Effect of Tumor Promoters on Ultraviolet Light-Induced Mutation and Mitotic Recombination in Saccharomyces Cerevisiae1

[CANCER RESEARCH 40, 2323-2329. July 1980] 0008-5472/80/0040-OOOOS02.00 Effect of Tumor Promoters on Ultraviolet Light-induced Mutation and Mitotic Recombination in Saccharomyces cerevisiae1

Bernard A. Kunz,2 Mohammed A. Hannan, and R. H. Haynes

Department of Biology, York University, Toronto, Ontario. Canada M3J 1P3 Â¡B.A.K.,R.H.H]. and Ephraim McDowell Community Cancer Network and Division ol Experimental Pathology, University of Kentucky, Lexington, Kentucky 40506 [M.A.H.Â¡

ABSTRACT result in the expression of such mutant genes could be part of the carcinogenic process (16, 23, 24, 44). Thus, it has been Recently, it has been suggested that mitotic recombination proposed that mitotic recombination, which can lead to homo- is involved in tumor promotion. On this basis, one might expect zygosity of recessive alÃeles,could be involved in promotion tumor promoters to be recombinagenic. D7 is a diploid strain (16). The potentiation of mutagenic and recombinagenic effects of yeast in which both mutation and mitotic recombination can of carcinogens by various mechanisms also could result in be measured. We have used this strain to assay the known enhanced fixation and expression of tumor mutations and thus tumor promoters, iodoacetate, anthralin, and 12-O-tetradeca- might play a role in cocarcinogenesis. noylphorbol-13-acetate, and the cocarcinogen, catechol, for Trosko ef al. (36) have demonstrated that the potent tumor mutagenicity, recombinagenicity, and the ability to enhance promoter TPA3 enhances UV-induced mutation to drug resist ultraviolet light (UV)-induced genetic events. In the absence of ance in mammalian cells without itself being mutagenic. TPA preirradiation with UV, iodoacetate was found to be recombin also has been found to potentiate the induction of mutation by agenic whereas catechol was mutagenic; however, in both chemical carcinogens in Salmonella typhimurium (32) and in cases, the effects were small. Iodoacetate, anthralin, and cat Chinese hamster cells (18). To date, no effect of particular echol potentiated UV-induced mitotic crossing-over, aberrant promoters or cocarcinogens on specific recombination events colony formation, and mutation, while catechol also increased has been described. However, Kinsella ef al. (15) have pre UV-induced gene conversion. We were unable to detect any sented evidence which suggests that TPA stimulates the seg mutagenic or recombinagenic effect of 12-O-tetradecanoyl- regation of ouabain resistance in hybrid mammalian cells het phorbol-13-acetate in either whole cells or spheroplasts. Our erozygous for the marker. In addition, TPA has been claimed results do not indicate any consistent correlation between to increase the frequency of spontaneous and X-ray-induced tumor-promoting activity and the ability of an agent to induce sister chromatid exchanges in mammalian cells (16, 22), al mitotic recombination in yeast. However, the ability to poten though these observations remain controversial (21). tiate UV-induced mutation and mitotic recombination may re In view of the above findings, we decided to examine the flect the cocarcinogenic activity of certain promoters. effects of tumor promoters on mitotic events in the yeast Saccharomyces cerevisiae, a simple eukaryote, in which both INTRODUCTION mutation and recombination can be monitored. We have as sayed the known tumor promoters, TPA, (12, 38), iodoacetate The 2-stage theory of carcinogenesis proposes that malig (9), and anthralin (3), and the cocarcinogen, catechol (37), for nant transformation is the result of 2 sequential processes termed "initiation" and "promotion" (1, 7). Initiation must take mutagenicity, recombinagenicity, and the ability to potentiate UV-induced genetic events. The results indicate that iodoace place before promotion. If a promoter is given without prior tate is recombinagenic and that catechol is mutagenic, al exposure to an initiator or is applied before the initiator, then though in both cases the effects are quite small. Iodoacetate, transformation normally does not occur (2, 26). Many tumor anthralin, and catechol are all capable of enhancing UV-in promoters have also been shown to possess "cocarcinogenic" duced mutation and mitotic recombination. In general, the activity (37). When applied concurrently with a carcinogen, magnitude of potentiation is similar to that seen by Trosko ef they are able to enhance transformation frequencies above al. (36) and Lankas ef al. (18) for TPA-enhanced induced that expected for treatment with the carcinogen alone. Initiation mutation in mammalian cells. However, we were unable to find is known to be caused by subcarcinogenic doses of carcino any significant genetic effect of TPA. gens; the effects of these doses are additive and are regarded as being irreversible (4). These findings suggest that initiators MATERIALS AND METHODS induce mutations, and in fact the initiation potency of several polycyclic hydrocarbons has been correlated with their muta Yeast Strain. The diploid strain D7 was kindly provided by genic potency (13). The molecular bases of promotion and Dr. F. K. Zimmermann (Institut fÃ¼rMikrobiologie, Technische cocarcinogenesis remain unknown. If one assumes that many Hochschule, Darmstadt, Federal Republic of Germany). The tumor mutations are recessive (8, 11, 33), it follows that they genotype is will not be expressed if present as single copies in normal a ade2-40 cyh2 trp5-12 ilv1-92 diploid cells. It has been suggested that genetic events that Ã±ade2- /19 CYH2 trp5-2 7 Hv1-92

