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Mutation Research, 62 (1979) 341--353

© Elsevier/North-Holland Biomedical Press

U L T R A V I O L E T MUTAGENESIS O F N O R M A L AND X E R O D E R M A PIGMENTOSUM V A R I A N T H U M A N F I B R O B L A S T S

BRIAN C. MYHR *, DUNCAN TURNBULL and JOSEPH A. DiPAOLO ** Laboratory of Biology, National Cancer Institute, Bethesda, MD 20205 (U.S.A.)

(Received 22 September 1978) (Revision received 17 April 1979) (Accepted 1 May 1979)

Summary The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts b y ultraviolet light (UV) were compared under conditions of m a x i m u m expression of the 6-thioguanine resistance (TG r) phenotype. Selection was with 20 pg TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions o f the UV dose b u t the slope of the line for normal cells extrapolated to zero induced mutants at 3 J / m 2. The postreplication repair-defective XP4BE cells showed a higher frequency of TG r colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TG r colonies was n o t a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than a b o u t 50%. The UV doses and induced m u t a t i o n frequencies corresponding to 37% survival of cloning abilities were 6.7 J / m 2 and 6.2 × 10 -s, respectively, for normal cells and 3.75 J / m 2 and 17.3 × 10 -s for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.

* P r e s e n t a d d r e s s : D e p a r t m e n t o f G e n e t i c s a n d Cell B i o l o g y , L i t t o n - B i o n e t i c s , I n c . , 5 5 1 6 N i c h o l s o n Lane, Kensington, MD 20795 (U.S.A.). ** S e n d R e p r i n t R e q u e s t s t o : D r . J . A . D i P a o l o , L a b o r a t o r y o f B i o l o g y , N a t l . C a n c e r I n s t . , B u i l d i n g 37, R o o m 2 A - 1 9 , B e t h e s d a , M D 2 0 2 0 5 ( U . S . A . ) . A b b r e v i a t i o n s : A Z G , 8 - a z a g u a n i n e ; CE, c l o n i n g e f f i c i e n c y ; F B S , f e t a l b o v i n e s e r u m ; H G P R T , h y p o -

xanthine--guanine phosphoribosyltransferase; LN, Lesch--Nyhan syndrome 6-thioguanine; TG r, TG s , thioguanine-resist ant, thioguanine-sensitive.

( H G P R T - cells); T G ,

342 Skin fibroblasts from xeroderma pigmentosum (XP) patients usually exhibit defective repair of UV-induced pyrimidine dimers in their DNA. In this genetic disease, the fibroblasts are deficient in excision repair [ 3,5 ] and accordingly are much more sensitive to the lethal action o f UV light than normal cells [1,2,15, 21]. Maher and McCormick [23] have reported that exposure to a given dose of UV induces a higher frequency of AZG-resistant colonies (presumptive H G P R T - mutants) than are induced in normal fibroblast cultures. The induced mutation frequencies of normal and XP cells were equal, however, when comparisons at equal survivals were made. These results suggested that excision repair in human cells is essentially error-free and that the UV-induced mutants arise from another, error-prone repair process. A variant form of XP exists in which excision repair occurs at normal rates [4,19], but postreplication repair is defective [20,22]. The joining of low molecular weight daughter DNA, newly synthesized from parental DNA containing pyrimidine dimers, into high molecular weight DNA occurs at a reduced rate. Maher et al. [26] have reported that these XP variant fibroblasts are more mutable per lethal event than normal fibroblasts. The implication is that postreplication repair is an error-prone process that in XP variant cells is altered to an even higher probability of error per lethal event. These results were obtained b y a mutagenesis assay procedure that could easily lead to an erroneous comparison of the mutabilities of XP and normal cells. Standard expression times corresponding to 3 population doublings after UV-light treatments were used in an in situ procedure in which the induction of mutants and their selection are performed in the same culture dish. We have previously shown with Chinese hamster cells [29] that metabolic crossfeeding between wildtype H G P R T ÷ and induced H G P R T - phenotypes [8,14] may seriously influence the m u t a n t frequencies obtained by this method. Reconstruction experiments with LN fibroblasts ( H G P R T - mutant p r o t o t y p e ) were performed to obtain an efficiency of mutant detection, b u t this correction factor can vary considerably between dishes [24] and does n o t account for losses from metabolic crossfeeding within mosaic colonies of H G P R T ÷ and H G P R T cells. Furthermore, recent reports of a requirement for 6--16 population doublings after mutagenic treatment o f diploid human lymphoblasts in order to achieve maximum expression of the TG r p h e n o t y p e [34,35], raises the question of whether XP and normal cells were compared under conditions of complete expression. This study re-examines, b y entirely different mutant selection conditions, whether XP variant cells are more mutable b y UV than normal fibroblasts. Cells were reseeded at various times after UV irradiation to avoid crossfeeding problems and to demonstrate complete expression, and presumptive mutants were selected with TG at a concentration of the purine analog 10-fold higher than generally used by other investigators for human fibroblasts [7,18,26]. Materials and m e t h o d s Cell strains. Human diploid fibroblasts derived from skin biopsies were obtained from the American Type Culture Collection (Rockville, MD). The normal strain is designated CRL 1220; the XP4BE variant, CRL 1162; and the

343 Lesch--Nyhan syndrome cells, CRL 1110. The cells were obtained at passage Nos. 7--8 and larger stocks at passages 9--10 were stored in liquid nitrogen for experimental use. All strains were supplied mycoplasma-free and additional culturing tests of our cell stocks b y Flow Laboratories (Rockville, MD) were negative for mycoplasma. Culture media. Cell stocks were cultured in Ham's F10 medium lacking hypoxanthine and thymidine (powder supplied b y GIBCO, Grand Island, NY). The medium was supplemented with 10% fetal bovine serum (GIBCO) and 100 unit/ml o f penicillin G and 100 pg/ml of streptomycin. Cultures were seeded at 106 cells in 100-mm culture dishes with 20 ml medium and incubated at 37°C in a humidified atmosphere containing 5% CO2. These cultures were generally refed at 3 days and subcultured at 4--5 days. Cells were dispersed for subculture b y washing with Hanks' balanced salt solution and treating with 0.05% trypsin and 0.01% EDTA for a b o u t 4 min; cell counts were determined b y hemocytometer. UV irradiation. Cells attached to plastic culture dishes were exposed to a single 15-W germicidal lamp (G.E. G15T8) at a distance of 70 cm. The lamp was aged at least 100 h, prewarmed 15--20 min before each use, and operated at a constant current of 250 mA. The incident fluence rate at the cell surface was 0.78 J/m2/sec as determined b y potassium ferrioxalate dosimetry [9]. Survival to UV. Survival to UV irradiation was measured as the colonyforming ability of irradiated cells relative to the cloning efficiency of untreated controls. Appropriate numbers o f cells (200--5000) were seeded in 100-mm dishes a b o u t 7--10 h prior to irradiation to allow attachment and spreading o f the cells. For each experiment, the medium was completely aspirated from a series of 5 dishes for each dose and the dish cover removed during the irradiation. Growth medium was replaced, the cultures returned to the incubator, and the colonies refed every 7 days until staining at 16--18 days. Cloning efficiencies for the cells ranged from 22 to 38% for the normal 1220 strain and from 27 to 41% for the XP4BE strain. UV mutagenesis and selection. Cells from 4

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts.

341 Mutation Research, 62 (1979) 341--353 © Elsevier/North-Holland Biomedical Press U L T R A V I O L E T MUTAGENESIS O F N O R M A L AND X E R O D...
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