Fanconi anemia cells have a normal gene structure for topoisomerase I

Cells from Fanconi anemia (FA) patients have defective DNA repair and are hypersensitive to DNA crosslinking agents such as mitomycin C (MMC). We examined the possibility that topoisomerase I is involved in the DNA crosslink repair system and is deficient in FA group A cells. FA cells and control cells were exposed to MMC with or without camptothecin (CPT), a topoisomerase I inhibitor. The cells did not show any increased sensitivity to killing by MMC with CPT, suggesting that the topoisomerase I is not involved in MMC-damaged DNA repair. However, FA cells showed increased sensitivity to CPT in comparison to control cells, raising the possibility of altered topoisomerase I in FA cells. Therefore, a mutation analysis was performed on topoisomerase I cDNA from FA cells by using chemical cleavage mismatch scanning and nucleotide sequencing. No mutation was detected from GM1309, a group A FA cell line. A base transition (C to T) at position 241, causing an amino acid change (His to Tyr), was found in GM2061, a FA cell line of unknown complementation group. However, allele-specific oligonucleotide hybridization analysis showed that this is a gene polymorphism. We conclude that FA cells have normal gene structure for topoisomerase I.     

Follow-up analysis of translocation and dicentric frequencies, measured by FISH-chromosome painting in breast cancer patients after partial-body radiotherapy with little bone marrow exposure

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (? = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (? = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.     

The Chinese hamster cell mutant V-H4 is homologous to Fanconi anemia (complementation group A)

V-H4, a mitomycin C (MMC)-sensitive Chinese hamster cell mutant, is phenotypically very similar to Fanconi anemia (FA) cells. Genetic complementation analysis shows that V-H4 belongs to the same complementation group as FA group A cells. Proliferating hybrid cell lines obtained after fusion of V-H4 with normal or FA group B cells show an increased resistance to MMC. Absence of complementation was noted in V-H4 x FA group A hybrid cell lines. This was shown not to be due to the absence of a specific human chromosome. The V-H4 mutant represents the first rodent mutant that is genotypically similar to FA complementation group A cells.     

Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

The fate of 8-methoxypsoralen-photoinduced DNA interstrand crosslinks in Fanconi's anemia cells of defined genetic complementation groups

The fate of 8-methoxypsoralen (8-MOP)-photoinduced DNA interstrand crosslinks was followed by alkaline elution in Fanconi's anemia (FA) fibroblasts belonging to complementation groups A (FA 150 and FA 402) and B (FA 145) in comparison to a normal (1 BR/3) and a heterozygote (F 311) cell line. Clonogenic cell survival to 8-MOP photoaddition was established in parallel for all cell lines. In comparison to normal cells, group A FA cells demonstrated a higher photosensitivity than group B cells (sensitivity index 2.3 and 1.5, respectively), the heterozygote cell line being only slightly more sensitive. FA cells from both groups A and B demonstrated an incision capacity of crosslinks, the kinetics and extent of which being, however, different from that of normal or heterozygote cells. The incision is slower in FA cells and, at 24 h of post-treatment incubation, the amount of crosslinks incised is clearly lower than that observed in normal cells for group A cells, whereas in group B cells incision approaches the level of normal cells. These results correlate with survival as well as with rates of DNA semi-conservative synthesis after 8-MOP photoaddition.     

Alteration of a DNA-dependent ATPase activity in xeroderma pigmentosum complementation group C cells.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks by fast protein liquid chromatography Mono Q column chromatography. Similar elution profiles were observed with the extracts from human cells normal in repair and xeroderma pigmentosum cells belonging to complementation groups A through G except for group C. An alteration in elution of one of the five ATPases, designated DNA-dependent ATPase Q1, was observed with a cell line of complementation group C. This alteration was observed with all tested cell lines that belonged to group C. ATPase Q1 in HeLa cell extracts exhibited about 2-fold higher activity with ultraviolet light-irradiated DNA as compared to that with non-irradiated DNA, whereas little difference in the effects of two DNAs was observed with the ATPase activities in the extract from group C cells.     

Chromosomal instability of fanconi anemia cells is not the consequence of a defective repair activity of the ribosomal protein S3.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder characterized by developmental defects, hypersensitivity toward oxygen and DNA crosslinking agents, and susceptibility to cancer. An increased level of reactive oxygen intermediates and an increased level of 8-oxoguanine in FA cells point to a defective oxygen metabolism. Recent investigations showed that FA cells from several complementation groups have a reduced capacity to repair oxidatively damaged DNA. One major enzyme involved in the repair of oxidative DNA lesions is the ribosomal protein S3. Previous reports implied a role for the ribosomal protein S3 in DNA repair in FA cells. However, a more detailed analysis of the ribosomal protein S3 in FA cells from complementation groups A-E could not confirm this. DNA analysis and Western blot analysis did not show significant differences in ribosomal protein S3 between FA cells and cells from healthy individuals. Furthermore, even the overexpression of the ribosomal protein S3 did not reduce the chromosomal instability of FA cells.     

Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.     

Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.     

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA

Summary Transfectants obtained by mouse DNA-mediated gene transfer in Fanconi anemia (FA) primary fibroblasts from the genetic complementation groups A and B were examined for the frequencies of chromosomal aberrations and cytotoxicity following treatments by cross-linking agents. Cells from group A (FA 150), which is the most sensitive to such agents, are partially corrected for both the chromosomal and cellular hypersensitivity to 8-methoxypsoralen photoaddition. In contrast, after treatment with mitomycin C (MMC), only the chromosomal sensitivity is re-established to a near normal level. The opposite is true for FA group B cells (FA 145), i.e. cell survival to MMC is partially corrected, whereas the frequency of MMC-induced chromosomal aberration remains close to that of the untransfected cells. The partial phenotypic correction of the two end points examined is interpreted as indicating either a gene dosage effect or the necessity of introducing more than one gene type in order to achieve complete recovery of a normal phenotype. The phenotypic dissociation between the clastogenic and cellular hypersensitivity to crosslinking agents may offer the opportunity of isolating separately the responsible gene(s) by conventional rescue techniques.     

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.     

Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Deficiency of DNA ligase activity in Fanconi's anemia

Summary A significant decrease in DNA ligase activity was observed in lymphocytes and fibroblasts of a patient with Fanconi's anemia (FA). This decrease is related to the observed DNA repair deficiency indicated by the delayed closing of repair DNA strands following UV irradiation. Other steps of DNA repair were analyzed in the FA fibroblasts, including endonucleolytic incision of DNA, repair DNA synthesis, and exonucleolytic removal of the photoproducts. No differences were found against control cells. The action of DNA ligase is delayed during replication in the FA cells, as seen by an accumulation of replicative intermediates.     

The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.