Etoposide and adriamycin but not genistein can activate the checkpoint kinase Chk2 independently of ATM/ATR.

We have investigated the effects of three unrelated topoisomerase 2 inhibitors, genistein, adriamycin, and etoposide, on phosphorylation/activation of the checkpoint kinase Chk2 in normal or ATM-deficient (ATM-) human fibroblasts and in cells overexpressing a catalytically inactive ATR kinase. We demonstrate that genistein activates Chk2 in a strictly ATM-dependent manner, whereas etoposide and adriamycin can trigger Chk2 activation in long-term cultures of ATM- cells. Moreover, these two latter genotoxic compounds were found to activate Chk2 in fibroblasts expressing the dominant negative form of ATR. We also report a significant decrease in the accumulation in G2-phase of ATM- cells when genistein did not activate Chk2. In conclusion, our results strongly support that activation of Chk2 could be dependent on the type and/or extent of DNA damage and under the control of either an ATM-dependent or an ATM and, maybe, an ATR-independent pathway.     

Caffeine inhibits the checkpoint kinase ATM.

The basis of many anti-cancer therapies is the use of genotoxic agents that damage DNA and thus kill dividing cells. Agents that cause cells to override the DNA-damage checkpoint are predicted to sensitize cells to killing by genotoxic agents. They have therefore been sought as adjuncts in radiation therapy and chemotherapy. One such compound, caffeine, uncouples cell-cycle progression from the replication and repair of DNA [1] [2]. Caffeine therefore servers as a model compound in establishing the principle that agents that override DNA-damage checkpoints can be used to sensitize cells to the killing effects of genotoxic drugs [3]. But despite more than 20 years of use, the molecular mechanisms by which caffeine affects the cell cycle and checkpoint responses have not been identified. We investigated the effects of caffeine on the G2/M DNA-damage checkpoint in human cells. We report that the radiation-induced activation of the kinase Cds1 [4] (also known as Chk2 [5]) is inhibited by caffeine in vivo and that ATM kinase activity is directly inhibited by caffeine in vitro. Inhibition of ATM provides a molecular explanation of the attenuation of DNA-damage checkpoint responses and for the increased radiosensitivity of caffeine-treated cells [6] [7] [8].     

p21CIP1 is dispensable for the G2 arrest caused by genistein in human melanoma cells

We have investigated the effect of genistein on cell cycle distribution of the human choroidal melanoma cell line OCM-1. We report that this isoflavonoid arrested cells in G2. This effect was correlated with the induction of the CDK inhibitor p21CIP1. However, while CDK1 activity was markedly reduced following genistein treatment, CDK2 activity was not affected. This was in agreement with the absence of G1 arrest that we observed but caused some doubt about the functionality of p21CIP1. Attempts to demonstrate mutation or post-translational modification of p21CIP1 from OCM-1 cells were unsuccessful. In fact, the level of p21CIP1 induced by genistein was shown to be insufficient to cause CDK2 inhibition. The role of p21CIP1 in the inhibition of CDK1 was questionable, as we demonstrated that genistein impaired Tyr15 dephosphorylation of CDK1 and because CDK1-cyclin B1 complexes from treated cells could be reactivated upon exposure to CDC25 phosphatase. Finally, we report that p21CIP1 was not absolutely required for the genistein-induced G2 arrest, as the isoflavone caused at least partial G2 arrest in p21-deficient Rat-1 fibroblasts as well as in p21-/- mouse embryo fibroblasts.     

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.     

Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive.

