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PubMed2002

Ku affects the ATM-dependent S phase checkpoint following ionizing radiation.

Following exposure to genotoxic stress, proliferating cells actively slow down DNA replication through an S phase checkpoint to provide time for repair. The ATM-dependent pathway plays an important role in the S phase checkpoint response following ionizing irradiation. We report that there is a stronger S phase checkpoint response in irradiated Ku80(-/-) cells as compared with their wild-type counterparts, which has no relationship to DNA-dependent protein kinase (DNA-PK) activity but correlates with a higher ATM activity and with more ATM bound to chromatin DNA in such cells. Wortmannin, a nonspecific inhibitor of ATM, not only reduces the higher activity of ATM kinase, but also abolishes the stronger S phase checkpoint response in Ku80(-/-) cells. Furthermore, a specific ATM antisense oligonucleotide abolishes the stronger S checkpoint response in Ku80(-/-) cells and renders these cells practically indistinguishable from Ku80(+/+) cells for this endpoint. These results in aggregate indicate that the stronger S checkpoint in irradiated Ku80(-/-) cells is due to the higher ATM kinase activity.

Androstadienes↗
PubMed2002

The radioprotective effect of the 24 kDa FGF-2 isoform in HeLa cells is related to an increased expression and activity of the DNA dependent protein kinase (DNA-PK) catalytic subunit.

We previously reported that overexpression of the 24 kDa basic fibroblast factor (or FGF-2) isoform provides protection from the cytotoxic effect of ionizing radiation (IR). DNA double-strand breaks (DSB), the IR-induced lethal lesions, are mainly repaired in human cells by non-homologous end joining system (NHEJ). NHEJ reaction is dependent on the DNA-PK holoenzyme (composed of a regulatory sub-unit, Ku, and a catalytic sub-unit, DNA-PKcs) that assembles at sites of DNA damage. We demonstrated here that the activity of DNA-PK was increased by twofold in two independent radioresistant cell lines, HeLa 3A and CAPAN A3, over expressing the 24 kDa FGF-2. This increase was associated with an overexpression of the DNA-PKcs without modification of Ku expression or activity. This overexpression was due to an up-regulation of the DNA-PKcs gene transcription by the 24 kDa FGF-2 isoform. Finally, HeLa 3A cells exhibited the hallmarks of phenotypic changes associated with the overexpression of an active DNA-PKcs. Indeed, a faster repair rate of DSB and sensitization to IR by wortmannin was observed in these cells. Our results represent the characterization of a new mechanism of control of DNA repair and radioresistance in human tumor cells dependent on the overproduction of the 24 kDa FGF-2 isoform.

Androstadienes↗
PubMed2003

Genetic analysis identifies putative tumor suppressor sites at 2q35-q36.1 and 2q36.3-q37.1 involved in cervical cancer progression.

We performed comparative genomic hybridization (CGH) and high-resolution deletion mapping of the long arm of chromosome 2 (2q) in invasive cervical carcinoma (CC). The CGH analyses on 52 CCs identified genetic losses at 2q33-q36, gain of 3q26-q29, and frequent chromosomal amplifications. Characterization of 2q deletions by loss of heterozygosity (LOH) in 60 primary tumors identified two sites of minimal deleted regions at 2q35-q36.1 and 2q36.3-q37.1. To delineate the stage at which these genetic alterations occur in CC progression, we analysed 33 cervical intraepithelial neoplasia (CIN) for LOH. We found that 89% of high-grade (CINII and CINIII) and 40% of low-grade (CINI) CINs exhibited LOH at 2q. To identify the target tumor suppressor gene (TSG), we performed an extensive genetic and epigenetic analyses of a number of candidate genes mapped to the deleted regions. We did not find inactivating mutations in CASP10, BARD1, XRCC5, or PPP1R7 genes mapped to the deleted regions. However, we did find evidence of downregulated gene expression in CFLAR, CASP10 and PPP1R7 in CC cell lines. We also found reactivated gene expression in CC cell lines in vitro after exposure to demethylating and histone deacetylase (HDAC) inhibiting agents. Thus, these data identify frequent chromosomal amplifications in CC, and sites of TSGs at 2q35-q36.1 and 2q36.3-q37.1 that are critical in CC development.

Antigens, Nuclear↗
PubMed2003

Multiple molecular mechanisms contribute to radiation sensitivity in mantle cell lymphoma.

