Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer

The high morbidity and mortality of colorectal cancer pose a significant public health problem worldwide. Here we assessed the pro-cancer efficacy and mechanism of action of CCNB1 in different colorectal cancer cells. We provided evidence that CCNB1 mRNA and protein level were upregulated in a subset of human colorectal tumors, and positively correlated with Chk1 expression. Repression of Chk1 caused a significant decrease in cell proliferation and CCNB1 protein expression in colorectal cancer cells. Furthermore, downregulation of CCNB1 impaired colorectal cancer proliferation in vitro and tumor growth in vivo. Specifically, suppression of CCNB1 caused a strong G₂/M phase arrest in both HCT116 and SW480 cells, interfering with the expression of cdc25c and CDK1. Additionally, CCNB1 inhibition induced apoptotic death in certain colorectal cancer cells. Together, these results suggest that CCNB1 is activated by Chk1, exerts its oncogenic role in colorectal cancer cells, and may play a key role in the development of a novel therapeutic approach against colorectal cancer.     

Combined inhibition of Chk1 and Wee1 as a new therapeutic strategy for mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive, incurable disease, characterized by a deregulated cell cycle. Chk1 and Wee1 are main regulators of cell cycle progression and recent data on solid tumors suggest that simultaneous inhibition of these proteins has a strong synergistic cytotoxic effect. The effects of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) have been herein investigated in a large panel of mature B-cell lymphoma cell lines. We found that MCL cells were the most sensitive to the Chk1 inhibitor PF-00477736 and Wee1 inhibitor MK-1775 as single agents. Possible involvement of the translocation t(11;14) in Chk1 inhibitor sensitivity was hypothesized. The combined inhibition of Chk1 and Wee1 was strongly synergistic in MCL cells, leading to deregulation of the cell cycle, with increased activity of CDK2 and CDK1, and activation of apoptosis. In vivo treatment with the drug combination of mice bearing JeKo-1 xenografts (MCL) had a marked antitumor effect with tumor regressions observed at non-toxic doses (best T/C%=0.54%). Gene expression profiling suggested effect on genes involved in apoptosis. The strong synergism observed by combining Chk1 and Wee1 inhibitors in preclinical models of MCL provides the rationale for testing this combination in the clinical setting.     

Acquired small cell lung cancer resistance to Chk1 inhibitors involves Wee1 up‐regulation

Platinum‐based chemotherapy has been the cornerstone treatment for small cell lung cancer (SCLC) for decades, but no major progress has been made in the past 20 years with regard to overcoming chemoresistance. As the cell cycle checkpoint kinase 1 (Chk1) plays a key role in DNA damage response to chemotherapeutic drugs, we explored the mechanisms of acquired drug resistance to the Chk1 inhibitor prexasertib in SCLC. We established prexasertib resistance in two SCLC cell lines and found that DNA copy number, messengerRNA (mRNA) and protein levels of the cell cycle regulator Wee1 significantly correlate with the level of acquired resistance. Wee1 small interfering RNA (siRNA) or Wee1 inhibitor reversed prexasertib resistance, whereas Wee1 transfection induced prexasertib resistance in parental cells. Reverse phase protein microarray identified up‐regulated proteins in the resistant cell lines that are involved in apoptosis, cell proliferation and cell cycle. Down‐regulation of CDK1 and CDC25C kinases promoted acquired resistance in parental cells, whereas down‐regulation of p38MAPK reversed the resistance. High Wee1 expression was significantly correlated with better prognosis of resected SCLC patients. Our results indicate that Wee1 overexpression plays an important role in acquired resistance to Chk1 inhibition. We also show that bypass activation of the p38MAPK signaling pathway may contribute to acquired resistance to Chk1 inhibition. The combination of Chk1 and Wee1 inhibitors may provide a new therapeutic strategy for the treatment of SCLC.     

