[CANCER RESEARCH 40, 2323-2329. July 1980]0008-5472/80/0040-OOOOS02.00

Effect of Tumor Promoters on Ultraviolet Light-induced Mutation andMitotic 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 olExperimental Pathology, University of Kentucky, Lexington, Kentucky 40506 [M.A.H.¡

ABSTRACT

Recently, it has been suggested that mitotic recombinationis involved in tumor promotion. On this basis, one might expecttumor promoters to be recombinagenic. D7 is a diploid strainof yeast in which both mutation and mitotic recombination canbe measured. We have used this strain to assay the knowntumor promoters, iodoacetate, anthralin, and 12-O-tetradeca-noylphorbol-13-acetate, and the cocarcinogen, catechol, for

mutagenicity, recombinagenicity, and the ability to enhanceultraviolet light (UV)-induced genetic events. In the absence of

preirradiation with UV, iodoacetate was found to be recombinagenic whereas catechol was mutagenic; however, in bothcases, the effects were small. Iodoacetate, anthralin, and catechol potentiated UV-induced mitotic crossing-over, aberrant

colony formation, and mutation, while catechol also increasedUV-induced gene conversion. We were unable to detect anymutagenic or recombinagenic effect of 12-O-tetradecanoyl-phorbol-13-acetate in either whole cells or spheroplasts. Ourresults do not indicate any consistent correlation betweentumor-promoting activity and the ability of an agent to inducemitotic recombination in yeast. However, the ability to potentiate UV-induced mutation and mitotic recombination may reflect the cocarcinogenic activity of certain promoters.

INTRODUCTION

The 2-stage theory of carcinogenesis proposes that malig

nant transformation is the result of 2 sequential processestermed "initiation" and "promotion" (1, 7). Initiation must take

place before promotion. If a promoter is given without priorexposure to an initiator or is applied before the initiator, thentransformation normally does not occur (2, 26). Many tumorpromoters have also been shown to possess "cocarcinogenic"

activity (37). When applied concurrently with a carcinogen,they are able to enhance transformation frequencies abovethat expected for treatment with the carcinogen alone. Initiationis known to be caused by subcarcinogenic doses of carcinogens; the effects of these doses are additive and are regardedas being irreversible (4). These findings suggest that initiatorsinduce mutations, and in fact the initiation potency of severalpolycyclic hydrocarbons has been correlated with their mutagenic potency (13). The molecular bases of promotion andcocarcinogenesis remain unknown. If one assumes that manytumor mutations are recessive (8, 11, 33), it follows that theywill not be expressed if present as single copies in normaldiploid cells. It has been suggested that genetic events that

1Supported by grants from the National Research Council of Canada and the

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.

result in the expression of such mutant genes could be part ofthe carcinogenic process (16, 23, 24, 44). Thus, it has beenproposed that mitotic recombination, which can lead to homo-

zygosity of recessive alíeles,could be involved in promotion(16). The potentiation of mutagenic and recombinagenic effectsof carcinogens by various mechanisms also could result inenhanced fixation and expression of tumor mutations and thusmight play a role in cocarcinogenesis.

Trosko ef al. (36) have demonstrated that the potent tumorpromoter TPA3 enhances UV-induced mutation to drug resist

ance in mammalian cells without itself being mutagenic. TPAalso has been found to potentiate the induction of mutation bychemical carcinogens in Salmonella typhimurium (32) and inChinese hamster cells (18). To date, no effect of particularpromoters or cocarcinogens on specific recombination eventshas been described. However, Kinsella ef al. (15) have presented evidence which suggests that TPA stimulates the segregation of ouabain resistance in hybrid mammalian cells heterozygous for the marker. In addition, TPA has been claimedto increase the frequency of spontaneous and X-ray-induced

sister chromatid exchanges in mammalian cells (16, 22), although these observations remain controversial (21).

In view of the above findings, we decided to examine theeffects of tumor promoters on mitotic events in the yeastSaccharomyces cerevisiae, a simple eukaryote, in which bothmutation and recombination can be monitored. We have assayed the known tumor promoters, TPA, (12, 38), iodoacetate(9), and anthralin (3), and the cocarcinogen, catechol (37), formutagenicity, recombinagenicity, and the ability to potentiateUV-induced genetic events. The results indicate that iodoace

tate is recombinagenic and that catechol is mutagenic, although in both cases the effects are quite small. Iodoacetate,anthralin, and catechol are all capable of enhancing UV-induced mutation and mitotic recombination. In general, themagnitude of potentiation is similar to that seen by Trosko efal. (36) and Lankas ef al. (18) for TPA-enhanced induced

mutation in mammalian cells. However, we were unable to findany significant genetic effect of TPA.

