Ku70 N-terminal lysines acetylation/deacetylation is required for radiation-induced DNA-double strand breaks repair.

Ku70 protein in hetero-trimeric complex with Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) represents a critical component of the nonhomologous-end-joining (NHEJ), the major machinery of DSBs repair in mammalian cells. It has been previously shown that modulation of Ku70 acetylation by histone deacetylases (HDAC) inhibitors induced sensitization of cancer cells to chemotherapeutic agents. However, the effects of such modulation on the repair of Ionizing Radiation (IR)-induced DSBs and the importance of dynamic equilibrium of acetylation/deacetylation have not been studied in details. To address these questions aceto-blocking and aceto-mimicking mutants were designed by replacing Ku70 lysine residues K317, K331 and K338 with arginine and glutamine respectively via site-directed mutagenesis. Transformed human embryonic lung fibroblasts MRC5VA were transfected to create stables cells lines over-expressing Ku70 mutant proteins. Clonogenic survival and γ-H2AX foci assays were performed to study the impact of these mutants on DNA repair proficiency of MRC5VA cells in response to IR. We report here that both Ku70 aceto-blocking and aceto-mimicking mutants rendered MRC5VA cells more susceptible to IR in terms of clonogenic survival and γH2AX foci. Moreover, modelling the possible interactions and structural impact of these aceto-blocking and aceto-mimicking mutants with DNA substrate showed that mimicking acetylation/deacetylation of K331 and K338 could directly compromise KU-DNA interactions, whereas K317 may have a more subtle role via forming a salt bridge with E330 thus optimising the positioning of the helix containing both K331 and K338 residues on the DNA. Our data indicates that dynamic equilibrium of acetylation/deacetylation of Ku70 lysine residues K317, K331 and K338 is critical for optimal repair of IR-induced DSBs, and may offer a novel therapeutic approach for cancer treatment.

Nonhomologous end joining and V(D)J recombination require an additional factor.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

Cellular and biochemical impact of a mutation in DNA ligase IV conferring clinical radiosensitivity.

DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who overresponded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show that wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase with distinct optimal requirements for adenylate complex formation versus rejoining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-sensitive activity to the protein consistent with the predictions of the structural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation with little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but confer marked radiosensitivity.

DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.

Defects in the kinetics of the repair of DNA double-strand breaks and inhibition of DNA synthesis in the ataxia telangiectasia AT5BI-VA cell line: comparison to a corrected hybrid, atxbc.

The nature of the primary biochemical defect in the human radiosensitive and cancer-prone syndrome, ataxia telangiectasia (AT), has remained obscure despite many efforts to elucidate it. In this study, AT complementation group D cells and a nearly isogenic corrected AT-hamster hybrid derivative have been analyzed for induction and repair of initial double-strand breaks (DSBs) after exposure to ionizing radiation, using a sensitive field-inversion electrophoresis technique. Results suggesting that initial levels of damage are the same in these two cell types, but indicating differences in the fast component of DNA repair, have been compared and correlated with those resulting from a study of the radioresistant DNA synthesis defect and its correction in the same cell lines. These measurements show that the radioresistant phenotype of the substantially corrected AT-hamster hybrid correlates with its higher level of fast-component DSB repair and higher level of inhibition of DNA synthesis, but that the initial damage induction does not contribute to the phenotype. We propose that the AT gene product(s) is likely to act early in a signaling pathway which controls both DNA repair and progression of cells through the phases of the cell cycle in response to ionizing radiation.

Relative contributions of levels of initial damage and repair of double-strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part II. Neutrons.

We have compared DNA double-strand break (dsb) induction and rejoining, using field-inversion gel electrophoresis, with survival in mutant (XR-V15B) and in wild-type parental (V79B) hamster cell lines after low dose neutron and X-irradiation. We found that neutrons do not appear to induce more dsbs than X-rays and deduce that increased sensitivity to neutrons is therefore not due to a higher initial yield of dsbs. Even with low doses of neutrons, there is a visible increase in the production of a smaller subset of DNA fragments which arise only after very high dose X-irradiation. In both cell lines, dsbs induced by neutrons are rejoined more slowly than those induced by X-rays. At long repair times (4 and 17 h) there are no significant differences in the fractions of unrejoined dsbs between neutrons and X-rays. We propose that neutron-induced dsbs have a higher probability of becoming lethal because they are more likely to be misrepaired during the slow stage of rejoining.

Relative contributions of levels of initial DNA damage and repair of double strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part I. X-rays.

In order to investigate the relationships between the induction and rejoining of DNA double-strand breaks (dsb) and their biological consequences it is necessary to measure these lesions uniquely and accurately, especially at relevant low doses of ionizing radiation. Differences in radiosensitivity between cell lines could be due to variations in dsb induction or to differences in the efficiency and/or accuracy of enzymatic repair of these lesions. We have used field-inversion gel electrophoresis to investigate dsb induction and rejoining in V79B parental and XR-V15B ionizing radiation-sensitive mutant cell lines. No difference has been found in the induction of dsb in XR-V15B cells compared with wild type cells; the assay sensitivity permits measurement of dsb induced by doses as low as 1 Gy (p < or = 0.05). The radiosensitivity of the mutant cells is manifested both in a lower fraction of dsb rejoined in the early, fast repair component and longer persistence of unrejoined dsb during post-irradiation incubation. The fraction of dsb remaining unrejoined after prolonged incubations (up to 17 h) correlates well with the higher radiosensitivity of the mutant (as judged by D10 values).

