Modulation of ultraviolet light mutational hotspots by cellular stress

J.

Mol. Biol. (1992) 228, 1031-1036

Modulation

of Ultraviolet Light Mutational by Cellular Stress Saraswathy

National

Hotspots

Seetharam

Laboratory of Molecular Carcinogenesis Cancer Institute, NIH, Bethesda, MD 20892, U.S.A.

and Michael 9900 Medical

M. Seidman-f

Otsuka Pharmaceuticals Center Drive, Rockville, MD 20850, U.S.A.

(Received 6 April

1992; accepted 26 August

1992)

Stressful treatments of cells provoke broad, transient, changes in cellular physiology and gene expression. In addition to these effects, DNA-damaging agents often induce permanent change in the form of mutations. Mutational patterns in target genes typically show hotspots and coldspots, the molecular basis of which appears to lie in the sequence context of the particular site. We determined the mutational pattern in an ultraviolet light-modified (in vitro) marker gene in a shuttle vector passaged through repair deficient (xeroderma pigmentosum) cells and compared it with patterns obtained from cells exposed to stress imposed by a DNA-damaging agent or a calcium ionophore. We found that the mutational hotspot pattern was altered by both stress treatments. We conclude that the cellular environment can influence the probability of mutagenesis at specific sites and propose that some of these effects on mutagenesis are mediated by alterations in cellular calcium levels.

Keywords: mutation

spectra;

shuttle

vector;

It is well established that mammalian cells can respond to changes in their environment, particularly those classified as stressful. Among the are physiological alterations, with responses pronounced effects on the concentrations of ions such as calcium (Trump & Berezesky, 1987), and the transcriptional activation of a large number of genes (Fornace et al., 1989; Resendez et al., 1985; Stein et al., 1989). The changes provoked by stress may last for hours to days and influence the biochemical and genetic milieu in which cellular processes occur. A stressful treatment that has been studied for many years is that provided by exposure of cells to DNA-damaging agents. In addition to the transitory effects on cell physiology and gene activation (Herrlich et al., 1984), DNA damage often has the permanent consequence of mutational change. Mutagenesis of damaged DNA is thought to occur when the replication apparatus inserts an a modified base. incorrect base across from Experiments over the last 30 years with many agents and many target genes have shown that mutagenesis is not random across a gene (Benzer, t Author to whom all correspondence should be addressed.

xeroderma

$08.00/O

hotspot

variability

1961; Miller, 1985). Instead, patterns of hotspots and coldspots are commonly observed. Since damage is also not randomly distributed throughout DNA (Brash & Haseltine, 1982), the simplest model would predict a direct correlation between modification intensity and mutation frequency at given sites. Studies of this issue reveal no direct relationship, and it is generally argued that the sequence context of the modified site must influence the probability of mutagenesis at that site (Fuchs, 1984; Brash et al., 1987). Although this is quite reasonable, discussion of the problem in these terms carries the implicit assumption that the dominant, if not exclusive, determinant of hotspots and coldspots is sequence context. However, much experimental mutagenesis, and all environmental mutagenesis, necessarily involves exposure of cells to the DNAdamaging agent. Consequently, the interaction of the replication apparatus and the modified base, which may result in a mutation, occurs im a cell responding to stress. The effect of this stress response on the process of mutagenesis has received limited attention. In t’he experiments discussed here we have asked if the altered cellular environment provoked by stress can influence the probability of mutagenesis 1031

002%2836/92/241031-06

pigmentosum;

