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Objective To investigate the effect of cetuximab (C225) combined with radiation on the expressions of Ku80 and ATM in CNE-2 nude mice xenograft tumor model.Methods The CNE2 nude mice xenograft tumor model was established and divided into control group,conventional radiotherapy (CRT) group,simulated intensity modulated radiation therapy (sIMRT) group,C225 group,CRT + C225 group and sIMRT + C225 group with 8 mice in each group.The dose of radiation was 20 Gy,and 1mg C225 per mice in abdominal cavity was applied for the drug treatment.The transcriptional levels of Ku80 and ATM were assayed by RT-PCR and Western blot.Results Compared with control group,the transcriptional and translational levels of Ku80 and ATM in CRT group,sIMRT group,CRT + C225 group and slMRT + C225 group were decreased (P ＜ 0.05).Compared with CRT group,the down-regulation of transcriptional and translational ATM level in sIMRT group was obvious (P ＜ 0.05),and further strengthened in the CRT + C225 group and sIMRT + C225 groups (P ＜0.05),but there was no significant difference of ATM expression between sIMRT + C225 group and CRT group (P ＞ 0.05).Conclusions C225 alone can restrain the transcriptional and translational level of ATM,and the transcriptional and translational level of Ku80 and ATM are also restrained after megadose.ATM is down-regulated by sIMRT but up-regulated by the combination of C225 and radiation.
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Objective To construct the KU80 inhibition cell model by RNAi in U2OS cell and to explore the relationship between the Ku80,telomeres and radiosensitivity in telomerase-negative tumor cells.Methods U2OS cells were transfected with the recombinant plasmids of pshRNA-K80 by the lipofectamine,and the stable transfected cell clones were selected by G418.After the selection,the cells were collected and analyzed by the flow cytometry.RT-PCR and Western blot were used to measure the expression of Ku80 and Real-time PCR was used to detect the length of telomeres.The radiosensitivity of U2OS was determined by clone formation array.Results The transfection efficiency of the positive cell clones detected by the flow cytometry was (83.23 ± 7.63) ％.The inhibition rate of the Ku80 gene transcription in the cell group with recombinant plasmid was(68.09 ± 1.16)％ and the inhibition rate of the Ku80 protein expression in the same group was (11.03 ± 2.45) ％.The results of Real-time PCR showed that the telomere length of the cell group with recombinant plasmid (1.07 ± 0.07) was significantly shorter than that of the control group (4.42 ± 1.30,F =38.58,P ＜ 0.05) and that of the empty plasmid group (4.11 ±0.84,F =38.58,P ＜ 0.05).Compared to the control group,the telomere length of the empty plasmid group did not changed(4.42 ±0.84 vs.4.11 ±0.84).U2OS cells with Ku80 expression suppressed had lower SF2 than that of the control cells (F =1089.61,P ＜0.05),and resulted in the SER of 1.47.Conclusions The Ku80 inhibition cell model in telomerase-negative U2OS cell line is successfully established which has the shorter telomere length,and is more sensitive to radiation.Telomere shortening caused by pshRNA-of Ku80 is likely to be one of the mechanisms of radiosensitization in this kind of cell model.
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Objective To investigate the inhibition effect of silencing Ku80 gene combined with irradiation on growth of esophageal cancer xenografs. Methods shRNA-Ku80 vector was constructed.The expression of Ku80 protein was inhibited by shRNA-Ku80 vector by using Western blotting. 20 BALB/c nude mice were randomly divided into 4 groups, including control group, radiation group, shRNA-Ku80 group and combined group. The growth of esophageal cancer xenografs was observed. The expression of Ku80 was examined in esophageal cancer xenografs by IHC. Results Effective target sequence was selected. The growth of esophageal cancer xenografs was inhibited by shRNA-H2 and radiation, especially in combined group. The inhibition rate of growth in three groups above was 32.0% , 39. 9% and 68. 9% ,respectively. The expression of Ku80 was reduced to 58% by shRNA-H2 in esophageal cancer xenografs ( t = 3.77, P ＜ 0. 05 ). Conclusions Combination of silencing Ku80 and radiation could enhance radiotherapeut effect of esophageal cancer xenografs.
