RNA干扰细胞周期检测点激酶CHK1和CHK2表达对食管癌细胞照射后细胞周期阻滞的消除作用
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摘要
我国是食管癌高发国家,发病人数约占全世界50%以上。临床就诊的食管癌病人多数为中晚期,需要接受放射治疗(放疗),放疗成为中晚期食管癌主要治疗方法之一。但食管鳞癌属于中度放射敏感肿瘤,单纯放疗后5年生存率较低,但肿瘤局部未控和复发率很高;如何提高食管癌细胞的放射敏感性,提高肿瘤局部控制率、减少复发而提高疗效,始终是研究的热点和难题。
DNA是放射线作用的主要靶分子,当放射线照射引起DNA损伤发生后,DNA损伤修复系统立即启动,并激活细胞周期检测点出现细胞周期阻滞,促进损伤DNA的修复。细胞周期检测点激酶1和2(CHK1和CHK2)是细胞周期检测点中非常重要的蛋白激酶,它们通过信号传导和放大,调节下游靶蛋白表达,使细胞周期出现阻滞。抑制CHK1和CHK2蛋白表达,可有效地消除肿瘤细胞在放射线或化疗药物作用后的细胞周期阻滞,增加放化疗的敏感性。但在食管癌细胞和食管癌组织中,CHK1和CHK2蛋白表达情况如何、照射后对细胞周期检测点如何调节,尚未见报道。
本研究首先观察食管癌细胞、食管癌组织以及癌旁不典型增生组织中CHK1和CHK2表达情况,并了解CHK1和CHK2表达与食管癌发生、发展以及单纯放疗疗效的关系;其次,观察体外培养的食管癌细胞在单纯照射或照射加药物wortmannin(以下简称WT)共同作用后,对食管癌细胞中CHK1和CHK2表达、细胞周期变化以及放射敏感性的影响;最后,通过脂质体介导的CHK1和CHK2反义寡核苷酸转染技术以及脂质体介导、质粒连接CHK1和CHK2短发卡状RNA(shRNA)的RNA干扰(RNAi)技术,探讨食管癌细胞转染前后,对CHK1和CHK2表达的变化以及照射后细胞周期的影响,为进一步进行体内和临床前期研究创造条件。
第一部分CHK1和CHK2在食管癌细胞和组织中的表达及其临床意义
目的:观察食管癌细胞、食管癌组织以及癌旁不典型增生组织中CHK1和CHK2的表达情况,了解食管癌组织中CHK1和CHK2表达与其发
Esophageal carcinoma is one of the most common malignant tumor in our country and more than 50 persent of patients who suffered from esophageal carcinoma aroud the world exist in China. Radiotherapy(RT) is one of the most important methods to therapied esophageal carcinoma, however, esophageal carcinoma has lower 5 years survival rate but higher failed control rate and recurrent rate after RT. It is still a challenged question how to enhance radiosensitivity of esophageal carcinoma cells, to decrease local recurrent rate, and to increase local contral rate and long-time survival..
At present, DNA is beleved the main target of irradiation, when irradiation induced DNA damage, system of DNA damage repair and checkpoint of cell cycle is actived immediately, arrest of cell cycle were induced to enhance repair for DNA lesions. Checkpoint kinase 1 and 2 (CHK1 and CHK2) are very important protein kinase for repair damaged DNA and arrest of cell cycle, through transfer and enlarge signal to down-stream proteins, retraded cell cycle course. CHK1 and CHK2 has not effect to growth of somatical cells, but inhibited protein expression of CHK1 and CHK2, it could release retarded cell cycle and increase sensitivity to killing tumor cells after irradiation or chemical drugs. To date, it is still not report about protein expression of CHK1 and CHK2 in esophageal carcinoma cells and tussis of esophageal carcinoma, and how to regulated cell cycle of esophageal carcinoma cells after DNA damage.
