Mutation Research, 284 (1992) 275-285 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

275

MUT 05186

Cellular responses to hematoporphyrin-induced photooxidative damage in Fanconi anemia, xeroderma pigmentosum and normal human fibroblasts S. Nocentini Institut Curie-Biologie, URA 1292 du CNRS, Paris, France (Received 15 July 1991) (Revision received 21 July 1992) (Accepted 23 July 1992)

Keywords: Antioxidant defense; Singlet oxygen; Fanconi anemia; Xeroderma pigmentosum

Summary Several observations reported in the literature suggest that singlet oxygen (10 2) might play a role in the clastogenic process in Fanconi anemia (FA) cells, and that the antioxidant status of xeroderma pigmentosum (XP) may also be altered. In order to test the ability of FA and XP cells, relative to normal ceils, to cope with 10 2 damage, the effects of photosensitization by hematoporphyrin (HP) have been determined (i) on host cell reactivation (HCR) of damaged infecting herpes simplex virus (HSV) or transfecting SV40 DNA, and (ii) on DNA template capability and clonogenicity of treated cells. Results showed no significant difference among the three types of cells, either for the survival of HP-photosensitized HSV, or for the yields of SV40 virus following transfection of cultures with damaged viral DNA. The treatment of cells with HP plus 365-nm light leads to a dose-dependent, homothetic reduction of 18S and 28S ribosomal RNA (rRNA) synthesis, presumably through a mechanism other than the formation of transcription termination sites. After a 24-h post-exposure incubation, the rate of rRNA synthesis was restored to higher than normal levels in all cell lines. Finally, two FA cell lines showed a higher survival to HP photosensitization than two normal cell lines. Another FA cell line and XP-A and XP-C ceils were in the range of sensitivity of the two normal strains for this treatment. These results indicate that FA cells possess an antioxidant defense system at least as efficient as that of normal cells for processing 102-induced damage.

Fanconi anemia (FA) is a rare autosomal recessive genetic disease characterized by physical malformations, growth retardation, progressive

Correspondence: Dr. S. Nocentini, Institut Curie-Biologie, URA 1292 du CNRS, 26 rue d'Ulm, F-75231 Paris Cedex 05, France.

pancytopenia, and a high cancer incidence (Fanconi, 1967; Glanz and Frazer, 1982). FA ceils have a high level of spontaneous chromosomal aberrations (Schroeder, 1982) and are very sensitive to the clastogenic and cytotoxic effects of bifunctional alkylating agents, such as mitomycin C (MMC) (Sasaki and Tonomura, 1973). The biochemical basis of this sensitivity has not yet

276

been elucidated. The inability to repair DNA interstrand cross-links was originally suggested to be the primary defect in FA (Fujiwara et al., 1977; Fujiwara, 1982) but this proposal was not supported, or only partially supported, by subsequent studies (Fornace et al., 1979; Poll et al., 1984; Papadopoulo et al., 1987; Rousset et al., 1990). A deficiency of D N A ligase and poly(ADP-ribose)polymerase previously described in FA cells (Hirsch-Kauffmann et al., 1978; Berger et al., 1982) has also not been confirmed by later studies (Mezzina et al., 1990; Scovassi et al., 1989). Several observations are consistent with a deficiency in the antioxidant defense system in FA cells. Chromosomal breakage in FA lymphocytes is decreased by exogenous superoxide dismutase and catalase (Nordenson, 1977) and by various antioxidants (Dallapiccola et al., 1985). Clastogenicity seems to depend upon the oxygen tension (Joenje et al., 1981; Joenje and Oostra, 1983). MMC-induced cell killing is reduced by exogenous superoxide dismutase in FA (Nagasawa and Little, 1983) but not in normal cells (Raj and Heddle, 1980). However, the non-enzymatic (atocopherol, vitamin C, sulfhydryl, etc.) or enzymatic (superoxide dismutase, catalase, glutathione peroxidase) antioxidant potential of FA is similar to or higher than that of normal fibroblasts (Gille et al., 1987). To explain these apparently contradictory findings, it was suggested that the elevated antioxidant defenses of FA cells were probably insufficient to cope with an observed abnormally high flux of endogenous activated oxygen species (Cerutti, 1985; Scarpa et al., 1985). Some indirect data indicate that ~O2 could be the critical contributor to the chromosomal breakage in FA (Joenje et al., 1987). This reactive oxygen species is formed during normal metabolism (Kanovsky, 1983; Wefers and Sies, 1983; Kanovsky et ai., 1988), and following oxidative stress simultaneously with, or as a consequence of, radical production (Krinsky, 1977). Furthermore, it is generated by natural cellular photosensitizers such as riboflavin (Joshi, 1985) and many xenobiotics such as porphyrins, tetracyclines and psoralens (Ito, 1978; Weishaupt et al., 1976; Hasan and Khan, 1986; de Mol et al., 1981)

