RNA Interfered Expression Of Checkpoint Kinase CHK1 And CHK2 To Release Retard Of Cell Cycle In Esophageal Carcinoma Cells After Irradiation

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: â‘ Expression 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. â‘¡Expression of CHK1 and CHK2 protein is not significant difference between tissues of ESCCs and its relevant precursor with semi-quantity analysis. â‘¢A fter RT, 1-year survival rate and median were 61.44% and 11.5 months respectively in 33 cases of ESCC, the evaluation intotal 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: â‘ D0 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. â‘¡Change 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. â‘¢In 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. â‘£Treated TE13 cells with 0-20μM WT for 1h previously, percent of G₀/G₁ 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 G₀/G₁ stage and increased percent of G₂/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 G₀/G₁ stage and decreased percent of G₂/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 G₀/G₁ stage and increased percent of G₂/M stage after irradiation(p<0.05). â‘¥During 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 wereobserved 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: â‘ ASON of CHK1 and CHK2 gene were transfected into TE13 and Eca109 cells with Oligofectamine reagent. â‘¡After 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. â‘¢After 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: â‘ After transfected with ASON of CHK1 and CHK2 genes, its...