Mutation Research, 254 (1991) 55-64 DNA Repair

55

Elsevier MUTDNA06408

The sensitivities of SV40-transformed human fibroblasts to monofunctional and DNA-crosslinking alkylating agents J u n j i M i y a k o s h i a, D o m i n i c A. S c u d i e r o c, J o a n A l l a l u n i s - T u r n e r b a n d R u f u s S. D a y III a a Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine and b Radiobiology Laboratory, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alb. T6G 1Z2 (Canada) and c Program Resources Incorporated, Frederick Cancer Research Center, P.O. Box B, Frederick, MD 21701 (U.S.A.)

(Received 14 June 1989) (Revision received 27 April 1990) (Accepted 7 June 1990)

Keywords: Fibroblasts, human, SV40-transformed; DNA-crosslinking alkylating agents; MNNG; MMS; 1,3-Bis-(2-chloroethyl)-l-

nitrosurea; 1-(2-Chloroethyl)-3-(2-hydroxyethyl)-l-nitrosourea;

SV40-transformed lines

Summary 4 repair-deficient (Mer-) and 2 repair-proficient (Mer ÷) lines of SV40-transformed human fibroblasts were assayed for colony-forming ability after treatment with MNNG, methyl methanesulfonate (MMS), 1,3-bis-(2-chloroethyl)-l-nitrosourea (BCNU), and 1-(2-chloroethyl)-3-(2-hydroxyethyl)-l-nitrosourea (HECNU). The sensitivities to MMS, BCNU and HECNU of these SV40-transformed lines were similar to those of comparably treated human tumor cells observed previously. However, unlike human tumor lines, whose post-MNNG survival is strongly dependent upon Mer phenotype, SV40-transformed lines showed a lack of dependence of post-MNNG colony-forming ability on Mer phenotype. No differences in glutathione levels that might explain these differences were detected. The amounts of SV40-specific DNA and RNA among the lines were found to vary widely, but no correlation with Mer phenotype was found.

Human cells repair m6Gua produced in DNA by SNl-type methylating agents (Lawley, 1976). This is accomplished by the protein m6MT, deficient in certain (Mer-) human tumor lines which

are specifically unable to support normally the growth of MNNG-treated adenoviruses (Day et al., 1980b; Yarosh et al., 1983, 1984). The Merphenotype is a property most usually of transformed human cells; normal fibroblasts and embryonic strains are generally Mer ÷ and able to

BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; GSH, glutathione; m6Gua, O6-methylguanine; HECNU, 1-(2chloroethyl)-3-(2-hydroxyethyl)-l-nitrosourea; Mer +, Mer-, able and unable to support the growth of MNNG-treated adenovirus; Mex +, Mex-, able and unable to repair m6Gua in DNA; MMS, methyl methanesulfonate; MNNG, N-methylN'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; m6MT, O6-methylguanine-DNA methyltransferase.

Present address: Dr. Junji Miyakoshi, Department of Experimental Radiology, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan).

Abbreviations:

Correspondence: Dr. Rufus S. Day III, Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alb. T6G 1Z2 (Canada).