In this report, firstly, expression of CHK1 and CHK2 were detected in esophageal carcinoma cells, tissus of esophageal carcinoma and its dysplasia, relationship between protein expression of CHK1 and CHK2 and development or prognosis after RT alone in esophageal carcinoma were observed. Secondly,
protein expression of CHK1 and CHK2, change of cell cycle and radiosensitivity were detected in esophageal carcinoma cells after radiation alone or combined with wortmannin(WT) in vitro. Finally, antisence oligodeoxynucleotide(ASON) and short hairpin RNA (shRNA)-plasmid vector of CHK1 and CHK2 gene were transfected into esophageal carcinoma cells with lipofectamine reagent, effect on mRNA and protein expression of CHK1 and CHK2 and cell cycle after radiation were analysed. Part I Study on expression of CHK1 and CHK2 in cells and tissue of esophageal carcinoma and its clinical significane Objective:To observed expression of CHK1 and CHK2 in esophageal carcinoma cells and tissues of esophageal carcinoma and its precucor, and relationship between expression of CHK1 and CHK2 in tissue of esophageal carcinoma and its development or prognosis after RT was analysized. Methods: In three human esophageal carcinoma cell lines of TE-1,TE-13 and Eca109, expression of CHK1 and CHK2 mRNA were detected with reverse transcriptase-polymerase chain reaction(RT-PCR), expression of CHK1 and CHK2 protein were measured with immunohistochemistry assay, western blotting and indirect immunofluorescence assay respectively. 33 cases of patient were biopsied with gastroscopy and diagnosed as esophageal squamous cell carcinoma(ESCC); among them, 12 cases of tissue neighbour tumor were collected and diagnosed as dysplasia simultaneously; protein of CHK1 and CHK2 in tissues of 33 cases of ESCC and 12 cases of dysplasia were detected with western blotting, prognostic related factors were analysis after single RT. Results: ①Expression of CHK1 and CHK2 in TE-1,TE-13 and Eca109 cells were observed in level of mRNA and protein in this report, expression of CHK1 and CHK2 protein were also observed in tissue of ESCC and its precursor. ②Expression of CHK1 and CHK2 protein is not significant difference between tissues of ESCCs and its relevant precursor with semi-quantity analysis. ③A fter RT, 1-year survival rate and median were 61.44% and 11.5 months respectively in 33 cases of ESCC, the evaluation in
total efficiency of clincial response is 100%(21 cases CR and 12 cases of PR). Age, tumor length of X-ray and CT, T stage,N stage, clinical stage and clinical response, were correlated with prognosis of ESCCs after RT alone with one-way survival analysis; but only clinical stage is the independent prognostic factor of ESCC with Cox multivariate analysis. Expression of CHK1 and CHK2 protein is not related with prognosis of ESCC after RT. Conclusion: Expression of CHK1 and CHK2 protein were universely exsited in esophageal carcinoma cells and tissues of esophageal carcinoma and its precursor, but expression of CHK1 and CHK2 protein is probably not correlated with development of ESCC and its prognosis after RT; clinical stage is the most important prognostic factor in ESCCs after RT in this report. Part II Effect on cell cycle and expression of CHK1 and CHK2 after irradiation alone or combined with wortnannin in esophageal cancer cells Objective: To observed effect on radiosensitivity, cell cycle and expression of CHK1 and CHK2 mRNA and protein after irradiation alone or combined with wortnannin(WT) in esophageal cancer cells in vitro. Methods Radiosensitivity were analysized with clonegentic assay and radiosensitized effect were observed with MTT methods; change of cell cycle were measured with flow cytometry(FCM) and expression of CHK1,CHK2, CHK1-S345,CHK2-T68,CDK1 and cyclin B1 protein were detected with western blotting after irradiation alone or combined with wortnannin in TE13 and Eca109 esophageal caicinoma cells in vitro. Result: ①D0 value were 2.70Gy and 2.15Gy and SF2 value were 0.785 and 0.748 respectively with clonegenetic assay in Eca109 and TE13 cells. 