in the presence of light. It may also be important to note that intracellular levels of formamidopyrimidine-DNA-glycosylase (FPG), a repair activity recently shown to recognize and incise DNA base modifications generated by ~O2, has not yet been determined in the different human cell lines (Muller et al., 1990). Xeroderma pigmentosum (XP), like FA, is an autosomal recessive genetic disease. It is clinically characterized by an extreme sunlight sensitivity and a high incidence of skin cancer (Kraemer and Slor, 1985). Fibroblasts isolated from XP patients are defective, to varying degrees, in excision repair (Cleaver, 1968; Cleaver and Bootsma, 1975) or in post-replication repair (Lehman et al., 1975) of lesions induced by UV or UV-mimetics. Although defective excision repair can account for much of the genotoxicity characteristic of XP, some data also implicate a role for anomalous repair of oxidative damage. In fact, XP cells are sensitive to bleomycin (Hurt and Robb, 1984) and asbestos (Yang et al., 1984) which are known to produce O2-reduced species. Moreover, XP skin biopsies demonstrate a progressive decline in catalase activity until the onset of tumor formation (Vuillaume et al., 1983), and XP fibroblasts, whether derived from embryos or infants, are markedly deficient in this enzyme (Vuillaume et al., 1986). The epidermis in XP patients also presents significant decreases in thioredoxin reductase and superoxide dismutase and an altered calcium transport (Schallreuter et al., 1991). The purpose of this study was to directly assess the capability of FA and XP fibroblasts, relative to normal human fibroblasts, to repair exogenously induced I o 2 damage. The generation of JO 2 was obtained by irradiating the biological material (cells, virus or viral DNA), with 365-nm light in the presence of hematoporphyrin (HP) as photosensitizer. Photoactivated HP modifies biomolecules by two possible competitive mechanisms: (i) the generation of ~O2, or (ii) the formation of substrate radicals (Girotti, 1983; Kessel, 1984). IO 2 undoubtly has a preponderant role in the phototoxicity of treated cells or tissues (Weishaupt et al., 1976; Moan et al., 1979; Kessel, 1984; Moan, 1986). The potential cellular targets are numerous: amino acids, DNA (guanosine residues, in particular) and lipids. The preferen-

277 tial oxidation of these different cellular constituents probably reflects the intracellular distribution of IO 2 (Moan and Christensen, 1981). The cellular targets thus vary according to the time of incubation with HP (Cozzani, 1981). The structures which are initially affected include the membranes of cellular organelles (i.e., mitochondria, lysosomes, microsomes), followed by the nuclear membrane. Lipid peroxidation and proteinprotein cross-linking have been implicated as the primary cause of membrane damage (Dubbelman et al., 1982; Moan and Vistnes, 1986). The most hydrophilic constituents, including cytosolic enzymes and nucleic acids, are attacked afterwards. In DNA, single-strand breaks, alkali-labile lesions, 8-hydroxyguanine, and DNA-protein cross-links have all been observed as has enzymatic inactivation (Boye and Moan, 1980; Moan et al., 1980; Musser et al., 1980; Moan and Boye, 1981; Dubbelman and Steveninck, 1982; Schneider et al., 1990). Chromosomal aberrations, specifically located in telomere and centromere regions, are also induced in HP-photosensitized cells (Evenson and Moan, 1982). Two approaches were employed here: (i) cell survival, and DNA template activity in vivo were measured following incubation of the ceils in the presence of HP plus 365-nm irradiation; (ii) in the host cell reactivation (HCR) assay, herpes simplex virus (HSV) or simian virus 40 (SV40) DNA was treated in vitro with HP plus UVA, and used to measure repair of oxidative damage following infection/transfection into untreated host cells. Material and methods