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Objective To study the role of KuS0 in the treatment of esophageal cancer through inhibiting the Ku80 expression by shRNA. Methods shRNA-KuS0 vector was constructed. The effectiveness and feasibility of RNA interference were confirmed by Western blot and RT-PCR methods. The cell sensitivity to ganuna-rays was studied by colony formation assay. The effects of shRNA-Ku80 on cell cycle were observed by flow cytometry analysis. Results ShRNA-Ku80 vector was constructed successfully. Inhibition of Ku80 expression by shRNA enhanced the sensitivity of esophageal cancer cells to gamma-rays, shRNA K3 or shRNA H2 showed higher percentage of cells in G2/M phase (61.8% vs 28.6% ;64.3% vs 28.6%). Conclusions Inhibitions of Ku80 expression by shRNA play a role in the treatment of esophageal cancer. Ku80 might be a new target of tumor treatment.
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Objective: To explore the correlation between the expressions of DSB (DNA double-strand break) repair protein (including Ku80, DNA-PKcs, and ATM) and radiosensitivity parameters of human tumor cell lines, and to reveal the value of the three proteins for the prognosis of the radiosensitivity of tumor cells. Methods: Eight tumor cell lines were selected including four human cervical carcinoma cell lines (HeLa, SiHa, C33A, and Caski), three human breast carcinoma cell lines (MCF-7, MDA-MB-231, MDA-MB453), and one human lung carcinoma cell line (A549). The expressions of Ku80, DNA-PKcs and ATM protein were measured by Western blotting. The apoptotic ratio of tumor cells was analyzed by flow cytometry after 48 h X-ray irradiation at 10 Gy of 6 MV. SF2 value (survival fraction at 2 Gy) and α and β values were obtained by clone formation assay. The correlation of protein expression with SF2, α/β value or apoptotic ratio was analyzed by Pearson linear correlation analysis. Results: The expression of same protein in different cell lines and the expression of the three proteins in the same cell line had significant difference. There was a positive correlation between the expression of DNA-PKcs and SF2 (r=0.723, P=0.043 0.05). The expression of the three proteins had no correlation with either apoptotic ratio or α/ β value (P>0.05). Conclusions: Tumor cells with higher expression of DNA-PKcs protein will have higher radioresistance. The expression level of DNA-PKcs protein in tumor cells may be an indicator for predicting the radiosensitivity of tumor cells.
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Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.
Subject(s)
Humans , Breast Neoplasms , Catalytic Domain , Cell Cycle , Cell Death , DNA , DNA Breaks, Double-Stranded , DNA Repair , DNA-Activated Protein Kinase , Drug Resistance , Drug Therapy , Homicide , Radiation, Ionizing , United NationsABSTRACT
Objective:To study the different Ku80 mRNA expression in normal lung tissue group, lung cancer group(non small cell lung cancer) without chemotherapy, lung cancer group with multiple chemotherapy(n≥2),to evaluate the relationship of tumor drug resistance with Ku80 mRNA expression in human lung cancer.Methods:The lung tissue gene level of Ku80 mRNA was measured by reverse transcription polymerase chain reaction(RT-PCR) in 25 normal lung tissues, 51 lung cancer tissue,taking ?-actin as inner reference.The data were analyzed by SPSS software.Results:Ku80 mRNA expression in normal lung tissue compared to lung cancer group without chemotherapy, lung cancer group with multiple chemotherapy(n≥2) had statistic significant meaning,respectively P
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Abstract Objective:To study the expression and its biologic specification of the Ku80 gene molecular cloning.Methods: Constructingrecombinant sense or antisense Ku80 expression vectors (Ku80/pCAGGS) and transferring into LCA .Those Ku80 mRNA and protein were de-teited in the Ku80s-LCA, Ku80as-LCA and by Northern-blotting and or Western-blotting.Results: Those Ku80 mRNA were detected in the Ku80s-LCA and Ku80as-LCA by Northern-blotting. Only the sense Ku80 protein was defected in the Ku80s-LCA , but wasn' t detected in the Ku80as-LCA by Western-blotting.Conclusion:The sense or antisense Ku80 were not only transferred in to LCA ,but also expressed in the mR-NA . Only the sense Ku80 protein was expressed in the Ku80s-LCA , but wasn' t expressed in the Ku80as-LCA . The Ku80 is a candidate tar-get for cancer gene therapy.