1μM WT decrease survival rates of Eca109 and TE13 cells measured with MTT methods after irradioation. ②Change of cell cycle in TE13 cells were mesaured with FCM, arrest of G2/M stage is gradually increased concomitant with boosted doses of irradiation at 24h after irradiated by 0-15Gy(p<0.05); at 48h after irradiation, arrest of G2/M stage is gradually released and it is negative related with doses of irradiation. ③In Eca109 cells, serious retardance of G2/M stage were observed at 12h after irradiated by 5Gy(p<
0.05), it has been obviously released at 24h and completely released at 48h after irradiation. ④Treated TE13 cells with 0-20μM WT for 1h previously, percent of G_0/G_1 stage were significantly decreased at 24h after irradiated by 5Gy(p<0.05); but treated Eca109 cells with 0-10μM WT for 1h previously, cell cycle were not influenced at 24h after irradiated by 5Gy(p>0.05); however, treated Eca109 cells with 20μM WT for 1h previously, markedly decreased percent of G_0/G_1 stage and increased percent of G_2/M stage at 24h after irradiated by 5Gy(p<0.05). ⑤Treated Eca109 cells with 10μM WT from 1h before irradiated by 5Gy until 12h later after irradiation, cell cycle did not influenced comparative with irradiated by 5Gy alone(p>0.05); but 20μM WT could increased percent of G_0/G_1 stage and decreased percent of G_2/M stage after irradiation(p<0.05). Treated Eca109 cells with 20μM WT from 1h brfore irradiated by 5Gy until 24h later after irradiated, it could decreased percent of G_0/G_1 stage and increased percent of G_2/M stage after irradiation(p<0.05). ⑥During 0-4h after irradiated by 5Gy, increased percent of S stage were observed in TE13 cells but not in Eca109 cells; treated with 10μM WT for 1h before irradiated by 5Gy, obvious increased percent of S stage were detected in Eca109 cells but not in TE13 cells. ⑦Apart from expression of cyclinB1 protein in cytoplasm were decreased at 24h after irradiated by 15Gy, expression of CHK1, CHK2, CDK1 and cyclinB1 in nucleoli and cytoplasm of TE13 and Eca109 cells were not any changed at 1h after irradiated by 0-15Gy, and at 24h after irradiated by 0, 5Gy or 15Gy, and during 0-4h after irradiated by 5Gy. Level of CHK2-T68 phosphated expression in cytoplasm were markedly increased after irradiation but not in nucleoli of TE13 and Eca109 cells; phosphated level of CHK2-T68 pretion in TE13 and Eca109 cells arrivaled at peak about 30min later, decreased at 1h and recovered to normal level at 24h after radiated by 5Gy. In TE13 and Eca109 cells, phosphated level of CHK2-T68 protein in nucleoli and CHK1-S345 in nucleoli and cytoplasm were not changed after irradiation .⑧Treated with 10 and 20μM WT for 1h then irradiated by 5Gy, decreased expression of CHK2 in nucleoli and phosphated level of CHK2-T68 protein in cytoplasm were