Cells and media 1BR/3, kindly provided by C. Arlett (University of Sussex, Falmer, UK), and Jac, obtained from D. Pham Din (Paris, France) are normal human skin fibroblasts. The FA skin fibroblast cell lines FA 71 and FA 150 (complementation group A) were obtained from Dr. R. Vos (Hadassah Hospital, Jerusalem, Israel); the FA strain FA 145 (complementation group D) was supplied by Dr. M. Buchwald (Hospital of Sick Children, Toronto, Canada). GM 5509, an XP complementation group A cell line, and GM 2995, an XP

complementation group C cell line, were from the Human Genetic Cell Repository (Camden, NJ, USA). Cells were maintained at 37°C in an atmosphere of 5% CO2-air in Eagle's modified essential medium (MEM, Gibco) supplemented with 15% fetal calf serum (Seromed) and 20 /zg/ml gentamycin. Experiments were performed using cells which were passaged 15-25 times. Subclones P and TC-7 of CV1 African green monkey kidney cells used in virus assays were grown under the same conditions as human cells, except that the medium contained 7% fetal calf serum.

Virus and viral DNA purification Stocks of the Shealy strain of HSV type I were prepared by low multiplicity inoculation (10 -2 plaque-forming units (pfu)/cell) of confluent CV1-TC7 cells. Viruses were harvested at 72-96 h post infection. Infectious SV40 virus stock was prepared by infecting CV1-P confluent monolayers with a multiplicity of infection of 10 pfu/cell. Viral DNA was extracted (Hirt, 1967) from infected cells and purified by equilibrium centrifugation in cesium chloride-ethidium bromide gradients. HP photosensitization of cells, HSV and SV40 DNA HP (Sigma) was treated as described previously (Cauzzo et al., 1977) and diluted in phosphate-buffered saline (PBS) prior to use. Cell monolayers were incubated in Petri dishes for 1 h at 37°C in the dark in the presence of HP, and were then exposed to 365 nm radiation from a HPW 125 Philips lamp equipped with a pyrex glass water-filter (HP-hu treatment). The contribution of wavelengths below 340 nm was negligible and total output was about 99% at 365 nm, which is the spectral region of maximal absorbance for HP. HSV stock solution at 10 7 pfu/ml was diluted (1/20) in PBS containing HP at the appropriate concentration, incubated for 20 min on ice in the dark and exposed for different times to 365-nm radiation. During the irradiation, virus suspension was maintained on ice with constant agitation. Samples were then further diluted in MEM 15% fetal calf serum in order to obtain a multiplicity of infection < 0.1.

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Purified SV40 D N A at 10 # g / m l in PBS was exposed, after 30 min equilibration time with 5 or 100 ~ g / m l HP, to 365-nm light. Irradiation was performed in 96-multiwell dishes with covers in place. For dosimetric purposes, the appropriate correction was made for absorption by the plastic. The dose rate was 8.4 or 10 j / m 2 - s as determined by a black-ray UV meter J 221 (Ultraviolet Products, San Gabriel, CA, USA).