Field-inversion gel electrophoresis analysis of the induction and rejoining of DNA double-strand breaks in cells embedded in agarose.

Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities. The sensitivity limits of the method are 1 Gy for the double-strand break induction experiments and 10 Gy for rejoining experiments. Under selected conditions, no significant degradation of DNA had been observed in rodent cell lines during repair incubation up to 17 h in either irradiated cells or unirradiated controls (background levels for neutron experiments, 2.2 +/- 0.3% at Time 0 compared to 2.3 +/- 0.5% after 17 h of incubation; background levels for X-ray experiments, 2.3 +/- 0.6% at Time 0 and 3.7 +/- 1.1% after 17 h of incubation). In preliminary experiments with the A549 human oat cell carcinoma cell line, DNA DSB background levels remained constant in unirradiated controls up to 4 h in the range reported for the rodent cell line.

Flow cytometry (FCM) of early radiation response in epidermoid carcinoma of uterine cervix.

DNA flow cytometry (FCM) investigation of tumor specimens before and after 30 Gy 137Cs radiation treatment was performed in 33 cases of epidermoid uterine cervix carcinoma. Distinct differences in the type of FCM response to radiation were seen when the results of DNA index (DI) in diploid and aneuploid tumors and proliferation index (PI) values in diploid tumors from pretreatment and 30 Gy irradiated specimens were compared. We observed partial or total reduction of PI in 12 of 17 diploid and near diploid tumors, and total reduction of the aneuploid population in 14 of 16 aneuploid tumors. No significant correlation was found between the type of FCM response and clinical stage of the disease or the histological degree of differentiation.

Flow cytometry analysis of ploidy and proliferation activity in classical and spermatocytic seminoma.

In a series of 49 cases of seminomas, namely 19 classical seminomas, 21 seminomas with syncytiotrophoblastic cells and 9 spermatocytic seminomas, DNA ploidy and S-phase cell fraction of the cell cycle were estimated in paraffin-embedded histopathological material. DNA aneuploidy was detected in 16/19 classical seminomas (84%), in all seminomas with syncytiotrophoblastic cells (100%) and in 6/9 spermatocytic seminomas (67%). In three cases two distinct aneuploid stemlines were detected, in four cases regional variations in ploidy level were observed, clearly proving cellular heterogeneity within the studied specimens. No significant differences in distribution of ploidy levels of aneuploid tumors were detected either between distinct groups of seminomas or in relation to the age of the patients. On the other hand, mean values of S-phase cell fractions in our material offer statistically highly significant differences between defined groups of tumors. Spermatocytic seminomas had the highest level of proliferation activity, which is in contrast with the clinicopathological observations (relatively slow growth, rare occurrence of metastases, local malignancy). The results of proliferation activity analysis and the relatively highest incidence of diploid tumors support the theory of different origin of spermatocytic seminomas in comparison with other germ cell tumors.

Flow cytometry (FCM) analysis of endometrial hyperplasia and carcinoma.

In a series of 52 biopsy specimens (31 endometrial carcinomas, 10 atypical endometrial hyperplasias, and 11 cases of normal endometrium), DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded material. DNA aneuploidy was detected in 2 of the 10 atypical endometrial hyperplasias and 7 of the 31 endometrial carcinomas. The majority of aneuploidy was found to be connected with the loss of tumor differentiation. No ploidy disturbances were found in normal endometrium. The S-phase cell fraction value of normal endometrium was significantly lower when compared with that of endometrial carcinoma. The broad variation in S-phase cell fraction values of the endometrial carcinomas and atypical endometrial hyperplasias was in contrast with the low variability of S-phase cell values of normal endometrium. Very low incidence of aneuploidy in the group of well differentiated endometrial carcinomas (Grade I) enables the suggestion that the presence of aneuploidy predicts a more aggressive disease and that the detection of an aneuploid stemline in atypical endometrial hyperplasia may already indicate the neoplastic transformation.

Growth, flow cytometric, and karyological characterization of L1210 cell sublines resistant to various Pt derivatives.

Growth, flow cytometric, and karyological characteristics were analyzed with respect to differences between sensitive L1210 cells and eight sublines resistant to cisplatin (DDP), 1,2-diaminocyclohexane(DACH)-Pt(II) citrate, DACH-Pt(II) glucarate, cis-dichloro-bis-(isopropylamine)-trans-dihydroxyplatinum (IV) (CHIP, iproplatin), and methotrexate (MTX). No great differences were found in growth properties. The sensitive and a majority of resistant sublines displayed similar flow cytometric and karyological characteristics, major differences were found only in the sublines resistant to CHIP. The relationship between the mechanism(s) of resistance and different pharmacokinetics of the drugs is discussed with special reference to DNA content and chromosome structure.

Flow cytometric analysis of primary cervical and vulvar carcinomas and their metastases.

In a series of 25 patients with cervical carcinoma and 35 patients with vulvar carcinoma, clinically classified (FIGO) as Stage I and Ib, respectively, DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded samples of the primary tumors and their metastases by means of flow cytometry (FCM). The two groups of patients were selected cases in whom lymph nodes removed at radical operation were histopathologically verified as metastatic ones. Prevailing part of primary tumors of both anatomic sites had diploid DNA content and low S-phase fraction. Irrespective of the DNA content and low proliferative activity the early stage carcinomas behaved aggressively as indicated by the presence of metastases, the DNA content of which corresponded in most of the cases with that of primary tumor. It appears that prediction of the tumor growth based only on cytogenetic and cytokinetic parameters may be connected with difficulties.