Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.     

In-silico screening for DNA-dependent protein kinase (DNA-PK) inhibitors: Combined homology modeling,docking, molecular dynamic study followed by biological investigation

DNA-dependent protein kinase (DNA-PK) is a key enzyme in non-homologous DNA end joining (NHEJ) repair pathway. The targeted inhibition of such enzyme would furnish a valuable option for cancer treatment. In this study we report the development of validation of enzyme homology model, and the subsequent use of this model to perform docking-based virtual screening against a database of FDA-approved drugs. The nominated highest ranking hits (Praziquantel and Dutasteride) were subjected to biological investigation. Additionally, molecular dynamic study was carried-out for binding mode exploration. Results of the biological evaluation revealed that both compounds inhibit the DNA-PK enzymatic activity at relatively high concentration levels with an IC₅₀ of 17.3 μM for praziquantel and >20 μM for dutasteride. Furthermore, both agents enhanced the anti-proliferative effects of doxorubicin and cisplatin on breast cancer (MCF7) and lung cancer (A549) cell lines. This result indicates that these two hits are good candidate as DNA-PK inhibitors and worth further structural modifications to enhance their enzyme inhibitory effects.     

DNA-PK基因沉默对卵巢癌细胞细胞凋亡的影响

目的 探讨DNA-PK基因对沉默卵巢癌细胞凋亡的影响及机制.方法 DNA-PK shRNA以脂质体介导转染卵巢癌细胞株Skov3,24h后接受2Gy X射线照射,继续培养48 h.收集细胞,标记Annexin Ⅴ/PI,流式细胞术分析细胞凋亡情况,同时,RT-PCR检测p53和p21 mRNA的表达量.结果 2Gy辐射引起2.40％±1.53％ Skov3细胞凋亡,高于对照组（t=2.618,P=0.026）,而转染DNA-PK shRNA的Skov3细胞经2Gy照射,凋亡细胞为18.74％±4.15％,明显高于单纯2Gy照射组（t=9.052,P＜0.001）及DNA-PK shRNA转染组（2.77％±1.73％）（t=8.869,P＜0.001）.RT-PCR检测结果显示,2Gy照射组,Skov3细胞p53 mRNA表达量高于对照组,而DNA-PKshRNA转染的Skov3细胞接受2Gy照射,其p53 mRNA表达量降低,接近正常水平,单独转染DNA-PK shRNA组p53 mRNA表达量无明显变化.转染DNA-PK shRNA的Skov3细胞经2Gy照射,其p21 mRNA表达量低于2Gy照射组接近正常组水平,单独转染DNA-PK shRNA组Skov3细胞p21 mRNA表达水平与正常对照组相当.结论 DNA-PK shRNA可增加辐射后巢癌细胞株的细胞凋亡,其可能机制是通过下调p53和p21 mRNA表达,而启动细胞凋亡.     

DNA依赖蛋白激酶与大肠癌淋巴转移相关性研究

Objective:To investigate DNA-dependent protein kinase(DNA-PK)expression,and its relationship with lymphatic metastasis in colorectal cancer.Methods:Tumor tissues from 60 patients,divided into two groups according to lymphatic metastasis,were immunohistochemically stained to detect the DNA-PK expression including Ku70,Ku80 and PKcs proteins.Results:Positivity of both Ku70 and Ku80 in colorectal cancer was negatively correlated with lymphatic metastasis with an rvalue of-0.57 and -0.38,respectively.Similar correlation was found between Ku expression,especially Ku70.and long-term survival.PKcs,however,displayed no significant correlation.Statistical analysis failed to detect any correlation between DNA-PK expression,and clinical characteristics,such as age,sex,tumor Jocation,tumor thickness and distant metastasis(P＞0.05).Conclusion:DNA-PK expression,especially Ku70 expression,is negatively correlated with lymphatic metastasis,and the survival of patients with colorectal cancer.Ku70 expression may be a potential indicator for the preoperative evaluation,and prognosis in colorectal cancer.     

Homologous recombinational repair vis-à-vis chlorambucil resistance in chronic lymphocytic leukemia

The objective of this study was to further define the role of homologous recombinational repair (HRR) in resistance to the nitrogen mustards in B-cell chronic lymphocytic leukemia (B-CLL). We have demonstrated previously that increased chlorambucil (CLB)-induced HsRad51 nuclear foci formation correlated with a CLB-resistant phenotype in B-CLL lymphocytes. In this report, we measured the protein levels of HsRad51 and Xrcc3 (an HsRad51 paralog) and correlated them with the in vitro CLB cytotoxicity (LD(50)) in lymphocytes from seventeen B-CLL patients. Both HsRad51 (r=0.75, P=0.0005) and Xrcc3 (r=0.52, P=0.03) protein levels correlated with the in vitro CLB LD(50). In addition, multiple linear regression analysis showed a significant correlation between Xrcc3 and Rad51 protein levels versus the CLB LD(50) (r=0.78, P=0.0014), suggesting that both proteins influence CLB cytotoxicity. Moreover, since HsRad51 expression varies in cell lines during the cell cycle, we determined proliferating cell nuclear antigen (PCNA) protein levels to assess possible differences in cell cycle progression. There was no correlation between PCNA protein levels and the CLB LD(50) (r=0.042, P=0.87) or with HsRad51/Xrcc3 protein levels. Our data suggest that HsRad51 and Xrcc3 protein expression may be predictive of the response in B-CLL patients to treatment with nitrogen mustards.     

Contribution of apoptosis in the cytotoxicity of the oxaliplatin-irinotecan combination in the HT29 human colon adenocarcinoma cell line

Interactions between the topoisomerase I inhibitor irinotecan (CPT-11) and the platinum derivative oxaliplatin (L-OHP) were investigated in HT29 colon cancer cell line. Synergism was observed when cells were simultaneously exposed to drugs or when cells were first exposed to CPT-11. Flow cytometric studies showed a G(2)/M accumulation when cells were exposed to the simultaneous and CPT-11-->L-OHP combinations whereas a persistent S phase delay was observed when cells were first exposed to L-OHP. We characterised the cytotoxic effect by assessing the induction of apoptosis. Irinotecan induced substantial DEVDase activity and poly(ADP-ribose) polymerase cleavage while this activity was moderate and delayed after exposure to L-OHP. Combination experiments showed a sequence-dependent onset of apoptosis, the CPT-11-->L-OHP schedule being the earliest and the most effective; on the other hand the apoptotic signaling generated by CPT-11 was partly inhibited in the simultaneous combination and in the L-OHP-->CPT-11 sequence. Cell death studies using a dual staining technique showed a shift from apoptosis to necrosis when combining these drugs at high concentrations. Synergistic interactions observed using CPT-11 before L-OHP may be linked to an early apoptotic signaling while the L-OHP-induced S phase block could account for the observed additive effect in the reverse sequence. An additional phenomenon might work towards synergism for the simultaneous combination.     

Ability to repair DNA double-strand breaks related to cancer susceptibility and radiosensitivity

Traditional radiobiology has aimed at elucidating the mechanism of radiosensitivity of cancer cells and normal cells. Because the mechanism of DNA double-strand break (DSB) repair, which is inherently important to radiosensitivity, was unknown, it has been difficult to obtain results applicable to clinical radiotherapy from traditional radiobiology research. Today, however, the molecular mechanism of DNA DSB repair has been elucidated because of the rapid advances in molecular biology. In DNA DSB repair, at least two major repair mechanisms, homologous recombination and nonhomologous end joining (NHEJ) have been reported. In the NHEJ pathway, DSBs are directly, or after processing of the DNA ends, rejoined at an appropriate chromosomal end. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA DSB repair by NHEJ. We have investigated how the ability of repair of DNA DSB influences cancer susceptibility and the radiosensitivity of tumors and normal tissues by focusing on the activity of DNA-PK. In the near future, research on DNA DSB repair mechanism will be able to be applied to research on carcinogenesis, prediction of radiosensitivity of tumors and normal cells, and sensitization of tumor cells.     

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥ 3 μM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 μM PM. Numbers of oocytes with ≥ 5 γH2AX foci were significantly increased both 1 and 8 days after exposure to ≥ 1 μM PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥ 30 μM) and increased γH2AX foci (≥ 3 μM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.     

The association of DNA-dependent protein kinase activity of peripheral blood lymphocytes with prognosis of cancer

Background:

Repair of various types of DNA damages is critical for genomic stability. DNA-dependent protein kinase (DNA-PK) has an important role in DNA double-strand break repair. We examined whether there may be a correlation between DNA-PK activity in peripheral blood lymphocytes (PBLs) and survival percentages in various cancer patients. We also investigated the changes of DNA-PK activity in PBLs after radiotherapy.

Methods:

A total of 167 of untreated cancer patients participated in this study. Peripheral blood was collected, separated, and centrifuged. DNA-PK activity was measured by DNA-pull-down assay. Chromosomal aberrations were examined by cytogenetic methods.

Results:

DNA-PK activity of PBLs in advanced cancer patients was significantly lower than that in early stage. The patients with lower DNA-PK activity in PBLs tended to have the lower disease-specific survivals and distant metastasis-free survivals than those with higher DNA-PK activity in advanced stages. There was also a tendency of inverse correlation between DNA-PK activity and excess fragments. The DNA-PK activity of PBLs in most patients decreased in response to radiation as the equivalent whole-body dose increased.

Conclusion:

Cancer patients in advanced stage, with lower DNA-PK activity of PBLs might have higher distant metastasis and exhibit poorer prognosis. Therefore, DNA-PK activity in PBLs could be used as a marker to predict the chromosomal instability and poorer prognosis.     

DNA-dependent protein kinase: effect on DSB repair,G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2?Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1?h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24?h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4?Gy, but decreased both at higher doses of 20?Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.