HSV HCR assay Stationary monolayers of the different human cell strains were infected at a multiplicity < 0.1 p f u / c e l l with untreated or HP-treated, irradiated or mock-irradiated HSV type I (0.2 m l / d i s h ) and kept in normal growth medium (2 ml). At the end of the virus cycle (20 h), cells were scraped in medium, and the intracellular virus were released by a 20-s ultrasonic treatment at 4°C. Virus yields produced from different cultures were determined by titering on CV1-TC7 ceils. Confluent monolayers were inoculated with appropriate dilutions of HSV. After virus absorption at 37°C for 1 h, the cultures were overlaid with a 1 : 1 dilution of 2 × M E M containing 4% fetal bovine serum and 2% methylcellulose. After 3 - 4 days of incubation at 37°C, the cells were fixed and stained with G i e m s a - e t h a n o l (1 : 1) and the plaques were counted. Surviving fractions were calculated and plotted as survival curves. Transfection of SV40 DNA and SV40 uirus assay Subconfluent cultures of human fibroblasts of different origin in 35-mm dishes were rinsed with 1 ml of TBS (8 g NaC1, 0.38 g KC1, 0.1 g N a z H P O 4 . H 2 0 , 0.114 g CaCI 2. 2 H 2 0 , 0.155 g MgCI 2 - 6 H 2 0 , 24.8 ml 1 M Tris, pH 7.5, per liter in water) and transfected with 10 ng SV40 D N A in 0.4 ml TBS containing 400 ~ g / m l DEAE-dextran (Pharmacia). Transfections were carried out at 37°C for 2 h. The D N A solution was removed, ceils were rinsed twice with 1 ml TBS, and incubated for 5 days in 2 ml of growth medium. Viruses were released by three cycles of freezing thawing, and the titer was determined on CV1-P cells. CV1-P cells were infected in 35-mm dishes with 0.2 ml of virus diluted in M E M without serum. The infection was carried out for 2 h at

37°C. Cultures were then rinsed with 2 ml PBS and overlaid with 3 ml per dish of 0.9% agar (bioagar, Difco) in medium containing 1% fetal bovine serum. After 7 - 8 days of incubation at 37°C, cells were stained with neutral red (0.01% in 2 ml of 0.9% agar in medium). Plaques were scored on the day of and the day after staining. Survival is expressed as pfu per ng damaged transfected DNA, divided by the value obtained for undamaged transfected DNA.

FPG protein assay HP-photosensitized or control SV40 D N A was precipitated ( e t h a n o l / s o d i u m acetate) and redissolved at 10 / z g / m l in 14 mM Hepes, 20 mM KCI, pH 7.6. Each sample containing 0.3 ~ g of D N A dissolved in 30/~1 was incubated for 1 h at 37°C with 1.2 ng of FPG protein (gift of C. Boiteux, Institut Gustave Roussy, Villejuif, France). Incubation was also carried out without enzyme, to determine strand breakage directly due to HP-hu treatment. Separation of the different conformations of SV40 D N A (i.e., supercoiled, open circular and linear forms) was performed by horizontal gel electrophoresis in 0.8% agarose gels with T A E buffer (0.04 M Tris-acetate, p H 8.0, 1 mM EDTA). The D N A bands were stained with ethidium bromide, irradiated from below with a UV transilluminator box, and photographed with Polaroid type 55 black and white film. MMC treatment Cells were incubated for 1 h at 37°C in culture medium without serum containing various concentrations of M M C (Sigma). The drug was removed by washing with PBS. Clonogenic surcg'al Treated and control cells were rinsed with PBS, trypsinized, and seeded at appropriate densities in 100-mm Petri dishes in M E M medium, without feeder cells. For each point, 9 - 1 2 dishes were plated. Colonies were counted after 3 weeks. Relative survival was expressed as percentage of the cloning efficiency of control cells. Transcriptional analysis Chemicals and buffers, radioactive labeling and chase, R N A extraction, and analysis by polyacryl-

279

amide-agarose gel electrophoresis have already been described in detail (Nocentini, 1982). Results

HCR of HP-hv-treated HSV The survival of HSV treated with HP plus 365 nm light was determined in control, FA (complementation groups A and D) and XP-A cells. Typical dose-response surves are presented in Fig. 1. Virus irradiated in the absence of HP showed 100% survival within experimental error in all strains, indicating that 365-nm UV alone does not

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affect the virus significantly in the range of doses studied. HSV was, however, damaged by HP-hu treatment as shown by its reduced ability to infect cell cultures. The inactivation curves show a shoulder, which is more or less pronounced according to HP concentration. The survival of the virus to this treatment is, however, very similar in all cell strains tested.

