strand specificity for mutations induced by (+)-anti bpde in the hprt gene in human t-lymphocytes

Mutation Research, 269 (t992) 129-140 129 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

MUT 05142

Strand specificity for mutations induced by ( + )-anti BPDE in the hprt gene in human T-lymphocytes

Bj6rn A n d e r s s o n *, S u s a n n F~ilt * a n d Bo L a m b e r t * Department of Clinical Genetics, Karolinska Hospital, S-104 Ol Stockholm, Sweden

(Received 11 December 1991) (Revision received 11 March 1992)

(Accepted 13 March 1992)

Keywords: Hprt; Benzo[a]pyrene diolepoxide; Mutational spectrum; Preferential repair

Summary

Mutations in the hprt gene in T-lymphocyte clones isolated from primary cultures treated with the (+)-anti enantiomer of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) in vitro, and from untreated control cultures, were characterized using polymerase chain reaction and direct sequencing of hprt eDNA and genomic fragments. The spectrum of BPDE-induced mutations was very specific and clearly different from the background spectrum, which comprised many different types of mutations. Of the BPDE-induced mutations, 20/22 were transversions of GC base pairs and 18/22 were GC > TA transversions, which is in agreement with what has been found in other mammalian systems. While no particular 'hotspot' was observed for BPDE in the hprt gene, a sequence context specificity was detected. Ten of the 14 BPDE-induced mutations in the coding region were located in the sequence context AGG, and 2 in AG dinucleotides, which indicates that such sequences are sensitive to BPDE mutagenesis. Nine of the 22 BPDE-induced mutations and 2/12 background point mutations caused mRNA splicing errors. Six of the BPDE-induced splicing errors were caused by GC > TA transversions in the AG dinucleotide of different splice aeceptor sites, which indicates that these sites may be frequent targets of BPDE mutagenesis. All mutated GC base pairs in the BPDE.induced spectrum were oriented so that the guanine was located on the non-transcribed strand. Assuming that the premutagenic lesion in these cases was covalent binding of BPDE to guanine and that BPDE bound randomly to both strands, the strand specificity of the BPDE-induced mutations indicates that preferential excision repair of BPDE adducts on the transcribed strand occurs in the hprt gene in human T-cells.

Correspondence: Dr. B. Andersson, Environmental Medicine Unit, CNT/NOVUM, S-141 57 Huddinge, Sweden.

* Present address: Environmental Medicine Unit, CNT/ NOVUM, S-141 57 Huddinge, Sweden.

The carcinogen 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (Weinstein et al., 1976; King et ai., 1976) is a metabolite of benzo[a]pyrene (B(a)P), a com- pound which is common in the environment since

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it is formed, e.g., during combustion of fossil fuelr, and is present in tobacco smoke (Brookes, 1977). The predominating form of BPDE formed in mammalian cells is the (+)-anti enantiomer (Yang et al., 1977), which has been shown to be the most mutagenie and carcinogenic form of BPDE (Brookes and Osborne, 1982). BPDE forms covalent adducts with DNA (Meehan et al., 1977), preferentially with the exocyclic N-2 amino group of guanine (Jeffrey et al., 1977). Other DNA adducts, both to guanine and to adenine, have also been demonstrated (Jeffrey et al., 1979). BPDE has been shown to iiJtercalate into DNA, and it has been hypothesized that the intercalation is required for covalent binding (Meehan et al., 1982; Geacintov, 1986; Wolfe et al., 1987), although the exact mechanism is not known. A strong stereoselectivity of BPDE damage to DNA has been demonstrated (Koreeda et al., 1978; Meehan and Straub, 1979), indicating that BPDE mutations could occur preferentially at certain DNA sequences.

Mutations induced by BPDE have been studied both in prokaryotic systems (Mizusawa et al., 1981; Chakrabarti et al., 1984; Eisenstadt et al., 1982), and in shuttle vectors and endogenous loci of CHO cells (Yang et al., 1987a, b, 1990; King and Brookes, 1984; Mazur and Glickman, 1988; Carothers et al., 1988; Carothers and Orun- berger, 1990). In these systems BPDE induced almost exclusively point mutations of GC base pairs, mostly OC > TA transversions. Other transversions as well as transitions and frameshift mutations have also been found, but to a lesser degree. A study of BPDE-induced mutations in the aprt gene in CHO cells indicated that runs of guanine bases flanked by adenines constitute 'hotspots' for BPDE mutagenesis (Mazur and Glickman, 1988), but this has not been seen in other systems. Chen et al. (1990) studied mutations induced by BPDE in the hprt gene in diploid human fibroblasts, and found a clustering of mutations in a run of 6 guanine bases, flanked by an adenine on the 5' side and a cytosine on the 3' side, in the third exon of the gene.