Mantle cell lymphomas (MCL) are characterized by their aggressive behavior and poor response to chemotherapy regimens. We report here evidence of increased in vitro radiation sensitivity in two cell lines that we have generated from two MCL patients (UPN1 and UPN2). However, despite their increased radiation sensitivity, UPN2 cells were totally resistant to apoptotic cell death, whereas UPN1 cells underwent massive apoptosis 6 h after irradiation. The frequency of induced chromosomal abnormalities was higher in UPN1 as compared to UPN2. Distinct mechanisms have been found to contribute to this phenotype: a major telomere shortening (UPN1 and UPN2), deletion of one ATM allele and a point mutation in the remaining allele in UPN2, mutation of p53 gene (UPN1 and UPN2) with absence of functional p53 as revealed by functional yeast assays. After irradiation, Ku70 levels in UPN1 increased and decreased in UPN2, whereas in the same conditions, DNA-PKcs protein levels decreased in UPN1 and remained unchanged in UPN2. Thus, irradiation-induced apoptotic cell death can occur despite the nonfunctional status of p53 (UPN1), suggesting activation of a unique pathway in MCL cells for the induction of this event. Overall, our study demonstrates that MCL cells show increased radiation sensitivity, which can be the result of distinct molecular events. These findings could clinically be exploited to increase the dismal response rates of MCL patients to the current chemotherapy regimens.

Antigens, Nuclear↗
PubMed2004

HIV-1 Tat increases cell survival in response to cisplatin by stimulating Rad51 gene expression.

Tat is an early regulatory protein of human immunodeficiency virus type 1, which plays a central role in the pathogenesis of AIDS by stimulating transcription of the viral genome and impairing several important cellular pathways during the progression of the disease. Here, we investigated the effect of Tat on cell response to DNA damage. Our results indicate that Tat production causes a noticeable increase in the survival rate of PC12 cells upon their treatment with genotoxic agents. Single-cell gel electrophoresis studies revealed reduced DNA breakage in PC12-Tat cells upon cisplatin treatment relative to the control cells. Furthermore, cytogenetic data exhibited less chromosomal damage in Tat-producing cells after recovery from cisplatin treatment, corroborating electrophoretic data. Examination of several proteins involved in the control of DNA repair showed elevated levels of Rad51, a key regulator of homologous recombination in cells expressing Tat. On the other hand, the level of Ku70, one of the components of the nonhomologous end-joining repair pathway, was slightly decreased in cells expressing Tat. Using a fluorescence-based assay, we demonstrated that repair of DNA double-strand breaks via homologous recombination is increased in Tat-producing cells. The results from in vitro nonhomologous end-joining assay revealed a reduced ability of protein extract from PC12-Tat cells compared to PC12 cells in rejoining linearized DNA. These observations ascribe a new role for Tat in host genomic integrity, perhaps by affecting the expression of genes involved in DNA repair.

Animals↗
PubMed2006

Ku80 and p53 suppress medulloblastoma that arise independent of Rag-1-induced DSBs.

Ku80 maintains the genome by repairing DNA double-strand breaks (DSBs) through nonhomologous end joining (NHEJ), a pathway that repairs nonspecific DSBs and Rag-1 Rag-2 (Rag)-specific DSBs. As a result, Ku80 deletion results in phenotypes characteristic of defective repair for both nonspecific DSBs (gamma-radiation hypersensitivity and genomic instability) and Rag-specific DSBs (immunodeficiency). ku80(-/-) mice also exhibit neuronal apoptosis, but we do not know the type of DSBs responsible for this response. In spite of genomic instability and immunodeficiency, cancer incidence is not increased in ku80(-/-) mice. However, deletion of the tumor suppressor, p53 greatly increases pro-B-cell lymphoma in ku80(-/-) mice due to IgH/c-Myc translocations suggesting that responses to Rag-specific DNA DSBs suppress cancer. Like suppression of pro-B-cell lymphoma, neuronal apoptosis requires p53 presenting the intriguing possibility that Rag-specific DSBs mediate neuronal development as they do lymphocyte development. Here we delete Rag-1 from ku80(-/-)p53(-/-) mice to differentiate the impact nonspecific vs Rag-specific DSBs have on ku80(-/-) mice. We find that deleting Rag-1 prevents pro-B cell lymphoma confirming Rag-induced DSBs induce this form of cancer. Both the triple mutant mice and the p53(-/-)rag-1(-/-) mice exhibit T-cell lymphoma and medulloblastoma; incidence of T-cell lymphoma is the same for both cohorts whereas incidence of medulloblastoma is higher for the triple-mutant cohort. Thus, p53-mediated neuronal apoptosis likely suppresses medulloblastoma in Ku80-deleted mice and Ku80 likely suppresses medulloblastoma by repairing nonspecific DNA DSBs instead of Rag-specific DSBs. Our observations are the first to show that Ku80 suppresses cancer caused by nonspecific DNA damage and we present a novel mouse model for medulloblastoma.

