Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's syndrome

Summary The cytogenetics of six cases of adult progeria (Werner's syndrome) from three Sardinian families were investigated. The overall increased incidence of chromosome breakage found in cultured lymphocytes and fibroblasts seems to be age-dependent. The occurrence of clonal variegated translocation mosaicism, previously found by other authors in fibroblast cell lines derived from Werner patients was demonstrated also in fibroblasts analyzed in situ on the outgrowth halos from primary skin explants; a strong indication that these aberrations are present in the in vivo precursors. The same type of clonal structural aberration was found for the first time also in 72h-cultured lymphocytes. These findings demonstrate that Werner's syndrome is indeed a further example of a chromosome rearrangement syndrome.     

Homology-directed repair is required for the development of radioresistance during S phase: interplay between double-strand break repair and checkpoint response

The S-phase-dependent radioresistance to killing uniformly seen in eukaryotic cells is absent in radiosensitive mutants with defects in genes involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination (homologous recombination repair: HRR). This implicates, for the first time, a concrete DNA repair process in the radiosensitivity of a specific cell cycle phase. The cell cycle-dependent fluctuations in radiosensitivity reflect a fundamental and well-documented radiobiological phenomenon that still awaits a detailed molecular characterization. The underlying mechanisms are likely to combine aspects of DNA repair and cell cycle regulation. Advances in both fields allow a first dissection in the cell cycle of the molecular interplay between DSB repair and DNA damage checkpoint response and its contribution to cell survival. Here we review the available literature on the topic, speculate on the ramifications of this information for our understanding of cellular responses to DNA damage, and discuss future directions in research. An effort is made to integrate relevant phenomena of radiation action, such as low-dose radiosensitivity and the G(2) assay in this scheme.     

Evaluation of Different Biomarkers to Predict Individual Radiosensitivity in an Inter-Laboratory Comparison–Lessons for Future Studies

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.     

On the mechanism of adaptive response in human cells

There was investigated one of the mechanisms of adaptive response, related to chromosome aberrations induced by gamma-rays, in lymphocytes of healthy donors and donors with hereditary diseases (Marfan's syndrome and homocystinurea) whose cells are repair-deficient. 3H-thymidine treatment was used as an adaptive dose in G1-period of cell cycle and 8-methoxypsoralen (8-MOP), activated with UV-light, was used as a challenge agents. Cells of healthy donors and cells of patients with Marfan's syndrome had normal adaptive response in relation to gamma-irradiation and photomutagenic action of 8-MOP. There was no induction of adaptive response in realation to gamma-irradiation and 8-MOP photomutagenic action in cells of patients with homocystinurea. The cells from donors characterised with normal repair system and lack of adaptive response 8-MOP photomutagenic action wasn't modified by 3H-thymidine. We have found parallelism of adaptive response protective effect against chromosome aberrations, induced by UV activated 8-MOP and gamma-rays in repair proficient cells of healthy donors and repair deficient cells of patients with Marfan's syndrome. These data lead us to conclusion that mechanism of adaption, at least in some cases has no connection with repair process modification.     

Clonal structural chromosomal rearrangements in lymphocytes of four patients with Werner's syndrome

Multiple numerical and structural chromosome abnormalities were found in cultured lymphocytes of four patients with Werner's syndrome. The proportion of metaphases with structural and/or numerical aberrations varied from 30 to 44% and several of them were clonal. These results confirm definitively that Werner's syndrome is a chromosome rearrangement syndrome and that these non-constitutional chromosome changes are not exclusive of cultured fibroblasts but present also in lymphocytes.     

Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced G2 delay.

Induction and repair of DNA double-strand breaks (dsb) was investigated in plateau phase Chinese hamster V79 cells and three radiosensitive mutant cell lines derived from them, irs-1, irs-2 and irs-3, using a pulsed-field gel electrophoresis assay, Asymmetric Field Inversion Gel Electrophoresis (AFIGE). There was no difference in the induction of DNA dsb per Gy and dalton between the radiosensitive mutant cells and wild-type V79 cells despite the wide differences in their radiosensitivity. Also, repair of DNA dsb proceeded in all cell lines with similar kinetics. In contrast to these observations at the DNA level, irradiation of exponentially growing cells showed a prolonged delay in G2 for irs-2 cells and a shortened delay in G2 for irs-1 cells, as compared to wild-type V79 cells. These results confirm previous observations suggesting that a deficiency in the rejoining of DNA dsb is unlikely to be the cause of the increased radiosensitivity of irs cells, and implicate alterations in postirradiation cell cycle progression as a possible cause for this phenomenon, although the mechanism is not known.     

Differentiated cells from BALB/c mice differ in their radiosensitivity

Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2–3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.     