MATERIALS AND METHODS

Yeast Strain. The diploid strain D7 was kindly provided byDr. F. K. Zimmermann (Institut fürMikrobiologie, TechnischeHochschule, Darmstadt, Federal Republic of Germany). Thegenotype is

a ade2-40 cyh2 trp5-12 ilv1-92

ñade2- /19 CYH2 trp5-2 7 Hv1-92

Media. YPD medium was used for routine growth and con-

' The abbreviation used is: TPA. 12-O-tetradecanoylphorbol-13-acetate.

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ß.A. Kunz et al.

tained (per liter): 10 g yeast extract (Difco Laboratories, Detroit,Mich.); 20 g Bacto-peptone (Difco); and 20 g glucose. Minimal

media contained (per liter): 6.75 g yeast nitrogen base (Difco);and 20 g glucose. Appropriate nutrients were added at theconcentrations suggested by Sherman ef al. (28). Supplemented minimal media contained less adenine (5 /ig/ml) toenhance coloring due to expression of the ADE2 alíeles(42).For solid media, 20 g agar (Difco) were added per liter. Agaroverlays for use with whole cells were as described for minimalmedia but contained 0.75% agar (w/v). Overlays used forspheroplasts were as described for minimal media but in addition contained 1 M sorbitol, 2% YPD broth (v/v), and 3% agar(w/v).4 lodoacetate and catechol were dissolved in sterile

distilled H2O, anthralin was dissolved in dimethyl sulfoxide, andTPA was dissolved in acetone. Appropriate solvent controlswere included in the genetic tests and were found to benegative. Except for TPA, the solutions were added to auto-claved media that had cooled to 50°; TPA in solution wasadded to agar overlays (43° or 48°) during the course of an

experiment. All solutions were prepared immediately prior totheir incorporation into media.

Detection of Mitotic Events. The diploid yeast strain D7,constructed by Zimmermann ef al. (43), is heteroallelic at theADE2 locus; ade2-40 causes an absolute adenine requirementand the formation of red colonies, while ade2-7 79 is leaky and

results in pink coloration. These alíelescomplement so that D7grows in the absence of adenine, forming white colonies.Reciprocal mitotic crossing-over between ADE2 and the centromere produces red-pink twin sectors (42). Other genetic

events such as monosomy, deletion, forward mutation, andgene conversion result in additional classes of aberrant colonies. However, in this latter case, specific colony types cannotbe ascribed unequivocally to a particular genetic process.There are also 2 noncomplementary heteroalleles at the TRP5locus. Gene conversion at this site is signaled by the emergence of tryptophan-independent colonies. In addition, this

strain is homozygous for a defect at the ILV1 locus and thus isunable to grow in the absence of isoleucine. Both true reversionand forward mutation to suppression can alleviate this requirement (43).

Preparation of Cell Suspensions. The following procedurewas used to avoid high initial titers of convenants and revert-ants. Five tubes, each containing 5 ml YPD broth, were inoculated with approximately 200 cells/ml. These cultures wereincubated at 30°with shaking for 3 days to reach stationaryphase (approximately 3 x 10B cells/ml). Aliquots from each

tube were then diluted 10-fold and plated on supplemented

minimal media lacking tryptophan or isoleucine. These plateswere incubated at 30°for 3 days, during which time the culturetubes were stored at 4°.The plates were then scored, and a

culture containing low numbers of spontaneous convertantsand revertants was selected. Cells from these cultures werewashed 3 times and resuspended in distilled sterile H2O priorto each experiment.

Irradiation. A 15-watt GÈ germicida! lamp was used; the

incident dose rate was adjusted to 4 ergs/sq mm/sec asmeasured with a Latarjet dosimeter. All experiments werecarried out under yellow light to avoid photoreactivation. Irra-

* R. Storms, personal communication.

dialed suspensions contained 2 x 106 or 5 x 106 cells/ml and

were agitated during UV exposure.UV Experiments. Washed cells (2 x 106 or 5 x 106 cells/

ml) were irradiated with various doses of UV. Aliquots werediluted and spread on appropriately supplemented minimalmedia, with or without a particular promoter. After 6 days ofincubation at 30° in the dark, the plates were scored for

viability, gene conversion, colored colony formation, mitoticcrossing-over, and mutation. For experiments involving TPA,

irradiated cells or spheroplasts were added to agar overlayscontaining TPA, and these overlays were then poured ontosupplemented minimal media plates.