Animals↗
PubMed2006

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism.

Ionizing radiation exposure results in the activation of several tyrosine kinase receptors that participate in radiation-induced DNA damage response and radioresistance. We previously showed that insulin-like growth factor 1 receptor (IGF-1R) inhibition enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) cells. In this paper, we demonstrate that in U1810 NSCLC cells gamma-radiation activates IGF-1R within 10 min, with a maximal activation effect 2 h post-irradiation. Impairment of IGF-1R tyrosine kinase activity enhances human lung cancer cells radiosensitivity by a mechanism that involves phosphatidylinositol 3-kinase (PI3-K) and p38 kinase. In an active form, IGF-1R binds and activates p38 kinase, promoting receptor signaling. Conversely, inhibition of IGF-1R phosphorylation results in IGF-1R/p38 complex disruption and p38 kinase inactivation. We have also demonstrated that in insulin-like growth factor-1-stimulated cells, Ku-DNA-binding activation is induced by ionizing radiation within 4 h, reaches a maximum level at 12 h and remains active up to 72 h. Blockade of IGF-1R activity or its downstream signaling through p38 kinase induces a decrease in radiation-mediated Ku-DNA-binding activation and downregulates the level of Ku86, without affecting Ku70 expression in the nucleus of U1810 cells. The IGF-1R signaling via PI3-K does not interfere with the p38 signaling, the Ku-DNA-binding activity or the level of Ku86. Our present study demonstrates for the first time that ionizing radiation activates IGF-1R. Inhibition of IGF-1R signaling via p38 kinase induces radiosensitivity by a novel mechanism involving nuclear Ku86.

Antigens, Nuclear↗
PubMed2001

Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells.

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5' distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

Antigens, Nuclear↗
PubMed2006

Effect of Ku proteins on IRES-mediated translation.

BACKGROUND INFORMATION: Ku is an abundant nuclear heterodimeric protein composed of 70 and 86 kDa subunits. As an activator of the catalytic subunit of DNA-PK (DNA-dependent protein kinase), Ku plays an important role in DNA repair and recombination. Ku is also involved in actions independent of DNA-PK, such as transcription regulation and telomere maintenance. Although Ku is localized in the cytoplasm under specific cellular conditions, no functions for Ku outside of the nucleus have as yet been reported. In addition to DNA binding, Ku binds specific RNA sequences with high affinity. However, no specific cellular mRNA targets for Ku have been identified. RESULTS: In a yeast three-hybrid system, Ku70 bound to an RNA bait that contained an IRES (internal ribosomal entry site) element. A single band with migration properties similar to those of Ku70 was immunoprecipitated with anti-Ku antibody, using UV cross-linked complexes formed by HeLa cell nuclear extracts and an IRES-containing RNA probe. IRES activity was reduced in Ku80(-/-) cells. Overexpression of Ku proteins stimulated IRES-dependent translation. CONCLUSIONS: The present study suggests that Ku binds IRES elements within RNA molecules, and that Ku plays a role in the modulation of IRES-mediated mRNA translation.

Animals↗
PubMed1999

The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes.

Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining. The assembly of Ku and DNA-dependent protein kinase at DNA ends is now understood in greater detail. Murine genetic knockouts for DNA ligase IV and XRCC4 are embryonic lethal, indicating that nonhomologous end joining is essential for viability. Interestingly, neurones, in addition to lymphocytes, are particularly vulnerable to an absence of NHEJ.

Aging↗
PubMed2001

Ku protein and DNA strand breaks in lip glands of normal and primary Sjögren's syndrome subjects: lack of correlation with apoptosis.

UNLABELLED: The aim was to examine tissue expression of Ku protein in lower lip salivary gland (LSG) biopsies from cases of primary Sjögren's syndrome (SS) and from normal subjects. METHODS: immunohistochemistry was used with antibodies to Ku70/86 and also Ki67, PCNA and p53. In addition, the Klenow method was applied in order to detect evidence of apoptosis. Sections of hyperplastic tonsil served as additional controls. RESULTS: in normal controls, LSG acinar cells stained negatively whereas LSG excretory duct cell nuclei stained positively with Ku and Klenow and occasionally with PCNA but negatively with Ki67 and p53. In LSG focal sialadenitis of SS cases, some lymphocytic cells showed staining with Ku, Ki67, PCNA, Klenow and p53. In addition to duct cell Ku and Klenow as well as PCNA staining which was not much different from normals, a few ductal epithelial and also mononuclear cells stained with p53. In focal sialadenitis, some acinar cells showed staining with PCNA as well as with Klenow. CONCLUSIONS: our findings in LSG biopsies of SS cases added little to an increased understanding about the pathogenetic mechanisms in the development of focal sialadenitis in SS. However, in normal LSG, ductal epithelial but not acinar cells seem to express a constitutively specific Ku protein and Klenow profile, suggestive of DNA strand breaks but not clearly associated with ongoing apoptotic events. It may reflect an enhanced stress response, which may be pathogenetically important in the early events of focal sialadenitis development in primary Sjögren's syndrome.