1 Supported by grants from the National Research Council of Canada and the Media. YPD medium was used for routine growth and con- Natural Sciences and Engineering Research Council of Canada. 2 To whom requests for reprints should be addressed. Received November 15, 1979; accepted April 8, 1980. ' The abbreviation used is: TPA. 12-O-tetradecanoylphorbol-13-acetate.

JULY 1980 2323

tained (per liter): 10 g yeast extract (Difco Laboratories, Detroit, dialed suspensions contained 2 x 106 or 5 x 106 cells/ml and Mich.); 20 g Bacto-peptone (Difco); and 20 g glucose. Minimal were agitated during UV exposure. media contained (per liter): 6.75 g yeast nitrogen base (Difco); UV Experiments. Washed cells (2 x 106 or 5 x 106 cells/ and 20 g glucose. Appropriate nutrients were added at the ml) were irradiated with various doses of UV. Aliquots were concentrations suggested by Sherman ef al. (28). Supple diluted and spread on appropriately supplemented minimal mented minimal media contained less adenine (5 /ig/ml) to media, with or without a particular promoter. After 6 days of enhance coloring due to expression of the ADE2 alÃeles(42). incubation at 30Â° in the dark, the plates were scored for For solid media, 20 g agar (Difco) were added per liter. Agar viability, gene conversion, colored colony formation, mitotic overlays for use with whole cells were as described for minimal crossing-over, and mutation. For experiments involving TPA, media but contained 0.75% agar (w/v). Overlays used for irradiated cells or spheroplasts were added to agar overlays spheroplasts were as described for minimal media but in ad containing TPA, and these overlays were then poured onto dition contained 1 M sorbitol, 2% YPD broth (v/v), and 3% agar supplemented minimal media plates. (w/v).4 lodoacetate and catechol were dissolved in sterile Spheroplast Formation. Approximately 109 stationary phase distilled H2O, anthralin was dissolved in dimethyl sulfoxide, and cells of D7 were washed twice in cold buffer (1 M KCI-100 HIM TPA was dissolved in acetone. Appropriate solvent controls EDTA-10 rriM Tris, pH 7.5) and resuspended in 2 ml of this were included in the genetic tests and were found to be buffer containing 12.5 jul mercaptoethanol and 4 mg zymolyase negative. Except for TPA, the solutions were added to auto- 5000 (Kirin Brewery Co. Ltd.) per ml. Samples were incubated claved media that had cooled to 50Â°; TPA in solution was at 37Â°for 20 min. The resulting spheroplasts were then washed added to agar overlays (43Â° or 48Â°) during the course of an 3 times with and resuspended in 1 M sorbitol. experiment. All solutions were prepared immediately prior to Chemicals, lodoacetate and catechol were purchased from their incorporation into media. Sigma Chemical Co., St. Louis, Mo. TPA was purchased from Detection of Mitotic Events. The diploid yeast strain D7, Consolidated Midland Corp., Brewster, N. Y. Anthralin (Pfaltz constructed by Zimmermann ef al. (43), is heteroallelic at the and Bauer Co., Stamford, Conn.) was a gift of Dr. C. Gairola ADE2 locus; ade2-40 causes an absolute adenine requirement (Tobacco and Health Institute, University of Kentucky). and the formation of red colonies, while ade2-7 79 is leaky and results in pink coloration. These alÃelescomplement so that D7 RESULTS grows in the absence of adenine, forming white colonies. Reciprocal mitotic crossing-over between ADE2 and the cen lodoacetate, Anthralin, and Catechol. If mitotic recombi tromere produces red-pink twin sectors (42). Other genetic nation plays a role in tumor promotion or cocarcinogenesis, events