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

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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 ¡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.¡ 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 alíeles,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 fürMikrobiologie, 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 ñade2- /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 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1980 American Association for Cancer Research. ß.A. Kunz et al. 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 alíeles(42). incubation at 30° 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 50°; TPA in solution was at 37°for 20 min. The resulting spheroplasts were then washed added to agar overlays (43° or 48°) 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 alíelescomplement 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.
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