Conversion of replicative intermediates in human DNA-repair defective cells

We have examined the conversion of intermediates of DNA replication in normal human skin fibroblasts and fibroblasts isolated from patients with genetic diseases caused by putative DNA repair defects. Experiments were performed in non-transformed, unchallenged cells using alkaline sucrose sedimentation analysis to demonstrate precursor low molecular weight (LMW) DNA molecules which converted into high molecular weight (HMW) DNA with time. Analyses of conversion of replicative intermediates were conducted in cells from patients with ataxia telangiectasia (AT), Fanconi anemia (FA), Bloom syndrome (BS), Cockayne syndrome (CS) and xeroderma pigmentosum (XP). Our studies show that conversion of replicative intermediates occurs in all cell strains examined. However, XP cells (complementation groups A and E) show evidence of abnormalities in the conversion of LMW replicative intermediates, with the most dramatic alterations shown by cells from complementation group A.     

Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts.

Six strains of Bloom syndrome (BlS) fibroblasts responded to co-cultivation with normal fibroblasts at a 1:2 ratio by a reduced rate of sister chromatid exchanges (SCE's) from a mean of 67.5 (range = 59--78) to 28.4 (range = 21--35). The response was dose-dependent in one strain tested at 1:2, 1:1, and 2:1 ratios. In addition, quadriradial exchange figures and other signs of increased chromosomal instability were not found in BlS cells following co-cultivation with control cells. Control cells did not respond to BlS cells and maintained a normal rate of SCEs. Culture medium conditioned for 48 hrs by normal fibroblasts could also reduce the rate of SCEs in BlS fibroblasts, but less than in co-cultivation. We suggest that the reduced rate of SCEs and the lack of chromosomal instability in BlS cells following co-cultivation represent a corrective effect that is related to the basic defect and not dependent on cell-to-cell contact.     

Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

Reduced uptake and incorporation of 3H-thymidine in fanconi anemia fibroblasts

Summary Uptake of ³H-thymidine and its incorporation into DNA was studied in fibroblastic cell lines derived from normal individuals, patients with Fanconi anemia, and those heterozygous for this genetic trait. Uptake and incorporation for the normal cells were about five and seven times higher, respectively, than for Fanconi anemia fibroblasts; mean values for heterozygotes were intermediate. This effect was dependent on the duration of cell exposure to ³H-thymidine and was not observed with other labeled compounds. Thus, a genetically-determined metabolic defect may exist in Fanconi anemia patients which can be readily studied at the cellular level. This finding may be relevant to the observed clinical, cytogenetic, biochemical, and biologic properties related to expression of the Fanconi anemia gene.     

Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.     

Transformation of DNA repair-deficient human diploid fibroblasts with a simian virus 40 plasmid

Fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and G, as well as Bloom syndrome (BS) and Fanconi anemia (FA) have been transfected with a plasmid, pSV7, containing the early region of Simian virus 40 (SV40). All of the cultures exhibited cytologic changes characteristic of transformed cells and expressed T-antigen. They also contained integrated copies of DNA derived from the vector, and in several cases, extrachromosomally replicated DNA. Not all of the transfected cultures became immortalized. The transformed xeroderma pigmentosum (XP) cultures retained their UV-sensitive phenotype in all but one case. The BS and FA cell lines retained their characteristic phenotype. All of the cultures, except the BS cells, can be readily transfected with the plasmids, pSV2neo and pSV2gpt.     

Diphtheria toxin resistance in human fibroblast cell strains from normal and cancer-prone individuals

Mutants resistant to diphtheria toxin (Dip^r) have been selected from a variety of human fibroblast cell strains derived from both normal subjects and individuals with known genetic predisposition to cancer such as xeroderma pigmentosum, Fanconi anemia and Bloom's syndrome. Treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) led to a marked increase in the frequency of Dip^r mutants in various cell strains. The increase in the frequency of Dip^r mutants occurred in a linear dose-dependent manner in response to MNNG and ethyl methanesulfonate, in one of the cell strains examined. The rate of muation to diphtheria toxin as determined by fluctuation analysis was very similar in various cell strains (1–3 × 10^?7 mutations/cell/generation), except for the strain GM1492 (8.8 × 10^?7 mutations/cell/generation) which is derived from a Bloom syndrome patient.     