In the same study (Chen et al., 1990), it was found that BPDE induced mainly G C > T A transversions, with the mutated G located on the non-transcribed strand, and it was suggested that

human fibroblasts preferentially repair BPDE adducts on the non-transcribed strand. Vrieling et al. (1989) found a strand specificity for UV-induced mutations in the hprt gene of Chinese hamster V79 ceils, and showed subsequently (Vr~eling et al., 1991) that the strand specificity was due to preferential repair of lesions on the transcribed strand.

To further elucidate the specificities of BPDE mutagenesis in human cells and to provide data for comparison with possible B(a)P-induced mutations in vivo, we have studied the spectrum of mutations induced by the (+)-anti BPDE enantiomer in primary human T-lymphocytes. The T- cell cloning assay (Albertini et al., 1982; Morley et al., 1983) was used for the isolation of 6-thioguanine-resistant T-cell clones from cell cultures treated with BPDE in vitro and from untreated control cultures. Point mutations were characterized using the polymerase chain reaction (PCR) (Saiki et al., 1988) and direct DNA sequencing (Wong et al., 1987; Gyllensten and Erlich, 1988) of hprt eDNA and genomic fragments. Here we show that the spectrum of hprt mutations induced by BPDE is very specific and differs greatly from the control spectrum. In contrast to the spectrum presented by Chen et al. (1990), no particular 'hotspot' was found. There seems to be a strong strand specificity for BPDE-induced mutations also in human lymphocytes.

Material and methods

Treatment of cells and T-cell cloning The T-cell cloning procedure used for selec-

tion of these mutants has been described previously (Andersson and Lambert, 1990). Freshly isolated lymphocytes from healthy male donors were stimulated with phytohemagglutinin (PHA) for 24 h before treatment. The cells were then treated with the specific (+)-anti enantiomer of BPDE for 24 h in medium containing fetal calf serum and PHA. Four T-cell cloning experiments were performed with BPDE concentrations rang- ing from 0.3 #M to 0.6/zM. After an expression phase of 9-12 days the cells were plated (30,000 cells/well) in a medium containing 2 /zg/ml 6- thioguanine (TO). TG-resistant T-cell clones were isolated from treated and untreated cultures in

all experiments as described (Andersson and Lambert, 1990).

Preparation of RNA and DNA Total cytoplasmic RNA was prepared as fol-

lows: approximately 5 x 106 cells were washed twice using sterile ice-cold isotonic NaCl solution, and resuspended in 250/zl 10 mM Tris-HCl, pH 7.8, containing 150 mM NaCl (buffer 1) and 10 mM vanadyl-ribonucleoside complex. The cells were lysed by the addition of 26 /zl 10% NP40 and incubation on ice for 60 s. After centrifugation for 2 min, 250/zl 40 mM Tris-HCI, pH 7.8, containing 40 mM EDTA and 700 mM NaC! (buffer 2), and 50/zl 10% SDS was added to the supernatant. After extraction with phenol/ CHCl3/isoamyl alcohol (iaa) three times and once with CHCl3/iaa the RNA was precipitated by addition of 1 ml 95% ethanol. The RNA was pelleted by centrifugation for 15 rain in an Ep- pendoff centrifuge, redissolved in 250/,~l buffer 1, 250 /.d buffer 2 and 50 ~l 10% SDS, and the extraction and precipitation procedures were re- peated.

For DNA preparation, approximately 1 x 107 cells were washed twice and lysed in 2 ml of a buffer containing 75 mM NaCI, 24 mM EDTA, 0.2 mg/ml proteinase K and 0.4% SDS for 4 h. Protein and RNA were precipitated by the addition of 750 ~l saturated NaCI and after a short vortexing the samples were centrifuged at 2500 rpm for 15 rain. The DNA was precipitated by the addition of 6.5 ml 95% ethanol, rcdissolved in TE buffer, precipitated a second time using 7.5 M ammonium acetate and isopropanol and finally dissolved in TE buffer.

