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.     

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.     

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.     

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.     

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.     

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.     

A unique human mutant B-lymphoblastoid cell line (ataxia telangiectasia) which exhibits increased siste-chromatid exchange retaining hypersensitivity to neocarzinostatin and bleomycin

Chromosome aberrations and sister-chromatid exchanges (SCEs) were examined in 4 ataxia telangiectasia (AT)-derived B-lymphoblastoid cell lines (B-LCLs) (AT-S, AT-SHI, AT-SHI B13A and AsHa) following treatments with neocarzinostatin (NCS) and bleomycin. All of these cell lines exhibited extremely high frequencies of chromosome aberrations with the NCS and bleomycin treatments. Among them, AsHa, a mutant B-LCL originating from an AT patient, showed high frequencies of SCEs under high bromodeoxyuridine (BrdU) concentrations retaining hypersensitivity to NCS and bleomycin with regard to chromosome aberrations. A clear BrdU dose-dependent increase in SCEs (9.85 SCEs/cell at 40 μg/ml, 36.65 SCEs/cell at 100 μg/ml on average) in this mutant was observed. When AsHa mutant cells were treated with NCS (0.02 μg/ml) and/or bleomycin (5.0 μg/ml) under 40 μg/ml BrdU (minimum BrdU concentration for sister-chromatid differential staining), SCE levels increased from 9.85 (baseline level) to 21.1 with NCS and 20.5 with bleomycin, in a dose-dependent manner. These observations indicate that AsHa is a unique AT-derived mutant cell clone with a high SCE character retaining the original hypersensitivity to bleomycin and NCS.     

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.     

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Paradoxical changes in SCE frequencies persistently elevated in vivo, on exposure to a mutagen in vitro

Lymphocyte cultures from 4 individuals with persistently significantly elevated frequencies of sister-chromatid exchange (SCE) were examined with no treatment, and with 2 concentrations of mitomycin C. In each of the 4 cases, the mean level of SCEs in the untreated lymphocytes exhibited a paradoxical reduction in SCE frequency when exposed to the lower (0.005 microgram/ml) of the two doses of mitomycin C. At the second higher dose of mitomycin C (0.025 microgram/ml) the mean level of SCE/cell exceeded the untreated mean. When the distributions of SCE/cell were examined it appeared that the untreated cultures had two or more populations of cells; one was in the normal SCE frequency range, while the second population was in an elevated SCE frequency range. The paradoxical reduction in SCE frequency was apparently due to elimination of, or mitotic inhibition of cells in the highest range of SCE frequency, while a small elevation in SCEs was initiated in the cells with a normal SCE frequency. Thus, mean levels of SCE/cell can be misleading. This data suggests that new exposure to the same or a different genotoxic agent might possibly result in a misleading lowering of the mean SCE frequency.     

Effects of theophylline on chromosomal breakage and sister-chromatid exchange

Frequencies of both sister-chromatid exchange (SCE) and chromosomal breakage (CB) were studied in the lymphocytes of normal individuals (10 and 7 individuals respectively). The cells were exposed in vitro to 3 different concentrations of theophylline (1, 10 and 100 micrograms/ml). A significant concentration effect of the drug was demonstrated for both SCEs and CB. Utilizing a Dunnett's test for individual comparisons, the 10 and 100 micrograms/ml concentrations both demonstrated a significant elevation of SCEs and CB compared to the untreated control cultures. This study suggests that in vitro concentrations of theophylline equal to or greater than 10 micrograms/ml, corresponding to serum levels attained during therapy, increase the frequency of SCEs and chromosome breakage in human lymphocytes.     

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.     

Transfection of BLM into Cultured Bloom Syndrome Cells Reduces the Sister-Chromatid Exchange Rate toward Normal

The gene BLM, mutated in Bloom syndrome (BS), encodes the nuclear protein BLM, which when absent, as it is from most BS cells, results in genomic instability. A manifestation of this instability is an excessive rate of sister-chromatid exchange (SCE). Here we describe the effects on this abnormal cellular phenotype of stable transfection of normal BLM cDNAs into two types of BS cells, SV40-transformed fibroblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Clones of BLM-transfected fibroblasts produced normal amounts of BLM by western blot analysis and displayed a normal nuclear localization of the protein by immunofluorescence microscopy. They had a mean of 24 SCEs/46 chromosomes, in contrast to the mean of 69 SCEs in controls transfected only with the vector. BLM-transfected fibroblast clones that expressed highest levels of the BLM protein had lowest levels of SCE. The lymphoblastoid cells transfected with BLM had SCE frequencies of 22 and 42 in two separate experiments in which two different selectable markers were used, in contrast to 57 and 58 in vector-transfected cells; in this type cell, however, the BLM protein was below the level detectable by western blot analysis. These experiments prove that BLM cDNA encodes a functional protein capable of restoring to or toward normal the uniquely characteristic high-SCE phenotype of BS cells.     