(Q 1992 Academic

Press Limited

at specific sites in modified DNA. We have distinguished effects on the DNA from those on the cells by introducing an ultraviolet light irradiated (in vitro) shuttle vector plasmid carrying a mutagenesis marker gene into cells exposed t,o stress. The and the mutational spectra were determined hotspot patterns compared with that obtained from control cells. The first goal in this study was to generate a u.v.? mutational spectrum that would be the basis for comparison with experiments with stressed cells. Xeroderma pigmentosum cells (XP12BESV40, complement,ation group A: Robbins et al., 1974) were used so as to eliminate repair act’ivity and simplifJ7 interpretation of the data. The U.V. irradiated shuttle vector, pZ189 (Seidman et aZ., 1985; Bredberg et al., 1986), was introduced into the XPA cells by lipofection with the lipid DOT;MA, as originally described by Felgner et al. (1987). After a 48 hour mutagenesis and replication period, plasmids with mutations in the suppressor tyrosyl tRNA (su@‘) marker gene were recovered at a frequency of about @3%, about 100-fold above the background. This was in good agreement with previous results with this vector (Bredberg et al., 1986). The sequences of 145 independent mutants were determined. The majority of the mutations were single base substitutions (86%) with some tandem mutations (14%). As in earlier work with these cells, the primary mut’ation was the G. C to 8.T transition (Table 1) (Bredberg et al., 1986). The mutation pattern (Fig. 1) showed strong hotspots at positions 155; 156, 168 and 169 (mutations at t’hese 4 sites accounted for almost 70% of all mutations). The similar frequency of event’s at 155 and 156 (17

t Abbreviations used: u.v.. ultraviolet light; DOTMA, 1~.[l-(2,3-dioleyloxy)propgl]-IY;,~,N-trimethviammonium chloride: MMS, methyl methane sulfonate.

and 18%) was of particular interest because these sites are the corresponding positions on opposite st’rands of an eight-base palindrome (positions 152 of u.v. modification at these to 159). The intensity

Tabie 1 &se

substitutionns in single and tandem

‘mutations

Treatment k&se change

DOTMA

DOTMA-IO

DOTMA-MMS

d. Transitions G.C+A.T A.T-tG.C s.

151 (92%) 0

41 0

(‘58%)

60 (900,;,) 3 (44 “,,b)

Transversions

G~G4.G G.C+T.A A-T->T.$ S.T-+C,G

1 (@f~%l 4 (2%j 8 (5%) I (@6”j,)

8 (134,) 11 (IS?/,) 0 0

1 (150/,) 1 (1.6%) 2 (3%) 0

Seroderma pigmentosum XPIBE (simiatl virus 40-transformed) fibroblasts (Robbins et al., 1974) were transfeeted with the pZlR9 shuttle vettor (Seidman et a.1..1985) irradiated with either 0 or 300 J/m’ using a germicidal lamp (254 nm). The plasmid preparations were introduced into the cells by lipofection (Felgner el al.. 1987) using S-( i-(2,3-diole$oxy)prapylichloride ,1’.~,N-trimethylammonium (DOTMA) CURL Gaithersburg. MD). according to the manufacturer’s prowcol. except that the transfection period was limited to 90 min. Tn the experiments with ionosphere (IO) t,reatment. the transfection was followed by a 6 h incubation with 6 pM-ionomycin (Calbiochem. San Diego, CA), after which time the cells were washed and fed with fresh medium. In other experiments, the cells were treated with 1 pg of met,hyl methane sulfonate (MM) for 2 h, washed and refed, and then transfected (using DOTK4) 24 h later. After 48 b transfecection. the progeny piasmids were harvested. purified and introduced into the E. coli indicator strain M13M7070 (Seidman et al., 1985). The bacteria were plated j-broml,-4-chloro-3-indolS’!on indicator plates containing P,D-gslactopiranoside and mutant colonies (white or light blue) selected and purified. The plasmids were extracted and the sequence of the mutant s?qP tRNA marker gene determined (Sequenase, USE. OH). Only the sequences of independent mutant plasmids were reported.