Spheroplast Formation. Approximately 109 stationary phase

cells of D7 were washed twice in cold buffer (1 M KCI-100 HIMEDTA-10 rriM Tris, pH 7.5) and resuspended in 2 ml of this

buffer containing 12.5 jul mercaptoethanol and 4 mg zymolyase5000 (Kirin Brewery Co. Ltd.) per ml. Samples were incubatedat 37°for 20 min. The resulting spheroplasts were then washed

3 times with and resuspended in 1 M sorbitol.Chemicals, lodoacetate and catechol were purchased from

Sigma Chemical Co., St. Louis, Mo. TPA was purchased fromConsolidated Midland Corp., Brewster, N. Y. Anthralin (Pfaltzand Bauer Co., Stamford, Conn.) was a gift of Dr. C. Gairola(Tobacco and Health Institute, University of Kentucky).

RESULTS

lodoacetate, Anthralin, and Catechol. If mitotic recombination plays a role in tumor promotion or cocarcinogenesis,then one might expect tumor promoters to be recombinagenicor to influence the levels of induced mitotic recombination. Toinvestigate these possibilities, we performed experiments toassay the recombinagenicity of iodoacetate, anthralin, andcatechol and to examine the ability of these agents to potentiateUV-induced mitotic recombination. Nonirradiated cells or cells

exposed previously to a single dose of UV were plated onmedia containing various concentrations of the 3 chemicals.Under the treatment conditions used, cell survival was usuallygreater than 90%, and the agents tested did not sensitize thecells to killing by UV (Tables 1 and 2). However, the rate ofcolony growth was retarded by increasing concentrations ofiodoacetate and catechol, and this resulted in very small colonies. In the absence of UV, iodoacetate induced gene conversion, while catechol induced mutation and appeared to stimulate the formation of aberrant colonies, although the number ofevents scored for aberrant colony formation was very small(Table 1). As shown in Table 3, the yields (number/cell plated)of convertants and mutants produced by iodoacetate and catechol, respectively, rose to a maximum and then declined athigher doses. These increases were approximately 2-fold andwere reproducible. During the course of our experiments, wefound no effect of anthralin on the frequencies of gene conversion or mutation. Although it appeared that anthralin caused adose-related decrease in the frequency of aberrant colonies,

the actual number of these colonies observed was so small thatno significance could be attached to this decline. Following UVirradiation, mitotic crossing-over, aberrant colony formation,

and mutation were enhanced on medium containing iodoacetate, anthralin, or catechol. Only catechol increased the recovery of UV-induced gene convertants. In general, potentiation

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Tumor Promoters and Mitotic Recombination

Table 1

Effects of increasing concentrations of iodoacetate. anthralin. and catechol on mitotic gene conversionand formation of aberrant colonies

Irradiated or nonirradiated cells were spread on medium containing iodoacetate, anthralin. or catechol atthe indicated concentrations.

UV dose (J/Agent sqm)Iodoacetate

000015151515Anthralin

000015151515Catechol

000015151515Concen

tration024602460.000.060.100.140.000.060.100.140.00.60.81.00.00.60.81.0Survivingfraction¡ig/ml1

.00(779)a0.97

(754)0.94(734)1.02(796)1

07(835)1.03(806)1.01

(800)0.97(755)tig

/ml100(890)0.97

(860)0.95(844)0.98

(876)0.99

(880)0.98(876)1.01(902)0.98(872)mg/mt1.00(740)0.97(717)0.95

(704)0.89(660)1.03(762)0.97(724)0.96

(708)0.83(616)trp

* / 105 survi

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)Aberrant

colonies/1 O4survi

vors2.6

(2)2.6(2)4.1(3)1.3

(1)106

(89)136(110)152d(122)1636

(123)4.5

(4)3.5(3)1.2(1)1.1

(1)118

(104)137(118)146(132)163rf

(142)7.5

(6)19(14)16(11)20

(13)1

05(80)145°(105)150C(106)167C

(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 insome instances the effect appeared to saturate at high doses.The highest concentrations of catechol and iodoacetate depressed the frequencies of UV-induced mitotic gene conversionand crossing-over, respectively, to values less than those of

the negative controls. The reasons for this effect are unknown,but a similar reduction has been reported for the enhancementby tobacco smoke condensate of UV-induced reversion in a