Southern blots Southern blotting was performed as described

(Andersson and Lambert, 1990). The hprt gene was analyzed by probing Pst I-digested DNA with a full-length hprt eDNA probe (courtesy of Dr. C.T. Caskey). Clonal identity was determined by analysis of the rearrangements of the genes coding for the T-cell receptor/3- and y-chains. For this purpose HindIII-digested DNA was hy- bridized with probes for the two T-cell receptor loci (courtesy of Drs T.W. Mak and T.H. Rabbits) and the clonal identity was determined by corn-

131

paring the different rearrangement patterns as described by Nicklas et al. (1986).

cDNA synthesis and PCR A 150-/zl aliquot of the RNA precipitate con-

taining approximately 2 /,tg total RNA was centrifuged and the pellet was washed using 70% ethanol and dried in vacuum. The RNA was dissolved in sterile water and added to a reaction tube containing 1 × PCR buffer (50 mM KCI, 10 mM Tris-HCl, pH 8.4, 2.5 mM MgCI2), 1 mM of each dNTP, 20 pmoles of the downstream hprt- specific PCR primer (see below) and 32.5 units RNasin (Promega) in a 20-~1 reaction. Fifty units Moloney murine leukemia virus reverse transcrip- tase (Pharmacia) was added and the sample was incubated at 42°C for 45 rain.

After the incubation, 8 p,l 10 × PCR buffer (as above except that the concentration of MgCl 2 was 15 mM), 50 pmoles of each PCR primer, H20 up to a final volume of 100 p,l and 2.5 units of Taq polymerase (Perkin-Elmer Cetus) were added to the eDNA reaction mix and PCR was performed as follows: an initial denaturation step at 94°C for 6 rain, 30 cycles (94°C for 30 s, 60°C for 30 s and 72°C for 30 s) and a final elongation step at 72°C for 8 rain. The primers used for amplification of human hprt cDNA were: 5'- TACGCCGGACGGATCCGTT-3' and 5'-AG- GACTCCAGATGTTTCCAA-3', flanking the coding region of the eDNA.

Genomic PCR PCR reactions were performed in 1 × PCR

buffer (as above with 1.5 mM MgCI 2) containing 200 p,M of each dNTP, 50 pmoles of each primer, 2.5 units Taq polymerase and 200-300 ng human DNA. For amplification of a fragment containing exon 1, 10% DMSO was added and only 20 pmoles of each primer was added in order to improve the specificity. The cycles were run as above for fragments containing exons 5, 6 and 9. For the fragments containing exons 7-8 and 1, annealing temperatures of 55°C and 57°C, respectively, and an elongation time of 1 rain was used. The primers used were: exon 1: 5'-TGGGACGTCTGGTCCAAGGAT-

TCA-3' (1225-1248) and 5'-CCGAACCCGG- GAAACTGGCCGCCC-3' (1851-1828)

132

exon 5: 5'-TACAGGCTTCCAAATCCCAG-3' (31,440-31,559) and 5'-GCTFACCT'ITAG- GATGGTGC-3' (31,707-31,688)

exon 6: 5'-CCTGCACCTACAAAATCCAG-3' (34,728-34,747) and 5'-TCTGCCATGCTAT- TCAGGAC-3' (35,281-35,262)

exons 7 and 8." 5'-CCCTGTAGTCTCTCTG- TATG-3' (39,768-39,787) and 5'-CTCCTTC- CAGCACCTCATAA-3' (40,181-40,200)

exon 9: 5'-AACCCTGACAACTAATAGTG-3' (41,353-41,372) and 5'-TGTIq'CACTCAA- TAGTGCAG-3' (41,762 -41,781 ).

The positions given are according to Edwards et al. (1990).

Single-strand DNA synthesis One twentieth of the PCR reaction was run on

a 3.75% polyacrylamide gel, from which the band containing the amplified hprt DNA was exc;sed. The gel slice was immersed in 20 /~1 H20 and frozen in dry ice-ethanol, thawed at room temperature, frozen a second time and thawed at + 4°C overnight. 2/zl of the gel slice solution was subjected to asymmetric PCR using, for amplified hprt eDNA, 50 pmoles of an internal primer and 1 pmole of one of the original PCR primer and for the genomic fragments, the same proportions of the original PCR primers, in 1 × PCR buffer (with 1.5 mM MgCIz), 200/zM of each dNTP and 2.5 units of Taq polymerase in a 100-/~1 reaction. PCR was run as above except that an annealing temperature of 55°C was used. Both strands were amplified in separate reactions with different internal primers in excess. The internal primers used for the asymmetric PCR of hprt eDNA were: 5'-ATTATGGACAGGACTGAA-3' and 5'-AAATCCAACAAAGTCTGGC.3'.

