Effects of caffeine-induced defective DNA replication on SCE and chromosome aberrations produced by alkylating agents

The effect of caffeine (CAF) pretreatment (during the first cell cycle) on the frequency of sister-chromatid exchanges (SCE) and chromosome aberrations induced by bifunctional(MC)- and monofunctional(M-MC)-mitomycin C, 4-nitroquinoline N-oxide (4NQO) and ethyl methanesulphonate (EMS) were examined by using a BrdU—Hoechst staining technique. When CAF was added to the cultures during the first cell cycle in the presence of BrdU and then the cultures treated with MC, M-MC, 4NQO or EMS during the second cell cycle, the effect of the CAF was synergistic, i.e., the SCE level achieved was much higher than that expected from a simple additive effect of the agents and CAF. These results do not support the concept that the process of SCE is a manifestation of CAF-sensitive post-replication repair of DNA damage (single-strand exchanges), but, instead, point to exchanges between the double-strands of the DNA duplex present in each chromatid. CAF at certain concentrations is known to significantly slow down the rate of DNA-chain growth, but not appreciably induce strand breaks. Inasmuch as CAF alone induced only a small increase in SCE rates, possible mechanisms which may induce SCE are not only related to the slowing down of the rate of DNA-chain growth, but may also involve breaks in the template strand permitting double-strand exchanges to occur. The mechanisms responsible for chemically induced SCE are also discussed.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.     

Three-way differentiation of sister chromatids in endoreduplicated (M3) chromosomes of Bloom syndrome B-lymphoid cell line

Summary The three-way differentiation of sister chromatids (3-way SCD) in M₃ endoreduplicated chromosomes in a Bloom syndrome (BS) B-lymphoid cell line, suggested that in addition to exchanges between sister chromatids (intra-exchanges), non-sister chromatid exchanges (inter-exchanges) also occur, especially in BS high SCE cells. In BS diploid chromosomes such inter-exchanges probably get confused with intra-exchanges when total SCEs are accounted for. Bloom syndrome high SCE cells probably do not follow the same bromodeoxyuridine (BrdU) uptake pattern over three cell cycles as normal cells. The 3-way SCD in M₃ endoreduplicated chromosomes can be explained on the basis of Schvartzman's second model (1979) as well as Miller's model (1976), depending on the pattern of uptake of BrdU over three cell cycles. An interference in the previous events of exchanges in the following cell cycle (i.e., cancellation of SCEs) in BS chromosomes was observed in some regions, though not in high numbers.     

Cell cycle progression and SCE rate of Bloom syndrome cells with/without co-cultivation in the presence/absence of normal cells

The present study was undertaken to examine cell cycle progression and SCE rate in three types of B-lymphoid cell line, viz., normal (KS-86), high-SCE Bloom syndrome (BS (BS2-2) and dimorphic BS (BS-SYW). In order to compare the dimorphic condition (BS-SYW) with artificial dimorphism (co-cultivation of BS2-2 with KS-86) these experiments were designed to test whether the BS B-lymphoid cell line cultures would influence the cell cycle progression and SCE rates of a normal B-lymphoid cell line, and vice versa. The present study resolved the controversy reported in the literature, by finding a definite time period under co-cultivation conditions when the SCE in normal cells was increased after 8 days of co-culture, whereas SCE in the BS cells decreased immediately with co-cultivation. In the dimorphic BS cell line (BS-SYW) the SCE frequency of a high-SCE cell population was also observed to be lower than that of a non-dimorphic BS cell line (BS2-2), thus corroborating the experimental observations under co-cultivation conditions. The decrease in BS SCE and increase in normal SCE (after a particular time period) is attributed to numerous causes discussed in relation to the cell cycle progression.     

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

Cell cycle rate and sister chromatid exchange profile in polyethylene glycol-exposed/unexposed Bloom syndrome and normal cells

Summary A delay in cell cycle progression and a decrease in sister chromatid exchange (SCE) frequency was observed in Bloom syndrome (BS) cells when exposed to polyethylene glycol (PEG) treatment followed by co-cultivation with unexposed normal cells, whereas the SCE rate of both normal and BS cells increased when PEG-exposed normal cells were co-cultured with unexposed BS high SCE cells. This indicates the role of normal cells, probably of cell membranes (which were disturbed or disrupted by PEG treatment), in complementing the deficiency of BS cells under co-culture conditions, and suggests that BS cells may be deficient in membrane bound factor(s) present in normal cells.     

Levels of sister-chromatid exchanges in hybrids between Bloom syndrome B-lymphoblastoid cells and various cell lines with lymphoid malignancy

The present study has been undertaken to examine the effect of cell hybridization of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) and various cell lines from lymphoid malignancies in order to clarify the relationship between sister-chromatoid exchange (SCE) and malignant conditions. Cell hybridization studies have shown that though BS high-SCE frequencies were completed by fusion with normal cells, fusion with various malignant cell lines did not result in complete normalization of BS SCEs, with 15-30 SCEs remaining per hybrid cell, demonstrating possibly common defects in DNA of BS and malignant cells. These findings strongly support the idea that the characteristic high SCE frequency in BS cells has some connection with the malignant condition, and that at least one step in carcinogenesis is either accompanied by the production of SCEs, or that SCEs themselves cause such a step to occur.     

Analysis of spontaneous sister-chromatid exchanges in vivo by three-way differentiation

By applying an adaptation of the method of three-way differentiation to murine bone marrow cells in vivo, the basal frequency of sister-chromatid exchange (SCE) per cell was evaluated. An SCE frequency directly proportional to the estimated relative incorporation of 5-bromodeoxyuridine (BrdU) to the chromosomes was observed for the 3 consecutive cell cycles, implying that the majority, if not all, of the SCEs in vivo were produced by the incorporated BrdU. This conclusion was supported by the finding that in the first cycle of division, a very high frequency of cells without SCE was observed. From these data, a spontaneous frequency of SCE as low as 0.15 SCE/cell/cell cycle was inferred.     

