPK in response to the DNA damage drug.     

The role of gangliosides in fenretinide-induced apoptosis of neuroblastoma

Fenretinide is thought to induce apoptosis via increases in ceramide levels but the mechanisms of ceramide generation and the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. In SH-SY5Y neuroblastoma cells, evidence suggests that acid sphingomyelinase activity is essential for the induction of ceramide and apoptosis in response to fenretinide. Downstream of ceramide, apoptosis in response to fenretinide is mediated by increased glucosylceramide synthase activity resulting in increased levels of gangliosides GD3 and GD2 via GD3 synthase. GD3 is a key signalling intermediate leading to apoptosis via the activation of 12-Lipoxygenase, and the parallel induction of GD2 suggests that fenretinide might enhance the response of neuroblastoma to therapy with anti-GD2 antibodies.     

Functional switching of ATM: sensor of DNA damage in proliferating cells and mediator of Akt survival signal in post-mitotic human neuron-like cells

Ataxia-telangiectasia(A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias.The gene mutated in this disease,ATM(A-T,mutated),encodes a 370-kDa Ser/Thr protein kinase.ATM not only mediates cellular response to DNA damage but also acts as an activator of Akt in response to insulin.However,despite intensive studies,the mechanism underlying the neuronal degeneration symptoms of human A-T is still poorly understood.We found that the topoisomerase inhibitors etoposide and camptothecin readily induced apoptosis in undifferentiated proliferating SH-SY5Y cells but could not induce apoptosis in neuronally differentiated SH-SY5Y cells.In addition,etoposide induced p53 phosphorylation and H2AX foci formation in proliferating SH-SY5Y cells but failed to do so in differentiated SH-SY5Y cells.Moreover,while inhibition of ATM in undifferentiated SH-SY5Y cells partially protected them from etoposide-induced apoptosis,the same treatment had no effect on cell viability in differentiated SH-SY5Y cells.These results suggest that DNA damage or defective response to DNA damage is not the cause of neuronal cell death in human A-T.In contrast,we discovered that Akt phosphorylation was inhibited when ATM activity was suppressed in differentiated SH-SY5Y cells.Furthermore,inhibition of ATM induced apoptosis following serum starvation in neuronally differentiated SH-SY5Y cells but could not trigger apoptosis under the same conditions in undifferentiated proliferating SH-SY5Y cells.These results demonstrate that ATM mediates the Akt signaling and promotes cell survival in neuron-like human SH-SY5Y cells,suggesting that impaired activation of Akt is the reason for neuronal degeneration in human A-T.     

Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.     

Distinct p53-independent apoptotic cell death signalling pathways in testicular germ cell tumour cell lines

The induction of apoptosis by diverse apoptotic stimuli was studied in a panel of 6 testicular germ cell tumour (TGCT) cell lines with defined p53 status. Although the sensitivity to a particular stimulus varied considerably among the TGCT cell lines, the differences in response were not associated with the presence of functional p53. Mutant (mt) p53-expressing NCCIT and S2 (no p53 protein) were both readily triggered into apoptosis by cisplatin and doxorubicin, while wild-type(wt)-p53-transactivation-competent 2102 EP cells failed to undergo drug-induced apoptosis. Moreover, transactivation-deficient NCCIT cells and wtp53-expressing NT2 cells were equally sensitive to cisplatin, doxorubicin, gamma radiation, and cell-permeable C2-ceramide. Our p53 data suggest that, at least in this panel of non-isogeneic TGCT cell lines, hypersensitivity to therapeutic agents is not associated with p53 status. Next, we examined the impact of p53 inactivation on apoptosis induction in isogeneic NT2 sublines expressing human papillomavirus E6 protein. Evidently, abrogation of p53 function did not affect the hypersensitivity to apoptotic stimuli. We noted that drug-sensitive S2 cells were highly resistant to radiation-induced apoptosis, indicating distinct signalling pathways for chemotherapy and irradiation. The impaired radiation-induced apoptotic pathway in S2 and 2102 EP could not be restored by addition of cell-permeable C2-ceramide, suggesting that the blockade is downstream of ceramide generation. Ligation of Fas/APO-1/CD95 by anti-Fas effectively induced apoptosis in Fas-antigen expressing S2, 2102 EP and 833 KE. The efficient Fas-mediated activation of apoptosis in drug-, radiation-, and ceramide-resistant 2102 EP cells further suggests that diverse apoptosis-inducing factors may use distinct signalling pathways. In summary, we demonstrated the presence of distinct p53-independent apoptotic pathways in TGCT cells.     