observed in Eca109 cells, but expression of CHK1 in Eca109 cells and expression of CHK1 and CHK2 in Eca109 and TE13 cells were not influenced; mRNA expression of CHK1 and CHK2 were also not influenced in Eca109 and TE13 cells after irradiated alone or combined with WT. Conclusion: Radiosensitivity is higher in TE13 cells than Eca109 cells and radiosensitivity is possibly some related with arrest of G2/M after radiation. Radiation only effect phosphated level of CHK2-T68 protein but not expression of CHK1 and CHK2 protein in esophageal cancer cells. WT has rediosensitized effect to TE13 and Eca109 cells, could influenced cell cycle in TE13 and Eca109 cells, and only effected expression of CHK2 in Eca109 cells. rediosensitized effect of WT could has not related with expression of CHK1 and CHK2 in TE13 cells, but it could has related with expression of CHK2 but not CHK1 in Eca109 cells. Part III Effect on expression of CHK1 and CHK2 and distribution fo cell cycle after irradiation in esophageal carcinoma cells with antisense oligodeoxynucleotide and RNA interference. Objective: To observed effect on expression of CHK1 and CHK2 and change of cell cycle after radiation in esophageal carcinoma cells with antisense oligodeoxynucleotide(ASON) and RNA interference. Methods: ①ASON of CHK1 and CHK2 gene were transfected into TE13 and Eca109 cells with Oligofectamine reagent. ②After short hairhip RNA(shRNA) of CHK1 and CHK2 gene were designed, synthesized and connected with vector of pENTER~(TM) plasmid, they were transformed into TOP10 E coli, then selected and enlarged culture of E coli; extracted and identified with sequences of plasmid DNA, then transfected plasmid DNA into TE13 and Eca109 cells with reagent of Liporfectamine~(TM) 2000. ③After transfected, expression of CHK1 and CHK2 protein and mRNA were detected with western blotting and RT-PCR respectively and distribution of cell cycle were measured with folw cytomitry after radiated by 5Gy, cell survival rate of 5Gy were evaluated with clonegenetic assay. Result: ①After transfected with ASON of CHK1 and CHK2 genes, its
DNA是放射线作用的主要靶分子,当放射线照射引起DNA损伤发生后,DNA损伤修复系统立即启动,并激活细胞周期检测点出现细胞周期阻滞,促进损伤DNA的修复。细胞周期检测点激酶1和2(CHK1和CHK2)是细胞周期检测点中非常重要的蛋白激酶,它们通过信号传导和放大,调节下游靶蛋白表达,使细胞周期出现阻滞。抑制CHK1和CHK2蛋白表达,可有效地消除肿瘤细胞在放射线或化疗药物作用后的细胞周期阻滞,增加放化疗的敏感性。但在食管癌细胞和食管癌组织中,CHK1和CHK2蛋白表达情况如何、照射后对细胞周期检测点如何调节,尚未见报道。
本研究首先观察食管癌细胞、食管癌组织以及癌旁不典型增生组织中CHK1和CHK2表达情况,并了解CHK1和CHK2表达与食管癌发生、发展以及单纯放疗疗效的关系;其次,观察体外培养的食管癌细胞在单纯照射或照射加药物wortmannin(以下简称WT)共同作用后,对食管癌细胞中CHK1和CHK2表达、细胞周期变化以及放射敏感性的影响;最后,通过脂质体介导的CHK1和CHK2反义寡核苷酸转染技术以及脂质体介导、质粒连接CHK1和CHK2短发卡状RNA(shRNA)的RNA干扰(RNAi)技术,探讨食管癌细胞转染前后,对CHK1和CHK2表达的变化以及照射后细胞周期的影响,为进一步进行体内和临床前期研究创造条件。
第一部分CHK1和CHK2在食管癌细胞和组织中的表达及其临床意义
目的:观察食管癌细胞、食管癌组织以及癌旁不典型增生组织中CHK1和CHK2的表达情况,了解食管癌组织中CHK1和CHK2表达与其发
Esophageal carcinoma is one of the most common malignant tumor in our country and more than 50 persent of patients who suffered from esophageal carcinoma aroud the world exist in China. Radiotherapy(RT) is one of the most important methods to therapied esophageal carcinoma, however, esophageal carcinoma has lower 5 years survival rate but higher failed control rate and recurrent rate after RT. It is still a challenged question how to enhance radiosensitivity of esophageal carcinoma cells, to decrease local recurrent rate, and to increase local contral rate and long-time survival..
At present, DNA is beleved the main target of irradiation, when irradiation induced DNA damage, system of DNA damage repair and checkpoint of cell cycle is actived immediately, arrest of cell cycle were induced to enhance repair for DNA lesions. Checkpoint kinase 1 and 2 (CHK1 and CHK2) are very important protein kinase for repair damaged DNA and arrest of cell cycle, through transfer and enlarge signal to down-stream proteins, retraded cell cycle course. CHK1 and CHK2 has not effect to growth of somatical cells, but inhibited protein expression of CHK1 and CHK2, it could release retarded cell cycle and increase sensitivity to killing tumor cells after irradiation or chemical drugs. To date, it is still not report about protein expression of CHK1 and CHK2 in esophageal carcinoma cells and tussis of esophageal carcinoma, and how to regulated cell cycle of esophageal carcinoma cells after DNA damage.