Transfecting activity of HP-hu-treated SV40 DNA The above results seem to indicate that HPhu-induced lesions in HSV are similarly processed by FA, XP and normal ceils. However, it is not known to what extent the different viral constituents (envelope lipoproteins, DNA, etc.) are damaged, and furthermore, which ones are important for inactivation. In particular, the possibility cannot be ruled out that photosensitized virions fail to penetrate host cells because of damage to the viral envelope. In order to avoid this problem, purified SV40 DNA was treated with HP-hu and then transfected into the different host cell lines. The occurrence of DNA damage was also determined. Fig. 2a shows that HP-h u treatment induces DNA modifications which are recognized and incised by FPG protein. The incisions are detected as a dose-dependent increase in the open circular form at the expense of the closed circular supercoiled form of DNA. This enzyme has been shown to release 8-hydroxyguanine (Tchou et al., 1991) produced in DNA by 10 2 following irradiation in the presence of methylene blue (Schneider et al., 1990). Fig. 2b shows that, for more severe photosensitizing treatments, the direct formation of singlestrand breaks in SV40 DNA is also observed. The survival of transfecting activity of HP-hudamaged SV40 DNA in the different cell lines is reported in Fig. 3. Inactivation of such activity was observed only for severe photosensitizing treatments, and is similar in all cell lines. At such high doses, 365-nm radiation alone probably induced some damage to viral DNA (see Fig. 2). Like HP-hu treatment, 365-nm light alone is believed to inactivate cell cultures through the generation of 10 2 (Tyrrel and Pidoux, 1989). However, no significant effect on transfecting activity of SV40 DNA irradiated in the absence of HP was observed.

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Fig. 2. Gel electrophoresis of SV40 DNA after exposure to HP and 365 nm light. (A) After irradiation tor up to 60 min (mn) at a dose rate of 8.4 J m2.s in the presence or absence of 5/zg/ml HP, DNA samples were ethanol-precipitated and resuspended in Hepes buffer. Half of the sample material was incubated for 1 h at 37°C in the presence of FPG protein. Electrophoresis in 0.8% agarose was carried out for 16 h at 30 V. (B) After photodynamic treatment (HP at 100 ~g/ml, irradiation 10 J m2.s for up to 180 min), DNA samples were directly loaded on 0.8% agarose gel and electrophoresed for 9 h at 30 V. sc, supercoiled form; n, open circular form; 1, linear form.

Template actiuity of DNA in HP-hu-treated cells T h e t e m p l a t e activity of D N A after H P photo-

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exposure to HP (100 /zg/ml) and 365 nm (10 J m2.s) in 1BR/3 ((3), FA 145 (e), FA 150 (A) and XP-A ([:3) cells. Data are mean values of three experiments.

sensitization of cells was investigated in o r d e r to m o n i t o r D N A d a m a g e a n d repair. T h e rationale of the m e t h o d is that lesions which constitute t e r m i n a t i o n sites for R N A polymerase impair t r a n s c r i p t i o n in a q u a n t i t a t i v e or qualitative m a n n e r (Michalke and Bremer, 1969). R e p a i r of damage leads to a recovery of R N A synthesis a n d to a r e e s t a b l i s h m e n t of a n o r m a l ratio b e t w e e n two transcriptionally linked genes. This m e t h o d was previously employed to p r o b e D N A d a m a g e a n d repair after irradiation with 254-nm UV, or following t r e a t m e n t with psoralens plus U V A (365 n m ) (Nocentini, 1976, 1978). In the p r e s e n t study, the rate of R N A synthesis was m e a s u r e d for the r R N A species 28S and 18S at different times after HP-hu t r e a t m e n t in normal, F A (complem e n t a t i o n group A) a n d X P - A fibroblasts. D a t a s u m m a r i z e d from two e x p e r i m e n t s are r e p o r t e d in Fig. 4. T h e synthesis of the two r R N A species is seen to decrease in a d o s e - d e p e n d e n t m a n n e r for all strains. T h e i n h i b i t i o n of 28S a n d 18S R N A is, however, h o m o t h e t i c a n d not differential as it should be if t r a n s c r i p t i o n t e r m i n a t i o n lesions