Cell cycle suspension: A novel process lurking in G2 arrest

Cell cycle checkpoint is a self-protective mechanism for cells to monitor genome integrity and ensure the high-fidelity transmission of genetic information to daughter cells. Insufficient function of cell cycle checkpoints has been demonstrated to partially account for tumor initiation, promotion and progression. In the ten melanoma cell lines that we tested in preliminary experiments, two human uveal melanoma cell lines, 92-1 and OCM-1, were found to be significantly different in terms of radiosensitivity but similar in DNA repair ability. Evident G₂ arrest was induced in both cell types and the maximum was reached at 16 h after irradiation regardless of X-rays or high-LET carbon beams. OCM-1 cells overrode the G₂ arrest and reentered the cell cycle right after reaching the maximum, whereas 92-1 could not. Upon 10 Gy of radiation, the cell cycle of 92-1 was suspended and remained unchanged for up to 5 d. The cell cycle suspension is a unique process lurking in G₂ arrest and related to cellular radiosensitivity. Its induction is dose-dependent and there is a dose threshold for it. The degradation of Cyclin B1 has been found related to the cell cycle suspension though, the mechanism of cell cycle suspension is still under investigation. Basing on our knowledge, this is the first report on cell cycle suspension and we present here a de novo mechanism to cellular radiosensitivity. Further clarification of the mechanism underlying cell cycle suspension is believed to be of significance in tumor radiosensitization or even direct tumor control.     

Chromosome instability in lymphocytes from a patient with Werner's syndrome is not associated with DNA repair defects

Different cellular parameters used to detect genetic instability were analyzed in lymphocytes from a patient affected by Werner's syndrome (WS). Cytogenetic studies indicated the presence of structural and numerical chromosomal abnormalities and the occurrence of variegated translocation mosaicism. The baseline mutation frequency was similar to that observed in normal donor samples. DNA repair investigations showing a normal capability to perform UV-induced DNA repair synthesis and a normal sensitivity to various mutagens (UVC light, mono- and bi-functional alkylating agents) indicate that different DNA repair mechanisms act normally in WS. In this feature, WS appears to differ from the other genetically determined syndromes in which chromosomal instability is associated with a marked hypersensitivity to specific DNA-damaging agents.     

On the response of a cell population to low-dose irradiation

The cell composition of a population of human blood lymphocytes was studied after irradiation at doses of 5 cGy, 1.0 Gy and 5 cGy + 1.0 Gy and the use of a cytokinesis block. The frequencies of uni-, bi- and multinucleate lymphocytes with and without micronuclei (MN) were taken into account. By the standard criterion the frequency of binucleate lymphocytes with MN among binucleate lymphocytes--the donors were characterized as follows: in with reduction of radiosensitivity after irradiation with 5 cGy + 1.0 Gy as compared to the values of radiosensitivity after irradiation with 1.0 Gy only (an adaptive response, AR); in with no change of radiosensitivity after exposure to these doses (no AR); and with an increased ofradiosensitivity after exposure to these doses (syndrome of increased radiosensitivity, IRS). It was found that upon exposure to 1.0 Gy and 5 cGy + 1.0 Gy in some donors with AR, without AR and with IRS the total numbers of damaged cells in the population and the number of binucleate cells with MN were equal. This result calls in question the involvement of the repair mechanism in the alteration of radiosensitivity of lymphocytes in these donors. It was also observed that in the same donors a simultaneous increase (or a decrease in the case of IRS) of the portion of undamaged binucleate cells in the population took place. Our results demonstrate the existence of a new, populational, mechanism involved in the alteration of radiosensitivity after exposure to the adaptive and challenge doses.     

Studies on the radiosensitivity of cells from patients with basal cell naevus syndrome.

No difference in survival was observed between cultured cells from basal cell naevus syndrome (BCNS) patients and normal controls following exposure of fibroblasts to ionizing radiation. Potential lethal damage repair in BCNS cells, measured by holding experiments, was also no different from normal. G0-irradiated lymphocytes from BCNS patients were found to have a significantly higher level of X-ray-induced chromosome aberrations compared with normals. This increase is, however, small, and, taken together with the survival data, suggests that increased cell killing as a measure of the unusual clinical radiosensitivity is not the major effect of the BCNS gene.     

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level.The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.     

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

Radiation could induce p53-independent and cell cycle--unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.     

Influence of endogenous glutathione level on X-ray induced cell cycle delay in human lymphocytes

Here, induction of chromosomal aberration after X-irradiation and the pattern of cell cycle kinetics have been investigated in human lymphocytes, after exogenous addition of reduced glutathione or by depleting levels of reduced glutathione endogenously. Involvement of cell cycle regulator proteins such as p53 and p21 has been investigated to elucidate their role in induction of delay in cell cycle progression after irradiation.     

The effect of aging on cell-cycle kinetics and X-ray-induced chromosome aberrations in cultured lymphocytes from patients with Down syndrome.

To evaluate the effects of aging on cytogenetic characteristics of lymphocytes from Down syndrome (DS), cell-cycle kinetics after PHA stimulation and chromosome-type aberration frequencies after X-ray exposure were investigated in vitro in the lymphocytes derived from 4 (or 3 for X-ray treatment) age groups of DS patients and age-matched controls. The results clearly showed higher mitotic and proliferation index levels in younger groups compared to older groups at the various culture intervals, whether the lymphocytes were from the DS patients or controls. The age-related changes of the proliferation index were mainly attributed to a delayed response to PHA as age increased. The changes of PHA responses seemed to be particularly marked during adolescence. Nonetheless, no significant differences were observed between the DS patients and age-matched controls for each age group. In all age groups, frequencies of both chromosome-type exchanges and deletions were elevated in the DS patients by about 1.3 times in comparison with the controls. The magnitude of radiosensitivity, however, seemed to decrease slightly in the 40-49-year group. To our knowledge, the present study is the first report in the literature to deal with the effect of aging on the greater radiosensitivity of DS lymphocytes.     

A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.