Cytogenetic studies using various clastogens in two patients with Werner syndrome and control individuals

Summary Chromosome studies were performed on peripheral lymphocytes from two patients with Werner syndrome and two healthy control individuals to detect spontaneous and/or mutagen-induced chromosomal instability of this disease. Diepoxybutane, isonicotinic acid hydrazide, 4-nitro-quinoline-1-oxide, and bleomycin were used as standard clastogens. While the spontaneous frequency of chromosomal breakage was much higher in lymphocytes from both patients than in the control cells, the basic rate of sister chromatid exchange (SCE) was found to be in the control range. The sensitivity to clastogens of the patients' cells, however, was not substantially increased as compared with the controls if the degree of multiplication of the spontaneous breakage rate or SCE frequency was taken as the basis for comparison. No indication of a greater inhibition of proliferation by the clastogens in the patients' cells than in normal cells was observed using BrdU-labelled lymphocytes. Thus, the lymphocytes from both patients of the present study lacked essential features of the classical chromosome instability syndromes.     

Cytogenetic analyses utilizing various clastogens in two sibs with Fanconi anemia,their relatives,and control individuals

Summary Structural chromosome damage, sister chromatid exchange (SCE), and proliferation kinetics were studied on lymphocyte cultures from the peripheral blood of two sibs exhibiting signs of Fanconi anemia, their relatives, and control individuals. While the rate of spontaneous chromosome breakage was at the lower limit of that known for Fanconi anemia in our patients, a distinctly greater increase than in controls of breakage frequency could be induced by isoniazid (INH), 4-nitroquinoline-1-oxide (NQO), and diepoxybutane (DEB) in their lymphocytes. Increased aberration frequencies as compared with controls were also observed in the clastogen-exposed lymphocyte cultures of the parents of both sibs, but in some experiments (NQO, DEB 24h) only in the cells of the healthy brother. There was an increase in the breakage rate of bromodeoxyuridine (BrdU)-labeled consecutive mitoses under the action of NQO, but a decrease with INH as the test clastogen.No significantly higher SCE frequency was found throughout the study in untreated and clastogen-exposed FA lymphocytes as compared with the respective controls. Proliferation was clearly inhibited by INH and NQO as indicated by a distinct increase of the percentage of BrdU-labeled first and a drastic decrease of third metaphases. The present test clastogens were shown not only to be suitable for ensuring the diagnosis of FA in patients with a low incidence of spontaneous breakage but also for determining clastogen-sensitive heterozygotes. According to these results cross-link repair cannot be the only mechanism affected by the basic defect of Fanconi anemia.Dedicated to Professor Dr. A. Barthelmess on the occasion of his 75th birthdayThis paper contains parts of the M.D. theses of D.K., H.M., and M.N.     

Repeated measurement of spontaneous and clastogen-induced sister-chromatid exchange.

Sister-chromatid exchanges (SCE), both spontaneous and chemically-induced [bleomycin (BLM), mitomycin-C (MMC), streptonigrin (SN), and 4-nitroquinoline-1-oxide (4NQO)], were studied in the lymphocytes of 24 normal individuals on 2 or 3 different occasions, separated by periods of up to 2 years. For all BLM-induced SCEs, the variation in SCE frequency among the samples from a single individual was significantly greater than the variation between replicate cultures on a given day. These results raise questions concerning the validity of conclusions based on a single observation of chemically-induced SCEs.     