Chk1蛋白激酶及其抑制剂的研究进展

Chk1(checkpoint kinase 1,Chk1)属丝氨酸/苏氨酸蛋白激酶家族成员,在DNA损伤反应（DNA damage response,DDR）中是细胞周期检测点的核心蛋白。很多研究表明,Chk1蛋白激酶具有促进肿瘤细胞增生的功能,它的缺乏能使肿瘤细胞对放疗或化疗更加敏感,且其抑制剂与其它分子靶向药物联合应用具有“合成致死”的效应,因此Chk1可作为今后肿瘤治疗的新靶点。本文就Chk1蛋白激酶及其在肿瘤治疗中的作用作一综述。     

下调Chk1和Chk2基因促进射线照射后HeLa细胞凋亡

目的 探讨下调Chk1和Chk2基因对HeLa细胞射线照射后细胞周期和凋亡的影响及其作用机制.方法 应用流式细胞仪检测不同剂量60Co照射引起的HeLa细胞周期和凋亡的动力学变化,以激光共聚焦显微镜和Western blot法检测转染Chk1和Chk2正义寡核苷酸(sODN)和反义寡核苷酸(AsODN)对Chk1和Chk2蛋白表达的影响,以AnnexinV-PI法、凋亡细胞的周期特异性检测法及透射电镜观察单独或联合转染Chk1和Chk2反义寡核苷酸对射线照射后HeLa细胞凋亡的影响.结果 不同剂量的60Co照射可引起Hela细胞小同程度的G2/M期阻滞,15 Gy的60Co照射48 h后,G2/M期细胞可达75.53%±3.72%.当细胞周期阻滞解除后,细胞凋亡明显增加.转染Chk1 AsODN组的早期凋亡、晚期凋亡和死亡细胞共有94.42%±4.78%,明显高于转染Chk1 sODN组(44.35%±2.08%,P<0.0001);转染Chk2 AsODN组的凋亡细胞共有93.08%±5.24%,明显高于转染Chk2 sODN组(48.98%±3.35%,P<0.0001);而共转染Chk1和Chk2 AsODN组的凋亡细胞共有94.26%±4.92%,明显高于共转染Chk1和Chk2 sODN组(42.46%±2.56%,P<0.0001);共转染Chk1和Chk2AsODN组与仅转染Chk1 AsODN或Chk2 AsODN组比较,差异无统计学意义(P>0.05).凋亡细胞的周期特异性检测结果显示,转染Chk1和Chk2 sODN引起的凋亡主要来自于G1期细胞,而转染Chk1 AsODN或Chk2 AsODN后,G1期、S期、G2/M期的凋亡细胞均较对应sODN转染组明显增加,尤其共转染Chk1和Chk2 AsODN组更为显著.结论 射线照射激活细胞周期检测点信号传导通路引起细胞自我保护,是临床上产生放疗抵抗的主要原因,而阻断该信号通路的关键激酶Chk1或Chk2,可显著增强细胞的放射敏感性,其机制在于改变了凋亡细胞的周期特异性,凋亡细胞可以来自G1、S、G2/M各周期的细胞.     

DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.     

The natural anticancer compound rocaglamide selectively inhibits the G1‐S‐phase transition in cancer cells through the ATM/ATR‐mediated Chk1/2 cell cycle checkpoints

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over‐expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide‐A (Roc‐A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1‐S phase. Roc‐A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc‐A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR‐Chk1/Chk2 checkpoint pathway. However, Roc‐A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc‐A by a time‐resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc‐A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc‐A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.     

Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL.     

Targeting ATR and Chk1 kinases for cancer treatment: a new model for new (and old) drugs

Trying to kill cancer cells by generating DNA damage is by no means a new idea. Radiotherapy and genotoxic drugs are routinely used in cancer therapy. More recent developments also explored the potential of targeting the DNA damage response (DDR) in order to increase the toxicity of radio- and chemo- therapy. Chk1 inhibitors have pioneered studies in this regard. Interestingly, early studies noted that Chk1 inhibitors were particularly toxic for p53-deficient cells. The model proposed for this observation was that this effect was due to the simultaneous abrogation of the G2 (Chk1) and G1 (p53) checkpoints. We here challenge this view, and propose a model where the toxicity of Chk1 inhibitors is rather due to the fact that these compounds generate high loads of replicative stress (RS) during S-phase, which are further boosted by the less restrictive S-phase entry found in p53-deficient cells. This new model implies that the particular toxicity of Chk1 inhibitors might not be restricted to p53-deficient cells, but could be extended to other mutations that promote a promiscuous S-phase entry. In addition, this rationale also implies that the same effect should also be observed for other molecules that target the RS-response (RSR), such as inhibitors of the Chk1-activating kinase ATR.     