0921-8777/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

56 repair m 6 G u a (Day et al., 1980b, 1987; Medcalf and Lawley, 1981; Day, Dobler and Honore, in preparation). Of the human tumor lines studied in our laboratories 21 of 100 have the M e r - phenotype (Day et al., 1980b; Scudiero et al., 1984a; and unpublished results). Relative to Mer + lines, Mer human tumor cell lines are hypersensitive to M N N G - p r o d u c e d cell killing (Day et al., 1980b; Scudiero et al., 1984a) and to both M N N G - i n duced mutation (Baker et al., 1979, 1980; Domoradski et al., 1984) and M N N G - i n d u c e d sister-chromatid exchanges (Wolff et al., 1977, used lines found later to differ in Mer phenotype; Day et al., 1980b). The situation with M e r - lines produced by SV40-transformation of Mer + 'parental' human fibroblasts is somewhat different. Unlike human tumor-derived lines, lines produced by SV40 transformation show sensitivities to M N N G - p r o duced cell killing that are unrelated to their Mer phenotype (Day et al., 1980b; Babich and Day, 1987, 1988, 1989). For example, the survival of a typical human tumor Mer line is 1% at 1 /.tM M N N G whereas the survival of a typical SV40transformed M e r - fibroblast is about 1% at 10 ~ M M N N G , much like that obtained for Mer + normal human fibroblasts (Scudiero et at., 1980, 1981, 1984b). Such resistance of SV40-transformed human fibroblast M e r - lines is not observed in all laboratories: both Domoradski et al. (1984) and Boyle et al. (1987) have observed in SV40-transformed Mer lines the extreme sensitivity found typical of Mer-- human tumor lines in our laboratory. Boyle et al. (1987) found two SV40 cell lines in which m 6 M T was absent (and which were presumably M e r - ) to be hypersensitive to killing by methylnitrosourea (MNU). This observed interlaboratory difference may, in part, be due to the known differences in activation of M N U and M N N G . Both are activated to form the toxic methyldiazonium ion (Lawley, 1976). However, unlike M N U , which is activated by hydroxyl ion (Veleminsky et al., 1970), M N N G is activated intracellularly by free thiol (Lawley and Thatcher, 1970). One explanation for the anomalously elevated survival of the M N N G - t r e a t e d SV40-produced M e r - lines, then, is that these lines are depleted in G S H and do not activate M N N G well (see Sedgwick and Robins, 1980). Thus, although

they would lack m6MT, there would be fewer m 6 G u a produced in the D N A by the M N N G treatment, and the survival would be as great as for Mer + cells with normal levels of GSH. To investigate further the differences between SV40-transformed human tumor Mer lines, we have studied the SV40-transformed lines using chemical inactivating agents other than M N N G . In addition we have investigated two hypotheses: (1) that the SV40 genomic copy number or the level of SV40 expression in a transformed line is related to the cellular Mer phenotype and (2) that low levels of cellular G S H in the SV40-transformed lines may explain the abnormal cellular resistance to M N N G of SV40 produced M e r lines. Materials and methods

Cell lines and cultures. Post-crisis SV40-transformed human fibroblast cell lines W18VA2, WI26VA4, WI38VA13, W98VA1, GM637, GM638, AT5BIVA2, SV80, XP12T703, IMR90830, IMR90-890, were purchased or were kind gifts as previously reported (Day et al., 1980b). The line termed XP12T703 here has been termed XP12 (Day et al., 1980b), XP12RO(SV) Boyle et al. (1987) and XP12ROSV Domoradzki et al. (1984), CRL1584, from the American Type Culture Collection (Rockville, M D ) was isolated as a post-crisis line from SV40 transformation of a human placental strain initiated by Dr. Janice Chou. Stocks of cells were cultured at 3 7 ° C in Dulbecco's modified Eagle Medium (Gibco) plus 10% ( v / v ) fetal calf serum plus 1% ( y / v ) of a solution containing 5000 units penicillin plus 5000 /~g streptomycin per ml (DMEM). Stocks were passaged once weekly at a ratio of 1:6. Lines previously determined to be Mer include IMR90-890, IMR90-830, SV80, XP12T703, GM638, WI38VA13 and W l 8 V A 2 (Day et al., 1980b). CRL1584 was determined to be deficient in host-cell reactivation of M N N G - d a m a g e d human adenovirus 5 (data not shown; see Assay for Mer phenotype). Thus, by definition (Day et al., 1980a) it has the M e r - phenotype. Lines determined previously to have the Mer ÷ phenotype were AT5BIVA2, W98VA1, WI26VA4, and GM637 (Day et al., 1980b).