We have investigated the mechanism of S-phase arrest elicited by the carcinogen benzo(a)pyrene dihydrodiol epoxide (BPDE) in p53-deficient cells. Inhibition of DNA synthesis after BPDE treatment was rapid and dose dependent (approximately 50% inhibition after 2 h with 50 nM BPDE). Cells treated with low doses (50-100 nM) of BPDE resumed DNA synthesis after a delay of approximately 4-8 h, whereas cells that received high doses of BPDE (600 nM) failed to recover from S-phase arrest. The checkpoint kinase Chk1 (but not Chk2) was phosphorylated after treatment with low doses of BPDE. High concentrations of BPDE elicited phosphorylation of both Chk1 and Chk2. Adenovirus-mediated expression of "dominant-negative" Chk1 (but not dominant-negative Chk2) and the Chk1 inhibitor UCN-01 abrogated the S-phase delay elicited by low doses of BPDE. Consistent with a role for the caffeine-sensitive ATM or ATR protein kinase in low-dose BPDE-induced S-phase arrest, both Chk1 phosphorylation and S-phase arrest were abrogated by caffeine. However, low doses of BPDE elicited Chk1 phosphorylation and S-phase arrest in AT cells (from ataxia telangiectasia patients), demonstrating that ATM is dispensable for S-phase checkpoint responses to this genotoxin. BPDE-induced Chk1 phosphorylation and S-phase arrest were abrogated by caffeine treatment in AT cells, suggesting that a caffeine-sensitive kinase other than ATM is an important mediator of responses to BPDE-adducted DNA. Overall, our data demonstrate the existence of a caffeine-sensitive, Chk1-mediated, S-phase checkpoint that is operational in response to BPDE.     

ATR-Chk1 Axis Protects BCR/ABL Leukemia Cells from the Lethal Effect of DNA Double-Strand Breaks

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (γ-H2AX). γ-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.     

Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.     

Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway

Claspin is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Here, we show that human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide caused activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin was cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.     

Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.     

DNA Damage-Induced Accumulation of Centrosomal Chk1 Contributes to its Checkpoint Function

The checkpoint kinase Chk1 is an established transducer of ATR- and ATM-dependent signalling in response to DNA damage. In addition to its nuclear localization, Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B-Cdk1 during unperturbed cell cycles. Here, we demonstrate that DNA damage caused by ultraviolet irradiation or hydroxyurea treatment leads to centrosomal accumulation of endogenous Chk1 in normal human BJ fibroblasts and in ATR- or ATM-deficient fibroblasts. Chemical inhibition of ATR/ATM by caffeine led to enhanced centrosomal Chk1 deposition associated with nuclear Chk1 depletion. In contrast to normal or ATM-deficient fibroblasts, genetically ATR-deficient Seckel-fibroblasts showed detectable constitutive centrosomal accumulation of Chk1 even in the absence of exogenous insults. After DNA damage, the centrosomal fraction of Chk1 was found to be phosphorylated at ATR/ATM phosphorylation sites. Forced immobilization of kinase-inactive but not wild-type Chk1 to centrosomes resulted in a G2/M checkpoint defect. Finally, both DNA damage, and forced centrosomal expression of Chk1 in the absence of genotoxic treatments, induced centrosome amplification in a subset of cells, a phenomenon which could be suppressed by inhibition of ATM/ATR-mediated signaling. Taken together, our results suggest that accumulation of phosphorylated Chk1 at centrosomes constitutes an additional element in the DNA damage response. Centrosomal Chk1 induces G2/M cell cycle arrest and may evoke centrosome amplification, the latter possibly providing a backup mechanism for elimination of cells with impaired DNA damage checkpoints operating earlier during the cell cycle.     

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3′,4′-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser⁴²⁸. This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser¹⁵) and Chk1 (Ser²⁹⁶) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo.     

Cleavage-mediated activation of Chk1 during apoptosis

The Chk1 kinase is highly conserved from yeast to humans and is well known to function in the cell cycle checkpoint induced by genotoxic or replication stress. The activation of Chk1 is achieved by ATR-dependent phosphorylation with the aid of additional factors. Robust genotoxic insults induce apoptosis instead of the cell cycle checkpoint, and some of the components in the ATR-Chk1 pathway are cleaved by active caspases, although it has been unclear whether the attenuation of the ATR-Chk1 pathway has some role in apoptosis induction. Here we show that Chk1 is activated by caspase-dependent cleavage when the cells undergo apoptosis. Treatment of chicken DT40 cells with various genotoxic agents, UV light, etoposide, or camptothecin induced Chk1 cleavage, which was inhibited by a pan-caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethyl ketone. The cleavage of Chk1 was similarly observed in human Jurkat cells treated with a non-genotoxic apoptosis inducer, staurosporine. We have determined the cleavage site(s), Asp-299 in chicken and Asp-299 and Asp-351 in human cells. We further show that a truncated form of human Chk1 mimicking the N-terminal cleavage fragment (residues 1-299) possesses strikingly elevated kinase activity. Moreover, the ectopic expression of Chk1-(1-299) in human U2OS cells induces abnormal nuclear morphology with localized chromatin condensation and phosphorylation of histone H2AX. These results suggest that Chk1 is activated by caspase-mediated cleavage during apoptosis and might be implicated in enhancing apoptotic reactions rather than attenuating the ATR-Chk1 pathway.     

p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 Arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.     

