Enhancement of recovery of chemical carcinogen-induced ouabain-resistant mutants in chinese hamster cells by the tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate

319

Mutation Research, 73 (1980) 319--329

© Elsevier/North-Holland Biomedical Press

ENHANCEMENT OF R E C O V E R Y OF CHEMICAL CARCINOGEN-INDUCED OUABAIN-RESISTANT MUTANTS IN CHINESE HAMSTER CELLS BY THE TUMOR-PROMOTING AGENT, 12-O-TETRADECANOYL-PHORBOL-13-ACETATE *

GEORGE

R. L A N K A S

**, C. S T U A R T

BAXTER

*** and R O B E R T

T. C H R I S T I A N

Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267 (U.S.A.)

(Received 21 March 1980) (Revision received 19 May 1980) (Accepted 1 July 1980) Summary The effects of a tumor-promoting agent on the frequency of mutation to ouabain resistance and survival of Chinese hamster cells treated with a chemical carcinogen have been investigated. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) significantly enhanced t h e mutation frequency induced b y the carcinogen, methylazoxymethanol acetate (MAM), w i t h o u t having similar effects on cytotoxicity, at concentrations of 2 #g/ml or less. The observed degree of enhancement of mutagenesis increased with p r o m o t e r concentration up to the point where the latter exhibited frank toxicity. Exposure of the cells to the promoter for a period of 2 or 6 h was found ineffective, b u t subsequently a significant enhancement was found after a 27-h exposure time. The maximum effect occurred after a 5-day exposure, with a increase in the mutation frequency of 250%. Treatment of cells with TPA alone resulted in no enhancement of spontaneous mutation rates, nor did treatment of cells prior to addition of carcinogen-induced mutagenesis. In contrast, TPA was found to be effective when applied as late as 6 weeks following carcinogen treatment. These results are consistent with the hypothesis that TPA owes its promoting activity towards chemically-induced mutagenesis and carcinogenesis to its ability to. enhance * S u p p o r t e d b y G r a n t s C A 2 4 0 2 2 a n d ES 0 0 1 5 S , a w a r d e d b y t h e N a t i o n a l Cancer Institute and the N a t i o n a l I n s t i t u t e o f E n v i r o n m e n t a l H e a l t h S c i e n c e s , r e s p e c t i v e l y , D H E W , a n d b y a g r a n t f r o m the C h e m i c a l I n d u s t r y Institute o f T o x i c o l o g y . ** P r e s e n t a d d r e s s : B i o d y n A m l e s , I n c . , D e p a r t m e n t o f T o x i c o l o g y , P . O . B o x 4 3 , M e t t i e r s R o a d , E a s t Millstone, NJ 08878 (U.S.A.) *** T o w h o m requests for r e p r i n t s s h o u l d b e m a d e . Abbreviations: TPA, tetradecanoylphorbol xymethanol acetate.

acetate (phorbol myrlstate acetate); MAM, methylazo-

320

expression of latent somatic genetic modifications by epigenetic mechanisms. They do not support mechanisms involving TPA-induced inhibition of DNArepair replication, or mutagenic activity of TPA per se. The notably similar qualitative response to TPA of several parameters in mouse-skin tumorigenesis and Chinese hamster cell mutagenesis suggest that the mechanism of action of the promoter is sir'nilar in the 2 diverse biological systems.