such as monosomy, deletion, forward mutation, and then one might expect tumor promoters to be recombinagenic gene conversion result in additional classes of aberrant colo or to influence the levels of induced mitotic recombination. To nies. However, in this latter case, specific colony types cannot investigate these possibilities, we performed experiments to be ascribed unequivocally to a particular genetic process. assay the recombinagenicity of iodoacetate, anthralin, and There are also 2 noncomplementary heteroalleles at the TRP5 catechol and to examine the ability of these agents to potentiate locus. Gene conversion at this site is signaled by the emer UV-induced mitotic recombination. Nonirradiated cells or cells gence of tryptophan-independent colonies. In addition, this exposed previously to a single dose of UV were plated on strain is homozygous for a defect at the ILV1 locus and thus is media containing various concentrations of the 3 chemicals. unable to grow in the absence of isoleucine. Both true reversion Under the treatment conditions used, cell survival was usually and forward mutation to suppression can alleviate this require greater than 90%, and the agents tested did not sensitize the ment (43). cells to killing by UV (Tables 1 and 2). However, the rate of Preparation of Cell Suspensions. The following procedure colony growth was retarded by increasing concentrations of was used to avoid high initial titers of convenants and revert- iodoacetate and catechol, and this resulted in very small colo ants. Five tubes, each containing 5 ml YPD broth, were inocu nies. In the absence of UV, iodoacetate induced gene conver lated with approximately 200 cells/ml. These cultures were sion, while catechol induced mutation and appeared to stimu incubated at 30Â°with shaking for 3 days to reach stationary late the formation of aberrant colonies, although the number of phase (approximately 3 x 10B cells/ml). Aliquots from each events scored for aberrant colony formation was very small tube were then diluted 10-fold and plated on supplemented (Table 1). As shown in Table 3, the yields (number/cell plated) minimal media lacking tryptophan or isoleucine. These plates of convertants and mutants produced by iodoacetate and cat were incubated at 30Â°for 3 days, during which time the culture echol, respectively, rose to a maximum and then declined at tubes were stored at 4Â°.The plates were then scored, and a higher doses. These increases were approximately 2-fold and culture containing low numbers of spontaneous convertants were reproducible. During the course of our experiments, we and revertants was selected. Cells from these cultures were found no effect of anthralin on the frequencies of gene conver washed 3 times and resuspended in distilled sterile H2O prior sion or mutation. Although it appeared that anthralin caused a to each experiment. dose-related decrease in the frequency of aberrant colonies, Irradiation. A 15-watt GÃˆ germicida! lamp was used; the the actual number of these colonies observed was so small that incident dose rate was adjusted to 4 ergs/sq mm/sec as no significance could be attached to this decline. Following UV measured with a Latarjet dosimeter. All experiments were irradiation, mitotic crossing-over, aberrant colony formation, carried out under yellow light to avoid photoreactivation. Irra- and mutation were enhanced on medium containing iodoace tate, anthralin, or catechol. Only catechol increased the recov * R. Storms, personal communication. ery of UV-induced gene convertants. In general, potentiation