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.     

Cytogenetic sensitivity of G0 lymphocytes of Fanconi anemia patients and obligate carriers to mitomycin C and ionizing radiation

It is well known that Fanconi anemia (FA) patients show a hypersensitivity to the effect of cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), while the sensitivity of these patients to ionizing radiation is still controversial. Fanconi anemia heterozygotes do not show a hypersensitivity to the above mentioned agents compared to normal individuals. To examine the radio-sensitivity of Fanconi anemia patients and heterozygotes, ten patients and 13 heterozygotes were enrolled in this study. Standard metaphase analysis for detection and verification of radio-sensitivity was used to establish the relationship between gamma-ray and chromosome breakages in these groups. Statistical analysis was used for the assessment of aberrations including chromatid and chromosome breaks and exchanges. Results of chromosome aberration yield that: (i) differentiation between obligate carriers and the control group after MMC treatment and gamma irradiation was not possible; (ii) homozygotes were clearly distinguishable from heterozygotes and controls after MMC treatment; (iii) FA patients don't show hypersensitivity to gamma irradiation compared to normal controls and heterozygous carriers.     

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.     

Roles of DNA interstrand crosslinking and its repair in the induction of sister-chromatid exchange and a higher induction in fanconi's anemia cells

The roles of DNA crosslink and its repair in the induction of sister-chromatid exchanges (SCEs) were studied in normal, xeroderma pigmentosum (XP) complementation group A, and Fanconi's anemia (FA) fibroblasts after treatment with mitomycin C (MC) or decarbamoyl mitomycin C (DMC) for 1 h. FA strains were 5—30-fold more sensitive to MC killing than normal cells, but normally responded to DMC killing. XP group-A cells were twice and only slightly more sensitive to DMC and MC killings, respectively, than normal cells. The induction rate of immediate SCEs by MC was 1.7 times higher, despite a normal SCE rate by DMC, in FA strains than that in normal cells. Alternatively, SCE rates by DMC and MC were 6 times and only 1.3 times higher, respectively, in XP cells than in normal cells. In normal cells, the reduction of MC-induced SCEs as a function of repair time followed a biphasic curve of the first rapid (half-life, 2 h) and the second slow (half-life, 14 h) components. Such components corresponded exactly to the first half-excision and the second slow repair processes of molecular crosslink repair. In MC-induced SCEs, FA17JTO cells exhibited only the slow reduction component without the first rapid component and a higher saturation level in the time-dependent reduction in SCEs. This indicates that SCEs are produced by crosslinks remaining unrepaired for long times (24—48 h) after treatment of FA cells. Conversely, XP group-A cells capable of the first half-excision manifested the first rapid reduction in SCEs, although the second component declined at the slowest rate (half-life, 48 h) owing to a defect in the second mono-adduct repair. The reduction in DMC-induced SCEs followed only the slow component. Thus, these results demonstrate that crosslink can be the lesion leading to SCE, and the MC-induced SCE frequency is higher in FA cells than in normal cells. In the FA20JTO strain, such a repair defect seemed to be less than in FA17JTO cells, judged from the survival and SCE characteristics.     

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.     

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.     

Heterozygous carriers for Bloom syndrome exhibit a spontaneously increased micronucleus formation in cultured fibroblasts

Summary Cultured fibroblasts of homozygotes and heterozygotes for Bloom syndrome exhibit an enhanced formation of micronuclei. The number of spontaneously occurring micronuclei permit clear separation of heterozygotes from either normal controls or homozygous patients without overlap between these groups. The observed differences could not be enhanced further by the addition of various mutagens. We conclude from the increased chromosomal damage that heterozygotes for Bloom syndrome may have a higher risk for malignant diseases.     