Communications

1033 GGGGGrGGrm

CCACCCC~~CGCC~CC~C~~GA~~A~C~A~A~~~C~~A~ I * 100

I

I 110

I 120

I 130

I 140

I 150

I 160

I 170

I 180

I

I

I

I

I

I

I

I

GGTGGGGITCCCGAGC~C~~~A~~~~T~~~CAT~~~~~TC~CCCCCAC~C~ AAAAC -AA w&l AA AA AA A!!3 A

3’ A A A A A A A A A A

GA

Figure 2. Mutation spectrum from cells transfected with u.v.-treated prior to transfection. There was no mutation at position 43 two sites is similar (Brash et al., 1987). The appearance of mutations at position 43, an infrequently mutagenized site, was noteworthy as well. The protocol followed in this experiment was designed to avoid deliberate stress to the cells, save whatever the transfection process might have imposed. Since mutagenesis more typically occurs in stressed cells, it was of interest to ask if treatment of the cells with a DNA-damaging agent prior to the transfection would affect the mutagenesis of the u.v.-treated plasmid. The XP cells were incubated with the alkylating agent methyl methane sulfonate (MMS). After 24 hours, by which time no reactive MMS would be present (Lawley, 1984), they were transfected with the U.V. irradiated pZ189 preparation as in the previous experiment. We recovered mutant plasmids at frequencies similar to those in the first experiment, indicating that the pretreatment did not have a pronounced effect on the mutation frequency. This was in agreement with an earlier study on mutation frequency and pretreatment of cells with mitomycin (Roilides et al., 1988). The sequence analysis of 54 plasmids with single or tandem mutations revealed a slight decrease in the frequency of single base changes (75%) and a corresponding increase in tandem mutations (25%). As with the control pattern, the collection was dominated by the G. C to A. T transition (Table 1). The mutational spectrum of the single and tandem mutations (Fig. 2) was similar to the control pattern with hotspots at positions 156 and 168. However, there were no mutations at position 155. The reduction of mutations at this site was reminiscent of the patterns we had obtained in experiments in which the cells were transfected with a calcium phosphate protocol (Bredberg et al., 1986;

5’

A A A A

‘l-l- AT -TTATT TT TT TT TT T-r T T T T T T T T T T T A

pZ189 following 2 h incubation

with MMS 24 h

Seetharam et al., 1990; Seetharam & Seidman, 1991). An increase in intracellular levels of calcium is a common result of stressful treatments o-f cells, and it has been proposed that these increases underlie much of the changes provoked by stress, including DNA damage (Trump & Berezesky, 1987). The possibility that some of the effects of the MMS might be linked to perturbation of the calcium levels led us to study the consequences of treating the cells with a calcium ionophore after the transfection of the u.v.-modified plasmid. The cells were transfected as before with the DOTMA procedure, incubated for six hours with ionomycin, and then the plasmids harvested 48 hours after the start of the transfection (Fig. 3). The ionophore treatment did not influence the mutation frequency of either unmodified or u.v.-treated plasmid. Analysis of 48 plasmids with single (75%) and tandem mutations (25%) revealed a higher frequency of transversions than in the other experiments (Table 1). The mutation spectrum showed hotspots at positions 156 and 168, as with the other patterns. Mutagenesis at position 155 (2%) was clearly reduced relative to the control pattern. In addition, there was a new hotspot, not seen in the other patterns, at the cytosine triplet at positions 108 to 110 (25% of all mutations, p < 0.001). In Table 2 we have summarized the results of the experiments discussed here as well as from previous work with XPl2BE cells. We have listed the total number of plasmids with single or tandem mutations and the number of mutations at selected sites from relevant experiments. Position -156 has been a hotspot in all experiments (and in all cell lines) regardless of manipulation, and serves as a useful internal marker for the relative intensity of

1034

14‘.Seetharam

and Jf. N. Seidman

CcACCCCAAGGGcrcGccGGl-rr I 110

1 100

5”

I 120

I 130

I 160

I 140

I 170

150 I I I I I 1 $ I GfXGGGGITCCCGAGCGGCCAAAGGCAAAGGGAGCAGACKl-MATCIGCCGl-CATCGACMXXAAGtLZ’TCGAATCCl-l’CCCCCACCACCA MA AA G TT TA a AA G TT A AA G TT A AA T-r A AA I!3

A

T-r

rr

A

AGE

A A

T T

&!I

A

c;r !z

A

A Figure 3. ;\‘Iut’ation spectrum from cells transfected 6 h. There was no mutation at position 43.