57

Chemicals and colony-forming ability. The alkylating agents used in this study were obtained as previously (Scudiero et al., 1984b). On the day prior to treatment, cells were trypsinized and plated at 50, 100, 500, 1000 and 5000 cells per 60-mm cell-culture plate in Ham's F12 medium (Gibco, 500 ml) to which had been added 100 ml fetal calf serum and 5 ml of a solution containing 5000 units/ml penicillin and 5 m g / m l streptomycin. On the next day, the medium on all plates to receive a given concentration of BCNU, M N N G or H E C N U , was removed and replaced with 4 ml of complete Ham's F12 to which had just been added, at most, 0.01 volume of an appropriate ethanol stock solution of chemical. Ethanol at this concentration did not affect colony-forming ability. Treatment with MMS was similar except MMS was diluted directly into complete Ham's F12: no ethanol stock was prepared. After a 1-h treatment, the medium containing the chemical was removed and replaced with fresh medium. Medium was replaced with fresh medium every 2-3 days until colonies contained 50 or more cells (2-3 weeks). At that time colonies were fixed, stained with Giemsa, and enumerated. Assay for Mer phenotype. The assay describing the M N N G treatment of purified adenovirus 5 virions for 30 min in pH 9.0 Tris-HC1 buffer, followed by inactivating unreacted M N N G with N-acetyl-L-cysteine and subsequent plaque assay has been published (Day et al., 1980b). Assay for GSH content. Intracellular GSH was measured using a modification of the technique described by Tietze (1969). Cells grown as monolayers were trypsinized, washed in phosphatebuffered saline, and counted. Duplicate samples of 106 cells per sample were aliquoted, pelleted and resuspended in 1 ml trichloroacetic acid (5% TCA in 0.01 N HC1 with 0.5 mM EDTA). The cell lysates were centrifuged to remove debris, aspirated, then frozen in glass tubes. Immediately before assay, cell lysates were extracted 5 times with equal volumes of ether. To prepare cells for spectrophotometric assay, a 200-/~1 aliquot of the lysate was added to a 2-ml cuvette containing N A D P H (200 /~M), DNTB (100 /tM) and GSH reductase (1.57 units/ml) in buffer (0.1 M

KHzPO4). The absorbance at 412 nm was measured in a Beckman spectrophotometer (DU-7). For each sample, GSH content was determined by comparison to standards containing known amounts of GSH. Each sample was assayed in duplicate, and the average G S H concentration expressed in fmoles GSH/cell.

Preparation and analysis of cellular DNA. We detected SV40 sequences by Southern blotting (Reed and Mann, 1985) using the BamHI SV40 containing fragment of pBRSV (obtained from the American Type Culture Collection, Rockville, MD, to probe electrophoresed HindlII, EcoRI or B a m H I digested genomic D N A s prepared (Scudiero et al., 1984a) from the SV40-transformed lines. To analyse membranes for the yactin (ACTG) gene, membranes were stripped and reprobed with the 2.1-kb BamHI fragment of pHFgA-1 containing the ACTG gene (Gunning et al., 1983; obtained from P. Gunning through C. Fregeau). Details of our procedure have been published (Miyakoshi et al., 1990). Preparation and analysis of cellular RNA. Total RNA was purified as described (Chirgwin et al., 1979). 20/~g cellular RNA was electrophoresed in 1% agarose gels with 0.66 M formaldehyde, transferred to Gelman Biotrace RP using 10 x SSC, baked, and analysed as above with the SV40 probe. Membranes were stripped and reprobed with the 7-actin probe. (For full details, see Fourney et al., 1988.) Results

Cellular studies. Sensitivities of selected SV40transformed Mer + and M e r - lines to inactivation by M N N G , BCNU, H E C N U and MMS are depicted in Fig 1. Each curve is the result of at least 6 determinations. The standard deviation associated with the determination of each point was usually less than 10% of the value of the point. The Mer ÷ lines are W98VA1 and WI26VA4 (open symbols); the M e r - lines are Wl8VA2, GM638, IMR90-830 and WI38VA13 (closed symbols). Fig. l a shows that the p o s t - M N N G survival depends little on Mer phenotype. The curves are either linear or show two components.

