High Levels Of Sister Chromatid Exchange Research Articles

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

Preclinical studies on NSC290205 aza‐steroid alkylator activity in combination with adriamycin against lymphoid leukaemia

Summary NSC290205 (A) is an hybrid synthetic antineoplastic ester that is a combination of a d-lactam derivative of androsterone and an alkylating derivative of N,N-bis(2-chloroethyl)aniline. We tested NSC290205 for synergistic antileukaemic activity with adriamycin (ADR), (i) in vitro against the human lymphoid leukaemia cell lines: CCRF-CEM, MOLT-4, and RPMI-8226, (ii) in vivo against P388 lymphocytic and L1210 lymphoid murine leukaemias (at incipient and advanced phase). Our results indicated significant cytostatic and cytotoxic synergy of NSC290205 and ADR in vitro. We further examined these results in vivo by replacing cyclophosphamide in the standard CHOP (cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone) regimen with NSC290205 (AHOP) and comparing the efficiency of these two regimens in vivo. Although treatment of P388 and L1210 with cyclophosphamide or NSC290205 alone yielded equivalent results, AHOP produced a clear benefit for survival compared with CHOP against advanced leukaemias, confirming the in vitro observations [higher percentage increase in median lifespan of treated animals over the untreated (control): 188% and 239% in L1210, 308% and 353% in P388, P < 0.01, for CHOP and AHOP respectively]. AHOP also proved to be more genotoxic and cytostatic than CHOP, inducing higher sister chromatid exchange levels and cell division delays on P388 cells in vivo. NSC290205 showed superior antineoplastic potential against lymphoid leukaemia and significant synergy with ADR, producing an excellent therapeutic outcome.

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

A role for PML and the nuclear body in genomic stability.

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells. The Bloom syndrome gene BLM encodes a RecQ DNA helicase, whose absence from the cell results in genomic instability epitomized by high levels of sister-chromatid exchange (SCE) and cancer predisposition. We show here that BLM co-localizes with PML to the NB. In cells from persons with Bloom syndrome the localization of PML is unperturbed, whereas in APL cells carrying the PML-RARalpha oncoprotein, both PML and BLM are delocalized from the NB into microspeckled nuclear regions. Treatment with retinoic acid (RA) induces the relocalization of both proteins to the NB. In primary PML-/- cells, BLM fails to accumulate in the NB. Strikingly, in PML-/- cells the frequency of SCEs is increased relative to PML+/+ cells. These data demonstrate that BLM is a constituent of the NB and that PML is required for its accumulation in these nuclear domains and for the normal function of BLM. Thus, our findings suggest a role for BLM in APL pathogenesis and implicate the PML NB in the maintenance of genomic stability.

Chromosome Breaks and Sister Chromatid Exchange as Predictors of Second Cancers in Hodgkin's Disease

Hodgkin's disease (HD) survivors face an increased risk of developing second cancers. We evaluated baseline cytogenetic biomarkers, sister chromatid exchange (SCE) and chromosome breaks [spontaneous (SCB) and bleomycin-induced (BIB)], as predictors of second cancer risk in a cohort of 105 adult HD patients. During follow-up, seven second cancers occurred. SCBs and BIBs showed no association with risk of second primaries. Multivariate Cox regression revealed that high levels of SCEs (relative risk (RR) = 11.3, p = 0.02) and age (RR = 1.08, p = 0.02) predicted second cancer risk. Histology, stage, and treatment were not associated with elevated risk. In conclusion, baseline SCE frequencies may be a useful biomarker for identifying HD patients at increased risk of developing second cancers. These results need to be verified in a larger cohort with a longer follow-up time.

CANCER ANTIGEN EXPRESSION AND CHROMOSOMAL CHANGES IN A CARCINOGEN-TRANSFORMED BLOOM-SYNDROME B-LYMPHOBLASTOID CELL-LINE

The phenomenon of sister chromatid exchange (SCE) is an interesting genetic event in metaphase chromosomes, even though its exact mechanism remains unknown. The fact that SCE can take place, whether 'spontaneously' or induced by various agents, is in itself important, for such an event, involving damage and possible repair of bilateral loci in chromosomes, presents opportunities for modification of the cell characters (e.g. oncogene activation). BS B- lymphoblastoid cell lines with high SCE levels were highly susceptible to malignant transformation by chemical carcinogenesis, in which cancer antigens are expressed on the cell surface with the disappearance of immunoglobulin (Ig), when the number of SCE exceeded 140 SCE per cell (over the baseline of 70 SCE per cell). The existence of an increased level of SCE which might cause unequal recombination between either sister chromatids or chromatids of homologous chromosomes may reflect phenomena secondary to malignant transformation or may have played a key role in initiation and maintenance of expression of the oncogenes and the antigens associated with cancers.

