Individual variability in the level of spontaneous sister chromatid exchanges

The contribution of genetic factors to spontaneous level of the sister chromatid exchanges (SCE) has been determined on the basis of the twin method of study. A close relation is shown to exist between the SCE tests in the group of the monozygotic twins which is a result of the common genotype. The SCE test with late BUdR introduction is under rigid genetic control.     

Demonstration of spontaneous sister chromatid exchanges in vivo

The frequency of sister chromatid exchanges (SCEs) was examined as a function of bromodeoxyuridine (BUdR) concentration in vivo. Oneyear-old Wistar rats were continuously infused with BUdR through the tail vein for 24 h, sacrificed, and mitotic preparations prepared from femur bone marrow. It was observed that from the minimum concentration of BUdR which permitted accurate scoring (1.9 μg/g wt/h) to a BUdR concentration of 7 μg/g wt/h, SCE frequency remained constant. Above 7 μg BUdR/g wt/h SCE frequency increased, saturating at higher BUdR concentrations. The stability of SCE frequency at low BUdR concentrations is interpreted to indicate the existence of spontaneous SCEs in vivo.     

A decreased frequency of sister chromatid exchanges in pregnant women

Sister chromatid exchanges (SCE) and cellular proliferation were studied in lymphocytes from 16 pregnant and 18 non-pregnant women. A lowered SCE frequency was found in lymphocytes obtained from pregnant women (9.41 +/- 0.39 vs. 11.07 +/- 0.42 SCE/metaphase in non-pregnant women). Lower proliferation rates were also common for cultures of pregnant women. Thus, physiological changes occurred in the organism of pregnant women may influence various cytogenetic indices registered in human peripheral blood cultures.     

Quantitative analysis of sister chromatid exchanges in the cell]

Assuming a random nature of distribution of sister chromatid exchanges (SCE) in a karyotype, the formulae have been obtained allowing the calculation of the number of SCE that are overlooked because of a limited resolving power of the SCE detection method. The results obtained mean that the actual number of SCE is more than the observed one, the part of overlooked exchanges being increased with the heightening of the SCE level. Taking into account overlook exchanges, the formula has been obtained that makes possible the calculation of the expected number of SCE observed in any group of chromosomes. These results were applied in the analysis of the SCE distribution among chromosomes. A better conformity has been obtained between the expected results and the observed ones, than under the assumption that the observed SCE are distributed in proportion to the lengths of chromosomes. The obtained formulae are of use in interpreting the lack of the observed SCE in small chromosomes and the excess of them in large ones.     

Reduced frequency of baseline sister chromatid exchanges in lymphocytes grown in antibiotics and serum-excluded culture medium

Peripheral venous blood from man, muntjac, and cattle were grown in medium (1) containing different serum (human AB+/FCS/autologous), (2) without serum or antibiotics (penicillin and streptomycin), or (3) without both serum and antibiotics to investigate to what extent certain essential culture components used in in vitro mammalian cell cultures affect the incidence of spontaneous sister chromatid exchanges (SCEs). The observation that exclusion of only serum from culture medium enhanced the frequency of SCEs whereas exclusion of both serum and antibiotics, which influence the cell cycle kinetics to a great extent, exhibited the lowest frequency of SCEs reported so far for lymphocyte cultures, indicates that the frequency of so-called spontaneous SCEs usually observed in normal lymphocyte cultures grown in medium supplemented with serum and antibiotics is in fact an elevated frequency.     

The frequency and distribution of sister chromatid exchanges in human chromosomes

Summary A detailed procedure is described for a rapid detection of phosphoglucomutase-2 (=phosphopentomutase; PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells.The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.     

Relationship of spontaneous chromosome instability and sister chromatid exchanges in Fanconi's anemia

The frequency of spontaneous instability of lymphocyte chromosomes of the first 2 mitoses, the rate of sister chromatid exchanges (SCEs), and the proliferative kinetics of lymphocytes were studied in a 6-year-old girl with Fanconi's anemia (FA) and in 4 healthy donors. The frequencies of aberrant cells and the total number of chromosome breaks in the FA patient decreased with cell transition from the first to the second mitosis. The FA lymphocytes had a slower proliferative kinetics and the level of SCEs was higher as compared with control. The probability of chromatid deletions at the sites of SCEs localization and in the dark and light stained chromatids was unequal. 33.8% of chromatid breaks were associated with SCEs. The data point to the relationship between SCEs and spontaneous chromosome instability in AF cells.     

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures

Summary Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.     

Statistical evaluation of sister chromatid exchanges

Summary Log-linear models are fitted to sister chromatid exchange (SCE) scores in order to test the significance of the differences in SCE scores observed between individuals or between experimental treatments. The analysis is performed at the level of chromosome groups. In each single test all measurements from all chromosome groups, both from the control and from the experimental sets, are utilized. By proceeding in this way full use is made of all the available information on the SCE scores at the level of chromosome groups and the shortcomings of the classical Student-t and chi-square tests are avoided.This work was supported by a grant Geconcerteerde Acties from the Belgian Government.     

Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes

Summary Lymphocytes from 20 normal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU—Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G groups had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.     

Deficiency of arginine and lysine causes increase in the frequency of sister chromatid exchanges

Summary An increase in the rate of sister chromatid exchanges (SCE) was found when V79 Chinese hamster cells were exposed to increasingly severe degrees of arginine and lysine deficiency. The data suggest a possible function of chromosomal proteins, and of histones in particular, in the maintenance of the low normal rate of SCE.     

Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes]

The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C-negative regions, as well as between G-bands and R-bands, has been produced.     

A family study of spontaneous sister chromatid exchange frequency.