58 U n l i k e the c a s e o f M N N G , s e n s i t i v i t i e s to the other chemicals show possible dependence on Mer p h e n o t y p e , a n d d e f i n i t e l y in the c a s e o f H E C N U . ( H E C N U is a D N A c r o s s l i n k i n g a g e n t s i m i l a r to B C N U , b u t has less c a r b a m o y l a t i n g a c t i v i t y bec a u s e o f a h y d r o x y e t h y l g r o u p r e p l a c i n g the c h l o roethyl group on the non-nitrosated urea nitrogen. See S c u d i e r o et al., 1984b.) I n the case o f B C N U ,

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Line

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Mer phenotype

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Fig. 1. Survival of colony forming ability of Mer + and Mer SV40-transformed human fibroblasts as a function of dose of 4 chemical agents. Cells were seeded at colony-forming densities, treated for 1 h with selected concentrations of MNNG, BCNU, HECNU or MMS and cultured to form colonies. Mer + lines: (©) W98VA1; (n) WI26VA4. Mer- lines: (O) W18VA2; (A) GM638; (v) IMR90-830; and (ll) WI38VA13. Dotted lines indicate range of survival of Mer + human tumor lines. Dashed lines indicate range of survival of Mer- human tumor cells (Scudiero et al., 1984a,b). In Fig. 1A, the area between the top dotted line and the hatched dot-dashed line indicates the range of survival of human fibroblasts to MNNG inactivation (Scudiero t:t al., 1981).

H E C N U a n d M M S , the show survival values very w i t h h u m a n t u m o r lines and Mer b y the d a s h e d

S V 4 0 - t r a n s f o r m e d lines s i m i l a r to t h o s e o b t a i n e d (Mer + shown by dotted lines, Fig. 1).

G S H content. C e l l u l a r G S H c o n t e n t was m e a s u r e d in o r d e r to test the h y p o t h e s i s t h a t the S V 4 0 - t r a n s f o r m e d M e r - lines m i g h t b e a n o m a l o u s l y r e s i s t a n t to M N N G d u e to d e p l e t i o n of G S H a n d c o n s e q u e n t loss of a b i l i t y to c o n v e r t MNNG i n t o a l e t h a l species (see L a w l e y a n d T h a t c h e r , 1970; S e d g w i c k a n d R o b i n s , 1980). H o w e v e r , the d a t a in T a b l e 1 i n d i c a t e a n a b s e n c e o f the c o r r e l a t i o n b e t w e e n G S H c o n t e n t a n d M e r p h e n o t y p e t h a t w o u l d be e x p e c t e d a c c o r d i n g to the idea. W h i l e the a v e r a g e G S H c o n t e n t o f the M e r + lines (5.0 _+ 2.2) is g r e a t e r t h a n t h a t o f the M e r - lines (3.5 _+ 2.6 f m o l e s / c e l l ) t h e d i f f e r e n c e is clearly insignificant. (The individual determinat i o n s are s u b j e c t to less e r r o r : _+ 10% o f the det e r m i n e d v a l u e as a s t a n d a r d d e v i a t i o n . ) SV40-specific m R N A in SV40-transformed lines. Fig. 2a s h o w s an a n a l y s i s e x t r a c t e d f r o m e a c h of the lines a f t e r e l e c t r o p h o r e s i s , hybridization with a probe

o f 20 /~g t o t a l R N A 12 S V 4 0 - t r a n s f o r m e d t r a n s f e r to m e m b r a n e , p r e p a r e d f r o m the en-

59 I m

I n

I ¢J1

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kb 9.57.54.42.41.4-

transcriptional control region characteristic of this line, although a lesser amount of material of this size is detected in all lines except W18VA2 and W98VA1 in this study. The R N A at 1.8 kb produced in great amounts by lines GM637 and IMR90-890 in particular, and less so in XP12T703, was also observed by Flint et al. (1983) in the SV80 line. The RNAs of greater sizes, seen for example in CRL1584, AT5BIVA2 and XP12T703 fall in the 6-10 kb range. These are larger than the size of the SV40 genome (5243 bp), and may thus represent transcriptional products that include transcripts of cellular DNA. There is no clear association between the pattern of steady stateSV40 mRNA levels and the Met phenotype of the cell line. Fig. 2b is a control autoradiograph prepared from the same membrane after removing

I

I

b.