Chk1/2对肿瘤细胞凋亡的调节作用

目的:观察顺铂作用下K562细胞及白血病骨髓单个核细胞(BMMNC)的细胞周期变化和转染Chk1/2反义寡核苷酸对顺铂诱导下K562细胞及白血病BMMNC凋亡的影响.方法:用流式细胞仪检测顺铂作用下K562细胞及白血病BMMNC的细胞周期变化.转染Chk1和Chk2反义寡核苷酸于K562细胞及白血病BMMNC,检测顺铂作用下转染细胞的凋亡率.结果:10 μmol/L顺铂作用下K562细胞和白血病BMMNC均出现S期阻滞,转染Chk1/2反义寡核苷酸可明显增加顺铂诱导下K562细胞凋亡,转染Chk1反义寡核苷酸可明显增加顺铂诱导下白血病BMMNC的凋亡率,但转染Chk2反义寡核苷酸未增加白血病BMMNC的凋亡率.结论:Chk1在肿瘤细胞凋亡中有重要调节作用,可作为肿瘤增敏治疗的有效靶点,Chk2在肿瘤细胞凋亡中的作用需进一步研究.     

Selective Chk1 inhibitors differentially sensitize p53-deficient cancer cells to cancer therapeutics

The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.     

Chk1在乳腺癌组织中的表达及临床病理意义

目的:检测乳腺癌组织中Chk1(checkpoint kinase1,Chk1)的表达水平,探索其与患者临床病理特征和预后的关系.方法:选取2012年01月至2017年01月在我院病理科存档的乳腺癌组织蜡块115例,应用免疫组织化学法检测其中Chk1蛋白的表达水平.采用卡方检验分析Chk1蛋白的表达情况与临床病理特征的...     

Chk1/2和Plk1蛋白在子宫内膜癌中的表达

目的 Chk1/2（checkpoint kinase 1、2）和Plk1（polo like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶,本研究检测3种激酶在子宫内膜癌及正常子宫内膜组织中的表达,探讨3种蛋白在两者之间的表达差异、与子宫内膜癌临床病理特征的关系及3种蛋白表达的相关性。方法 应用免疫组化SP法检测44例子宫内膜癌组织和21例正常子宫内膜组织中Chk1、Chk2和Plk1蛋白的表达情况。结果 Chk1、Chk2和Plk1蛋白在子宫内膜癌患者中的阳性率分别为47.7％、75.0％和31.8％,在正常子宫内膜中的阳性率分别为61.9％、61.9％和4.8％;Plk1蛋白在子宫内膜癌组织中的表达显著高于正常子宫内膜组织（P＜0.01）,而Chk1、Chk2的表达差异无统计学意义（P>0.05）。Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的子宫内膜癌患者中差异无统计学意义（P>0.05）;但Chk1的表达在不同分化程度的子宫内膜癌患者中差异有统计学意义（P<0.01）。Spearman等级相关分析,在44例子宫内膜癌患者中,Chk2与Plk1间的表达呈正相关（r＝0.482,P＝0.001）。结论 Plk1可能成为子宫内膜癌比较理想的治疗靶点,而CHK1/2在子宫内膜癌中表达及意义还有待进一步研究。     

Targeting the cell cycle for cancer therapy

Most if not all neoplasias show a directly or indirectly deregulated cell cycle. Targeting its regulatory molecules, the cyclin-dependent kinases, as a therapeutic mode to develop new anticancer drugs, is being currently explored in both academia and pharmaceutical companies. The development of new compounds is being focused on the many features of the cell cycle with promising preclinical data in most fields. Moreover, a few compounds have entered clinical trials with excellent results maintaining the high hopes. Thus, although too early to provide a cell cycle target based new commercial drug, there is no doubt that it will be an excellent source of new anticancer compounds.     

Activation of ATM/Chk1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells

Curcumin has been shown to inhibit the growth of various types of cancer cells; however, at concentrations much above the clinically achievable levels in humans. The concentration of curcumin achieved in the plasma after oral administration in humans was estimated to be around 1.8 μM. Here, we report that treatment of BxPC-3 human pancreatic cancer cells with a low and single exposure of 2.5 μM curcumin for 24 h causes significant arrest of cells in the G2/M phase and induces significant apoptosis. Immunoblot studies revealed increased phosphorylation of H2A.X at Ser-139 and Chk1 at Ser-280 and a decrease in DNA polymerase-β level in curcumin-treated cells. Phosphorylation of H2A.X and Chk1 proteins are an indicator of DNA damage whereas DNA polymerase-β plays a role in the repair of DNA strand breaks. Normal immortalised human pancreatic ductal epithelial (HPDE-6) cells remained unaffected by curcumin treatment. In addition, we also observed a significant increase in the phosphorylation of Chk1 at Ser-345, Cdc25C at Ser-216 and a subtle increase in ATM phosphorylation at Ser-1981. Concomitant decrease in the expressions of cyclin B1 and Cdk1 were seen in curcumin-treated cells. Further, curcumin treatment caused significant cleavage of caspase-3 and PARP in BxPC-3 but not in HPDE-6 cells. Silencing ATM/Chk1 expression by transfecting BxPC-3 cells with ATM or Chk1-specific SiRNA blocked the phosphorylation of ATM, Chk1 and Cdc25C and protected the cells from curcumin-mediated G2/M arrest and apoptosis. This study reflects the critical role of ATM/Chk1 in curcumin-mediated G2/M cell cycle arrest and apoptosis in pancreatic cancer cells.     