with

other hotspots. Position 168 has been a hotspot in all experiments in the XPl2BE cells, although it is variable in other cells (Bredberg et al., 1986; Seetharam et al., 1990). Compared with the DOTMA pattern, mutagenesis at position 155 was reduced in cells pretreated with MMS (p < O.OOl), in the cells incubated with the ionophore (p < OOl), and in earlier work in these cells with calcium transfection protocols (Seetharam & Seidman, 1991; Bredberg et al., 1986: p < O*OOl). In contrast, and in agreement with the DOTMA experiment, position 155 was a hotspot in the pattern from cells transfected with DEAE-Dextran (Seetharam & Seidman, 1991). The Table shows the frequency of mut#ations at position 43. Although mutagenesis at this site is rare under any circumstances, analysis of the combined data indicates that position 43 was mutagenized only in experiments in which calcium or deliberate stress was avoided @ < 0.001). We have called attention to this site because it is one of the most heavily modified by ultraviolet light (1.3 times the adduct frequency found at position 156, with both dimer and 6-4 photoproducts, Brash et al., 1987). Apparently, the sequence context and structure of

u.v.-treated

Treatment

Mutants

CaPO,

Ionophore MMS

DOTMB DDex

of mutations

3”

T T T

pZ189 and incubated with 6 PM-ionomycin

for

this site is such that mutagenic passage of the relevant polymerase through adducts is much less likely t,han through adducts at other sites such as position 168 (a relatively lightly modified site; Brash et al., 1987). The data in Table 2 suggest that inhibition of mutagenesis at position 43 can be modulated somewhat by the cellular condition. The outcome of the encounter between the replication apparatus and the lesion can show substantial quantitative variation from site to site in a gene. Hotspot patterns have been a feature of’mutagenesis experiments for many years (Benzer, 1961: Miller, 1985). The explanation that this is simply a function of non-uniform modificat’ion across the marker appears to be inadequate. The work of Fuchs (1984), and our own previous studies (Brash et al., 1987), called attention to the observation t,ha,t the probability of forming a mutation at sites of modificat’ion was not’ proportional to addwct frequency. Instead, some adduct sites in a gene were much more likely to be mutagenized than others. Fuchs (1984) suggested that the basis for the different mutational activity at different sites lay in the sequence context, which would influence the

at specijic

hotspots

43

155

156

168

164

0

6

49

38

48 54

0 0

1

I1

li

0

10

17

266

0

7

70

66

145

4 3

24 13

26 13

13

7

37

39

35

75 220

I

T

Table 2 Frequency

180

12

Reference

Bredberg et al. (1986) Seetharam et al. (1991) This report This report

This report Seetharam et al. (1991)

Dipyrimidine photoproducts may give rise to either single or tandem mutations. The total number of mutants in each data set refers to the total number of plasmids with mutations. Thus, a single or tandem mutation is each counted as 1. Only single mutations were found at positions 43. 155 and 156. Although both single and tandem mutations were found at position 168, only the single mutations are listed so as to facilitate comparison with positions 155 and 156. Some unpublished data ape included in the totals from the calcium phosphate transfection experiments. DDex. DEAE-Dextran.

Communications

structure of the modified sequence. We proposed a pass/fail model of options available to the replicative apparatus at the time of encounter with the lesion, and suggested that the frequency of mutations at a particular site was a function of the frequency of modification at the site coupled with the pass/fail ratio of the site (Brash et al., 1987). We interpret the results of the experiments described here as indicating that the pass/fail ratio at particular sites is not fixed. Instead, the probability of the replication apparatus inserting a mutation at particular modified sites can be influenced by the conditions in the cells at the time of the interaction of the replication apparatus with the lesion. This was the implication of our earlier observations of different hotspots in different cell lines (Bredberg et al., 1986; Seetharam et al., 1990, 1991) and is demonstrated more directly here. The stress response is complex and includes changes in cellular physiology as well as gene expression. Consequently, the molecular basis of the hotspot variability could be due to differences in the physiological environment and/or the complement of relevant gene products, which obtain at the time of the mutagenic event. The results with calcium phosphate transfection, as well as with the stressed cells in this report, imply that in the stress experiments, and the calcium transfection experiments, the cellular conditions at the time of the collision of the replication apparatus with the relevant photoproducts were similar in so far as events at position 155 were concerned. It seems reasonable to suggest that there was a role for cellular calcium levels in establishing these conditions. It might be asked if the putative physiological effects on the pass/fail ratio can be distinguished from the possible influence due to alterations in gene products following stress. An important consideration relevant to this question is the timing of the critical replication of the modified templates during which the mutagenesis occurs. The physiological recovery of cells from ionophore treatment is relatively rapid and internal calcium levels are restored to normal within minutes. However, the effects on gene expression and other cellular functions may persist for hours to days (Hesketh et al.; 1983). Consequently, if the critical replication occurred after the six hour ionophore exposure of our experiment, then it is most likely that alterations in gene products would affect directly or indirectly the mutational patterns. A recent study, which suggests that replicated plasmid is recoverable between 12 and 21 hours after transfection, would seem to support the gene product mechanism (Mah et al., 1991). However, the time at which replicated plasmid can be recovered is not necessarily the same as the time of the first rounds of replication, and so the question still requires resolution. Whatever the basis of the changes in the patterns, it is important to note that different sites in the marker gene were affected differently by the stress. In contrast to the inhibition at position 155, the frequency of events at other sites, such as 156 and