histidine auxotroph of S. cerev/siae (10).We then chose single concentrations of each of the 3 chem

icals to examine their ability to potentiate the induction ofmitotic recombination and mutation by a range of UV doses.Table 4 shows that, without prior UV irradiation, catechol andiodoacetate again increased significantly the frequencies ofmutation and gene conversion, respectively. Iodoacetate, anthralin, and catechol enhanced UV-induced mitotic crossing-

over, mutation, and formation of aberrant colonies, and catechol also potentiated induced gene conversion, thus confirmingour previous observations. In general, the degree of enhancement of a specific genetic event by a particular chemical wassimilar for all UV doses used.

TPA. To perform experiments with TPA, we modified theabove procedure by adding concomitantly yeast cells and TPAto appropriately supplemented agar overlays, which were thenpoured onto media selective for the various end points to be

scored. The overlays used to detect convertants and mutantscontained minute quantities (0.5 /¿g/ml) of tryptophan andisoleucine, respectively. Thus, all cells experienced limitedgrowth in the overlays, and this was accompanied by anincrease in the frequencies of spontaneous gene conversionand mutation. These frequencies were 2-fold higher than those

obtained by plating cells from the same cultures on medialacking tryptophan or isoleucine (data not shown).

Under conditions where the cells were able to grow anddivide several times in the presence of TPA, we found that thephorbol ester had no effect on gene conversion, aberrantcolony formation, or mutation (Table 5). When cells were irradiated with UV and then exposed to TPA, there was no poten-tiation of the UV-induced genetic events. On the contrary, the

highest concentrations of TPA caused a decline in the frequency of induced aberrant colonies to a value less than thatfor UV alone. Thus, we chose a dose of 1 jug TPA per ml to usefor further experiments, inasmuch as this dose did not have adetrimental effect on UV-induced aberrant colonies. We then

examined the ability of this single dose of TPA to increase therecovery of gene convertants, mutants, and aberrant coloniesproduced by a range of UV doses (Table 6). There was nosignificant enhancement of the UV-induced genetic end points.

However, yeast possesses a thick cell wall, which may havehampered penetration of the promoter into the cells. To elimi-

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S. A. Kunz et al.

Table 2Effects 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 atthe indicated concentrations.

UV doseAgent (J/sqm)Iodoacetate

000015151515Anthralin

000015151515Catechol

000015151515Concen

tration024602460.000.060.100.140.000.060.100.140.00.60.81.00.00.60.81.0Crossovers/

104 sur-

Surviving fractionvivors¡ig/ml1.00

(4712)a1

.03(4860)1

.00(4724)0.95(4484)1.17(5520)1.13(5308)1

.07(5064)1.16(5452)fig

/ml1.00(4736)0.98

(4644)0.98(4648)0.96(4568)1.09(5152)1.04(4916)1

.02(4840)1

.07(5072)mg/ml1

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

(4800)0.94(4576)0.95

(4640)NO"NDNDND56

(31)75(40)87(44)40(22)NDNDNDND52

(27)79(39)81(39)79(40)NDNDNDND71

(32)106e(51)11

4e(52)112e (52)ilv

* / 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)

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 3Effects of increasing concentrations of iodoacetate and catechol on the yields

of mitotic gene convenants and mutants, respectively

Iodoacetate(jig/ml)0

24

6(rpVcell

plated"(x10"Ì9

(70)"

23(182)14(108)12 (95)Catechol

(mg/ml)0.0

0.60.81.0ilv

' /cell plated3(X10')7.5

(82)12 (132)16 (179)

7.5 (82)

Yields calculated from data in Tables 1 and 2.' Numbers in parentheses, actual numbers of colonies scored.

nate this possible barrier, the cell wall was removed by enzymedigestion, and the resulting spheroplasts were added to aregeneration agar overlay containing TPA. Spheroplaststreated this way are known to continue protein and RNAsynthesis at near normal rates (14). In the regeneration overlay,they are able to synthesize new cell walls (34) and then growto form colonies. Preliminary experiments demonstrated thatyeast spheroplasts were more sensitive to UV than were wholecells; thus, the UV dose was reduced to 10 J/sq m. Whenspheroplasts were treated with TPA, there was no effect onsurvival, spontaneous mutation, or gene conversion (Table 7).Furthermore, TPA was unable to potentiate UV-induced con

version, mutation, or formation of aberrant colonies. Althoughit appeared that TPA caused a decrease in the frequency ofinduced aberrant colonies, the actual number of coloniesscored in this case was small, and the decline in frequency wasnot significant. The results obtained with the spheroplastsshould be interpreted cautiously, as cells "traumatized" by

wall removal do not exhibit entirely normal behavior. Spheroplasts are more sensitive to UV, DNA synthesis rates can be50% lower (14), and the frequencies of induced gene conversion and mutation are reduced compared to those for similardoses of UV applied to whole cells (compare Tables 6 and 7).