2.4-

r,

1.4" Fig. 2. Analysis of SV40-specific RNA in SV40-transformed human fibroblast lines. For each line 20 #g total RNA was electrophoresed, transferred to a membrane, and (a) hybridized with a probe prepared from the entire SV40 genome from pBRSV or (b) the membrane was stripped and rehybridized with a v-actin specific probe prepared from the 2.1-kb BamHI fragment of pHFgA-1 containing the A C T G gene. The + or - preceding the cell line designation indicates the Mer phenc~ type of the line.

kb 12.28,17.16.15.04.0-

tire SV40 genome (selected so that all SV40specific RNA would be detected), followed by a 3-day autoradiographic exposure. The RNAs detected at about 2.5 and 2.9 kb were resolved as a doublet with overnight exposure (not shown) and appeared somewhat smaller in WI38VA13 than in the remaining lines. These RNAs likely correspond to the spliced early m R N A species encoding the large T- and small T-antigens (2310 and 2590 bases in length, not taking into account the length of the poly A tract added post-transcriptionally; see Appendix A of Tooze, 1981). Our pattern of SV80 mRNAs is very similar to that observed for the SV80 line by Flint et al. (1983). Flint et al. (1985) attribute the m R N A of 3.8 kb in SV80 to the transcription of a duplicated early

3.0-

2,0-

1.61.0-

0.5060.39-

Fig. 3. Analysis of SV40-spceific D N A in SV40-transformed human fibroblast lines. 5/xg DNA from each line was digested with EcoRI, electrophoresed, transferred and hybridized with a probe prepared from the entire SV40 genome from pBRSV followed by overnight autoradiography. The band detected in the DNA of CRL1584 at about 6 kb is more clearly visible with a 5-h exposure. The + or - preceding the cell line designation indicates the Mer phenotype of the line.

60 the SV40-probe and rehybridizing with the T-actin probe and shows that approximately equal amounts of T-actin specific R N A s were applied to the membrane. S V40 DNA in transformed cell lines. Fig. 3 shows an autoradiograph of a membrane onto which EcoRI-restricted genomic D N A s from the lines were transferred and probed using the entire SV40 genome. The lines GM637, IMR90-890 and AT5BIVA2 contain so much SV40 D N A that D N A sizes are difficult to determine even at reduced exposures. (The longer exposed autoradiograph is shown so that bands of relatively low intensity may be seen.) CRL1584, XP12T703, IMR90-830 gave rise to bands of near genomic size indicating the possibility of circular cellular SV40 D N A or tandem integration. Half of the lines (SV80, W98VA1, G M 3 8 , WI38VA13, WI26VA4 and W18VA2) did not contain a band of the length of the SV40 genome, and thus neither contain circular genomes nor more than two SV40 genomes tandemly integrated. D N A dot blots (not shown) employing known amounts of SV40 D N A mixed with 5 /~g of herring sperm D N A as an internal standard gave an estimate of < 1 to 2 SV40 g e n o m e s / t r a n s f o r m e d cell for these lines, 2 - 8 in CRL1584, XP12T703 and IMR90-830, and > 100 for AT5BIVA2, GM637, and IMR90-830. No correlation with the amount of SV40 D N A in the cells with the Mer phenotype was evident.

Discussion Survival studies. M e r - human tumor lines are up to 50-fold more sensitive than are Mer + human lines to inactivation by M N N G (Day et al., 1980b; Scudiero et al., 1984a; Day and Babich, 1987, 1988, 1989). By comparison, the 4 M e r SV40-transformed lines studied here were much more resistant to M N N G inactivation. The sensitivities of the most resistant of these, W18VA2 and 1MR90-830, lie near those of normal human fibroblasts (Scudiero, 1980; Scudiero et al,, 1981, 1984a) shown by the area between the upper dotted and the hatched dot-dashed lines of Fig. 1. The lack of dependence of the M N N G sensitivities of the SV40-transformed lines on their Mer phenotype is not due to differences in their G S H con-