The frequency of spontaneous sister chromatid exchanges (SCEs) was determined in PHA-stimulated peripheral lymphocytes of 52 individuals, comprising 12 complete 2-generation pedigrees. Neither intraindividual variation between replicate cultures established from the same blood sample nor variation among samples from the same individual initiated at different times was significant. However, familial factors affecting mean SCE frequencies were indicated by detection of significant differences among, but not within, families. Although sample sizes were small, a genetic contribution to the SCE frequency was suggested by the observed pattern of familial correlations.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

Frequency of mutagen-induced sister chromatid exchanges in the course of 3 cell cycles

The induction of SCE by fotrine (0.125 and 0.250 microgram/ml) and thiophosphamide (5 micrograms/ml) during the first three cell cycles was studied in the Chinese hamster cells. No increase in the SCE number was observed after treatment with thiophosphamide and fotrine at the G2 stage (the first stage from the moment of fixation) as compared with the control variants. The maximal sensitivity of the cells to the SCE induction by the mutagens is marked at the G1 stage of the first cell cycle before the moment of fixation. The level of SCE remains approximately the same in the second cell cycle before the moment of fixation (20-32 h) and decreased down to the control level at the G1 stage of the third cell cycle (48-52 h).     

Thiophosphamide induction of sister chromatid exchanges at various phases in the cell cycle of a Chinese hamster cell culture]

Influence of three concentrations of thiophosphamide (thioTEPA) on the formation of sister chromatid exchanges (SCE) has been studied at different phases during 2 cell cycles in cultured Chinese hamster cells. It is shown that the frequency of SCE does not differ from the control level under the effect of the mutagen on cells in the G2 phase of the first cell cycle from the moment of harvesting. Thiophosphamide induces the same number of SCE at S, G1 stages of the first cell cycle and G2 of the second one till the moment of harvesting. The number of SCE correlates in a direct proportion with a concentration of thiophosphamide. A scheme of forming SCE is proposed.     

Effects of temperature on sister chromatid exchanges

Summary The influence of temperature on sister chromatid exchanges was investigated, and the results are discussed in connection with factors possibly involved in temperature-induced SCE-formation.Whereas the SCE frequency increased with increasing growth temperature in a cell line of Xenopus laevis (EAX), which permits the examination of great temperature differences, a Chinese hamster cell line (V-79) revealed a U-shaped temperature-response curve. In addition, it was found that cold treatment at 4°C caused an induction of SCEs in the V-79 cell line.Different BrdU concentrations had no effect on the temperature-induced SCE frequencies and mitomycin C led to an induction of SCEs parallel to the base-line values at different temperatures.     

Effect of incubation temperature on the frequency of sister chromatid exchanges in Bloom's syndrome lymphocytes

Summary The effect of incubation temperature on the frequency of sister chromatid exchange (SCE) has been studied in blood cultures from three Bloom's syndrome (BS) patients, three controls, and three BS heterozygotes. All cell types show slight increases of SCE at 39°C while at 35°C and 32°C, SCE is reduced considerably in BS and slightly increased in normal cells. Prolonging lymphocyte culture to 140 h and adding BUdR for the last two S periods causes a similar decrease in the percentage of SCE in normal and BS cells but, while the latter show a further reduction if they are incubated at 32°C during BUdR labelling, the normal cells show an increase. Therefore, BS and control lymphocytes respond similarly to changes in incubation time and differently to changes in incubation temperature. The possibility that the discrepant behaviour of the BS and control cultures may be due to different growth kinetics of their B and T lymphocytes has been discussed but considered unlikely. Since low temperature lengthens the cell cycle, it has been suggested that our findings and those published by others on co-cultivation experiments (except those of Tice et al. 1978) can be explained by assuming that slow growth reduces SCE in BS cells. This, and unpublished observations (Giannelli et al. 1981), suggest that some imbalance in the factors responsible for DNA replication may exist in BS and possibly account for the high level of SCE.     

Trex2 enables spontaneous sister chromatid exchanges without facilitating DNA double-strand break repair

Trex2 is a 3' → 5' exonuclease that removes 3'-mismatched sequences in a biochemical assay; however, its biological function remains unclear. To address biology we previously generated trex2(null) mouse embryonic stem (ES) cells and expressed in these cells wild-type human TREX2 cDNA (Trex2(hTX2)) or cDNA with a single-amino-acid change in the catalytic domain (Trex2(H188A)) or in the DNA-binding domain (Trex2(R167A)). We found the trex2(null) and Trex2(H188A) cells exhibited spontaneous broken chromosomes and trex2(null) cells exhibited spontaneous chromosomal rearrangements. We also found ectopically expressed human TREX2 was active at the 3' ends of I-SceI-induced chromosomal double-strand breaks (DSBs). Therefore, we hypothesized Trex2 participates in DNA DSB repair by modifying 3' ends. This may be especially important for ends with damaged nucleotides. Here we present data that are unexpected and prompt a new model. We found Trex2-altered cells (null, H188A, and R167A) were not hypersensitive to camptothecin, a type-1 topoisomerase inhibitor that induces DSBs at replication forks. In addition, Trex2-altered cells were not hypersensitive to γ-radiation, an agent that causes DSBs throughout the cell cycle. This observation held true even in cells compromised for one of the two major DSB repair pathways: homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Trex2 deletion also enhanced repair of an I-SceI-induced DSB by both HDR and NHEJ without affecting pathway choice. Interestingly, however, trex2(null) cells exhibited reduced spontaneous sister chromatid exchanges (SCEs) but this was not due to a defect in HDR-mediated crossing over. Therefore, reduced spontaneous SCE could be a manifestation of the same defect that caused spontaneous broken chromosomes and spontaneous chromosomal rearrangements. These unexpected data suggest Trex2 does not enable DSB repair and prompt a new model that posits Trex2 suppresses the formation of broken chromosomes.