In this report, firstly, expression of CHK1 and CHK2 were detected in esophageal carcinoma cells, tissus of esophageal carcinoma and its dysplasia, relationship between protein expression of CHK1 and CHK2 and development or prognosis after RT alone in esophageal carcinoma were observed. Secondly,
protein expression of CHK1 and CHK2, change of cell cycle and radiosensitivity were detected in esophageal carcinoma cells after radiation alone or combined with wortmannin(WT) in vitro. Finally, antisence oligodeoxynucleotide(ASON) and short hairpin RNA (shRNA)-plasmid vector of CHK1 and CHK2 gene were transfected into esophageal carcinoma cells with lipofectamine reagent, effect on mRNA and protein expression of CHK1 and CHK2 and cell cycle after radiation were analysed. Part I Study on expression of CHK1 and CHK2 in cells and tissue of esophageal carcinoma and its clinical significane Objective:To observed expression of CHK1 and CHK2 in esophageal carcinoma cells and tissues of esophageal carcinoma and its precucor, and relationship between expression of CHK1 and CHK2 in tissue of esophageal carcinoma and its development or prognosis after RT was analysized. Methods: In three human esophageal carcinoma cell lines of TE-1,TE-13 and Eca109, expression of CHK1 and CHK2 mRNA were detected with reverse transcriptase-polymerase chain reaction(RT-PCR), expression of CHK1 and CHK2 protein were measured with immunohistochemistry assay, western blotting and indirect immunofluorescence assay respectively. 33 cases of patient were biopsied with gastroscopy and diagnosed as esophageal squamous cell carcinoma(ESCC); among them, 12 cases of tissue neighbour tumor were collected and diagnosed as dysplasia simultaneously; protein of CHK1 and CHK2 in tissues of 33 cases of ESCC and 12 cases of dysplasia were detected with western blotting, prognostic related factors were analysis after single RT. Results: ①Expression of CHK1 and CHK2 in TE-1,TE-13 and Eca109 cells were observed in level of mRNA and protein in this report, expression of CHK1 and CHK2 protein were also observed in tissue of ESCC and its precursor. ②Expression of CHK1 and CHK2 protein is not significant difference between tissues of ESCCs and its relevant precursor with semi-quantity analysis. ③A fter RT, 1-year survival rate and median were 61.44% and 11.5 months respectively in 33 cases of ESCC, the evaluation in
total efficiency of clincial response is 100%(21 cases CR and 12 cases of PR). Age, tumor length of X-ray and CT, T stage,N stage, clinical stage and clinical response, were correlated with prognosis of ESCCs after RT alone with one-way survival analysis; but only clinical stage is the independent prognostic factor of ESCC with Cox multivariate analysis. Expression of CHK1 and CHK2 protein is not related with prognosis of ESCC after RT. Conclusion: Expression of CHK1 and CHK2 protein were universely exsited in esophageal carcinoma cells and tissues of esophageal carcinoma and its precursor, but expression of CHK1 and CHK2 protein is probably not correlated with development of ESCC and its prognosis after RT; clinical stage is the most important prognostic factor in ESCCs after RT in this report. Part II Effect on cell cycle and expression of CHK1 and CHK2 after irradiation alone or combined with wortnannin in esophageal cancer cells Objective: To observed effect on radiosensitivity, cell cycle and expression of CHK1 and CHK2 mRNA and protein after irradiation alone or combined with wortnannin(WT) in esophageal cancer cells in vitro. Methods Radiosensitivity were analysized with clonegentic assay and radiosensitized effect were observed with MTT methods; change of cell cycle were measured with flow cytometry(FCM) and expression of CHK1,CHK2, CHK1-S345,CHK2-T68,CDK1 and cyclin B1 protein were detected with western blotting after irradiation alone or combined with wortnannin in TE13 and Eca109 esophageal caicinoma cells in vitro. Result: ①D0 value were 2.70Gy and 2.15Gy and SF2 value were 0.785 and 0.748 respectively with clonegenetic assay in Eca109 and TE13 cells. 