It has recently been reported from this and other laboratories (Lankas et al., 1977, 1978; Trosko et al., 1977) that carcinogen-induced mutation frequencies in cultured mammalian cells are enhanced by tumor-promoting agents. The latter are intrinsically noncarcinogenic compounds which are able to considerably potentiate the tumorigenic effects of certain chemicals. Experiments in topical treatment of mouse skin (Berenblum, 1954; Boutwell, 1974) first led to the recognition of the phenomenon of promotion, and also to the proposal that is represented the second step in a general 2-stage model for chemical carcinogenesis, the first step being termed initiation. Subsequently experiments involving administration to rats and mice (Armuth and Berenblum, 1972, 1974; Peraino et al., 1975) and transplacental exposure of mouse fetuses (Armuth and Berenblum, 1977) demonstrated that 2-stage carcinogenesis and tumor promotion were not peculiarities of mouse skin. The transformation of cultured mammalian cells treated with subeffective concentrations of carcinogenic initiators and subsequently to the promoting agent TPA (Lasne et al., 1974. Kennedy et al., 1979; Little et al., 1979) or the proposed promoter saccharin (Mondal et al., 1978} further suggests that a 2-stage mechanism of carcinogenesis may be widely operational. The finding that the majority of carcinogenic agents are mutagenic in bacteria (Ames et al., 1975) and many in mammalian cells (Huberman and Sachs, 1976) led to the suggestion that initiation involved irreversible mutational changes, and although this theory remains controversial, an excellent correlation between mutagenicity and initiating activity has been demonstrated in mammalian cells (Huberman, 1975). The mechanism of promotion is as yet also unclear, but has been proposed to involve reversible modulation of expression of the modified genome (Boutwell, 1974). Should initiation, as the first stage in carcinogenesis, be a mutation event, it is an intriguing proposition that agents which are active in tumor promotion should also enhance chemically-induced mutagenesis. In support of this proposition it has been found that promoters enhance at several loci frequencies of mutation induced by ultraviolet light or various chemical carcinogens, including methylazoxymethanol acetate (MAM), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene. Promoting agents of different chemical structure have been shown to be active in this respect, examples being phorbol derivatives (Lankas et al., 1977; Trosko et al., 1977), and several linear alkanes (Lankas et al., 1978) and saturated fatty acid esters (Wey and Baxter, 1979). These agents were i n n o instance found to possess mutagenic activity per se. Our finding that tumor-promoting agents of diverse chemical structure were active in the above cell culture system, together with the observation that linear alkanes with between 6 and 16 carbon atoms showed very similar chain length-

321 dependent relative activities both as topical t u m o r promoters in vivo (Sick, 1966) and enhancers of chemically-induced mutagenesis in vitro (Lankas et al., 1978), suggested that this latter property could be exploited to gain further insight into the mechanism of action of promoters in vivo. Work with the highly p o t e n t p r o m o t e r 12-O-tetradecanoylphorbol-13acetate (TPA) in 2-stage mouse-skin carcinogenesis had led to the delineation of certain characteristics of the mode of action of this, and, to a lesser extent, other promoting agents. The degree of activity of TPA was thus found to be highly dependent on the dose of promoter, the duration of exposure to p r o m o t e r and time of administration relative to application of carcinogen. TPA is also found to be without carcinogenicity per se in the vast majority of systems studied. An apparent exception is the hairless mouse (Iversen and Iversen, 1979), a system probably having many unrecognized characteristics compared to normal counterparts. Therefore, the purpose of this study was to determine if our original observations on the enhancement of mutagenesis in vitro by t u m o r promoters (Lankas et al., 1977, 1978) could be extended further the include the classical properties, mentioned above, associated with t u m o r promotion in vivo. In this way we hoped to establish whether or n o t this cell-culture system could provide a relevant model system for the study of t u m o r promotion. Materials and methods Chinese hamster V79 cells (a gift of Dr. R. Hart, Ohio State University, Columbus, OH) were grown in a modified minimum essential medium (Eagle's) supplemented with 1.5 times the standard concentration of vitamins and amino acids and 10% fetal-calf serum. Cells were seeded at a density of 2 × l 0 s cells per plate in 10 ml medium and 3 h allowed for cell attachment. Plates were then dosed with MAM (Aldrich Chemical Co., Milwaukee, WI) dissolved in medium without serum. Cells were exposed to MAM (24 #g/ml for 24 h), unless otherwise stated, after which medium was replaced with fresh material. TPA (Consolidated Midland Corp., Brewster, NY) was made up and stored as a stock solution acetone at a concentration of 1.0 mg/ml. Addition of the above promoter solution to the cells in fresh medium was carried o u t at various times following exposure to MAM, and the length of exposure to the promoters was also varied, as described in the text. Unless otherwise stated, 30 h after termination of mutagen exposure the culture medium was replaced with the same volume containing 1 mM ouabain (Sigma Chemical Co., St. Louis, MO), and 2 weeks later the plates were fixed with ethanol and stained with Giemsa and the number of viable colonies determined. A minimum of 10 plates were used per point and all experiments were repeated at least 3 times to verify the consistency of the results obtained. Concurrent with the mutagenesis assays, toxicity assays were carried out to determine cell survival following the various chemical treatments. Cells were seeded at a density of 200 cells per plate and treated as described in mutation assays, with the exception that ouabain was n o t included in the medium.

