In vitro chemiluminescence assay to measure excision repair in cell extracts.
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Animals↗
Biomedical subjects
B Salles
Publications and source records attributed to B Salles.
[DNA-dependent protein kinase: a major protein involved in the cellular response to ionizing radiation].
DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a regulatory sub-unit, the heterodimeric Ku protein (composed of a 70- and a 86-kDa subunit) which binds DNA ends and targets the catalytic sub-unit, DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks induced in DNA after exposure to ionizing radiation as shown by the extreme radiosensitivity of cells with mutations in Ku86, Ku70 or DNA-PKcs genes. Cells deficient in DNA-PK activity also exhibit hypersensitivity to genotoxic drugs such as cisplatin and nitrogen mustards. In the first part of this review, the current knowledge on the biochemical characteristics of DNA-PK, its mechanism of action in DNA repair and the phenotype of DNA-PK deficient cells is summarized. These results suggest that DNA-PK might play a role in the acquisition of a resistant phenotype of human tumors to radiotherapy, chemotherapy using genotoxic drugs or to both treatments. In the second part of this review, the studies devoted to inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.
DNA Damage↗
Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.
We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).
Animals↗
Decreased expression of topoisomerase IIbeta in poly(ADP-ribose) polymerase-deficient cells.
Recent studies with poly(ADP-ribose) polymerase (PARP)-deficient mice have highlighted the role of this enzyme in genomic stability and response to various genomic insults. In the absence of DNA damaging treatment, we report here that a PARP-deficient cell line (PARP-/-) established from knockout mice displays a decrease in topoisomerase II (topo II) activity as measured by decatenation of kinetoplast DNA. Immunoblotting of whole and nuclear cell extracts showed that reduced activity was associated with decreased amount of the 180 kDa topo IIbeta protein but not of the 170 kDa topo IIalpha. The decreased topo IIbeta expression did not stem from transcriptional regulation of gene expression since levels of topo IIbeta mRNA were similar in PARP (-/-) compared with the parental PARP (+/+) cells. The decreased topo II activity was associated with cell resistance to VP16, a topo II inhibitor. These observations indicate that PARP may play a role in the stabilization and/or distribution of topo IIbeta.
Animals↗
Overexpression of DNA polymerase beta in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs.
DNA polymerase beta (pol beta) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol beta cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol beta. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol beta overexpression in cancer predisposition and tumor progression during chemotherapy.
Animals↗
DNA-Dependent protein kinase activity correlates with clinical and in vitro sensitivity of chronic lymphocytic leukemia lymphocytes to nitrogen mustards.
The objective of this study is to investigate the role of DNA-dependent protein kinase (DNA-PK) in the chronic lymphocytic leukemia (CLL) lymphocyte response to nitrogen mustard therapy. DNA-PK is a nuclear serine/threonine kinase that functions in DNA double-strand break repair and in the joining process in recombination mechanisms. In a series of 34 patients with B-CLL, either untreated (n = 16) or resistant to chlorambucil (n = 18), the kinase activity of the complex, as determined by its capacity to phosphorylate a peptide substrate in vitro, is increased in the resistant samples as compared with the untreated ones (24.4 +/- 2.6 arbitrary units [a.u.] [range, 12.7 to 55.8 a.u.] versus 8.1 +/- 2.8 a.u. [range, 0.9 to 44.5 a.u.], respectively (P < .0001]), independent of other clinical and biological factors. Linear regression analysis shows an excellent correlation between the level of DNA-PK activity and the inherent in vitro sensitivity of CLL lymphocytes to chlorambucil (r = .875, P =.0001). The regulation of DNA-PK activity was associated with increased DNA-binding activity of its regulatory subunit, the Ku heterodimer, in resistant samples. These results suggest that this activity is a determinant in the cellular response to chlorambucil and participates in the development of nitrogen mustard-resistant disease. The increase in DNA-PK activity might contribute to the enhanced cross-link repair that we previously postulated to be a primary mechanism of resistance to nitrogen mustards in CLL.
Aged↗
Interactions of the transcription/DNA repair factor TFIIH and XP repair proteins with DNA lesions in a cell-free repair assay.
We have studied the interactions between DNA damage and human proteins involved in the early steps of nucleotide excision repair (NER) reaction under in vitro conditions with human protein extracts. By using a new assay, we have detected a long-lived DNA/protein complex involving XPA and TFIIH in the course of the NER process. The formation of this complex is exclusively limited to DNA lesions that are substrates of the human excinuclease. We show that, while XPA binding to damaged DNA is ATP-independent, stable association of TFIIH with DNA lesions is promoted by ATP hydrolysis and is dependent on the integrity of XPA and XPC proteins in the cell extract. In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA. Finally, the co-binding of XPA and TFIIH to DNA damage is correlated to a dose-dependent titration of TFIIH and not XPA from the free protein fraction.