2324 CANCER RESEARCH VOL. 40

Table 1 Effects of increasing concentrations of iodoacetate. anthralin. and catechol on mitotic gene conversion and formation of aberrant colonies Irradiated or nonirradiated cells were spread on medium containing iodoacetate, anthralin. or catechol at the indicated concentrations. colo UV dose (J/ * / 105 survi nies/1 O4survi tration024602460.000.060.100.140.000.060.100.140.00.60.81.00.00.60.81.0Surviving m)IodoacetateAgent sq fractionÂ¡ig/ml1 vors0.92.461.5"1.2262728330.80.91.00.8272626280.80.60.50.8303537d17(70)(182)(108)(95)(220)(216)(226)(248)(71)(80)(81)(72)(238)(228)(238)(242)(57)(45)(33)(54)(228)(253)(259)(103)Aberrantvors2.6

.00(779)a0.97 000015151515Anthralin (2)2.6 (754)0.94 (2)4.1 (734)1.02(796)1 (3)1.3 (1)106

07(835)1 (89)136 .03(806)1.01 (110)152d (800)0.97 (122)1636 (755)tig (123)4.5 /ml1 000015151515Catechol 00(890)0.97 (4)3.5 (860)0.95(844)0.98 (3)1.2 (1)1.1 (876)0.99 (1)118

(880)0.98 (104)137 (876)1.01 (118)146 (902)0.98(872)mg/mt1.00(740)0.97(717)0.95(132)163rf (142)7.5

000015151515Concen (6)19 (14)16 (704)0.89 (11)20 (660)1.03(762)0.97(724)0.96 (13)1

05(80)145Â° (105)150C (106)167C (708)0.83(616)trp (103)

Numbers in parentheses, actual numbers of colonies scored. 6 Genetic frequencies highly significant, p < 1%, compared to control cells not exposed to a promoter or cocarcinogen. c Genetic frequencies significant at the 1% level. " Genetic frequencies significant at the 5% level. increased with the concentration of drug in the plates, but in scored. The overlays used to detect convertants and mutants some instances the effect appeared to saturate at high doses. contained minute quantities (0.5 /Â¿g/ml) of tryptophan and The highest concentrations of catechol and iodoacetate de isoleucine, respectively. Thus, all cells experienced limited pressed the frequencies of UV-induced mitotic gene conversion growth in the overlays, and this was accompanied by an and crossing-over, respectively, to values less than those of increase in the frequencies of spontaneous gene conversion the negative controls. The reasons for this effect are unknown, and mutation. These frequencies were 2-fold higher than those but a similar reduction has been reported for the enhancement obtained by plating cells from the same cultures on media by tobacco smoke condensate of UV-induced reversion in a lacking tryptophan or isoleucine (data not shown). histidine auxotroph of S. cerev/siae (10). Under conditions where the cells were able to grow and We then chose single concentrations of each of the 3 chem divide several times in the presence of TPA, we found that the icals to examine their ability to potentiate the induction of phorbol ester had no effect on gene conversion, aberrant mitotic recombination and mutation by a range of UV doses. colony formation, or mutation (Table 5). When cells were irra Table 4 shows that, without prior UV irradiation, catechol and diated with UV and then exposed to TPA, there was no poten- iodoacetate again increased significantly the frequencies of tiation of the UV-induced genetic events. On the contrary, the mutation and gene conversion, respectively. Iodoacetate, an highest concentrations of TPA caused a decline in the fre thralin, and catechol enhanced UV-induced mitotic crossing- quency of induced aberrant colonies to a value less than that over, mutation, and formation of aberrant colonies, and cate for UV alone. Thus, we chose a dose of 1 jug TPA per ml to use chol also potentiated induced gene conversion, thus confirming for further experiments, inasmuch as this dose did not have a our previous observations. In general, the degree of enhance detrimental effect on UV-induced aberrant colonies. We then ment of a specific genetic event by a particular chemical was examined the ability of this single dose of TPA to increase the similar for all UV doses used. recovery of gene convertants, mutants, and aberrant colonies TPA. To perform experiments with TPA, we modified the produced by a range of UV doses (Table 6). There was no above procedure by adding concomitantly yeast cells and TPA significant enhancement of the UV-induced genetic end points. to appropriately supplemented agar overlays, which were then However, yeast possesses a thick cell wall, which may have poured onto media selective for the various end points to be hampered penetration of the promoter into the cells. To elimi-