Chromosomal aberrations in patients with primary biliary cirrhosis

Summary Chromosomal aberrations in untreated lymphocyte cultures, bleomycin (BLM)-induced aberrations and sister chromatid exchanges (SCE) in the peripheral blood lymphocytes of 11 patients suffering from primary biliary cirrhosis (PBC) and 14 matched control individuals were analysed. The lymphocytes of the PBC patients had on average a lower mitotic index (2.3) compared with controls (3.5) in the untreated cultures. The mean baseline rate of aberrations of the cultured lymphocytes of the patients was 5.3 aberrations per 100 metaphases (%); this was significantly different (P=0.0291) from that of the controls with a mean of 2.3%. In lymphocytes of the patients and controls, most of the aberrations observed took the form of gaps; there was an almost equal breakage rate in both groups (0.5% and 0.4%, respectively). The average number of mitoses with aberrations in the PBC patients studied was double that of the controls (4.9% and 2.3% respectively, P=0.0323). The mean number of the BLM-induced aberrations was 54.0% and 27.7% for the lymphocytes of the patients and controls, respectively. The mean number of the aberrant mitoses in the BLM cultures was 6 times higher than that of the untreated cultures for both groups, 25.7% and 14.6% respectively (P=0.018). The chromosomal distribution of baseline and induced aberrations was not random. The PBC patients had a mean number of 8.7 SCE per mitosis, which was significantly higher than the SCEs in the controls (6.3 SCE per mitosis; P=0.0156). The evidence suggests that the chromosomes of the lymphocytes of PBC patients may be less stable than those of the control individuals in this study.     

Opposite responses in two DNA repair capacity tests in lymphocytes of head and neck cancer patients

Genomic instability has long been recognized as the main feature of neoplasia and a factor modulating individual cancer susceptibility. There are attempts to find effective assays of both individual DNA repair capacity and genetic instability, and their relation to the cancer risk. Genetic predisposition plays an important role in the etiology and development of head and neck squamous cell carcinoma (HNSCC). The aim of our study was to search for a correlation between chromosomal instability and DNA repair capacity in HNSCC patients and healthy controls. The chromosomal instability was measured by the number of bleomycin (BLM)-induced chromosomal aberrations and diepoxybutane (DEB)-induced sister chromatid exchanges. The DNA repair capacity was assessed using the DEB-induced adaptive response (AR). The HNSCC patients in our study showed a significant increase in chromosomal instability after a preterminal exposure of their lymphocytes to either BLM for the last 5 h or DEB for the last 24 h of incubation. However, the AR was higher in HNSCC patients than in the control group, suggesting an increase in the DNA repair capacity in the cancer patients as compared to the control. There is no correlation between the DNA repair capacity estimated on the basis of preterminal exposures to BLM and DEB and the DNA repair capacity estimated on the basis of the adaptive response to DEB. The preterminal exposure and the adaptive response test may activate different DNA repair mechanisms.     

Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients

Summary The frequency of spontaneous 6-thioguanine (TG)-resistant peripheral blood lymphocytes in five unrelated Werner syndrome (WS) patients was determined using an autoradiographic labeling assay. The average frequency of TG-resistant lymphocytes was eightfold higher in WS patients than in sex- and age-matched normal control donors. This finding and previous identification of increased spontaneous chromosomal rearrangements and deletions in WS cells or cell lines suggest that WS is a human genomic instability or mutator syndrome.     

Effect of oxidants and antioxidants on chromosomal breakage in Fanconi anemia lymphocytes

Summary Peripheral blood lymphocytes from eight Fanconi anemia (FA) patients, 14 FA heterozygotes, and nine normal subjects have been tested for their susceptibility to chromosomal breakage induction by diepoxybutane (DEB) and by two peroxides. In addition, the effect of five antioxidants was investigated in standard cultures and in cultures stressed either with DEB or with butylhydroperoxide (BHP) or with hydrogen peroxide (H₂O₂). DEB, BHP, and H₂O₂ dramatically increased the chromosomal breakage levels in homozygous and heterozygous FA cells. A partial correction of chromosomal instability was obtained by treating the patients' lymphocytes with antioxidants. A protective effect was also noted in the DEB or peroxide-stressed lymphocytes of patients and heterozygotes, grown in the presence of antioxidants.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

No evidence of chromosomal instability in nevoid basal-cell carcinoma syndrome

The nevoid basal-cell carcinoma syndrome (NBCCS) is a rare multisystem disorder characterized by several alterations of the skin, skeletal, nervous, endocrine system. These patients are more susceptible to cancer and suggestions have been made about an association with the chromosomal breakage syndromes. We studied the induction of chromosomal aberrations by mitomycin C (MMC) and bleomycin and that of sister-chromatid exchanges by MMC and 4-nitroquinoline N-oxide in lymphocytes of 4 NBCCS patients. The frequencies of both spontaneous and induced cytogenetic effects were within normal ranges, suggesting that there is no chromosomal instability in NBCCS patients.     