Sister chromatid exchange analysis in familial groups of malignant melanoma patients

Sister chromatid exchange (SCE) analysis was carried out on peripheral blood lymphocytes of 20 familial malignant melanoma (FMM) and 39 sporadic malignant melanoma (SMM) untreated patients, belonging to 10 and 39 families, respectively. The study was extended to 39 unaffected close relatives of FMM patients, to 187 unaffected close relatives of SMM patients, and to 20 unaffected unrelated individuals (control group), all examined under the same conditions. The mean SCE rates/cell were significantly higher in MM families than in the control group, and in melanoma patients than in their close relatives. The mean SCE levels of FMM and SMM patients, (8.4 ± 0.8 and 8.0 ± 0.3, respectively) were similar, and so were the distributions of individuals in classes of increasing SCE values (with a modal value at 7–8 SCEs/cell). The mean SCE levels of close relatives of FMM and SMM patients were also similar (5.4 ± 0.2 and 5.4 ± 0.1, respectively, with a modal value at 4–5 SCEs/cell), and slightly higher than in the control group (4.7 ± 0.2 SCEs/cell). More than 7 SCEs/cell were observed in the majority (41 of 59) of FMM or SMM patients, in a smaller fraction (25 of 227) unaffected relatives, and in none of 20 unrelated unaffected individuals. These observations favor the hypothesis that higher SCE levels may be an expression of constitutional lesions predisposing to this neoplastic disease.

Sister chromatid exchange in methotrexate resistant and sensitive C3H10T1/2 mouse cells

Sister chromatid exchange (SCE) induction by methotrexate (MTX) was analyzed in C3H10T1/2 clone 8 mouse cells and in two MTX-resistant subclones with numerous double minute chromosomes (DM) present in the majority of cells. Significantly higher SCE levels were found, as expected, in sensitive cells after treatments with 10(-2) or 10(-5) M MTX but not in resistant cells permanently growing in the presence of a high concentration of MTX (2 x 10(-3) M) and characterized by a markedly lower cell cycle replication index (R.I.), i.e. in conditions that are known to otherwise favour SCE induction. These observations suggest, for the MTX-resistant cells under study, the existence of conditions limiting SCE formation.

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ehtyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3–9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.

Malignant transformation of Bloom syndrome B-lymphoblastoid cell lines by carcinogens.

Three types of Bloom syndrome B-lymphoblastoid cell lines, as well as one derived from a normal person, treated with 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine (0.3 micrograms/ml for 24 hr), were studied for tumorigenicity in nude mice, colony formation in soft agar, cytogenetic changes, and immunoglobulin markers. When normal and Bloom syndrome cells with normal sister chromatid exchange (SCE) levels and karyotypes (type I) were treated with carcinogens, no significant changes occurred in the immunoglobulin profile and karyotype, only rare colony formation was seen, and no tumors were produced. In contrast, when Bloom syndrome cells with high SCE levels (type II with normal karyotype and type III with an abnormal karyotype) were treated with carcinogens, tumors were produced in 22 of 53 nude mice injected; a high rate of colony formation in soft agar was seen; the cells exhibited virtual loss of immunoglobulin markers; and structural changes in chromosomes 1, 2, 3, 4, 5, 7, 11, 14, and 15 were found in the tumors in addition to the original chromosome abnormalities present in the injected cells. It appears that Bloom syndrome B-lymphoblastoid cell lines with high levels of SCE are highly susceptible to the action of carcinogens, as evidenced by tumor formation in nude mice and colony formation in agar. Apparently, the carcinogens were capable of transforming only those cells that had a critical level of SCE (approximately 140 per cell) and not those with only mildly increased levels (approximately 13 per cell).

Sister chromatid exchange (SCE) induced by laser-UV-microirradiation: correlation between the distribution of photolesions and the distribution of SCEs.