Wogonin potentiates the antitumor action of etoposide and ameliorates its adverse effects

Wogonin, a flavone in the roots of Scutellaria baicalensis, reduced etoposide-induced apoptotic cell death in normal cells, such as bone marrow cells and thymocytes. On the other hand, wogonin potentiated the proapoptotic or cytotoxic action of etoposide in tumor cells, such as Jurkat, HL-60, A549, and NCI-H226. These contradictory actions of wogonin on apoptosis are distinguished by normal or cancer cell types. Wogonin had no effect on apoptosis induced by other anticancer agents in the tumor cells. Thus, the potentiation effect of wogonin was observed only in etoposide-induced apoptosis in tumor cells. In a functional assay for P-glycoprotein (P-gp), wogonin suppressed excretion of calcein, a substrate for P-gp, in these tumor cells. Moreover, wogonin decreased the excretion of radiolabeled etoposide and accordingly increased intracellular content of this agent in the cells. P-gp inhibitors showed a similar potentiation effect on etoposide-induced apoptosis in these tumor cells. Thus, wogonin is likely to potentiate the anticancer action of etoposide due to P-gp inhibition and accumulation of this agent. These findings suggest that wogonin may be a useful chemotherapeutic adjuvant to potentiate the pharmacological action of etoposide and ameliorate its adverse effects.     

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.     

Differential sensitivity of malignant glioma cells to methylating and chloroethylating anticancer drugs: p53 determines the switch by regulating xpc, ddb2, and DNA double-strand breaks

Glioblastoma multiforme is the most severe form of brain cancer. First line therapy includes the methylating agent temozolomide and/or the chloroethylating nitrosoureas [1-(2-chloroethyl)-1-nitrosourea; CNU] nimustine [1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea; ACNU], carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU], or lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CCNU]. The mechanism of cell death after CNU treatment is largely unknown. Here we show that ACNU and BCNU induce apoptosis in U87MG [p53 wild-type (p53wt)] and U138MG [p53 mutant (p53mt)] glioma cells. However, contrary to what we observed previously for temozolomide, chloroethylating drugs are more toxic for p53-mutated glioma cells and induce both apoptosis and necrosis. Inactivation of p53 by pifithrin-alpha or siRNA down-regulation sensitized p53wt but not p53mt glioma cells to ACNU and BCNU. ACNU and BCNU provoke the formation of DNA double-strand breaks (DSB) in glioma cells that precede the onset of apoptosis and necrosis. Although these DSBs are repaired in p53wt cells, they accumulate in p53mt cells. Therefore, functional p53 seems to stimulate the repair of CNU-induced cross-links and/or DSBs generated from CNU-induced lesions. Expression analysis revealed an up-regulation of xpc and ddb2 mRNA in response to ACNU in U87MG but not U138MG cells, indicating p53 regulates a pathway that involves these DNA repair proteins. ACNU-induced apoptosis in p53wt glioma cells is executed via both the extrinsic and intrinsic apoptotic pathway, whereas in p53mt glioma cells, the mitochondrial pathway becomes activated. The data suggest that p53 has opposing effects in gliomas treated with methylating or chloroethylating agents and, therefore, the p53 status should be taken into account when deciding which therapeutic drug to use.     

Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells

Temozolomide (TMZ) produces O(6)-methylguanine in DNA, which in turn mispairs with thymine, triggering futile DNA mismatch repair (MMR) and ultimately cell death. We found previously that in p53-proficient human glioma cells, TMZ-induced futile DNA MMR resulted not in apoptosis but rather in prolonged, p53- and p21-associated G(2)-M arrest and senescence. Additionally, p53-deficient cells were relatively more TMZ resistant than p53-deficient glioma cells, which underwent only transient G(2)-M arrest before death by mitotic catastrophe. These results suggested that prolonged G(2)-M arrest might protect cells from TMZ-induced cytotoxicity. In the present study, we therefore focused on the mechanism by which TMZ induces G(2)-M arrest and on whether inhibition of such G(2)-M arrest might sensitize glioma cells to TMZ-induced toxicity. U87MG glioma cells treated with TMZ underwent G(2)-M arrest associated with Chk1 activation and phosphorylation of both cdc25C and cdc2. These TMZ-induced effects were inhibited by the Chk1 kinase inhibitor UCN-01. Although not in itself toxic, UCN-01 increased the cytotoxicity of TMZ 5-fold, primarily by inhibiting cellular senescence and increasing the percentage of cells bypassing G(2)-M arrest and undergoing mitotic catastrophe. In addition to enhancing TMZ-induced cytotoxicity in p53-proficient cells, UCN-01 also blocked TMZ-induced Chk1 activation and transient G(2)-M arrest in p53-deficient U87MG-E6 cells and similarly enhanced TMZ-induced mitotic catastrophe and cell death. Taken together, these results indicate that Chk1 links TMZ-induced MMR to G(2)-M arrest. Furthermore, inhibition of the cytoprotective G(2) arrest pathway sensitizes cells to TMZ-induced cytotoxicity and may represent a novel, mechanism-based means of increasing TMZ efficacy in both p53 wild-type and p53 mutant glioma cells.