Antibody Specificity↗
PubMed1998

Rejoining of DNA double-strand breaks in vitro by single-strand annealing.

Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error-prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End-joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single-strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase epsilon, FEN-1 endonuclease, and exonuclease activities of 5'-3' and 3'-5' directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA-dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error-prone NHEJ pathway that creates deletions by microhomology-driven SSA.

Animals↗
PubMed2003

Differential modulation of Ku70/80 DNA-binding activity in a patient with multiple basal cell carcinomas.

Ku70/80 nonhomologous end-joining activity is essential for resolving random DNA double-strand breaks, and the Ku70/80 protein complex has been proposed as "caretaker" of genomic stability. We studied the Ku70/80 heterodimer activity in a patient affected by multiple basal cell carcinomas with a personal history of moderate exposure to ionizing radiation. The Ku70/80 DNA-binding activity was analyzed, by electrophoretic mobility shift assay, in five tumor biopsies from different sites and at distinct clinical stages, and in three matched normal skin samples from the same patient. As control normal tissues from healthy individuals were also tested. The five basal cell carcinomas were classified as "non aggressive" and "aggressive" on the basis of morphologic parameters and expression of the molecular markers bcl-2, Ki67/MIB1, and p53. A 62% increase in the Ku70/80 DNA-binding activity was found in normal skin from the patient, compared to unexposed individuals (p<0.0001). The nuclear activity of the heterodimer was further increased in nonaggressive basal cell carcinomas compared to both matched normal skin from the patient (31%, p=0.0001) and tissues from healthy controls (73%, p=0.0001). Strikingly, the two aggressive basal cell carcinomas tested showed very low Ku70/80 DNA-binding activity with a reduction of 87% compared to normal skin from the patient (p<0.0001) and 64% compared to controls (p=0.001). Although these results are limited to only one patient, together with other recent studies they support the hypothesis that downregulation of the nonhomologous end-joining pathway may be associated with tumor progression.

Aged↗
PubMed2000

Expression of Ku70 correlates with survival in carcinoma of the cervix.

Cervical carcinoma affects around 3400 women in the UK each year and advanced disease is routinely treated with radiation. As part of a programme to establish rapid and convenient methods of predicting tumour and patient responses to radiotherapy, we have examined the relationship between the pre-treatment expression of the Ku components of the DNA damage recognition complex DNA-PK and patient survival in cervical carcinoma. Using immunohistochemistry of formalin-fixed sections of tumour biopsies, antibodies to Ku70 and Ku80 stained identical regions of tumour and there was a high degree of correlation between the mean number of cells stained positive for the two components in 77 tumours (r = 0.82, P<0.001). In 53 tumours there was a borderline significant correlation between measurements of tumour radiosensitivity (surviving fraction at 2 gray: SF2) and Ku70 expression (r = 0.26, P = 0.057) and no correlation for Ku80 (r = 0.18, P = 0.19). However, all tumours with a low number of Ku70 or Ku80 positive cells were radiosensitive. Furthermore, using log-rank analysis there was significantly higher survival in the patients whose tumours had a low Ku70 expression (P = 0.046). This difference was also reflected with Ku80, but did not reach statistical significance (P = 0.087). The study suggests that lack of Ku protein leads to radiosensitivity in some tumours and that other factors are responsible for radiosensitive tumours with high Ku expression. It is likely that the most accurate prediction of treatment outcome will lie in assessing the expression of several proteins involved in the recognition and repair of DNA damage, one of which will be Ku.

Antigens, Nuclear↗
PubMed2002

Ku86 is essential in human somatic cells.

Ku86 plays a key role in nonhomologous end joining in mammals. Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability. In contrast, no human patient has been described with mutations in either Ku86 or Ku70. This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans. To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus. Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation. Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time. These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis. These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.

Antigens, Nuclear↗
PubMed2005

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1.

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.

Animals↗