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.     

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.     

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).     

Different mutations responsible for the elevated sister-chromatid exchange frequencies in Bloom syndrome and X-irradiated B-lymphoblastoid cell lines originating from acute leukemia

Cell hybridization and co-cultivation protocols have been used to determine whether the increased rates of sister-chromatid exchanges (SCEs) exhibited by Bloom syndrome (BS) and a human mutant cell line (CCRF-SB-T1), originating from an X-irradiated acute leukemia-derived B-lymphoblastoid cell line, have the same or different bases. Cell fusion of CCRF-SB-T1 with each of 4 different BS B-lymphoblastoid cell lines (LCLs), retaining a high-SCE character, exhibited low (normal level) numbers of SCEs, signifying complementation. Co-cultivation of CCRF-SB-T1 and BS B-LCLs also resulted in a significant reduction in SCE level, from 70 to 35, in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential staining (SCD) from 15 micrograms/ml (0.05 mM) to 2.0 micrograms/ml (0.01 mM) and resulted in a completely normal level of SCE in CCRF-SB-T1 cells. This strongly suggests that the defects in the 2 cell types are different. In the assay of cell extracts, the 4 BS cell lines appear to have lost thymidylate (TMP) synthetase activity (about 50% reduction from that of normal cells), whereas CCRF-SB-T1 cells show a 20% increase of TMP synthetase activity compared to normal cells.     

Restriction endonucleases do not induce sister-chromatid exchanges in Chinese hamster ovary cells

Bacterial restriction enzymes offer the unique opportunity to determine the biological and cytogenetic consequences of DNA double-strand breakage. To examine the role of various types of breaks in sister-chromatid exchange (SCE) formation, we used restriction enzymes with different recognition sequences and different cutting frequencies to generate DNA double-strand breaks in Chinese hamster ovary (CHO) cells. The restriction enzymes were introduced by electroporation into exponentially growing cells during the second replication cycle in bromodeoxyuridine, and SCEs were analyzed at mitosis. Contrary to results reported by others, we found no increase in SCE frequency in cells exposed to restriction enzymes despite the presence of numerous cells with chromatid aberrations. These data suggest that DNA double-strand breaks do not lead to SCE formation.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

Bloom syndrome B-lymphoblastoid cells are hypersensitive towards carcinogen and tumor promoter-induced chromosomal alterations and growth in agar.

The effects of the carcinogens (4NQO, 4-nitroquinoline-N-oxide; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; AFLG1, aflatoxin G1; AFLB1, aflatoxin B1; BNU, butylnitrosourea; MNU, methylnitrosourea) and the tumor promoter (TPA, 12-O-tetradecanoylphorbol-13-acetate) on sister chromatid exchanges (SCE), chromosome aberrations and colony formation (CF) were examined in three types of Bloom syndrome (BS) B-lymphoblastoid cell lines (B-LCLs); type I with normal SCE and normal karyotype; type II with high SCE and normal karyotypes; type III with high SCE and abnormal karyotypes. BS type I cells had the same SCE and CF response as normal cells to these carcinogens and TPA. In BS type II and III cells treated with carcinogens the SCE frequency increased to 140/cell from a baseline of 70/cell versus an increase of only 10/cell in normal cells. Colony formation occurred at the concentrations that caused the highest SCE. TPA caused a significant SCE increase and highly enhanced CF with dose dependency only in type III cells, suggesting that type III cells may be already in a pre-malignant state; type II cells appear to be one step behind those of type III in the process of becoming malignant. BS type II and III cells may be usable to establish a sensitive system to detect SCE-inducing agents.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

The interrelation between changes in the structural organization of replicon clusters, a retarded fork displacement rate and the high level of spontaneous SCEs in form II of xeroderma pigmentosum]

A cytogenetic observation, that the sister chromatid exchanges (SCE) occur 3 times more frequently in a special form of xeroderma pigmentosum--XPII than in the norm, prompted a study of DNA replication in this rare disease. Using DNA fiber autoradiography, the rate of fork movement and the frequency of initiation in the adjacent clusters of replicons were estimated. The rate of fork movement was significantly slower than that in classical XP and in normal cells. Here evidence was provided on another defect in DNA replication in XPII that involves a significantly decreased number of simultaneously operating adjacent clusters of replicons, which results in a decreased rate of DNA chain-growth. According to the Painter replication model for SCE, the exchanges arise due to double-strand DNA breaks occurring on the border between two adjacent clusters, respectively, completely and partially replicated. A retarded fork-displacement rate together with a decreased rate of DNA-chain growth may cause this situation to persist longer than in the norm. Thus, our data provide a further support of the replication model for SCE. A similar combination of cytogenetic and molecular defects has been obtained earlier in the Bloom syndrome cells.     

Racial differences in alcohol sensitivity: A new hypothesis

Summary Cocultivation of fibroblast cells from a male patient with Bloom syndrome (BS) and a female control reduced the rate of sister chromatid exchanges in the BS cells from a mean of 54 SCE per metaphase (range 42–65) to 41 (range 24–59). Medium used to culture control cells for 48 h also reduced the rate of SCE (from 40–65 to 33–54), whereas medium used for only 24h altered the SCE rate only slightly (to 39–61). Dialyzed medium concentrate with molecular cutoff at 15,000 did not alter the SCE rate. These initial studies suggest that normal cells produce an agent, presumably lacking in BS cells, that is capable of mitigating the chromosomal manifestation of the BS mutation (bl) in bl/bl cells.