tent. The unexpectedly high survival is not observed in all laboratories (Domoradski et al., 1984; Boyle et al., 1987) but is in others (Erickson et al., 1980a), possibly leaving only interlaboratory differences in experimental conditions as an explanation. The laboratory conditions that give rise to the difference in p o s t - M N N G survival of Mer tumor lines and Mer SV40-transformed lines were very likely operating during the experimentation presented here. On no occasion have we observed in Mer tumor lines the high p o s t - M N N G resistance characteristic of Mer SV40 lines (Day et al., 1980; Babich and Day, 1987, 1988, 1989; and the high resistance observed in this study). Neither have we observed in M e r - SV40-transformed lines the extreme p o s t - M N N G sensitivity of Mer tumor cells (Day et al., 1980a, b; Scudiero et al., 1984a; and indicated by the heavy dashed lines of Fig. 1). The large p o s t - M N N G survival differences between M e r - tumor and Mer SV40transformed lines have been observed in our laboratories in four studies (Day et al., 1980; Babich and Day, 1987, 1988, 1989). The postM N N G survival of Mer + fibroblasts and M e r tumor lines measured with similar materials within months of the M N N G curves of Fig. 1 showed sensitivities within those indicated for them in Fig. 1. Further, to our knowledge, there has been no report from any laboratory that the p o s t - M N N G sensitivity of M e r - human tumor lines approaches that of normal human fibroblasts. The heterogeneity in response of GM638 to M N N G inactivation is unlikely due to this line's being composed of a mixture of Mer + and M e r cells. The shape of the survival curve of M N N G treated adenovirus 5 indicates one Mer- population (see Day et al., 1980b). When treated with MMS, BCNU or H E C N U , the SV40-transformed Mer ÷ and M e r - lines behaved as Mer + and M e r - human tumor cells did (Fig. 1, Scudiero et al., 1984b) showing few differences in survival with MMS and B C N U but easily discernable differences with H E C N U . Both H E C N U and B C N U are 1-(2-chloroethyl)-lnitrosoureas. U p o n breakdown in water they produce at least two products: a 2-chloroethyldiazonium ion and an isocyanate (see Scudiero et al., 1984b). The 2-chloroethyldiazonium ion reacts

61 at the O6-position of guanine to form O6-chloro ethylguanine (Parker et al., 1987; Tong et al., 1982, 1983) repairable by m6MT (Robins et al., 1983) and a precursor to the interstrand crosslink lethal to M e r - cells (Erickson et al., 1980b). The chloroethylisocyanate from BCNU either produces DNA damage or inhibits DNA repair (Kann et al., 1974) whereas the hydroxyethyl-isocyanate from H E C N U likely reacts with itself and becomes inactive (see Scudiero et al., 1984b). That M e r - SV40-transformed fibroblasts are inactivated with a similar dependence on dose with both H E C N U and BCNU is consistent with the idea that H E C N U and BCNU inactivate these cells by the same mechanism, that is by D N A : D N A intrastrand crosslinking. The fact that the Mer ÷ cell lines are more resistant to H E C N U than to BCNU is thus consistent with the idea that BCNU inactivates cells both by DNA crosslinks and by chloroethylisocyanate damage and that H E C N U inactivates primarily through DNA crosslinks. The MMS survival curves are quantitatively very similar to the BCNU survival curves if a scale change is applied to the abscissa. The same is true of Mer + and M e r - human tumor lines (Scudiero et al., 1984b). This may be either fortuitous or be because MMS inactivates cells by modes of action similar to those of BCNU. MMS is not known to produce crosslinks in DNA, so that the former explanation holds favor.

GSH. Our measurements of G S H argue against the possibility that SV40-transformed M e r - cells are resistant to M N N G due to a deficiency in GSH. The Tietze (1969) assay that we used to measure GSH also determines GSSH as GSH. For M N N G not to be activated, the majority of the GSH would have to be present as GSSH. We consider this highly unlikely for 3 reasons: (1) GSSH is continually reduced (normally present intracellularly at < 1% of GSH) to GSH by an NADPH-dependent GSH reductase (Meister, 1983) not known to be altered in transformed cells; (2) M N N G produces as many SCE in the SV40-transformed line WI38VA13 as it does in human tumor M e r - fines (Day et al., 1980b); and (3) no obviously low methylation of the DNA of 5 lines of [14C-Me]MNNG-treated

