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Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high frequency of skin cancer but, in contrast to the other forms of XP, have normal post-ultraviolet (UV) DNA excision repair and nearly normal post-UV survival. However, like excision repair-deficient XP cells, the XP variant cells are UV hypermutable. We used a UV-treated plasmid shuttle vector, pZ189, to examine the DNA repair defect in lymphoblastoid cells from an XP variant patient, XPPHBE, and a normal control. Plasmid repair, mutagenesis, and replication occur within transfected cells in a process dependent on the cells' repair capacity. With the XP variant cells post-UV, plasmid survival was normal with but there was an abnormally increased post-UV plasmid mutation frequency. Sequence analysis of the mutated plasmids revealed an increased frequency of plasmids with single base substitution mutations with the XP variant cells. As in earlier studies with UV mutagenesis, there was a predominance of G:C-->A:T base substitution mutations with plasmids recovered from both cell lines. The frequency of G:C-->C:G transversions was significantly higher with plasmids recovered from the XP variant cells than from normal cells. The location of mutations in the marker gene was non-random with different mutagenic hotspots found in plasmids recovered from the XP variant cells and from the normal cells. This study suggests that plasmid UV hypermutability in the presence of normal UV survival may be related to the increased UV skin cancer susceptibility of XP variant patients.

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ18, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control. XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E. coli . An earlier report on this data [Seetharam et al., (1990) J. Mol. Biol., 212, 433] indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line. In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C → A:T transitions with both lymphoblastoid cell lines. This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions). Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms. These results were compared to similar data from a fibroblast line from the same patient [Bredberg et al., (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 8273]. The frequency of G:C → A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts. There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts. This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar.

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.

Xeroderma pigmentosum (XP), is a rare autosomal recessive disorder with increased sun sensitivity, multiple pigmentary abnormalities and a more than 1000-fold increased frequency of cancer of the sun exposed portions of the skin and eyes. Cultured cells from XP patients are hypersensitive to the cytotoxic and mutagenic effects of UV radiation and have defective DNA repair (reviewed in Robbins et al, 1974; Kraemer and Slor, 1985; Kraemer, 1987; Cleaver and Kraemer, 1989).

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultra-violet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m2-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01–1.0 μg/ml) for 3 different UV-A doses (3000–15000 J/m2). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks. These results suggest that the cytotoxic effects of photoactivated 8-MOP in human lymphoblastoid cells may depend on DNA interstrand crosslinks.

The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. Reduction in the rate of DNA synthesis was not associated with the appearance of the lysine-rich histone variant H1°. We conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins.

The present studies explore some of the limitations and possibilities of using cultured mammalian cells in the analysis of xenobiotic metabolism by microsomal monooxygenases. A large variety of cells in culture contain monooxygenase activity which is inducible severalfold by polycyclic hydrocarbons. Presently the application of cultured cells to studies of drug activation is restricted mainly by two factors: Selection of cells with specific sets of enzymes involved in xenobiotic metabolism may be facilitated by somatic cell hybridization techniques. A beginning has been made with the preliminary assignment of a locus required for aryl hydrocarbon hydroxylase expression to human chromsome 2 in hybrids of RAG cells, an established mouse line, and normal human bone marrow cells.

Oral 8-methoxypsoralen (8-MOP) plus high-intensity long-wavelength ultraviolet radiation (UV-A) is used clinically to induce remissions of psoriasis and mycosis fungoides. Leukocytes in 8-MOP containing blood receive UV-A exposure when circulating through the dermis during therapy. The present study utilizes an in vitro assay system to permit quantitation and correlation of multiple biological and physical alterations in human lymphoid cells induced by 8-MOP plus UV-A treatment. Additive inhibition of lymphoid cell DNA synthesis by 8-MOP (0.01 to 1 μ g/ml) plus UV-A (1,000 to 29,000 J/m 2 ) was accompanied by a synergistic potentiation of cell killing in the therapeutic exposure range. Reduction in tritiated thymidine ( 3 HTdR) incorporation to 65–70% of control value was associated with normal survival; while 3 HTdR incorporation of less than 50% of control induced by any 8-MOP plus UV-A combination tested was associated with less than 10% survival, 8-MOP-DNA-cross-links were detected by the alkaline elution assay only when 3 HTdR incorporation was reduced to less than 50% of control. The relative number of crosslinks increased proportionately with further 8-MOP plus UV-A-induced reduction in 3 HTdR incorporation. 8-MOP plus UV-A induced at most approximately a 2-fold increase in sister chromatid exchanges (SCE) per chromosome in lymphocytes or lymphoblastoid cells. Increasing 8-MOP plus UV-A exposure resulted in marked toxicity with few cells progressing to second division metaphases and no further increase in SCE's per chromosome. Addition of 13-cis retinoic acid (1 μ g/ml) to the lymphoblastoid cells prior to 8-MOP plus UV-A treatment did not significantly alter the 3 HTdR incorporation or cell survival. These studies dermonstrate that in vitro exposure of human lymphoid cells to therapeutic levels of 8-MOP and UV-A may decrease cellular DNA synthesis, produce DNA 8-MOP interstrand cross-links, reduce cell viability and induce small increases in sister chromosome exchanges.

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which < 200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37 degrees with 5% CO2 at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appreciably after 2--3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.