SeoNencing The entire asymmetric PCR reaction mix was

run on a 3.75% polyacrylamide gel and the band containing the single-stranded DNA, which mi- grates slower than double-stranded DNA, was cut out without staining with ethidium bromide. In- stead the position of the band was estimated by ethidil~m bromide staining a part of the gel containing a size marker. This was done because ethidium bromide staining of the single-stranded

DNA was found to cause unspecific terminations in the sequencing reactions (our own unpublished data). After addition of 50 /zl H20 the freeze- thaw procedure described above was used in order to extract the DNA. An aliquot of 15/zl of the gel slice solution was dried and dissolved in 7 /zl H20. After addition of 2 #1 Sequenase buffer and 10 pmoles of a sequencing primer (either one of the original PCR primers or one of the internal primers) primer-template annealing was performed at 65°C for 5 min, after which a sequencing reaction was performed using Sequenase (United States Biochemicals) and 35S-dATP according to the manufacturers' instructions. The sequencing products were separated on a 6% denaturir~g polyacrylamide gel using a base run- ner (International Biotechnologies, Inc.) and au- toradiographed for approximately 18 h using hy- per-film /3-max (Amersham). It was very difficult to obtain single-stranded DNA from the exon 1 containing fragment amplified from genomic DNA. Therefore, a recently described protocol for sequencing double-stranded PCR products via a cyclic reaction using Taq polymerase (Lee, 1991) was modified by the addition of 10% DMSO in the reaction mixes and used for the sequencing of the splice donor site in intron 1.

Results

Number of independent mutant clones Treatment of PHA-stimulated T-lymphocytes

with 0.3-0.6/zM BPDE resulted in a survival of on average 15% and an 8-54-fold elevation of the hprt mutant frequency compared to untreated control cultures, as reported earlier (Andersson and Lambert, 1990). The treatment was performed in the presence of serum, which may have reduced the effective BPDE concentration. Southern blot analysis of the hprt locus was performed on Pst I-digested DNA from 44 hprt mutant clones from BPDE-treated cultures and on 41 clones from the untreated cultures. Clonal identity was determined by Southern blot analysis of the loci for the T-cell receptor ~1- and ),-chains using HindlII-digested DNA. No alterations which were detectable on Southern blot were found among 33 independent BPDE-induced mutants while 4 out of 23 independent control mu-

TABLE 1

CONTROL MUTATIONS

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Mutant Position Exon Sequence Type of Protein mutation change

Coding region 91B8 3 1 TATG__GCG GC ~ AT

95A4 58-59 2 C'ITG__A_ATIT 2-bp del. 2817G2 118 2 CATG..QGAC GC ~ TA 256H2 124 2 CTAA_TI'A AT --, TA 91G10 134 2 ACAG.G.G.G.G.G.G.G.G.Ggta GC -~ AT 92H 11 229-232 3 GCTGACCTGC 4-bp del. 93D8 269-276 3 GTGATAGATCCATT Duplication

of 8 bp 4215B7 440 6 TG _C'TrTC TA ~ CG 2812F12 580 8 CTTG_G_ACT GC --* AT 92E12 617 9 TTFG_QTGT GC --* TA

Splice sites lntron 91D2 Exon 8: - 7 7 tgattct_ tttt 3-bp del. 92F1 Exon 8: - 1 7 ttagTI'G GC - , AT

Large alterations seen on Southern blot 289H6: deletion of the entire gene 4218D4: loss of the band containing

exon 1, and the appearance of novel shorter band

252F3: loss of the, bands containing exons 2-6

Defective translation start

frameshift gly39 --* stop ile41 ~ phe arg44 ~ iys frameshift frameshift

leu146 --~ pro asp193 ~ asn cys205 --, phe

cDNA change exon 8 loss loss of the first 21

bp of exon 8 and the entire ¢xon 8

lntron sequences are written in lower case.

tants had visible alterations (Andersson and Lam- bert, 1990). These alterations were: a deletion of the entire gene, a loss of the band containing exon 1 coupled with the appearance of a novel, shorter band and two losses of the bands containing exons 2-6 (Table 1).

Five of the control clones and 4 of the BPDE- induced clones, which were thought to be independent mutants, were by sequence analysis found to have hprt mutations identical to other clones isolated from the same original T-ceU cultures. When the T-ceU receptor Southern blot patterns were reexamined it was found that it could not be excluded that these 9 clones were in fact not independent, but instead were sibling clones of other mutants. The reason for the original misin- terpretation of the T-cell receptor patterns, caus-

ing the definition of some sibling clones as being independent, is probably the presence of normal cells in some mutant clones. This mix of cells made the T-cell receptor patterns appear different from that of their uncontaminated siblings. Five BPDE-induced mutants on which no South- ern blot analysis could be performed were analyzed by PCR and direct sequencing of hprt cDNA, and unique point mutations were found in all of these. The total numbers of individual mutants analyzed for hprt mutations in this study are therefore 18 control mutants and 34 BPDE- induced mutants.

Most of the background mutants probably de- rive from in vivo mutants, although a small proportion could have formed during the expression phase after the treatment. Approximately 95% of

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the mutants from the BPDE-treated cultures should be induced by BPDE judging from the average increase in mutant frequency in the treated cultures compared to the untreated cultures (Andersson and Lambert, 1990).