Suboptimal action of NF-kappaB in Fanconi anemia cells results from low levels of thioredoxin

Electrophoretic mobility shift assays (EMSA) revealed that under standard cell culture conditions NF-kappaB was induced in Fanconi anemia fibroblasts in contrast to control cells. Dithiothreitol, a potent synthetic redox potential-delivering compound, when added to growing cells, prevented this induction of NF-kappaB and, simultaneously, chromosomal instability was reduced. Fanconi anemia cells possess low endogenous levels of the naturally occurring antioxidant thioredoxin. Transfection of Fanconi anemia cells with thioredoxin cDNA containing a nuclear localization signal prevented both spontaneous as well as mitomycin C-induced chromosomal instability. A promotor construct with two NF-kappaB binding sites in front of the CAT gene induced little CAT expression in cells with low thioredoxin content in spite of induced NF-kappaB. In cells with higher thioredoxin content CAT expression was increased. Cotransfection of the NF-kappaB-dependent CAT plasmid with the Trx/nuc-plasmid into FA fibroblasts increased the CAT expression to almost that of control cells, indicating that in this model system with diminished thioredoxin content NF-kappaB requires thioredoxin for binding to its specific promotor. Since Fanconi anemia cells have low thioredoxin contents, NF-kappaB-dependent genes are expressed insufficiently. This explains part of the pathophysiological processes observed in Fanconi anemia.     

UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes

UV-induced DNA repair synthesis, as measured by autoradiography as well as by isopycnic centrifugation methods, was studied in a large number of cell strains from patients with the classic form of xeroderma pigmentosum (XP) or the De Sanctis-Cacchione syndrome (DSC) and several of their heterozygous parents. On the basis of the kinetics of repair synthesis in the cultured skin fibroblasts we have recognized four distinct groups of XP patients: (1) classic XP patients with low residual repair capacities, (2) classic XP patients with intermediate, but dose-dependent, levels of repair synthesis relative to the normal level, (3) patients, diagnosed as having classic XP, with a normal or only slightly reduced repair capacity and (4) DSC patients with a complete deficiency of repair synthesis. Complementation studies reported elsewhere have shown that different mutations are responsible for the detect in at least three of these groups. Cell strains of each of the four XP types were able to rejoin single-strand DNA breaks induced by X-rays. Most of the cell strains derived from heterozygotes showed normal repair activities. However, in the parents some of the of DSC-patients a significant reduction of the level of repair synthesis was found.     

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.     

Suppressing effect of tannic acid on the frequencies of mutagen-induced sister-chromatid exchanges in mammalian cells

The effects of tannic acid (m-galloyl gallic acid) and 7 of its analogues on the frequencies of sister-chromatid exchanges (SCEs) were investigated in cultured Chinese hamster cells. SCEs induced by UV-light or mitomycin C (MMC) were suppressed by post-treatment with tannic acid and 5 of its analogues. These effects were independent of the extension of the cell cycle. The compounds which showed an SCE-suppressing effect have a common structure of 3 neighboring hydroxy or methoxy groups substituted on the phenyl group in benzoic acid or ester. These decreasing effects of tannic acid were observed in the G1 phase but not in the S or G2 phase of the cell cycle and a greater decline of the frequencies of UV-induced SCEs during liquid holding was seen in the presence of tannic acid. However, cells irradiated with X-rays were not influenced by tannic acid. In cells from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient, and a normal human embryo, MMC-induced SCEs were also decreased by post-treatment with tannic acid. Tannic acid reduced the SCE frequencies in UV-irradiated FA and normal human cells but not in UV-irradiated XP cells. Our results suggest that tannic acid modifies DNA-excision repair and that the decrease in the amount of unrepaired DNA damage might cause the reduction of induced SCEs.     

Mutagen-induced sister chromatid exchange rate in Bloom syndrome remains unaltered in the presence of Bloom corrective factor

Summary Fibroblasts of a patient with Bloom syndrome (GM-1492) were cultured in the presence of either mitomycin C, ethylmethanesulfonate, or 4-nitroquinoline-1-oxide, (4-NQ1-O) and sister chromatid exchange was determined. The mutagens enhanced the sister chromatid exchange rate to different degrees, 4-NQ1-O being the most potent substance. Bloom corrective factor, which is present in normal cell-conditioned culture medium, reduced the spontaneously increased SCE in Bloom syndrome cells by about 20 SCE per metaphase but failed to reduce the additional mutagen-induced SCE increase. These findings indicate that only spontaneously, but not mutagen-indeuced, SCE in Bloom syndrome fibroblasts can be decreased by the Bloom corrective factor.