Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome.

The hereditary dysplastic nevus syndrome (DNS) is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS have been shown to be hypermutable to UV radiation (M.I.R. Perera et al., Cancer Res., 46: 1005-1009, 1986). To examine the mechanism involved in this UV hypermutability, we used a shuttle vector plasmid, pZ189, which carries a 160-base pair marker gene, supF, and can replicate in human cells. pZ189 was treated with UV radiation and transfected into DNS6BE, a lymphoblastoid cell line from a patient with hereditary DNS. Plasmid survival after UV was similar with the DNS6BE line and with a lymphoblastoid cell line from a normal donor. Plasmid mutation frequency was greater with the DNS line in accord with the DNS cellular hypermutability. Base sequence analysis was performed on 69 mutated plasmids recovered from the DNS line. There were significantly more plasmids with single base substitution mutations (P less than 0.01) in comparison to UV-treated plasmids passed through normal fibroblasts. pZ189 hypermutability and an increased frequency of single base substitutions was previously found with a cell line from a melanoma-prone xeroderma pigmentosum patient. These differences may be related to the increased melanoma susceptibility in both DNS and xeroderma pigmentosum.

Hereditary dysplastic nevus syndrome: lymphoid cell ultraviolet hypermutability in association with increased melanoma susceptibility.

The hereditary dysplastic nevus syndrome (DNS) is a well-characterized disorder in which affected individuals have increased numbers of premalignant (dysplastic) nevi and a markedly increased risk of developing cutaneous melanoma. Seeking evidence of a systemic disorder in DNS, we examined the effect of ultraviolet radiation on cultured lymphoid cells. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS had similar survival values following treatment with 2.3 to 9.0 J of 254-nm ultraviolet radiation per m2 as did lines from control individuals. Mutagenesis at the hypoxanthineguanine phosphoribosyltransferase locus was assessed by measuring the induction of resistance to thioguanine using a microtiter well assay. Three lymphoblastoid cell lines from patients with hereditary DNS and melanoma had a 2- to 3-fold greater frequency of induced mutants per clonable cell than three normal lines following exposure to 4.5 to 9.0 J of ultraviolet radiation per m2. Expanded clones of mutated DNS lymphoblastoid cell lines had less than 6% of normal hypoxanthine-guanine phosphoribosyltransferase activity. Inhibition and recovery of DNA synthesis following ultraviolet exposure were similar in 2 DNS and 2 normal lines. Repair by DNS lines of ultraviolet-induced DNA damage was in the normal range as measured by alkaline elution. Thus, hereditary DNS exhibits in vitro hypermutability which may reflect increased susceptibility to ultraviolet-induced somatic mutations in vivo. This abnormality may be related to the increased melanoma susceptibility of patients with hereditary DNS.

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.

Ultraviolet hypermutability of a shuttle vector propagated in xeroderma pigmentosum variant cells.

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.

Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts.

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ189, 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.

Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells.

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.

Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells.

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.

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation.

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultraviolet 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 survival or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01-1.0 microgram/ml) for 3 different UV-A doses (3000-15 000 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.

Effects of psoralens plus ultraviolet radiation on human lymphoid cells in vitro.

Photochemotherapy with oral 8-methoxypsoralen (8-MOP) plus long wavelength UV radiation (UVA) has been shown to affect lymphoid cells circulating through the skin. An in vitro assay was developed to mimic some of the therapeutic parameters of 8-MOP concentration and UVA exposure estimated to impinge upon lymphoid cells. In vitro treatment with these presumed therapeutic levels of 8-MOP plus UVA induced a level of inhibition of lymphoid cell DNA synthesis similar to that observed in vivo. Furthermore, the DNA synthesis inhibition was associated with DNA interstrand cross-link induction, reduced cell survival, and impaired immune reactivity. This assay predicts that such effects would be induced in vivo. A lymphoblastoid cell line from a patient with Cockayne's syndrome was shown to be hypersensitive to killing by radiation (280-320 nm) from a fluorescent sunlamp (UVB) but to have normal survival after treatment with 8-MOP plus UVA. Thus there is at least one major UVB recovery pathway in human cells that is different from the recovery pathway for 8-MOP plus UVA damage and leads us to believe that combined treatment with UVB and 8-MOP plus UVA may have a greater effect than either treatment alone.