281

Cell survival Results of clonogenic survival experiments with FA (complementation groups A and D), XP (complementation groups A and C) and normal fibroblasts photosensitized with HP, or treated with MMC, are presented in Fig. 5. The two normal cell strains tested had rather different sensitivities to HP-hu treatment (D37 of 1.7 k J / m 2 for Jac cells and of 2.5 k J / m 2 for 1 B R / 3 cells). XP-C, XP-A and FA 71 cells had sensitivities in the range of those of normal cells. FA 145 and FA 150 cells were the lines most resistant to this treatment (D37 of 3.1 k J / m 2 and 3.8 k J / m 2, respectively). However, and in accord with results obtained by others (Fujiwara et al., 1977; Fornace et al., 1979; Poll et al., 1984; Diatloff-Zito et al., 1986), cell lines FA 145 and especially FA 71 and FA

were induced randomly in the polycistronic ribosomal DNA by HP-hu treatment. Because of the respectively proximal and distal positions of 18S and 28S RNA genes with regard to the common promotor, the sensitivity of the 28S RNA species should in fact be greater than that of the 18S RNA species. This is not the case, and the results suggest that effects on RNA synthesis are essentially due to a block of initiation, rather than to a block of RNA chain elongation. The inhibition of 28S and 18S RNA synthesis is very similar in all strains. After 24 h of post-exposure incubation, the rate of RNA synthesis is observed to recover in all cell lines. Regardless of the initial inhibition, the synthesis of both 28S and 18S RNA largely surpasses the level of synthesis of untreated controis.

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Fig. 4. Rates of 28S and 18S R N A synthesis in HP-treated cells as a function of exposure to 365-nm radiation. Confluent monolayers of 1 B R / 3 (o), Jac ( r, ), F A 150 ( • ) and XP-A G M 5509 ( o ) fibroblasts were treated with HP (0.5 # g / m l for 1 h) and irradiated (10 J m . s ) at 15 min (continuous lines) or at 24 h (dashed lines) before labeling with 2 5 / , C i / m l of [3Hluridine for 1 h at 37°C. Total R N A was then extracted and analyzed with 1.7% polyacrylamide-0.5% agarose gel electrophoresis (4 h, 8 V / c m ) . Gels were sliced and the radioactivity (dpm) in the 28S and 18S R N A peaks of the absorbance profiles was determined. Values are expressed as percentage of 28S and 18S R N A synthesis in controls. Data are mean values of two separate experiments.

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Fig. 5. Survival of 1 B R / 3 (o), Jac (D), FA71 (ll), FA 145 ( • ) , FA 150 (e), XP-A G M 5509 ( a ) and XP-C G M 2995 ( ,7 ) fibroblasts photosensitized with HP (left panel) or treated with M M C (right panel). Cells were incubated for 1 h at 37°C in M E M without serum containing 0.5 p~g/ml HP followed by irradiation at 365 nm (5 J m2.s). The mean values of two (Jac and XP-C cells) or three experiments are reported ( 1 B R / 3 , FA 71, FA 145, FA 150 and XP-A cells). T r e a t m e n t with M M C was for 1 h at 37°C. Each point represents the mean of at least nine dishes from the same experiment ( 1 B R / 3 , FA 150 and XP-A cell lines), or the mean from two experiments (Jac cells) or from three experiments (FA 71, FA 145 or XP-C