DISCUSSION

Tumor promoters have been found to cause a myriad ofchemical, biological, and histological effects (for reviews, seeRefs. 4, 27, 31, and 37). However, few attempts have beenmade to explain tumor promotion or cocarcinogenesis in genetic terms. Kinsella and Radman (16) proposed that aberrantmitotic segregation events, induced by promoters, could resultin the expression of recessive genetic or epigenetic chromosomal changes that might lead to tumor formation. It alsoseemed reasonable to us that cocarcinogenic activity could be

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Tumor Promoters and Mitotic Recombination

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 concentrationsindicated.

UV dose (J/Agent sqm)NoneIodoacetate

(4^g/ml)Anthralin

(0.14^g/ml)Catechol

(0.6mg/ml)0•102030010203001020300102030Surviving

fraction1.00(3792)"1.02(3886)0.93(3516)0.94

(3550)0.99(3738)0.95(3616)0

99(3760)0.89(3386)1

05(3992)0.98(3714)0.93

(3530)0.93(3546)1

.04(3964)098(3706)0.84

(3198)0.87(3294)trp

' 1 10s1.21444912.1"1445931.21647870.924694"1736survivors(225)(273)(780)(1599)(392)(260)(850)(1578)(229)(295)(833)(1541)(186)(450)(1509)(2846)Aberrant

colonies/10" survi

vors1.0741852908.691218357o1.082224327°2.0147"371*599°(1)(72)(163)(254)(8)(82)(205)(302)(1)(76)(198)(290)(2)(136)(297)(493)Crossovers/

104

survivorsNDC44

(17)122(43)131

(46)NO55

(20)154(58)213b(72)ND58

(20)161(57)211°

(75)ND92

(34)172(55)249fc

(82)/Vv*/1060.616871930.7336116°281"0.52261056257°1.1*28b152U297"survivors(104)(105)(516)(1140)(136)(202)(737)(1608)(91)(140)(624)(1347)(275)(177)(820)(1652)

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 ontosupplemented minimal media.

UV dose (J/sqm)000015151515TPA(ng/ml)01101000110100Survivingfraction1.00(541)"0.94

(507)0.87(473)1

.05(569)1.03(555)0.85

(458)1.01(544)0.93

(504)trp

' / 10ssurvivors1.5

(83)1.3(68)1.6(75)1.4

(81)41

(228)45(204)35

(192)•44

(220)Aberrant

colonies/ 10" survi

vors3.7

(2)4.0(2)2.1(1)1.8

(1)135

(75)175(80)110(60)101

(51)ilv

' / 10esurvivors0.6

(31)0.8(40)0.7(32)07

(40)104

(288)119(219)107(291)107

(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 ontosupplemented minimal media.

UV dose (J/sqm)01020300

+TPA10+TPA20+TPA30+ TPASurviving

fraction1.00(788)"0.93

(727)0.91(718)0.93(732)0.94

(743)0.97(768)0.85(670)0.85

(673)frpVl

O5survivors1

.6(246)20(147)68

(450)126(921)1.5(229)25

(189)74(493)136

(918)Aberrant

colonies/104survivors1.3

(1)54(39)148(106)232

(170)2.7

(2)64(49)190(127)239

(161)i/v*/106

survivors0.4

(62)19(46)69

(167)123(302)0.4

(59)15(39)69

(154)137(310)

a Numbers in parentheses, actual numbers of colonies scored.

due to an increased frequency of expression of recessivegenes via potentiation of the mutagenic or recombinageniceffects of carcinogens.

Iodoacetate has been found to break DNA in Anacystisnidulans and in human T-cells and to inhibit the repair of y-ray-and X-ray-induced single strand breaks (6, 17). Chromosome

fragmentation by catechol has been described in Allium cepa(19, 20). Anthralin has been reported to bind to DNA in vitro

and to be a general inhibitor of both DNA synthesis and repairreplication (5, 25, 35). These properties of the 3 chemicals areconsistent with an ability to induce mutation and mitotic recombination and/or to enhance such genetic events induced byDNA-damaging agents.