Poh~t mutations in clones from untreated cultures Using PCR and direct sequencing, unique point

mutations were found in 12 of the 14 independent mutants without Southern blot alterations. In 2 mutants no eDNA alteration was found

although the entire coding sequence was exam- ined. This is possibly due to overgrowth of non- mutant cells during the expansion of these clones.

The point mutations consisted of 5 transitions, 3 transversions. 3 small deletions and a duplication of 8 base pairs (Table 1).

The base substitutions affected both GC and AT base pairs and resulted in 5 amino acid substitutions, 1 stop codon, 1 mutated start codon and a splicing defect (see below). The duplication and 2 of the small deletions (of 2 and 4 base

TABLE 2

BPDE-1NDUCED MUTATIONS

Mutant Position Exon Sequence Type of Protein mutation change

Coding region 284H2 47 2 CAGGTTA GC --, TA 285G3 88 2 GCTG_AGG GC ~ TA 255CI 1 119 2 ATGG_.ACT GC ~ TA 283A3 133 2 GACA_Ggt AT --, TA 421 H5 134 2 ACA_Qgta GC -.-, TA 281D10 134 2 ACA_Ggta GC -.* TA 255E5 166 3 AAGGAGA GC --~ TA 282E I0 208 3 AAGG_GGG GC -'., CG 95E8 380 4 CTGG_AAA GC -'* TA 283F6 393 5 CTTGA1"T GC -'* TA 282H 11 479 6 AGGICGC del. TA 254A9 481 6 GTCGCAA GC -.* CG 254B5 485 6 CAAGgta GC -~ TA 423G2 600 8 CAG~.GAT GC -~ TA

Splice sites lntron 251DI Exon h + I I GTGgtga GC --* TA

283D2 Exon 5: - 1 4 ctagAAT GC --, TA

25 IC1 Exon 5: + 5 5 taagttc GC -+ TA

284H8 Exon 6: - 1 5 aaa_gGAT GC -,* TA

251G4 Exon 7: - 1 6 acagCTT GC ~ TA

282A2 Exon 8: - 1 7 ttagTTO GC ~ TA

284Fi2 Exon 9: - ! 8 atagCAT GC --* TA

251EI 1 Exon 9: - 1 8 atagCAT GC -.-, TA

glyl5 --* val glu29 ~ stop gly39 --. val arg44 --* trp arg44 ~ met arg44 --* met glu55 ~ stop gly69 -~ arg gly126 -* val leu 130 --, phe frameshift alal60 --* pro exon 6 loss arg199 --, ser

eDNA change inclusion of 49 bp of intron i into the cDNA exon 5 loss

inclusion of 67 bp of intron 5 and a loss of exons 4 + 5 exert 6 loss

exon 7 loss

loss of the first

2 i bp of exon 8 and of the entire exon 8 loss of the first

17 bp of exon 9 loss of the first

17 bp of exon 9

Intron sequences are written in lower case.

pairs, respectively) caused frame shifts, while the third deletion mutation caused a splicing error. The 2 splicing mutants showed eDNA PCR products of abnormal size although the hprt Southern blot pattern was normal. By PCR and direct sequencing of genomic DNA fragments it was found that both mutations were located in the intronic splice acceptor site of intron 7. In clone 92F1, base substitution in the last base of intron 7 caused skipping of the first 21 base pairs of exon 8 and of the entire exon 8 (2 PCR products were obtained from this mutant). In clone 91D2 a 3-bp deletion 6-10 base pairs from exon 8 caused skipping of the entire exon 8. The mutations affecting mRNA splicing and the mechanisms of splicing errors have been discussed elsewhere (Andersson et al., 1992).

BPDE-induced mutations No alterations in the hprt Southern blot pat-

tern were found in any of the BPDE-induced mutants. Alterations of the hprt cDNA sequence were detected in 22 independent mutants. Six of the 34 independent mutants were not analyzed due to PCR contaminations. In 2 mutants, no eDNA alteration was found (possibly due to growth of normal cells in the clone) and from 4 mutants no eDNA PCR product was obtained, possibly due to poor quality of the cells.

Types of mutations The BPDE-induced mutations are listed in

Table 2. One deletion of a TA base pair and 21 single-base substitutions were found. All the base substitutions were transversions. One AT > TA transversion, 2 GC > CG transversions and 18 GC > TA transversions were found. Thus, there is a clear predominance of transversions of GC base pairs and particularly of GC > TA transversions, which constitute 82% of the total number of BPDE-induced mutations. The base substitutions caused 9 amino acid exchanges, 2 stop codons and 9 splicing errors. The 1-bp deletion, which was positioned in exon 6, caused a frameshift and a stop codon further downstream.