1035

168, was not affected. Finally, there was a new hotspot at positions 108 to 110 in the ionophore experiment. This was composed of tandem mutations in contrast to the single base substitution mutations of most of the other sites. According to the adenine insertion rule (Tessman, 1976; Strauss et al., 1982), tandem mutations would be expected to occur at dicytosine sites and the mutations at positions 108 to 110 are in accord with this feature of the rule. (In contrast, the adenine insertion p-rediction is not fulfilled in many of the mutations at this site.) However, many other mutagenizable dicytosine sites, such as 172 to 176, were not hotspots in this experiment. Consequently, the hotspot variability is a function of a particular sequence in the context of the cellular environment. We have little insight as to the nature or length of the relevant sequence, save that in the case of events at positions 155 and 156, those determinants must lie outside the eight base-pair palindrome. It has been established that the Klenow fragment of Escherichia coli polymerase I has contact with 19 bases of template DNA (Allen et al., 1989). It is likely that the relevant mammalian replication apparatus is in contact with a longer template region. The range of the sequence context that might influence the pass/fail ratio of particular modified sites could be much greater than the bases in the palindrome around the 155 and 156 hotspots. In previous publications we have emphasized hotspot variability in different cell lines. In light of the observations discussed here, it is clearly important to ask if that variability could now be explained by variations in the experimental manipulation of the cells. However, a review of the earlier data and the results presented here indicates that this explanation is not valid for the two major hotspots that were variable in different cell lines. As indicated in Table 2, position 168 has been a hotspot in every experiment with the XP12BE cells, regardless of the transfection method. Furthermore, the same transfection procedures were used with the cells in which position 168 was not a hotspot (Seetharam et al., 1989, 1990). Similar conclusions apply to another variable hotspot, at position 159. There may be multiple effecters of mutagenesis in mammalian cells, only some of which can be linked at this time to stress or cellular manipulation. We thank Dr encouragement.

Harry

Gelboin

for

support

and

References Allen

D. J., Darke, I?. L. & Benkovic, S. J. (1989). Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I. Biochemistry, 28, 4601-4607. Benzer, S. (1961). On the topography of the genetic fine structure. Proc. Nat. Acad. Xci., U.S.A. 47, 4-03-416. Brash, 9. E. C Haseltine, W. A. (1982). UV--induced mutation hotspots occur at DNA damage h,otspots. Nature (London), 298, 189-192.

S. Seetharam

1036

and Iti. X. Seidman

Brash, D. E., Seetharam, S., Kraemer, K. H., Seidman, M. M. & Bredberg, A. (1987). Photoproduct frequency is not the major determinant of UV base substitution hotspots or coldspots in human cells. Proc. Nat. Acad. Sci., U.S.A. 84, 3782-3786. Bredberg, A.; Kraemer, K. H. & Seidman, M. M. (1986). Restricted mutational spectrum in an UV-treated shuttle vector propagated in xeroderma pigmentosum cells. Proc. Nat. Acad. Sci., U.S.A. 83, %2738277. Felgner, P. L., Gadek, T. R.; Holm, M., Roman! R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. & Danielsen, M. (1987). Lipofectin: a highly efficient, lipid mediated DNA transfection procedure. Proc. -Qat. Acad. Sci., U.S.A. 84. 7413-7417. Fornace, A. J., Alamo, I., Hollander. M. C. & Lamoreaux, E. (1989). Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells. Expt. Cell. Res. 182, 61-74. Fuchs, R,. P. P. (1984). DP;A binding spectrum of the N-2-acetylaminofluorene N-acetoxy carcinogen significantly differs from the mutation spectrum. J. Mol. Biol. 177, 173-180. Herrlich, P., Mallick, U.: Ponta, H. & Rahmsdorf, H. J. (1984). Genetic changes in mammalian cells reminiscent of an SOS response. Hum. Genet. 67, 360-368. Hesketh, T. R., Pozzan; T.; Smith, G. A. & Metcalfe, J. C. (1983). Limits to the early increase in free cytoplasmic calcium concentration during the mitogenie stimulation of lymphocytes. Biochem. J. 212, 685-690.