Adenosine Triphosphate↗
Potentiation of chlorambucil cytotoxicity in B-cell chronic lymphocytic leukemia by inhibition of DNA-dependent protein kinase activity using wortmannin.
In this study, we examined the ability of wortmannin to modulate chlorambucil (CLB) cytotoxicity in lymphocyte samples from patients with B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested previously that enhanced cross-link repair is a primary mechanism of resistance to nitrogen mustards (NMs) in B-CLL. DNA-dependent protein kinase (DNA-PK) is involved in the repair of double-strand breaks and in rejoining steps in recombination mechanisms. Mutants defective in this process are hypersensitive to alkylating agents. We have recently demonstrated that the activity of DNA-PK is a determinant in the cellular response of B-CLL to CLB. The DNA-PK gene has homology to the P110 phosphatidylinositol 3-kinase (PI 3-K). Wortmannin, an inhibitor of P110 PI 3-K, also inhibits DNA-PK activity in vitro. We investigated the effect of wortmannin on DNA-PK activity and CLB toxicity in the lymphocytes from 11 patients with B-CLL. Our results demonstrate that DNA-PK activity is decreased after exposure to wortmannin in a dose-dependent manner. Wortmannin, at nontoxic concentrations, synergistically sensitized B-CLL lymphocytes to the effects of CLB. Moreover, we observed a significant correlation when we compared the fold decrease in DNA-PK activity and the synergistic value (I), obtained when wortmannin was used at 0.1 microM. In the resistant B-CLL lymphocyte samples, there was a highly significant correlation between the ability of wortmannin at 0.1 and 0.25 microM to decrease the level of DNA-PK activity and to increase CLB sensitivity. In a model of primary human tumor cells, our findings suggest that the inhibition of DNA-PK activity may be a powerful way to overcome resistance to NMs such as CLB and point to new possibilities to improve the effectiveness of NM therapy.
Androstadienes↗
Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein.
The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK[CS]), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts. The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population. Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells. These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.
Antigens, Nuclear↗
UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.
DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion.
Androstadienes↗
TATA binding protein discriminates between different lesions on DNA, resulting in a transcription decrease.
DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position -29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage.
Adenoviridae↗
Regulation of DNA-dependent protein kinase activity in leukemic cells.
The DNA-dependent protein kinase (DNA-PK) complex is composed of a catalytic (DNA-PKcs), and a regulatory subunit (Ku70/Ku86 heterodimer). The expression and function of DNA-PK subunits was investigated in purified blood lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL) either refractory to chemotherapy or untreated. Variations in DNA-PK activity were found amongst CLL samples by comparison to human cell lines. It was noticeable that the low DNA-PK activity was associated with samples from untreated patients that exhibited a sensitivity phenotype, determined in vitro, to the radiomimetic agent neocarcinostatin by comparison to samples from refractory patients. The regulation in DNA-PK activity was associated with Ku heterodimer expression while DNA-PKcs was unaffected. Moreover, the presence of an altered form of the Ku86 subunit was identified in samples with low DNA-PK activity. These results suggest a regulation process of the DNA-PK activity in fresh human cells.
Aged↗
A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype.
The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.
Cell Cycle↗
DNA repair activity in protein extracts from rat tissues.
Among DNA repair pathways, nucleotide excision repair (NER) is able to recognize and process a wide variety of DNA lesions. The NER mechanism can be summarized in two stages: incision/excision of the lesion and DNA repair synthesis. Here, we have assessed the repair synthesis activity of protein extracts from different rat tissues by an in vitro biochemical assay that reproduces the entire NER reaction. The protein extraction procedure was adapted to rat tissues and the biochemical parameters of the assay (high salt concentration, addition of EGTA) in order to minimize non-specific nuclease activity which allows the measurement of repair activity. Using this repair assay we detected a small increase in the extent of repair synthesis in liver compared to brain and lung tissue protein extracts. Similar results were obtained using a derivative assay that allows the measurement of the incision activity of tissue protein extracts with lower incision activity in lung tissue extract.
Animals↗
Deficient apoptotic process in cisplatin-resistant L1210 cells cannot account for the cellular response to various drug treatments.
Apoptosis is a major determinant of the effectiveness of antitumor chemotherapy since most of the drugs used in cancer treatment provoke cell death by this process. We selected L1210/0.7R (7-fold) and L1210/3R (16-fold) murine leukemia cells resistant to cisplatin (CDDP) by adaptation of parental L1210/S cells to increasing drug concentration. L1210/0.7R exhibited a decreased apoptosis response to CDDP compared to parental L1210/S, while it was totally defective in L1210/3R as analyzed by cell morphology, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. This default in apoptosis did not result from differential expression of the antiapoptotic protein bcl-2 or from altered expression of p53. L1210/3R was resistant to other cross-linking agents and sensitive to topoisomerase II inhibitors and microtubule poisons. Whatever the drug sensitivity phenotype to these agents, L1210/3R was totally defective in apoptosis in response to drug treatment, showing that apoptosis control cannot be directly involved in the resistance process of these cell lines.
