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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.

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.