Effect of poly(ADP-ribose)polymerase inhibitors on the frequency of sister-chromatid exchanges in Bloom syndrome cells

The effect of inhibitors of poly(ADP-ribose)polymerase, benzamide (Bam) and m-aminobenzamide (m-AB), on sister-chromatid exchanges (SCEs) and cell growth, was examined in lymphoblastoid cell lines from a normal adult (KS-64) and from a Bloom syndrome patient (BS1-2). The presence of Bam and m-AB increased the levels of SCEs in KS-64 and BS1-2 lymphoblastoid cells. Though the net increase was similar in the two types of cell, the relative increase was much lower in the BS1-2 cells. Bam and m-AB increased the number of SCEs in BS1-2 cells to levels of 95.4 +/- 3.24 and 98.1 +/- 3.23 per cell, respectively, as compared with the baseline level of 75.5 +/- 2.16. On the other hand, when KS-64 cells were treated with Bam and m-AB, the number of SCEs increased to 27.1 +/- 1.98 and 28.6 +/- 2.71 per cell, respectively, compared with the baseline number of 6.7 +/- 0.41 per cell. These inhibitors of poly(ADP-ribose)polymerase also inhibited cell growth at concentrations which induced SCEs in KS-64 as well as in BS1-2 cells. No significant decrease in the poly(ADP-ribose)polymerase activity or in the amount of poly-(ADP-ribose) was detected in BS1-2 cells as compared with KS-64 cells. The mechanism by which SCEs are increased in BS1-2 cells is discussed.     

Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells

The basal levels of superoxide dismutase (SOD) activity and chromosome aberration (CA) and sister-chromatid exchange (SCE) frequencies were examined in cultured fibroblasts or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs). These cells were derived from patients with chromosome instability syndromes (CISs) including Bloom's syndrome (BS), Fanconi's anemia (FA) and ataxia telangiectasia (AT). Embryonal fibroblasts and LCLs from normal subjects served as controls. Although LCLs tended to exhibit a higher SOD level than fibroblasts due to an elevation of Cu/Zn-SOD activity, BS and FA fibroblasts with increased frequencies of CAs and/or SCEs showed abnormally elevated SOD activity due to the manifold increase of Mn-SOD levels compared with control cells. However, BS and AT LCLs with almost control levels of CA and SCE frequencies showed no, or a slightly elevated, SOD activity, suggesting a possible selection of such cells during EBV transformation. The observed parallelism between the SOD activity and the cytogenetic manifestation may imply an involvement of active oxygen species, especially superoxide radicals, in the increased chromosome damage of CIS cells.     

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.     

In vitro SCE discrepancy between embryonic and extraembryonic mouse tissues

In vitro sister-chromatid exchange (SCE) background levels and cytokinetics were compared in embryonic (whole embryo cell suspensions) and extraembryonic (yolk sac and amnion, placenta) cells of inbred and outbred strains at various gestational stages (days 12-17). Results indicate a tissue origin (embryonal, extraembryonal) related variation in the formation of baseline SCE frequencies and cytokinetics. The significant higher SCE levels in extraembryonic tissues (maximum increase of 2 X the background values of the embryo cells) were independent of mouse strain and gestational stage. An average of 4-5 SCEs/cell in embryo cells is contrasted by 7-9 SCEs/cell in extraembryo cells. Mitotic index was generally lower and average generation time longer (by 2-3 h) in extraembryonic tissue cells. No significant differences in SCE frequencies and no changes in cytokinetics were detected at the BrdU concentrations used (1.2-4.8 micrograms/ml). The reason for the inter-tissue differences in baseline SCE is still not clear.     

Anthramycin-induced sister-chromatid exchange and caffeine potentiation in the chromosomes of Indian muntjac

Cell-cycle kinetics, sister-chromatid exchange (SCE) and chromosome aberrations have been studied from the skin fibroblasts of the Indian muntjac after treatment with 100 micrograms/ml of caffeine and 0.05 microgram/ml of anthramycin. The cultures were incubated for a period which was sufficient for the completion of two consecutive cell cycles and both the drugs appeared to produce a slight inhibitory effect. When anthramycin-treated cells were however post-treated with caffeine, the cells did not proceed beyond one cycle and exhibited a mitotic block. The SCE frequency in the control and the experiments with caffeine and anthramycin was 8.63, 18.32 and 34.88 per cell respectively. The SCEs were randomly distributed amongst all chromosomes unlike a non-random distribution within the X chromosomes. Caffeine and anthramycin produced only 0.5% and 3.1 cells with chromosome aberrations respectively. Potentiation of chromosome aberrations was observed when the anthramycin-treated cells were post-treated with caffeine. Caffeine potentiation presumably results from an inhibition of the cells to cycle and a failure to repair the effect of the mutagen on DNA.     

Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines

Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells.Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.