Mutations affecting mRNA splicing It was found that 9 out of the 22 BPDE-in-

duced mutations (41%) affected mRNA splicing.

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Eight mutations were GC > TA transversions in introns, and one was a GC > TA transversion in the last base pair of exon 6. In the control spectrum the proportion of splicing mutants was only 17% (2 out of 12 mutants with cDNA alterations), but a larger number of mutants needs to be analyzed in order to determine whether there is a true difference in the proportion of splicing mutants between the 2 spectra.

All 9 BPDE-induced splice site mutations were GC > TA transversions. One was located in the last base of an exon, 1 in the fifth base of an intron, 1 in the first base of an intron and 6 in the last base of introns (introns 4, 5, 6, 7 and 2 mutations in intron 8). This indicates that GC > TA transversions in the essential AG dinucleotide of splice acceptor sites are the predominating splice site mutations induced by BPDE. The alterations in splicing pattern caused by these mutations have been discussed in detail elsewhere (Andersson et al., 1992).

Strand specificity of mutations There is a strong strand specificity of the

BPDE-induced mutations. All of the 13 mutants which had base substitutions in the coding region had the purines (1 adenine and 12 guanines) of the affected base pairs on the non-transcribed strand. All of the 8 GC > TA transversions in intron sequences which caused splicing deficien- cies had the guanine on the non-transcribed strand. In the splice sites, however, the guanine bases in the GC base pairs that are most impor- tant for splicing are located on the non-transcribed strand, which could be the cause of the strand bias among the splicing mutants.

Discussion

Background spectrum of mutations The types of mutations found in the mutant

clones isolated from the untreated cultures are in agreement with published in vivo spectra in the human hprt gene (Rossi et al., 1990: Recio et al., 1990a). The most frequent single-base substitutions were GC > AT transitions (4/8, 50%). Dif- ferent mechanisms for frameshift mutagenesis have been suggested (for a review, see Ripley, 1990). The simplest mechanism involves misalign-

136

ment of the 2 strands of DNA during replication due to direct repeats, thus causing aeletions or duplications. Such slippage during replication could account for the 8-bp duplication (clone 93D8), where the two 5'-most base pairs of the duplicated segment (on each strand, depending on the set of 8 base pairs that were duplicated) and the first 2 base pairs after this segment are AT and TA. Alternatively, more complex mechanisms are necessary in order to explain the formation of the small deletions in clones 92Hll, 95A4 and 91D2, of which 2 and possibly all are flanked by TA base pairs. The proportion of mutations affecting mRNA splicing (14%, 2/14, of clones with normal Southern blot pattern) is lower than that in the in vivo spectra, but it is difficult to draw any conclusions from this due to the small number of clones analyzed.

BPDE-induced spectrum of mutations

Mechanisms of mutagenesis We have shown previously that there was a

8-54-fold induction of hprt mutations by the BPDE treatment in these experiments (Anders- son and Lambert, 1990). It can be assumed from comparisons with other reports on BPDE-induced mutagenesis in mammalian cells that a large majority of the mutations from the BPDE- treated cultures in the present work were induced by BPDE adducts. The possibility that some mutations were induced by secondary mutagens formed by the reaction of BPDE with the medium during the long treatment time used in the present experiments (24 h) is unlikely, due to the specificity of the BPDE-induced mutational spectrum.

The spectrum of BPDE-induced mutations consisted of almost exclusively transversions of GC base pairs, mainly GC > TA transversions, which is in agreement with results from other mammalian systems (Mazur and Glickman, 1988; Carothers and Grunberger, 1990; Yang et al., 1990a, b; Chen et al., 1990). All BPDE enan- tiomers bind covalently to DNA, but the adduct formed when (+).anti BPDE binds to the N-2 position of guanine, which is by far the most mutagenic BPDE adduct, has a position outside the DNA helix while other BPDE adducts stay

intercalated in the DNA helix. It has been hypothesized that the (+)-anti BPDE adduct assumes a conformation where it lies along the minor groove of DNA (Weinstein et al., I976; Jeffrey et al., 1979), but recent work has indicated that the adduct is mobile so that it assumes different positions, and that it is positioned outside the helix part of the time (reviewed in Gr~islund and Jernstr6m, 1989).