14-3-3σ and p21 synergize to determine DNA damage response following Chk2 inhibition

DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21, and 14-3-3σ have each been shown to be independent effectors of the G2 DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21-/-, 14-3-3σ-/- cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G2 checkpoint defects. Pharmacologic inhibition of Chk2 in p21-/-, 14-3-3σ-/- cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3σ synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in human cancers.     

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL,Jak2-V617F,or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.     

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

Berberine, a genotoxic alkaloid, induces ATM-Chk1 mediated G2 arrest in prostate cancer cells

Berberine has been shown to possess anti-tumor activity against a wide spectrum of cancer cells. It inhibits cancer cell proliferation by inducing cell cycle arrest, at G1 and/or G2/M, and apoptosis. While it has been documented that berberine induces G1 arrest by activating the p53-p21 cascade, it remains unclear what mechanism underlies the berberine-induced G2/M arrest, which is p53-independent. In this study, we tested the anti-proliferative effect of berberine on murine prostate cancer cell line RM-1 and characterized the underlying mechanisms. Berberine dose-dependently induced DNA double-strand breaks and apoptosis. At low concentrations, berberine was observed to induce G1 arrest, concomitant with the activation of p53-p21 cascade. Upon exposure to berberine at a higher concentration (50μM) for 24h, cells exhibited G2/M arrest. Pharmacological inhibition of ATM by KU55933, or Chk1 by UCN-01, could efficiently abrogate the G2/M arrest in berberine-treated cells. Downregulation of Chk1 by RNA interference also abolished the G2/M arrest caused by berberine, confirming the role of Chk1 in the pathway leading to G2/M arrest. Abrogation of G2/M arrest by ATM inhibition forced more cells to undergo apoptosis in response to berberine treatment. Chk1 inhibition by UCN-01, on the other hand, rendered cells more sensitive to berberine only when p53 was inhibited. Our results suggest that combined administration of berberine and caffeine, or other ATM inhibitor, may accelerate the killing of cancer cells.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.     

Changes in the activity of cyclin-kinase complexes governing cell transition from G1 phase to DNA replication phase in E1A + c-Ha-ras transformants transfected with the bcl-2 gene

The antiproliferative effect of human bcl-2 gene transferred to E1A + c-Ha-ras-transformed rat embryo fibroblasts, which are characterized by the absence of cell cycle checkpoints after damage and by a high proapoptotic sensitivity was studied. Ionizing irradiation, adriamycin treatment, and serum starvation were shown to induce G1/S arrest in E1A + c-Ha-ras-transformants. Bcl-2 antiproliferative effect in E1A + c-Ha-ras-transformants was not associated with alterations in Cdk2, cyclin E and A contents. G1/S arrest following irradiation or serum starvation was accompanied by a decrease in kinase activity associated with cyclin E-cdk2, whereas G1/S arrest in tetraploid subpopulation after adriamycin treatment did not correlate with a decrease in cyclin E-associated kinase activity. Cyclin A-associated kinase activity did not decrease after any used treatment. Transfection of bcl-2 in E1A + c-Ha-ras-transformants resulted in elevated expression of cyclin-cdk complexes inhibitor p21/Waf-1, but not p27/Kip. Damaging agents caused p21/Waf-1 and p27/Kip accumulation, but bcl-2 overexpression did not restore functions of these inhibitors, since p21/Waf-1 and p27/Kip were unable to suppress cyclin-cdk complexes activity after damage. These results suggest that bcl-2 transfection in E1A + c-Ha-ras-transformants is likely to result in irradiation- or serum starvation-induced G1/S arrest accomplished by a selective decrease in cyclin E-associated kinase activity. Adriamycin-induced G1/S arrest seems to be realized via cyclin-cdk complexes activity-independent way involving antiproliferative targets downstream of cyclin E-cdk2 and cyclin A-cdk2 complexes.