cells). 150 have a greatly increased sensitivity to MMC

treatment (D37 of 0.05 and about 0.016 /~g/ml, respectively, versus 0.2 /~g/ml for 1 B R / 3 and 0.13 for Jac cells). XP-A and XP-C cells display similar, intermediate sensitivities to M M C treatment (D37 of about 0.08 /~g/ml). Discussion A plausible interpretation of previous observations, and in particular those of Joenje et al. (1987), is that IO 2 damage in FA cells is not repaired in a normal manner. In the present study, HP photosensitization was employed to generate ~O2 in order to analyze more directly the biological consequences of the resulting lesions in normal, FA and XP cells. The effects of

this treatment were investigated at different levels. H C R is a process whereby cellular activities repair damage in exogenous DNA. Here, intact HSV as well as purified SV40 D N A were employed as substrates. In the case of HSV infection, the importance of damage to the different viral macromolecules is not known. Lewin et al. (1980) reported that, for irradiations of HSV in the presence of HP derivatives resulting in a complete loss of plaque-forming ability, inactivated virions neither adsorb nor penetrate susceptible cells, probably due to envelope modifications. However, for milder photodynamic treatments than those used by Lewin et al., it cannot be excluded that 10 2 may diffuse and damage the D N A in virions still able to penetrate host cells. In transfecting purified SV40 DNA, damage photosensitized by HP was revealed as F P G proteinsensitive sites and single-strand breaks. The biological effects of ~O 2 were demonstrated by the photosensitization-dependent loss of HSV infecting, or SV40 D N A transfecting activities. However, no marked difference was observed in H C R among the various cell lines. It may be noteworthy that, in this assay using HP-photosensitized SV40 DNA, there appears to be no close correlation between induced lesions and the loss of biological activity. Evidently, even a high proportion of SV40 D N A molecules bearing F P G protein-sensitive sites does not result in a significant decrease of transfecting activity, indicating an efficient repair or tolerance mechanism for this type of damage. In the clonogenic cell survival assay, FA cells demonstrate a similar or moderately increased level of resistance to HP-hu treatment compared to both normal and XP-A cells. If one assumes similar HP uptake and compartmentalization, and identical deposition of damage in the different cell lines, it can be concluded that HP-hu treatment induces lesions that are repaired equally or somewhat more efficiently in FA than in normal or XP cell lines. Such relatively low differences in repair capability were not observed in the H C R assay. A possible explanation for these findings could be that, since none of the cell lines tested was actually severely deficient in the repair of this type of damage, and because of the low multiplicity of HSV infection ( < 0.1) or the lim-

283

ited number of transfecting SV40 DNA molecules, the extent of HP-hu-induced damage is likely quite low for all cells and, thus, insufficient to reveal differences in HCR. On the other hand, the photosensitization of whole ceils may overwhelm oxidative defense systems and demonstrate relatively low cellular differences. However, the damage to the virus or to naked DNA is not necessarily the same as that for whole cells. It is admitted, for example, that cell lethality is mainly mediated by membrane damage, especially to lysosomes and mitochondria. Transcription analysis was employed to test the functional state of template DNA. Results demonstrate that the inhibition of RNA synthesis is not due to transcription-terminating lesions induced by HP-hu treatment on cell DNA, but rather to a reduction of initiation, or to a general slowdown of synthesis rate. The direct inactivation of R N A polymerase by HP photosensitization, or a reduction of energy supply due to mitochondrial damage might thus be involved in the impairment of R N A synthesis. This emphasizes the importance of HP-hu-induced extragenomic lesions for the general physiology of the cells, even though RNA synthesis was restored to normal levels in all cell lines tested. In conclusion, these results do not indicate an abnormal processing of ~O2-induced damage in FA ceils. The sensitivities of XP-A and C ceils are also in a normal range. The complex involvement of I o 2 in the effects of oxygen tension and of polyfunctional alkylating agents in FA cells still remains to be elucidated.

Acknowledgements This work was financially supported by the CNRS, the CEA (Saclay, France), the ARC (Villejuif, France) and the INSERM. I thank Mrs. M. Guggiari for skilled technical assistance and Dr. Elliot Drobetsky for critically reading the manuscript.

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