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.     

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-wildtype 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-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A 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-wildtype 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-wildtype 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.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.     

p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.     

The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest

Poly(ADP)-ribose polymerase (PARP) inhibitors modify the enzymatic activity of PARP1/2. When certain PARP inhibitors are used either alone or in combination with DNA damage agents they may cause a G2/M mitotic arrest and/or apoptosis in a susceptible genetic context. PARP1 interacts with the cell cycle checkpoint proteins Ataxia Telangectasia Mutated (ATM) and ATM and Rad3-related (ATR) and therefore may influence growth arrest cascades. The PARP inhibitor PJ34 causes a mitotic arrest by an unknown mechanism in certain cell lines, therefore we asked whether PJ34 conditionally activated the checkpoint pathways and which downstream targets were necessary for mitotic arrest. We found that PJ34 produced a concentration dependent G2/M mitotic arrest and differentially affected cell survival in cells with diverse genetic backgrounds. p53 was activated and phosphorylated at Serine15 followed by p21 gene activation through both p53-dependent and -independent pathways. The mitotic arrest was caffeine sensitive and UCN01 insensitive and did not absolutely require p53, ATM or Chk1, while p21 was necessary for maintaining the growth arrest. Significantly, by using stable knockdown cell lines, we found that neither PARP1 nor PARP2 was required for any of these effects produced by PJ34. These results raise questions and cautions for evaluating PARP inhibitor effectiveness, suggesting whether effects should be considered not only on PARP's diverse ADP-ribosylation independent protein interactions but also on homologous proteins that may be producing either overlapping or distinct effect.     

Mitotic DNA damage response: Polo-like Kinase-1 is dephosphorylated through ATM-Chk1 pathway

DNA damage during the cell division cycle can activate ATM/ATR and their downstream kinases that are involved in the checkpoint pathway, and cell growth is halted until damage is repaired. As a result of DNA damage induced in mitotic cells by doxorubicin treatment, cells accumulate in a G2-like phase, not in mitosis. Under these conditions, two mitosis-specific kinases, Cdk1 and Plk1, are inhibited by inhibitory phosphorylation and dephosphorylation, respectively. G2-specific phosphorylation of Cdc25 was increased during incubation after mitotic DNA damage. Inhibition of Plk1 through dephosphorylation was dependent on ATM/Chk1 activity. Depleted expression of ATM and Chk1 was achieved using small hairpin RNA (shRNA) plasmid constructs. In this condition, damaged mitotic cells did not accumulated in a G2-like stage, and entered into G1 phase without delay. Protein phosphatase 2A was responsible for dephosphorylation of mitotic Plk1 in response to DNA damage. In knockdown of PP2A catalytic subunits, Plk1 was not dephosphorylated, but rather degraded in response to DNA damage, and cells did not accumulate in G2-like phase. The effect of ATM/Chk1 inhibition was counteracted by overexpression of PP2A, indicated that PP2A may function as a downstream target of ATM/Chk1 at a mitotic DNA damage checkpoint, or may have a dominant effect on ATM/Chk1 function at this checkpoint. Finally, we have shown that negative regulation of Plk1 by dephosphorylation is important to cell accumulation in G2-like phase at the mitotic DNA damage checkpoint, and that this ATM/Chk1/PP2A pathway independent on p53 is a novel mechanism of cellular response to mitotic DNA damage.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.     

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.     

Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites

Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM-/- cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.     

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.     

Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.     

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)     

Activation of the Budding Yeast Securin Pds1 But Not Rad53 Correlates with Double-Strand Break-Associated G2/M Cell Cycle Arrest in a mec1 Hypomorphic Mutant

Budding yeast Mec1, encoded by the yeast ATR/ATM homolog, negatively regulates cell cycle progression by activating Rad53 (Chk2) and Chk1, two parallel downstream checkpoint pathways. Chk1 phosphorylates Pds1 (securin), which prevents Pds1 degradation. We determined whether activation of both downstream pathways is required to establish G2 arrest in response to double-strand breaks (DSBs). In a hypomorphic mec1 mutant, Rad53 activation was not required to establish G₂ arrest triggered by a single HO endonuclease-generated DSB. However, Pds1 phosphorylation did correlate with G₂ arrest and mec1-21 pds1 cells did not arrest in G₂ after exposure to ionizing radiation. The G₂ checkpoint genes, CHK1 and PDS1, did confer radiation resistance in mec1-21, indicating that CHK1-mediated pathway is functional in the mec1 hypomorph. Thus, phosphorylation of Pds1 but not Rad53 correlates with G₂ arrest in response to DSBs in the mec1 hypomorphic mutant.     

53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer

53BP1 is a conserved nuclear protein that is implicated in the DNA damage response. After irradiation, 53BP1 localizes rapidly to nuclear foci, which represent sites of DNA double strand breaks, but its precise function is unclear. Using small interference RNA (siRNA), we demonstrate that 53BP1 functions as a DNA damage checkpoint protein. 53BP1 is required for at least a subset of ataxia telangiectasia-mutated (ATM)-dependent phosphorylation events at sites of DNA breaks and for cell cycle arrest at the G2-M interphase after exposure to irradiation. Interestingly, in cancer cell lines expressing mutant p53, 53BP1 was localized to distinct nuclear foci and ATM-dependent phosphorylation of Chk2 at Thr 68 was detected, even in the absence of irradiation. In addition, Chk2 was phosphorylated at Thr 68 in more than 50% of surgically resected lung and breast tumour specimens from otherwise untreated patients [corrected]. We conclude that the constitutive activation of the DNA damage checkpoint pathway may be linked to the high frequency of p53 mutations in human cancer, as p53 is a downstream target of Chk2 and ATM.     

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.     

Cell cycle checkpoints and their impact on anticancer therapeutic strategies

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia-telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53-defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN-01. These inhibitors preferentially abrogate cell cycle arrest in p53-defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21(waf1) and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies.