Loss of Ku70/Ku80 expression occurs more frequently in hereditary than in sporadic colorectal tumors. Tissue microarray study

Ku70 and Ku80 proteins take part in the repairing of DNA double-strand breaks by their function as a regulatory subunit of the DNA-dependent protein kinase. In this study, expression of both genes was analyzed in colorectal carcinoma tissue arrays applying immunohistochemistry. Expression of both genes decreased along with pT stage. Significant differences in Ku70 and Ku80 expression were found between pT3 and pT4 as well as between pT2 and pT3 tumors, respectively. Loss of Ku70/Ku80 expression was more frequently observed in hereditary than in sporadic tumors. We conclude that expression of Ku70/Ku80 genes is down-regulated in colorectal carcinoma and that defects of these genes are more frequently observed in hereditary than in sporadic tumors.     

Expression and heterodimer-binding activity of Ku70 and Ku80 in human non-melanoma skin cancer

BACKGROUND: Experimental data suggest that exposure to ultraviolet radiation may indirectly induce DNA double-strand breaks. AIM: To investigate the contribution of the non-homologous end-joining repair pathway in basal and squamous cell carcinomas. METHODS: Levels of Ku70 and Ku80 proteins were determined by immunohistochemical analysis and Ku70-Ku80 heterodimer-binding activity by electrophoretic mobility shift assay. Matched pathological normal margins and skin from healthy people were used as controls. RESULTS: A significant increase in Ku70 and Ku80 protein levels was found for both tumour types as compared with normal skin (p<0.001). Squamous cell carcinoma showed increased immunostaining as compared with basal cell tumours (p<0.02). A direct correlation was found between Ku70 and Ku80 protein levels and expression of the proliferation markers Ki-67/MIB-1 (p<0.02 and p<0.002, respectively) in basal cell carcinoma. DNA binding activity was increased in basal cell carcinoma samples as compared with matched skin histopathologically negative for cancer (p<0.006). In squamous cell carcinomas, however, the difference was significant only with normal skin (p<0.02) and not with matched pathologically normal margins. CONCLUSIONS: Overall, an up regulation of the Ku70 and Ku80 protein levels seems to correlate only with tumour proliferation rate. As non-homologous end joining is an error-prone mechanism, its up regulation may ultimately increase genomic instability, contributing to tumour progression.     

硝普钠及SOD降低大鼠心肌细胞DNA蛋白激酶二聚体和Ku70/80表达

目的观察硝普钠(SNP)及自由基清除剂超氧化物歧化酶(SOD)、过氧化氢酶(CAT)对大鼠心肌细胞H9C2和心肌组织DNA-PKcs和Ku70/80表达的影响。方法体外培养H9C2心肌细胞按SNP浓度和加入的自由基清除剂SOD和CAT浓度分组;SD大鼠随机分为5组,每组8只,分别采用0.9%氯化钠溶液、SNP、SNP+SOD、SNP+CAT和SNP+SOD+CAT行腹腔注射,1周后收集心肌组织。用Western blot法检测各组细胞DNA-PKcs和Ku70/80表达,免疫组织化学法检测DNA-PKcs和Ku70/80表达。结果心肌细胞的DNA-PKcs和Ku70/80蛋白相对表达量均随SNP剂量增加呈递增趋势;加入SOD或CAT或二者合用,均显著降低DNA-PKcs和Ku70/80表达(P0.05)。SNP组、SNP+SOD组、SNP+CAT组和SNP+SOD+CAT组大鼠心肌组织DNA-PKcs和Ku70/80表达水平均显著高于对照组(P0.05)。结论自由基清除剂SOD和CAT能够通过降低心肌细胞DNA-PKcs和Ku70/80的表达拮抗NO对心肌细胞的损伤。     

Characterization of Ku-deficient cells derived from Ku70-/- and Ku80-/- mice

ＴｏｄｅｔｅｒｍｉｎｅｔｈｅｐｈｙｓｉｏｌｏｇｉｃａｌｒｏｌｅｓｏｆＫｕ70ａｎｄＫｕ80 ,ｗｅｇｅｎｅｒａｔｅｄＫｕ70 - /-ａｎｄＫｕ80 - /-ｋｎｏｃｋｏｕｔｍｉｃｅｖｉａｔａｒｇｅｔｅｄｇｅｎｅｄｉｓｒｕｐｔｉｏｎ (Ｎｕｓｓｅｎｚｗｅｉｇｅｔａｌ Ｎａｔｕｒｅ 382 ,1 996;Ｏｕｙａｎｇｅｔａｌ ＪＥｘｐＭｅｄ 1 86,1 997) ＴｈｅｓｅｍｕｔａｎｔｍｉｃｅｓｈｏｗｐｒｏｆｏｕｎｄｄｅｆｉｃｉｅｎｃｙｉｎＶ(Ｄ)Ｊｒｅｃｏｍｂｉｎａｔｉｏｎ ,ｉｍｐａｉｒｅｄｌｙｍｐｈｏｃｙｔｅｄｅｖｅｌｏｐｍｅｎｔ,ａｎｄｉ…     

Polymorphisms in nonhomologous end-joining genes associated with breast cancer risk and chromosomal radiosensitivity

As enhanced chromosomal radiosensitivity (CRS) results from non- or misrepaired double strand breaks (DSBs) and is a hallmark for breast cancer and single nucleotide polymorphisms (SNPs) in DSB repair genes, such as non homologous end-joining (NHEJ) genes, could be involved in CRS and genetic predisposition to breast cancer. In this study, we investigated the association of five SNPs in three different NHEJ genes with breast cancer in a population-based case-control setting. The total patient population composed of a selected group of patients with a family history of the disease and an unselected group, consisting mainly of sporadic cases. SNP analysis showed that the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] has a significant, positive odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30-6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the selected patient group. For the c.-1310 C>G SNP (XRCC6Ku70)[corrected] a significant OR of 1.85 (95%CI: 1.01-3.41) was found for the He genotype in the unselected patient group. On the contrary, the HV genotype of c.1781G>T (XRCC6Ku70) [corrected] displays a significant, negative OR of 0.43 (95%CI: 0.18-0.99) in the total patient population. The He+HV genotypes of the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] also showed high and significant ORs in the group of "radiosensitive," familial breast cancer patients. In conclusion, our results provide preliminary evidence that the variant allele of c.-1310C>G (XRCC6Ku70) [corrected]and c.2099-2408G>A (XRCC5Ku80) [corrected] are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC6Ku70) [corrected] on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.     

反义Ku80在人肺癌细胞的表达及对放射线的敏感性研究

目的 :研究反义Ku80在人肺癌细胞的表达及对放射线的超敏效应。方法 :采用Western blotting法证明 ,Ku80 LCA中Ku80蛋白的表达情况 ,采用体外放射线敏感实验证明 ,反义Ku80在人肺癌细胞的表达及对放射线的超敏效应 ,用细胞存活克隆率和剂量效应曲线表示。结果 :(1)对照组和导入正义Ku80的LCA细胞可见Ku80蛋白表达印迹 ,导入反义Ku80的LCA细胞未见Ku80蛋白表达印迹。 (2 )导入反义Ku80的LCA细胞对放射线的敏感性明显增强 (P <0 0 1) ,并呈剂量依赖性。结论 :反义Ku80封闭了肿瘤细胞的Ku80蛋白表达后 ,对放射线呈超敏效应 ,为提高肿瘤放疗的特异性和靶向基因治疗奠定了基础。     

Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis

Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.     

Ku80分子克隆在人肺癌细胞的表达及生物学意义

目的：研究Ku80基因分子克隆在人肺癌细胞的表达及生物学意义。方法：用分子克隆的方法构建重组的正义,反义Ku80基因表达质粒,导入人肺癌细胞后,用Northern-blotting法和Western-blotting法证明Ku80正义,反义基因在mRNA和蛋白水平的表达情况。结果：（1）正义或反义Ku80克隆阳性的LCA细胞在mRNA水平均有正义或反义Ku80的表达印迹。（2）仅在正义Ku80克隆阳性的LCA细胞有正义Ku80蛋白表达印迹而反义Ku80克隆阳性的LCA细胞无Ku80蛋白表达印迹,结论：正义或反义Ku80基因的分子克隆可在人肺癌细胞的mRNA得到表达,正义Ku80克隆阳性的LCA细胞有正义Ku80蛋白表达,反义Ku80基因可封闭肿瘤细胞的正义Ku80蛋白的表达,此结论为将Ku基因作为肿瘤靶向基因治疗提供了实验依据。     

Downregulation of the retinoblastoma gene expression in the progression of malignant melanoma.

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.     

反义Ku70在人肺癌细胞的表达及对放射线的敏感性研究

目的：研究反义Ku70在人肺癌细胞的表达及对放射线的超敏效应。方法：采用Western-boltting法证明,Ku70－LCA中Ku70蛋白的表达情况,采用体外放线敏感实验症明,反义Ku70在人肺癌细胞的表达及对放射线的超敏效就,其结果以细胞存活克隆率和剂量效应曲线表示,结果：（1）对照组和人正义Ku70基因的LCA组细胞可见Ku70蛋白表达印迹,导入反义Ku70基因LCA组细胞未见Ku70蛋白表达印迹,（2）导入反义Ku70基因的LCA组细胞对放线的敏感性明显增强（P＜0．01）,并呈剂量依赖性,结论：反义Ku70封闭了肿瘤细胞的正义Ku70蛋白表达后,对放坶呈超敏感效应,为提高肿瘤放疗的特异性的靶向基因治疗奠定了基础。     

人非小细胞肺癌组织中Ku80 mRNA表达评价化学治疗敏感性的研究

目的：通过观察Ku80 mRNA在人正常肺组织、未经化疗和多次化疗的非小细胞肺癌组织的定量表达情况，探讨Ku80是否可以预测和评价肺癌化学治疗的敏感性。方法：采用RT-PCR方法，以B-actin为内参照，检测、比较25例正常肺组织和51例肺癌患者肺组织中Ku80 mRNA的半定量表达，以SPSS统计软件进行分析。结果：比较人正常肺组织组与未经化疗肺癌组和多次化疗肺癌组Ku80 mRNA表达量的差别具有统计学意义，分别为P〈0．05和P〈0．01。未经化疗肺癌组与多次化疗肺癌组（n≥2）的Ku80 mRNA的表达量相比具有统计学意义（P〈0．05）。结论：正常肺组织和肺癌组织在Ku80 mRNA表达量上具有统计学差异，并且Ku80 mRNA表达量在肺癌组织中随化疗次数增加明显上升，提示Ku80 mRNA定量表达可能成为对肺癌化疗的敏感性的预测和评价提供新的依据。     

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.     

Cell adhesion and communication proteins are differentially expressed in melanoma progression model

Cutaneous melanoma is an aggressive cancer derived from skin melanocytes. Tissue microarrays are being used to evaluate the roles of numerous proteins implicated in some of the pathways involved in melanoma pathogenesis. Based on a previous study using a complementary DNA microarray platform, the aim of this study was to evaluate the immunohistochemical expression of the adhesion and communication molecules connexin 43, desmocollin 3, cytokeratin 5, kallikrein 6, and kallikrein 7 in a melanoma progression model. We analyzed 59 common nevi, 22 atypical nevi, and 162 invasive and 29 metastatic melanomas on tissue microarrays using digital microscopy. The expression of desmocollin 3 and connexin 43 was higher in melanomas (P < .001). Kallikrein 6 expression was higher in melanomas than in common nevi (P < .006). The expression of cytokeratin 5 and kallikrein 7 was higher in atypical nevi than in melanomas (P < .001) and was higher in melanomas than in common nevi (P < .001). The expression of desmocollin 3 and connexin 43 in melanomas indicates loss of cell-cell interactions, which starts in the early steps of the melanoma progression model. Keratin expression in melanomas may play a particular role during melanocyte development. The expression of kallikrein 7 and kallikrein 6 in melanomas may be responsible for the loss of cell-cell adhesion.     

Distinct MHC gene expression patterns during progression of melanoma

Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas. © 2009 Wiley‐Liss, Inc.     

DNA repair and replication proteins as prognostic markers in melanoma

Aims: Elevated expression of DNA repair and replication genes has been reported in thick, non‐fixed primary melanomas that subsequently went on to metastasize, when compared to non‐recurrent primary tumours. This increased expression could contribute to the extreme resistance shown by melanoma to DNA‐damaging chemotherapeutics. We have investigated the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in melanoma. Methods and results: We used a tissue microarray containing samples from all stages of melanomagenesis to investigate the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in a larger, more representative and readily available set of fixed primary melanomas. High expression of topoisomerase IIα (TOP2A), that relieves torsional stress during DNA replication, and XRCC5 (Ku80), required for DNA double‐strand break repair, were associated with significantly worse survival. Conclusions: Two (XRCC5 and TOP2A) of seven DNA repair and replication proteins studied were prognostic for melanoma.     

Ku70 mRNA表达与人非小细胞肺癌化学治疗耐受性关系的研究

目的：分析Ku70 mRNA在人正常肺组织、未经化疗和多次化疗的非小细胞肺癌组织的定量表达水平与化学治疗耐受性的关系。方法：采用RT-PCR方法对26例正常肺组织和56例肺癌组织中的Ku70 mRNA水平进行半定量测定,分析Ku70 mRNA表达水平与化学治疗耐受性关系。结果：肺癌组织中Ku70 mRNA表达水平明显高于正常肺组织（P〈0.01）;经多次化疗的肺癌组织中Ku70 mRNA的表达水平明显高于未经化疗的肺癌组织（P〈0.01）。结论：随化疗次数增加,Ku70 mRNA在肺癌组织中表达量的明显增高可能与人肺癌组织对化学治疗耐受性相关。