A possible mechanism for the specific GC > TA transversions is that the BPDE binding causes the guanine to assume a conformation which is able to base-pair with adenine. Such a G to A base pairing has been reported for guanine-bound 2-aminofluorene (Norman et al., 1989). Different models for G to A base pairing have been suggested (Brown et al., 1989; Topal and Fresco, 1976). Another possibility is the pref~=rential insertion of adenine opposite a site where the purine is either removed or too badly damaged to be instructional (Strauss et al., 1982). It has been shown that alkali-labile sites caused by BPDE in human cells are not apurinic sites (Moran and Ebisuzaki, 1991), which indicates that the non-instructional lesions, if they occur, are probably formed by a mechanism other than purine loss. Nevertheless, since the great majority of the BPDE-induced mutations presented in this work are GC > TA transversions, it is likely that the majority of the observed induced mutations were caused by BPDE adduets to guanine. The mechanisms of the formation of the AT > TA transversion in clone 283A3 and the - A T deletion in clone 282H11 are unknown.

Sequence context Several studies have indicated a DNA se-

quence preference for BPDE mutagenesis. In E. coil (-1) frameshifts in runs of GC base pairs, and also different 1-bp substitutions were found to be induced by BPDE (Mizusawa et al., 1981; Bernelot-Moens et al., 1990). In shuttle vectors (Yang et al., 1987a, b), the majority of the mutations were GC > TA transversions. Also in these studies runs of guanines were implicated as hotspots for BPDE mutagenesis. Mazur and Glickman (1988) studied mutations induced by BPDE at the adenine phosphoribosyltransferase (aprt) gene in CHO cells and found clustering of

137

mutations, predominantly GC > TA transversions, in the sequence context 5'-AG(G)nA-3', i.e., runs of guanine bases flanked by adenine residues. Mutations induced by BPDE in the dihydrofolate reductase (dfhr) gene in the same cell type were studied by Carothers and Grun- berger (1990), who suggested a less strict sequence specificity for BPDE mutagenesis by showing that mutations clustered in 5'-AGG-3' and 5'-GGA-3' sequences and that the mutations could occur at different sites within these sensitive regions. This sequence preference could possibly be due to the necessity of stereospecific intercalation of BPDE into DNA, preceding the formation of the covalent adduct.

Of the 21 BPDE-induced single-base substitutions that are presented in this work 11 were found in either one of the G residues or, in one case, the A residue, of 5'-AGG-3' or 5'-GGA-3' sequences. In the coding region 10/14 mutations were located in this context. Only 2 mutations were located in 5'-AG(G)nA-3' sequences. Six such sequences are present in the non-transcribed strand of the hprt coding region and 3 of these are located in codons which are particularly evolutionarily conserved (Lambert et al., 1992), but they do not seem to be more sensitive to BPDE-induced mutagenesis than 5'-AGG-Y or 5'-(3GA-Y sequences themselves, Eight of the remaining 10 base substitutions were located in the (3 residue of 5'-GA-Y or 5'-AG-Y dinucleotides, 5 of which constituted the last 2 base pairs of introns. Only 2 base substitutions (one GC > CG and one GC > TA transversion) did not occur in AGG or A(J sequences. Taken to- gether these results support the findings of Carothers and Grunberger (1990) that 5'-AGG-3' and 5'-GGA-3' sequences are sensitive to BPDE mutagenesis, and that 5'-AG(G),A-3' sequences are not more sensitive than the 5'-GGA-3' and 5'-AGG-Y sequences themselves.

In mutants obtained from human fibroblasts treated with BPDE in the early G1 phase, Chert et al. (1990) found 29% of the mutations to be clustered in a run of 6 guanine.5 flanked by an adenine on the 5' side and a cytosine on the 3' side, in the third exon of the hprt gene. All mutated guanines in this spectrum were found to be on the non-transcribed strand. This implicated

the run of guanines as a hotspot for BPDE in the hprt gene in cells which have time for strand- specific excision repair after BPDE treatment. In the present BPDE-induced T-cell spectrum, where the same high degree of strand specificity was found, only one mutation was detected in the same stretch of 6 guanines, and that mutation was found in the 5'-most codon of the 6-G stretch, rather than in the adjacent downstream codon where 6 /7 of the mutations in the 6-G stretch detected by Chen et al. (1990) were located. It was proposed that the sensitivity of the 6-G stretch to BPDE mutagenesis could be due to inefficient repair of BPDE adducts in this region during G1 phase (Chert et al., 1990). If that proves to be correct, perhaps the repair of this region is different in T-lymphocytes compared to fibroblasts, which could possibly account for the difference between the 2 spectra.