Animals↗
Evaluation of DNA repair inhibition by antitumor or antibiotic drugs using a chemiluminescence microplate assay.
A recently derived in vitro chemiluminescence assay (Salles et al. [1995] Anal. Biochem., 232, 37-42) has been used to investigate the effects of a panel of twenty-two anticancer drugs and certain antibiotics on the excision repair activity of cell-free extracts from the human cell line, HeLa. This methodology, termed the 3D (Damaged DNA Detection) assay, based on the in vitro excision repair assay previously developed (Wood et al. [1988] Cell, 53, 97-106) has provided data indicating definite in vitro inhibition of DNA repair by actinomycin D, aphidicolin, doxorubicin, distamycin A and mithramycin A. This assay therefore offers the potential for identifying agents with the ability to inhibit DNA repair.
Anti-Bacterial Agents↗
Elderly patients with aggressive non-Hodgkin's lymphoma: disease presentation, response to treatment, and survival--a Groupe d'Etude des Lymphomes de l'Adulte study on 453 patients older than 69 years.
PURPOSE: To clarify disease characteristics and optimal treatment for elderly patients with non-Hodgkin's lymphoma (NHL), we performed a randomized trial in 453 patients older than 69 years with aggressive lymphoma. PATIENTS AND METHODS: Two hundred twenty patients received cyclophosphamide 750 mg/m2, teniposide (VM-26) 75 mg/m2, and prednisone 40 mg/m2/d for 5 days (CVP) and 233 patients received CVP plus pirarubicin (THP-doxorubicin) 50 mg/m2 (CTVP), each for six courses every 3 weeks. RESULTS: The median age was 75 years. Most patients had clinically aggressive disease; 30% had one and 53% two or three adverse prognostic parameters as defined by the International Prognostic Index. More patients on the CTVP arm had an elevated lactic dehydrogenase (LDH) level, but the two groups were otherwise well balanced. CTVP treatment was more frequently associated with leukopenia, thrombocytopenia, and infectious complications. Death during chemotherapy occurred in 16% and 21% of patients on the CVP and CTVP arms, respectively (not significant). Forty percent of patients achieved a complete response (CR): 47% on CTVP and 32% on CVP (chi2 = 20.98, P = .0001). The median time to treatment failure (TTF) was 7 months for CTVP versus 5 months for CVP (log-rank test, P < .05). The median survival time was 13 months in both groups; however, the 5-year survival rate was 26% with CTVP versus 19% with CVP (chi2 = 4.68, P < .05). Lymphoma progression was the primary cause of death. CONCLUSION: Elderly patients with aggressive lymphoma have an aggressive disease with adverse prognostic parameters at the time of diagnosis. Slightly longer survival was observed for patients treated with an anthracycline-containing regimen.
Aged↗
Preferential repair incision of cross-links versus monoadducts in psoralen-damaged plasmid DNA by human cell-free extracts.
Upon UVA irradiation psoralens covalently bind to DNA as monoadduct and interstrand crosslink. Psoralen photoadducts are processed via an excision repair reaction that has been reproduced in vitro with transcriptionnally active cell-free extracts. A derived in vitro assay that allows direct quantification of the incised sites has been set up and used to compare the efficiency of the incision reaction on monoadducts and interstrand cross-links. The incision reaction was performed with HeLa cell-free extracts on angelicin or 8-methoxypsoralen (8-MOP)-modified plasmid DNA substrates carrying known amounts of mono- and biadducts, within various relative ratios. In the case of 8-MOP modified plasmids consisting in a mixture of mono- and biadducts on the same DNA molecule, the incision signal was mainly due to the presence of interstrand cross-links. The extent of incision was linear with the number of cross-links up to about 4 cross-links per plasmid and then reached a plateau. The sensitivity of incision defined as the increase of incision by 2-fold over the background level corresponded to about 1 cross-link per plasmid molecule, and about 7% of the total cross-links were repaired under our assay conditions. The incision activity on angelicin monoadducts yielded only 27% when compared to that on 8-MOP cross-links. Furthermore, 8-MOP cross-links lowered the incision extent of angelicin monoadducts when the two photoadducts were present on distinct plasmid DNA molecules. These data are in line with the more rapid excision of psoralen interstrand cross-links vs monoadducts observed in vivo.
Cell-Free System↗