Repeated measurement of spontaneous and clastogen-induced sister-chromatid exchange

Sister-chromatid exchanges (SCE), both spontaneous and chemically-induced [blomycin (BLM), mitomycin-C (MMC), streptonigrin (SN), and 4-nitroquinoline-1-oxide (4NQO)], were studied in the lymphocytes of 24 normal individuals on 2 or 3 different occasions, separated by periods of up to 2 years. For all BLM-induced SCEs, the variation in SCE frequency among the samples from a single individual was significantly greater than the variation between replicate cultures on a given day. These results raise questions concerning the validity of conclusions based on a single observation of chemically-induced SCEs.     

Chromosomal instability in B-lymphoblasotoid cell lines from Werner and Bloom syndrome patients

Werner’s syndrome (WS) and Bloom’s syndrome (BS) are rare autosomal genetic diseases that predispose to cancer and are associated with genomic instability. To characterize the genomic instability of WS and BS, we analyzed and compared the cytogenetics of B-lymphoblastoid cell lines (LCLs) from WS and BS patients and healthy donors. Although, similar spontaneous frequencies of micronuclei (MN) and sister chromatid exchanges (SCE) were observed in LCLs from WS patients and healthy donors, they were much higher in BS-LCLs. We also examined the cells’ cytotoxic and cytogenetic formation (MN) response to camptothecin (CAM), etoposide (ETO), 4-nitroquinoline 1-oxide (4NQO), and mitomycin C (MMC). Compared to healthy donor LCLs, BS-LCLs but not WS-LCLs tended to be resistant to cytotoxicity and sensitive to MN induction by 4NQO and MMC. Spectrum karyotyping analysis revealed that most WS- and BS-LCLs generated “variegated translocation mosaicism” at high frequencies during cell culture. These findings support the idea that the basis of genomic instability in WS is different from that in BS.     

Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells.

Cells from persons with Bloom syndrome feature an elevated rate of sister-chromatid exchange (SCE). However, in some affected persons a minority of blood lymphocytes have a normal SCE rate. Persons who inherit the Bloom syndrome gene BLM identical by descent from a common ancestor very rarely exhibit this high-SCE/low-SCE mosaicism; conversely, mosaicism arises predominantly in persons who do not share a common ancestor. These population data suggested that most persons with Bloom syndrome in whom the exceptional low-SCE cells arise are not homozygous for a mutation at BLM but instead are compound heterozygotes. Following this clue, we carried out a genotype analysis of loci syntenic with BLM in 11 persons who exhibited mosaicism. In five of them, polymorphic loci distal to BLM that were heterozygous in their high-SCE cells had become homozygous in their low-SCE cells, whereas heterozygous loci proximal to BLM remained heterozygous. These observations are interpreted to mean that intragenic recombination between paternally derived and maternally derived mutated sites within BLM can generate a functionally wild-type gene and that low-SCE lymphocytes are progeny of a somatic cell in which such intragenic recombination had occurred.     

Cytogenetical characterization of UV-sensitive repair-deficient CHO cell line 43-3B. II. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by 4NQO, mono- and bi-functional alkylating agents

An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO).     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Analysis of spontaneous and bleomycin-induced chromosome damage in peripheral lymphocytes of long-haul aircrew members from Argentina