Small, medium, and large nuclear areas comprising approximately 5, 30, or 80% of the total area of the interphase nuclei of Chinese hamster cells (M3-1) cultivated in vitro were irradiated with a laser-UV-microbeam of wavelength 257 nm. The DNA of the cells was substituted with 5-bromodeoxyuridine (BrdUrd) for 1 cell cycle in one set of experiments. After microirradiation the cells were grown for a second cycle in medium without BrdUrd (protocol A). In a second set, cells with nonsubstituted DNA were microirradiated and grown for 2 additional cycles, the first in the presence, the second in the absence of BrdUrd (protocol B). In situ chromosome preparation and differential chromatid staining was subsequently performed. The induction of sister chromatid exchanges (SCEs) was found to be dependent on both the ultraviolet (UV) dose and the spatial distribution of the UV energy within the cell nucleus. Following both protocols the average number of chromosomes with SCEs was significantly higher after microirradiation of a large nuclear area as compared to microirradiation of a small nuclear area. In the latter case, multiple SCEs were noted on individual chromosome arms at the first postirradiation mitosis (protocol A). In other cells, especially at higher doses, protocol A resulted in shattering of a few closely neighbored chromosomes which were surrounded by intact ones with normal SCE levels. Microirradiation of medium-sized nuclear areas produced high levels of SCEs over a number of chromosomes which still appeared spatially related in a part of the metaphase spread. Finally, high SCE levels could be observed over most or all chromosomes when a large nuclear area (up to 100%) was exposed to the microbeam. Following protocol B the increase of SCEs was much less pronounced. Microirradiation of a small part of the cytoplasm in addition to the nuclei did not induce SCEs. Our results support the concept (i) that interphase chromosomes occupy distinct nuclear domains and indicate (ii) that the induction of SCEs by UV light is restricted to microirradiated chromatin.

Value and significance of SCE in human leukemia and cancer.

The introduction and refinement of sister chromatid exchange (SCE) techniques generated much enthusiasm, interest, and promise for their application in human neoplasia. In particular, the demonstration of high SCE levels in the cells of Bloom syndrome (BS) patients (1,2), a condition with a very high susceptibility to the development of cancer (primarily lymphoma or leukemia), and the application of SCE as a sensitive and specific index of carcinogenic and/or mutagenic activity (3,4), generated much excitement for the application of SCE studies in human neoplasia. It was hoped that unique changes, akin to those at the chromosome level, would characterize certain malignant conditions and states (5–7). Thus, the general concept was developed that individuals or families with high risk of cancer might show SCE changes characteristic of these groups and that cells involved in certain neoplastic conditions would display unique SCE patterns different from those of normal cells and allow for differential delineation of some neoplastic processes.

Normalisation of sister chromatid exchange frequencies in Bloom's syndrome by euploid cell hybridisation

BLOOM'S syndrome (BS) is a rare autosomal recessive genetic disorder characterised by stunted growth, Sun-sensitive telangiectatic erythema of the face and an abnormally high risk of cancer1. Cultured lymphocytes and fibroblasts from BS patients have a high frequency of spontaneous structural chromosome aberrations and characteristically high levels of sister chromatid exchanges (SCE)2. The primary defect at the molecular level is unknown, but the cytological findings are compatible with a deficiency in a DNA repair function3. The presence of a defect of DNA replication is also suggested by the demonstration that the rate of fork motion during replication in BS is slower than normal4. German et al.5 have reported the coexistence of cells with both high and normal levels of SCE, suggesting that the defect in BS is regulatory. Tice et al.6 concluded from co-cultivation experiments that SCE frequency is mediated by a diffusable agent(s) present in the medium of BS fibroblasts. Other investigators, however have shown suppression of SCE in conditions of co-cultivation7,8. We have clarified the conflict between these results by examining euploid somatic cell hybrids between BS and normal human fibroblasts.

Sister chromatid exchanges in the peripheral blood of cigarette smokers and in lung cancer patients; and the effect of chemotherapy.

Peripheral blood sister chromatid exchange (SCE) rates in chronic cigarette smokers and in subjects with cancer do not differ from those in healthy nonsmokers. SCE patterns were normal in 69 chronic cigarette smokers, including 62 patients with untreated lung cancer. In three chronic smokers with lung cancer, high SCE levels were related to recent intravenous chemotherapy.