In our experiments the T-iymphocytes had been stimulated with PHA for 24 h at the time of BFDE treatment. A large proportion of the cells should be near S phase and have little time for strand-specific repair before initiation of replication. The spectrum would thus be expected to be similar to the spectrum of mutations obtained after treatment of human fibroblasts in S phase with BPDE (Chen et al., 1990), in that the strand specificity of the mutations should be less obvious (see below) and that no accumulation of mutations in the 6-G stretch should be seen. The strong strand specificity of mutations in the present BPDE-induced spectrum indicates that the cells had time for strand-specific repair of BPDE adducts, but still no accumulation of mutations at the 6-G-stretch was found. A possible explana- tion for the unexpectedly strong strand specificity of the BPDE-induced mutations in the present spectrum may be that the cell growth was delayed by the BPDE block on replication. The delay would allow time for cells to repair BPDE adducts on the transcribed strand. Another possibility is that, since lymphocyte populations are never syn- chronized, there was a selection so that cells in G1 survived the BPDE treatment while cells in S phase did not. Survival was on average 15% in the treated cultures. A third possibility is that there is a higher rate of excision repair in T- lymphocytes than in fibroblasts, so that less time

138

is needed for the repair of BPDE adducts on the transcribed strand in T-lymphocytes. Also, BPDE may preferentially form adducts to the non-transcribed strand, specifically in lymphocytes and not in fibroblasts, but this is a very unlikely expla- nation.

Mutations affecting mRNA splicing Six of the 8 BPDE-induced splice site muta-

tions were GC > TA transversions located in the AG dinucleotide of splice acceptor sites. In only one of the sites was the mutated G located in an AGG sequence (intron 5). Mutations of single AG dinucleotides did not dominate the spectrum in the coding region, as only 2 mutations in AG dinucleotides were found there. This possibly indicates that the AG dinucleotides in splice acceptor sites may be particularly sensitive to BPDE mutagenesis. It is not known if there are any sequence motifs in the splice acceptor sites be- sides the AG dinucleotides that contribute to the high frequency of BPDE-induced mutations at these sites, in the dfhr locus of CHO cells treated with benzo[c]phenanthrene 42% of the induced mutations caused mRNA splicing defects (Car- others et al., 1990), which can be compared to 41% in the BPDE-induced spectrum in the present study. Splice mutations did not predominate to the same extent in the spectra of BPDE.in. duced mutations in the dthr and aprt loci in CHO cells (Carothers and Grunberger, 1990; Mazur and Glickman, 1988), or in the hprt gene in human fibroblasts (Chen et al., 1990).

Strand specificity In the hprt coding region, the ratio between

the guanine bases in which a mutation will cause an amino acid change in the transcribed strand and in the non-transcribed strand is 38 : 62. There are several GGA and GA sequences on the transcribed strand, and some of them are located in conserved codons (Lambert et al., 1992). Thus, assuming that BPDE binding to guanine is the predominant premutagenic lesion and that these lesions occur to the same extent on both strands, mutations due to mutated guanines on both strands would have been found unless strand- specific repair of BPDE adducts had been active

after BPDE treatment of the T-cells. It has been shown that BPDE binds preferentially to active chromatin (Obi et al., 1986), but it is not known whether there is a strand specificity of BPDE adduct formation. Human fibroblasts treated with BPDE in S phase were found to exhibit less obvious strand specificity (24% mutations in guanines on the transcribed strand) while cells treated in early G1 showed 100% strand specificity (Chen et al., 1990). This cell cycle dependence is similar to what was found for UV-induced mutations in the same cell type. In addition, in excision repair-deficient xeroderma pigmentosum cells the majority of the UV-induced mutations were found to be located on the transcribed strand, for cells irradiated both in G1 and in S phase, suggesting that the cell cycle-dependent strand specificity of mutations is due to preferential excision repair (McGregor et al., 1991). A similar result was found for UV-induced mutations in the hprt gene of CHO cells, where it was found that in repair- proficient cells the majority of the mutagenic lesions had been located on the non-transcribed strand while most of the mutagenic lesions had been on the transcribed strand in repair-deficient cells. The latter observation may possibly be due to differences in the mutability of the leading and lagging strands during replication (Menichini et al., 1991).

In conclusion, our data are in accord with previous studies in other systems and strongly suggest that the observed strand specificity of the BPDE-induced T-cell mutations is caused by preferential excision repair of BPDE adduets to guanine bases on the transcribed strand,

Acknowledgements

The authors wish to thank Dr. Bengt Jernstr~m for kindly supplying BPDE, Dr. Harry Vrieling for providing primer sequences and Drs. C.T. Caskey, T.W. Mak, T.H. Rabbits and R.J. Alber- tini for the DNA probes used. This study was supported by the OK Environment Foundation, The Swedish Tobacco Company, The Swedish Cancer Society, The Swedish Environmental Pro- tection Board, The Nilsson-Ehle Fund and the Marcus Borgstr~m Fund.

139

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