Spontaneous and bleomycin (BLM)-induced chromosomal aberrations in G0 and G2 stages of the cell cycle have been analyzed in peripheral lymphocytes of 21 long-haul aircrew members from Argentina in order to assess BLM-induced clastogenesis as a first approach to determine the DNA repair capacity and thereby the susceptibility to environmental cancers in aircrew. The possibility that occupational exposure of flight personnel to cosmic radiation can induce an adaptive response in their peripheral lymphocytes that can be detected by a subsequent in vitro treatment with BLM was also investigated. For comparison, aberrations were also scored in the lymphocytes of 15 healthy volunteers matched by age, health, sex, drinking and smoking habits to the flight personnel group. Aircrew exhibited a higher frequency of spontaneous dicentrics and ring chromosomes than the control population (p<0.05). BLM sensitivity test showed that aircrew and controls are equally sensitive to BLM G2 clastogenic effects, since both groups exhibited a similar frequency of chromatid breaks per cell (p>0.05). However, the aircrew sampled population was almost two times more sensitive to BLM G0 clastogenic effects than controls (p<0.05). Therefore, our data suggest that chronic exposure of aircrew to cosmic radiation increases the in vitro chromosomal sensitivity of their peripheral lymphocytes to BLM (at least in the G0 stage of the cell cycle), and that occupational exposure of flight personnel to cosmic radiation does not induce an adaptive response to this radiomimetic compound. Our results justify further studies aimed at determine if those aircrew members hypersensitive to BLM are more prone to develop environmental cancer than BLM-insensitive individuals.     

BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.     

Effects of 50-Hertz electromagnetic fields on proliferation and on chromosomal alterations in human peripheral lymphocytes untreated or pretreated with chemical mutagens

Cultivation of human peripheral lymphocytes (HPL) in the presence of 50Hz electromagnetic fields (EMFs) does not alter the spontaneous frequencies of sister-chromatid exchanges (SCE) and of chromosomal aberrations (CA), but leads to an enhancement of the cell cycle progression of HPLs in vitro. Pretreatment of HPLs with trenimon (TRN), diepoxybutane (DEB), or methylnitrosournea (MNU) in the G₀ phase of the cell cycle results in dose-dependent elevations of the SCE frequencies. In some cases culturing of HPLs pretreated with MNU or TRN in the presence of EMFs led to significantly higher frequencies of SCEs when compared to cells cultivated in the absence of EMDs. Since we did not use multiple fixation times these data may rather result from differential influences on HPL subsets than from EMF exposure.     

Chromosomal instability in a woman with infertility and two unaffected brothers: a new familial chromosomal breakage syndrome?

Repeated chromosomal analysis of peripheral blood lymphocytes and skin fibroblasts from a woman referred for amenorrhoea, streak gonads, hyperthyroidism, adiposity and elevated α-fetoprotein levels but no other manifestations of known chromosomal breakage syndromes demonstrated an increased spontaneous chromosomal breakage rate (ISCBR). Chromatid and chromosomal breaks were more numerous than sporadic rearrangements and dicentric chromosomes. Exposure of the cells to mitomycin C, diepoxybutane, X-rays or UV irradiation induced an increase in chromosomal and chromatid abnormalities over that in controls. A micronucleus assay demonstrated an increase in the incidence of formation of micronuclei and the population doubling time of the fibroblasts of the proposita was delayed. Chromosomal analysis was performed on lymphocytes of the parents and of five sibs of the proposita. Two brothers had chromosomal abnormalities identical to those of the patient and elevated α-fetoprotein levels, however, without any clinical abnormalities. The parents were affected by only a moderate ISCBR whereas two brothers and one sister were chromosomally normal. The clinical, chromosomal and biochemical findings in this family represent a novel chromosomal instability syndrome. Received: 30 October 1996 / Accepted: 27 March 1997     

The effect of low-level 60-Hz electromagnetic fields on human lymphoid cells. II. Sister-chromatid exchanges in peripheral lymphocytes and lymphoblastoid cell lines

Dividing human peripheral lymphocytes from 10 normal adults (5 males and 5 females) as well as lymphoid cell lines from patients with the chromosomal instability syndromes were exposed to low-level 60-Hz sinusoidal electromagnetic fields (EMF). The current density of the electrical field was 30 microA/cm2 while the strength of the magnetic field was either 1 or 2 gauss. The cytological endpoints measured included the frequency of sister-chromatid exchanges per chromosome; the distribution of first-, second-, and third-division cells and chromosome breakage (lymphoblastoid cells only). No statistically significant differences, indicative of EMF effects were observed between the treated and control cells regarding SCE frequency, cell cycle progression or chromosome breakage.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.