Lawley, P. D. (1984). In Chemical Carcinogens (Searle, C. E., ed.), p. 325, American Chemical Society, Washington DC. Mah, M. C. MM.,Boldt, J., Gulp, S. J.? Maher, V. M. & McCormick, J. J. (1991). Replication of acetylaminofluorene-adducted plasmids in human cells: spectrum of base substitutions and evidence of excision repair. Proc. Nat. Acad. Sci., U.S.A. 88, 10193-10197. Miller, J. H. (1985). Mutagenic specificity of ultraviolet light. J. Mol. Biol. 117, 525-567. Resendez, E., Attenello, J. W., Grafsky, A.; Chang, C. S. & Lee; $. S. (1985). Calcium ionophore A23187 induces expression of glucose regulated genes and their heterologous fusion genes. Mol. Cell. Biol. 5, 1212-1219. Robbins, J. H.; Kraemer? K. H., Lutzner; M. A., Festoff, B. W. & Coon, H. G. (1974). Xeroderma pigmen-

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tosum, an inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann. Int. Med. 80; 221-248. Roilides, E., Munson; P. J., Levine, A. S. & Dixon K. (198%). Use of simian virus 40 based shuttle vector to analyze enhanced mutagenesis in Mitomycin C treat)ed monkey cells. fMol. Cell. Biol. 8%3943-3946. Seetharam; S. & Seidman, M. M. (1991). Modulation of an ultraviolet mutational hotspot in a shuttle vector in xeroderma cells. Nucl. Acids Res. 19, 1601-1604. Seetharam, S.; Waters, H.. Seidman, M. M. & Kraemer. K. H. (1989). Ultraviolet mutagenesis in a plasmid vector replicat,ed in lymphoid cells from a patient with the melanoma prone disorder dysplast’ic nevus syndrome. Cancer Res. 49? 5918-5921. Seetharam, S., Kraemer, K. H., Waters, H. & Seidman, M. X. (1990). Mutational hotspot variability in an ultraviolet treated shuttle vector plasmid propagat,ed in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts. J. 1Mol. Biol. 212. 433-436. Seethara,m, S., Kraemer, K. H.. Waters, H. L. & Seidman, M. M. (1991). Ultraviolet mutational spect,rum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts. Mutat. Res. 254, 97-105. Seidman, M. M., Dixon, K., Razzaque; A.; Zagursky: R. J. & Berman, M. L. (1985). A shuttle vector plasmid system for studying carcinogen induced point mutations in mammalian cells. Gene, 3%. 233-237. Stein, B., Rahmsdorf, H. J., Steffen, A.; Litfin, M. & Herrlich, P. (1989). UV-induced DKA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein. Mol. Cell. Biol. 9. 51695181. Strauss, B., Rabkin, S.; Sagher, D. & Moore , P. (1982). The role of DKA polymerase in base substitution non-instructional mutagenesis on t,emplates. Biochinzie, 64. 829-838. Tessman, I. (1976). A mechanism of UV-reactivation. In Abstracts of the Bacteriophmge Meeting (Bulchari, A. I. 8t Ljungquist, E., eds), p. 87; Cold Spring Ha,rbor Laboratory Press, Cold Spring Harbor, NY. Trump, B. F. & Berezesky, I. K. (1987). Ion regulation, cell injury and carcinogenesis. Carcinogenesis, 8; 1027-1031.

by J. N. Sfilley