SV40-transformed M e r - cells was observed in a study in which DNA concentrations were not quantified (Day et al., 1980b). As a result of the sum of the evidence, a possible interpretation is that M e r - SV40-transformed human fibroblast lines receive as much M N N G damage as do other M e r - lines but survive better. In accounting for all the data we conclude that conditions other than GSH content influence p o s t - M N N G survival of SV40-transformed human fibroblasts. The range of cellular GSH measurements is in accord with measurements made by us previously: two human tumor lines, ME180 and HEp3, gave 6.4 and 7.8 fmoles/cell respectively (Allalunis-Turner et al., 1988). While the average values we present here are somewhat lower, they do not at all support the idea that differences in G S H content underly the anomalous sensitivity of MNNG-treated SV40transformed Mer ÷ and M e r - lines. In addition GSH content is constant within the stages of the cell cycle (Lee et al., 1988) so that GSH-coupled cell cycle effects on survival are unlikely. In vitro studies have shown that GSH blocks the formation of crosslinks in CNU-treated DNAs (Ali-Osman, 1989). The GSH concentrations employed approximated average intracellular concentrations. Our data indicate that the intracellular G S H concentration does not protect cells from the lethal effects of H E C N U nearly so well as does the Mer repair system, presumably m6MT.

SV40-specific nucleic acids. Although there is no correlation of number of copies of the SV40 genome with Mer phenotype, there may be an association between high expression of the 1.8-2.0 kb RNA species with high SV40-genomic copy number in the cases of GM637, IMR90-890 and XP12T703 (but not AT5BIVA2). The significance of this m R N A species is not known. One could imagine that SV40 produces the M e r - phenotype by site-specific integration into a 'Mer gene' followed by loss of remaining functional Mer gene information on the homologous chromosome (loss of heterozygosity). The chromosomal location of the SV40 genome in transformed human cells has been determined for several transformants: chromosome 7 in LN-SV (also called GM847), W18VA2 (Croce et al., 1973) and GM177 (Croce, 1977); chromosome 5

62

(GM639, Hwang and Kucherlapati, 1980); chromosome 8 and 12 (GM637, Kucherlapati et al., 1978; Croce, 1981); and chromosome 17 in GM54VA (Croce, 1977; GM54VA is almost certainly the same line as GM638, Dr. Arthur E. Greene, Coriell Institute, personal communication), indicating that transformation-related integration of SV40 into the human genome may be preferentially into chromosome 7. To our knowledge, except for GM637 and W18VA2 (and probably GM638), the lines we have used have not been assessed for chromosomal location of SV40 information. There is a wide divergence both in size and in quantity of the SV40-specific RNAs produced by the cell lines we have studied. None correlates with the Mer phenotype of the SV40-transformed lines. Neither does the amount of SV40-specific m R N A correlate with morphological transformation. For example, SV80 appears to be one of the ' m o s t transformed' of the lines by criteria of both morphology and high saturation density, but there is no great abundance of SV40-specific R N A produced in SV80 as compared with GM637 and IMR90-890. The results suggest another basis for the relationship of SV40 transformation and the Mer phenotype. The hypothesis that SV40 T-antigen transforms cells in part by overwhelming the retinoblastoma (RB) gene product (Ludlow et al., 1989) or p53 gene product (Wang et al., 1989; Baker et al., 1989) is consistent with this idea. SV40-transforms human cells in two steps: first is the appearance of morphologically transformed cells which have a limited life span. When these cells are cultured further, 'immortalized' lines may appear while the majority of the culture fails to grow ('crisis', Girardi et al., 1965). The cells which survive crisis likely differ by at least one mutation from the cells which die in crisis. Such a mutation may give rise to the Mer phenotype and to a cellular biochemistry which either elevates T-antigen synthesis or decreases proper maturation of the RB a n d / o r p53 product. Consistent with this idea is that none of 6 precrisis cultures of SV40transformed human fibroblasts we have tested are M e r - (Day, unpublished data). We have found in preliminary studies that these SV40 lines all express approximately equal levels of the RB gene 4.7-kb m R N A . In addition the I F N A and I F N B

genes absent in certain Mer + and M e r - brain tumor cell lines (Miyakoshi et al., 1990) are present in all SV40 lines studied here.

Acknowledgements This study was supported by an Establishm e n t / S c h o l a r s h i p G r a n t from the Alberta Heritage Foundation for Medical Research ( A H F M R ) to R. Day and A H F M R and Alberta Cancer Board fellowships to J. Miyakoshi. We thank Viki Bjerkelund and Shelby Hunt for expert assistance with the manuscript.

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