Chk1/2和Plk1蛋白在宫颈良恶性病变组织中的表达及其意义

背景与目的：Chk1/2（checkpoint kinase 1/2）和Plk1（polo-like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶.本研究主要探讨3种激酶蛋白在宫颈良恶性病变中的表达差异、与宫颈癌临床病理特征的关系及3种激酶在宫颈癌组织中表达之间的相互关系.方法：应用免疫组化SP法检测43例宫颈癌组织和20例慢性宫颈炎性组织中Chk1、Chk2和Plk1蛋白的表达情况.结果：Chk1、Chk2和Plk1蛋白在宫颈癌患者中的阳性率分别为76.7%、60.5%和32.6%,在慢性宫颈炎患者中的阳性率分别为30.0%、35.0%和0;Chkl和Plk1蛋白在宫颈癌组织中的表达显著高于慢性宫颈炎组织（P＜0.01）,而Chk2的表达差异无显著性（P＞0.05）.Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的宫颈癌患者中差异无显著性（P＞0.05）;但Chk1和Plkl的表达在不同分化程度的宫颈癌患者中差异有显著性（P＜0.05）.Spearman等级相关分析,在43例宫颈癌患者中,Chk1与Chk2的表达呈正相关（r=0.492,P=0.001）.结论：Chk1和Plk1可能成为比较理想的宫颈癌治疗靶点.     

G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer

Rigorous quality control steps, termed checkpoints, tightly regulate progression through the cell cycle. DNA-damaging chemotherapy and radiation activate functional cellular checkpoints. These checkpoints can facilitate DNA repair and promote cell death in unrepaired cells. There are at least three DNA damage checkpoints - at G1/S, S, and G2/M - as well as a mitotic spindle checkpoint. Most cancer cells harbour mutations in tumour suppressors and/or oncogenes, which impair certain cell checkpoints. Inhibiting the remaining cell checkpoints - particularly after exposure of cancer cells to chemotherapy and/or radiation - allows cell death, a strategy now being employed in cancer therapeutics. With our increasing knowledge of cell cycle regulation, many compounds have been developed to inhibit specific checkpoint components, particularly at the G2/M transition. One such target is checkpoint kinase-1 (Chk1). We review here the molecular framework of the cell cycle, the rationale for targeting Chk1, the preclinical concepts related to the development of Chk1 inhibitors, and the efficacy and safety results from Chk1 inhibitors now in phase I/II trials.     

Mangrove dolabrane‐type of diterpenes tagalsins suppresses tumor growth via ROS‐mediated apoptosis and ATM/ATR–Chk1/Chk2‐regulated cell cycle arrest

Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti‐cancer drugs. In this study, we show that a group of dolabrane‐type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS‐mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S‐G2 phase via activation of the ATM/ATR—Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T‐cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs.     

Chk2-mediated G2/M cell cycle arrest maintains radiation resistance in malignant meningioma cells

In continuation to our studies on radioresistance in meningioma, here we show that radiation treatment (7 Gy) induces G2/M cell cycle arrest in meningioma cells. Phosphorylation of Chk2, Cdc25c and Cdc2 were found to be key events since interference with Chk2 activation and cyclin B1/Cdc2 interaction led to permanent arrest followed by apoptosis. Irradiated cells showed recovery and formed aggressive intracranial tumors with rapid spread and morbidity. Nevertheless, knock down of uPAR with or without radiation induced permanent arrest in G2/M phase and subsequent apoptosis in vitro and in vivo. In conclusion, our data suggest that combination treatment with radiation and uPAR knock down or other inhibitors resulting in non-reversible G2/M arrest may be beneficial in the management of meningiomas.