JULY 1980 2325

Table 2 Effects of increasing concentrations of iodoacetate, anthralin, and catechol on mitotic crossing-over and mutation Irradiated or nonirradiated cells were spread on medium containing iodoacetate, anthralin, or catechol at the indicated concentrations. UV dose 104 sur- m)IodoacetateAgent (J/sq tration024602460.000.060.100.140.000.060.100.140.00.60.81.00.00.60.81.0Crossovers/SurvivingvivorsÂ¡ig/ml1.00 fraction * / 106survivors0.70.70.70.85688e88e77e0.80.90.90.86781d90e81"0.71.3e1.7e0.87491e106e89d(78)(78)(75)(81)(384)(587)(555)(526)(85)(92)(89)(85)(435)(501)(543)(514)(82)(132)(179)(82)(440)(546)(605)(521)

(4712)a1 000015151515Anthralin .03(4860)1 .00(4724)0.95 (4484)1.17(5520)1.13(5308)1

(31)75 (40)87 .07(5064)1.16(5452)fig (44)40 (22)NDNDNDND52 /ml1 000015151515Catechol .00(4736)0.98 (4644)0.98 (4648)0.96 (4568)1.09(5152)1.04(4916)1

(27)79 (39)81 .02(4840)1 (39)79 .07(5072)mg/ml1 (40)NDNDNDND71

000015151515Concen .00(4872)0.95 (4652)0.99 (4832)0.91 (4440)0.98(4772)0.98

(32)106e (4800)0.94 (51)11 (4576)0.95 4e(52)11 (4640)NO"NDNDND562e (52)ilv Numbers in parentheses, actual numbers of colonies scored. 6 ND, in the absence of UV, the frequency of crossovers involving ADE2 was so low that no red-pink twin sectors were detected. 0 Genetic frequencies highly significant, p < 1%, compared to control cells not exposed to a promoter or cocarcinogen. a Genetic frequencies significant at the 1% level. e Genetic frequencies significant at the 5% level.

Table 3 version, mutation, or formation of aberrant colonies. Although Effects of increasing concentrations of iodoacetate and catechol on the yields it appeared that TPA caused a decrease in the frequency of of mitotic gene convenants and mutants, respectively plated" induced aberrant colonies, the actual number of colonies Iodoacetate (mg/ ' /cell plated3 (jig/ml)0 (x10"ÃŒ9 ml)0.0 (X10')7.5 scored in this case was small, and the decline in frequency was (70)" not significant. The results obtained with the spheroplasts (82) should be interpreted cautiously, as cells "traumatized" by 24 23(182) 0.6 12 (132) 14(108) 0.81.0ilv 16 (179) wall removal do not exhibit entirely normal behavior. Sphero 6(rpVcell 12 (95)Catechol 7.5 (82) plasts are more sensitive to UV, DNA synthesis rates can be Yields calculated from data in Tables 1 and 2. ' Numbers in parentheses, actual numbers of colonies scored. 50% lower (14), and the frequencies of induced gene conver sion and mutation are reduced compared to those for similar doses of UV applied to whole cells (compare Tables 6 and 7). nate this possible barrier, the cell wall was removed by enzyme digestion, and the resulting spheroplasts were added to a DISCUSSION regeneration agar overlay containing TPA. Spheroplasts treated this way are known to continue protein and RNA Tumor promoters have been found to cause a myriad of synthesis at near normal rates (14). In the regeneration overlay, chemical, biological, and histological effects (for reviews, see they are able to synthesize new cell walls (34) and then grow Refs. 4, 27, 31, and 37). However, few attempts have been to form colonies. Preliminary experiments demonstrated that made to explain tumor promotion or cocarcinogenesis in ge yeast spheroplasts were more sensitive to UV than were whole netic terms. Kinsella and Radman (16) proposed that aberrant cells; thus, the UV dose was reduced to 10 J/sq m. When mitotic segregation events, induced by promoters, could result spheroplasts were treated with TPA, there was no effect on in the expression of recessive genetic or epigenetic chromo survival, spontaneous mutation, or gene conversion (Table 7). somal changes that might lead to tumor formation. It also Furthermore, TPA was unable to potentiate UV-induced con seemed reasonable to us that cocarcinogenic activity could be