1μM WT decrease survival rates of Eca109 and TE13 cells measured with MTT methods after irradioation. ②Change of cell cycle in TE13 cells were mesaured with FCM, arrest of G2/M stage is gradually increased concomitant with boosted doses of irradiation at 24h after irradiated by 0-15Gy(p<0.05); at 48h after irradiation, arrest of G2/M stage is gradually released and it is negative related with doses of irradiation. ③In Eca109 cells, serious retardance of G2/M stage were observed at 12h after irradiated by 5Gy(p<
0.05), it has been obviously released at 24h and completely released at 48h after irradiation. ④Treated TE13 cells with 0-20μM WT for 1h previously, percent of G_0/G_1 stage were significantly decreased at 24h after irradiated by 5Gy(p<0.05); but treated Eca109 cells with 0-10μM WT for 1h previously, cell cycle were not influenced at 24h after irradiated by 5Gy(p>0.05); however, treated Eca109 cells with 20μM WT for 1h previously, markedly decreased percent of G_0/G_1 stage and increased percent of G_2/M stage at 24h after irradiated by 5Gy(p<0.05). ⑤Treated Eca109 cells with 10μM WT from 1h before irradiated by 5Gy until 12h later after irradiation, cell cycle did not influenced comparative with irradiated by 5Gy alone(p>0.05); but 20μM WT could increased percent of G_0/G_1 stage and decreased percent of G_2/M stage after irradiation(p<0.05). Treated Eca109 cells with 20μM WT from 1h brfore irradiated by 5Gy until 24h later after irradiated, it could decreased percent of G_0/G_1 stage and increased percent of G_2/M stage after irradiation(p<0.05). ⑥During 0-4h after irradiated by 5Gy, increased percent of S stage were observed in TE13 cells but not in Eca109 cells; treated with 10μM WT for 1h before irradiated by 5Gy, obvious increased percent of S stage were detected in Eca109 cells but not in TE13 cells. ⑦Apart from expression of cyclinB1 protein in cytoplasm were decreased at 24h after irradiated by 15Gy, expression of CHK1, CHK2, CDK1 and cyclinB1 in nucleoli and cytoplasm of TE13 and Eca109 cells were not any changed at 1h after irradiated by 0-15Gy, and at 24h after irradiated by 0, 5Gy or 15Gy, and during 0-4h after irradiated by 5Gy. Level of CHK2-T68 phosphated expression in cytoplasm were markedly increased after irradiation but not in nucleoli of TE13 and Eca109 cells; phosphated level of CHK2-T68 pretion in TE13 and Eca109 cells arrivaled at peak about 30min later, decreased at 1h and recovered to normal level at 24h after radiated by 5Gy. In TE13 and Eca109 cells, phosphated level of CHK2-T68 protein in nucleoli and CHK1-S345 in nucleoli and cytoplasm were not changed after irradiation .⑧Treated with 10 and 20μM WT for 1h then irradiated by 5Gy, decreased expression of CHK2 in nucleoli and phosphated level of CHK2-T68 protein in cytoplasm were
observed in Eca109 cells, but expression of CHK1 in Eca109 cells and expression of CHK1 and CHK2 in Eca109 and TE13 cells were not influenced; mRNA expression of CHK1 and CHK2 were also not influenced in Eca109 and TE13 cells after irradiated alone or combined with WT. Conclusion: Radiosensitivity is higher in TE13 cells than Eca109 cells and radiosensitivity is possibly some related with arrest of G2/M after radiation. Radiation only effect phosphated level of CHK2-T68 protein but not expression of CHK1 and CHK2 protein in esophageal cancer cells. WT has rediosensitized effect to TE13 and Eca109 cells, could influenced cell cycle in TE13 and Eca109 cells, and only effected expression of CHK2 in Eca109 cells. rediosensitized