The results reported here are summarized qualitatively inTable 8. We have found that iodoacetate is recombinagenicand that catechol is mutagenic although the observed re-

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Table 7

Effects of TPA on spheroplasts

Irradiated or nonirradiated spheroplasts were added to agar overlays, with orwithout TPA (1 fig/ml), which were poured onto supplemented minimal media.Spheroplasts were able to regenerate cell walls and then grow to form colonies.The regeneration frequency was 33/102 spheroplasts added to the overlay.

UV dose(J/sqm)00

+TPA1010

+ TPASurvivingfraction1.00(621)1.03(639)0.66(413)0.64(400)(rp*/106

sur

vivors2.3(242)a2.3(249)9.7

(80)11 (90)Aberrant

colonies/10*survivorsNO0NO41

(17)27(11);/vV106

sur

vivors0.7(71)08(81)4.1

(34)5.7(46)

Numbers in parentheses, actual numbers of colonies scored.' ND. no aberrant colonies were detected

Table 8

Summary ofeffectslodoacetateAnthralinTPACatecholUV

dose(J/sqm)0

150

150

15015Gene

conversion00

00

00Aberrant

colony formation000

00Crossing-

overNDNDNS

NSNDMutation0000+

+ , a significant increase above control values; 0. no significant change fromcontrol values; ND. not detected; NS, not scored

sponses are small, lodoacetate, anthralin, and catechol enhance UV-induced mutation, mitotic crossing-over, and aber

rant colony formation, while catechol also increases the recovery of induced gene convertants. Thus, the agents tested areable to potentiate a wide range of genetic events. Moreover,the observed magnitude of enhancement is similar to that seenfor the TPA-increased recovery of induced mutants in mam

malian cells (18, 36) and for the potentiation of induced mutation and gene conversion in yeast by tobacco smoke condensate (10).

Although TPA is able to inhibit DMA repair synthesis (5, 25),we were unable to demonstrate any notable effect of thephorbol ester in either whole cells or spheroplasts, whethersubjected to UV irradiation or not. A possible exception is thedose-related decline in the frequency of UV-induced aberrant

colonies. This is similar to the decrease in the frequencies ofUV-induced mitotic gene conversion and crossing-over caused

by catechol and iodoacetate, respectively. The reason for thiseffect is unknown, but such a reduction has also been observedduring a study of the potentiation of UV-induced mutation and

mitotic recombination by tobacco smoke condensate (10).Although TPA may have had a slight genetic influence on thecells, it was not recombinagenic. Other investigators, usingdifferent strains and treatment conditions, also have not detected any effect of TPA on mitotic recombination in yeast(30).5 The structure of TPA and recent findings in mammalian

cells suggest that this promoter may function at the membranelevel (29, 39-41). It is possible that particular membrane

receptors for TPA exist in mammalian cells and that these are

' E. Hecker, personal communication.

lacking in yeast. Alternatively, S. cerevisiae may be capable ofmetabolizing TPA to intermediates which are not recombinagenic or mutagenic.

Of the 3 tumor promoters tested here (TPA, iodoacetate, andanthralin), only iodoacetate is recombinagenic. Thus, our results do not indicate any consistent correlation between tumor-

promoting activity and the ability of an agent to induce mitoticrecombination in yeast. However, this does not rule out thepossibility that mitotic segregation of recessive alíelesplays animportant role in carcinogenesis. If promoters do in fact act byinducing mitotic recombination in mammalian cells, it wouldappear that this effect is achieved by a mechanism whichcannot be provoked consistently in yeast. On the other hand,the potent cocarcinogen, catechol, which lacks tumor-promoting activity (37), increased the recovery of UV-induced mutants

and mitotic recombinants. These genetic events are also potentiated by iodoacetate and anthralin (but not TPA). Thus,capacity to enhance the mutagenic and recombinagenic effectsof UV may be related to the cocarcinogenic activity of certainpromoters.

ACKNOWLEDGMENTS

We thank Dr. B. J. Barclay for many helpful discussions and Ingeborg Vranesicfor her excellent typing assistance.

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1980;40:2323-2329. Cancer Res   Bernard A. Kunz, Mohammed A. Hannan and R. H. Haynes  cerevisiae

SaccharomycesMutation and Mitotic Recombination in Effect of Tumor Promoters on Ultraviolet Light-induced

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