2326 CANCER RESEARCH VOL. 40

Table 4 Effects of iodoacetate. anthralin. and catechol on genetic events induced by a range of UV doses Irradiated or nonirradiated cells were spread on minimal medium containing iodoacetate, anthralin, or catechol at the concentrations indicated. colo UV dose (J/ nies/10" survi 104 m)NoneIodoacetateAgent sq fraction1 ' 1 10s1.21444912.1"1445931.21647870.924694"1736survivors(225)(273)(780)(1599)(392)(260)(850)(1578)(229)(295)(833)(1541)(186)(450)(1509)(2846)Aberrantvors1.0741852908.691218357o1.082224327Â°2.0147"371*599Â°(1)(72)(163)(254)(8)(82)(205)(302)(1)(76)(198)(290)(2)(136)(297)(493)Crossovers/survivorsNDC44 .00(3792)"1.02(3886)0.93(3516)0.94 (17)122 (43)131 (3550)0.99(3738)0.95(3616)0 (46)NO55

^g/ml)Anthralin(4 (20)154 99(3760)0.89 (58)213b(72)ND58 (3386)1

^g/ml)Catechol(0.14 05(3992)0.98(3714)0.93 (20)161 (3530)0.93 (57)211Â° (75)ND92 (3546)1

mg/ml)0â€¢102030010203001020300102030Surviving(0.6 .04(3964)0 98(3706)0.84 (34)172 (3198)0.87 (55)249fc (82)/Vv*/1060.616871930.7336116Â°281"0.52261056257Â°1.1*28b152U297"survivors(104)(105)(516)(1140)(136)(202)(737)(1608)(91)(140)(624)(1347)(275)(177)(820)(1652) (3294)trp Numbers in parentheses, actual numbers of colonies scored. ' Genetic frequencies were highly significant, p < 1%. compared to control cells not exposed to a promoter or cocarcinogen. ND, in the absence of UV, the frequency of crossovers involving ADE2 was so low that no red-pink twin sectored colonies were detected.

Table 5 Effects of increasing concentrations of TPA on mitotic gene conversion, formation of aberrant colonies, and mutation Irradiated or nonirradiated cells were added to agar overlays, with or without TPA, which were poured onto supplemented minimal media. colo UV dose (J/ nies/ 10" survi ' / 10ssurvivors1.5 ' / 10esurvivors0.6 sqm)000015151515TPA(ng/ml)01101000110100Survivingfraction1.00(541)"0.94 vors3.7 (83)1.3 (2)4.0 (31)0.8 (507)0.87 (68)1.6 (2)2.1 (40)0.7 (473)1 (75)1.4 (1)1.8 (32)07 .05(569)1.03(555)0.85 (81)41 (1)135 (40)104

(228)45 (75)1 (288)119 (458)1.01 (204)35 75(80)1 (219)107 (544)0.93 (192)â€¢44 10(60)101 (291)107 (504)trp (220)Aberrant (51)ilv (271) Numbers in parentheses, actual numbers of colonies scored.