effect of WT could has not related with expression of CHK1 and CHK2 in TE13 cells, but it could has related with expression of CHK2 but not CHK1 in Eca109 cells. Part III Effect on expression of CHK1 and CHK2 and distribution fo cell cycle after irradiation in esophageal carcinoma cells with antisense oligodeoxynucleotide and RNA interference. Objective: To observed effect on expression of CHK1 and CHK2 and change of cell cycle after radiation in esophageal carcinoma cells with antisense oligodeoxynucleotide(ASON) and RNA interference. Methods: ①ASON of CHK1 and CHK2 gene were transfected into TE13 and Eca109 cells with Oligofectamine reagent. ②After short hairhip RNA(shRNA) of CHK1 and CHK2 gene were designed, synthesized and connected with vector of pENTER~(TM) plasmid, they were transformed into TOP10 E coli, then selected and enlarged culture of E coli; extracted and identified with sequences of plasmid DNA, then transfected plasmid DNA into TE13 and Eca109 cells with reagent of Liporfectamine~(TM) 2000. ③After transfected, expression of CHK1 and CHK2 protein and mRNA were detected with western blotting and RT-PCR respectively and distribution of cell cycle were measured with folw cytomitry after radiated by 5Gy, cell survival rate of 5Gy were evaluated with clonegenetic assay. Result: ①After transfected with ASON of CHK1 and CHK2 genes, its
引文
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31 Zhang P, Wang J, Gao W, et al. CHK2 kinase expression is down-regulated due to promoter methylation in non-small cell lung cancer. Mol Cancer, 2004, 3(1):14-21.
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19 Darbon JM, Penary M, Escalas N, et al. Distinct CHK2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells. J Biol Chem, 2000, 275(20):15363-15369.
20 Hirose Y, Berger MS, Pieper RO. Abrogation of the CHK1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res, 2001, 61(15):5843-5849.
21 Yu Q, Rose JH, Zhang H, et al. Antisense inhibition of CHK2/hCDS1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells. FEBS Lett, 2001, 505(1):7-12.
22 Tort F, Hernandez S, Bea S, et al. Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms. Leukemia, 2005, 19(1):112-117.
23 Tort F, Hernandez S, Bea S, et al. CHK2-decreased protein expression and infrequent genetic alterations mainly occur in aggressive types of non-Hodgkin lymphomas. Blood, 2002, 100(13):4602-4608.
24 Aktas D, Arno MJ, Rassool F, et al. Analysis of CHK2 in patients with myelodysplastic syndromes. Leuk Res, 2002, 26(11):985-987.
25 Hofmann WK, Miller CW, Tsukasaki K, et al. Mutation analysis of the DNA-damage checkpoint gene CHK2 in myelodysplastic syndromes and acute myeloid leukemias. Leuk Res, 2001, 25(4):333-338.
26 Shigeishi H, Yokozaki H, Oue N, et al. Increased expression of CHK2 in human gastric carcinomas harboring p53 mutations. Int J Cancer, 2002, 99(1):58-62.
27 Menoyo A, Alazzouzi H, Espin E, et al. Somatic mutations in the DNA damage-response genes ATR and CHK1 in sporadic stomach tumors with microsatellite instability. Cancer Res, 2001, 61(21):7727-7730.
28 Kimura K, Shinmura K, Yoshimura K, et al. Absence of Germline CHK2 Mutations in Familial Gastric Cancer Jpn J Cancer Res, 2000, 91(9):875-879.
29 Bertoni F, Codegoni AM, Furlan D,et al. CHK1 Frameshift Mutations in Genetically Unstable Colorectal and Endometrial Cancers. Genes Chromosomes Cancer, 1999, 26(1):176–180.
30 Haruki N, Saito H, Tatematsu Y, et al. Histological Type-selective, Tumor-predominant Expression of a Novel CHK1 Isoform and Infrequent in Vivo Somatic CHK2 Mutation in Small Cell Lung Cancer. Cancer Res, 2000, 60(17):4689-4692.
31 Zhang P, Wang J, Gao W, et al. CHK2 kinase expression is down-regulated due to promoter methylation in non-small cell lung cancer. Mol Cancer, 2004, 3(1):14-21.