Table 6 Effects of TPA on genetic events induced by a range of UV doses Irradiated or nonirradiated cells were added to agar overlays, with or without TPA (1 fig/ml), which were poured onto supplemented minimal media. colonies/ 104survivors1.3 survivors0.4 UV dose (J/sqm)01020300 fraction1 O5survivors1 .00(788)"0.93 .6(246)20 (1)54 (62)19 (727)0.91 (147)68 (39)148 (46)69 (718)0.93(732)0.94 (450)126 (106)232 (167)1 (921)1.5(229)25 (170)2.7 23(302)0.4

+TPA10 (743)0.97 (2)64 (59)1 +TPA20 (768)0.85 (189)74 (49)190 5(39)69 +TPA30 (670)0.85 (493)136 (127)239 (154)137 + TPASurviving (673)frpVl (918)Aberrant (161)i/v*/106 (310) a Numbers in parentheses, actual numbers of colonies scored.

due to an increased frequency of expression of recessive and to be a general inhibitor of both DNA synthesis and repair genes via potentiation of the mutagenic or recombinagenic replication (5, 25, 35). These properties of the 3 chemicals are effects of carcinogens. consistent with an ability to induce mutation and mitotic recom Iodoacetate has been found to break DNA in Anacystis bination and/or to enhance such genetic events induced by nidulans and in human T-cells and to inhibit the repair of y-ray- DNA-damaging agents. and X-ray-induced single strand breaks (6, 17). Chromosome The results reported here are summarized qualitatively in fragmentation by catechol has been described in Allium cepa Table 8. We have found that iodoacetate is recombinagenic (19, 20). Anthralin has been reported to bind to DNA in vitro and that catechol is mutagenic although the observed re-

JULY 1980 2327

Table 7 lacking in yeast. Alternatively, S. cerevisiae may be capable of Effects of TPA on spheroplasts metabolizing TPA to intermediates which are not recombina Irradiated or nonirradiated spheroplasts were added to agar overlays, with or without TPA (1 fig/ml), which were poured onto supplemented minimal media. genic or mutagenic. Spheroplasts were able to regenerate cell walls and then grow to form colonies. Of the 3 tumor promoters tested here (TPA, iodoacetate, and The regeneration frequency was 33/102 spheroplasts added to the overlay. anthralin), only iodoacetate is recombinagenic. Thus, our re col sults do not indicate any consistent correlation between tumor- UV dose(J/sq sur onies/10*survivorsNO0NO41 sur m)00 vivors2.3 vivors0.7(71)08(81)4.1promoting activity and the ability of an agent to induce mitotic (242)a2.3(249)9.7 recombination in yeast. However, this does not rule out the +TPA1010 possibility that mitotic segregation of recessive alÃelesplays an important role in carcinogenesis. If promoters do in fact act by (80)1 (17)27(11);/vV106(34)5.7 + TPASurvivingfraction1.00(621)1.03(639)0.66(413)0.64(400)(rp*/106 1 (90)Aberrant (46) inducing mitotic recombination in mammalian cells, it would appear that this effect is achieved by a mechanism which Numbers in parentheses, actual numbers of colonies scored. ' ND. no aberrant colonies were detected cannot be provoked consistently in yeast. On the other hand, the potent cocarcinogen, catechol, which lacks tumor-promot Table 8 ing activity (37), increased the recovery of UV-induced mutants effectslodoacetateAnthralinTPACatecholUVSummary of and mitotic recombinants. These genetic events are also po tentiated by iodoacetate and anthralin (but not TPA). Thus, dose con colony for capacity to enhance the mutagenic and recombinagenic effects (J/sqm)0 version00 mation000 overNDNDNS of UV may be related to the cocarcinogenic activity of certain

150 promoters.

150 00 ACKNOWLEDGMENTS

We thank Dr. B. J. Barclay for many helpful discussions and Ingeborg Vranesic 15015Gene 00Aberrant 00Crossing- NSNDMutation0000+ for her excellent typing assistance.

REFERENCES

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1980 American Association for Cancer Research. Effect of Tumor Promoters on Ultraviolet Light-induced Mutation and Mitotic Recombination in Saccharomyces cerevisiae

Bernard A. Kunz, Mohammed A. Hannan and R. H. Haynes

Cancer Res 1980;40:2323-2329.

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