322 Results In our earlier report (Lankas et al., 1977) we demonstrated that the effect of the promoter increased with increasing doses of mutagen. Therefore, in the experiments reported here we chose the highest dose of MAM (24 pg/ml) to maximize the effects of the promoter. In order to more precisely determine the o p t i m u m time of addition of the TPA, experiments were conducted on the dependence of m u t a t i o n frequency on the time of addition of the promoter, relative to addition of MAM as a mutagen. The results are shown in Table 1. in all cases where TPA was added, the cells were exposed to it for the duration of the experiment. Although the highest recovery of mutants occurred with the TPA was added 24 h after addition of the mutagen, this value did n o t differ significantly from those found in the other TPA-treated groups within the time periods examined, nor in experiments in which TPA was present specifically during selection only. All groups receiving TPA had higher m u t a t i o n frequencies compared to the control group which was n o t treated with promoter. Addition of the promoter 69 h after mutagen t r e a t m e n t (13 h after the start of ouabain selection) gave the lowest response to the promoter. This was expected since cells potentially sensitive to TPA would be selected against in the ouabain medium. Concurrent toxicity tests indicated that the 20% survival rate in MAM-treated plates was unaffected by TPA treatment. Following the determination of the o p t i m u m time of promoter addition, the effect of promoter concentration on carcinogen-induced mutation frequencies was determined. Cells were treated with various concentrations of TPA after mutagen treatment and subsequently selected for ouabain resistance as described in Materials and Methods. Table 2 shows that, in the first instance, TPA at a concentration of 2 pg/ml did n o t affect the degree of survival of cells

TABLE 1 EFFECT OF TIME OF ADDITION OF TPA ON MUTATION FREQUENCY Time of addition o f T P A (h) a Control (no TPA) 0 4 12 24 45 69

Survival (%)

Mean number of mutants/plate b

Mutants/106 Survivors c

23 27 22 24 22 23 23

17.7 26.2 26.1 24.6 28.2 27.3 22.8

385 595 593 513 641 593 496

-+ 3.9 -+ 4.1 ± 6.3 ± 2.2 ± 1.8 -+ 4 . 3 ± 4.5

a M A M t r e a t m e n t b e g u n at t i m e 0 a n d c o n t i n u e d for 2 4 h; a t 53 h o u a b a i n s e l e c t i o n m e d i u m a d d e d t o all p l a t e s and T P A r e a d d e d at 1 ~ g / m l w h e r e a p p r o p r i a t e , so t h a t o n c e a d d e d e x p o s u r e t o T P A c o n t i n u e d for the duration of the experiment. b 95% confidence interval (Student's t test). c A1 g r o u p s e x p o s e d t o T P A h a d a s i g n i f i c a n t l y g r e a t e r m u t a t i o n f r e q u e n c y c o m p a r e d to c o n t r o l (P 0.05).

323 TABLE 2 EFFECT OF TPA CONCENTRATION FOLLOWING MAM TREATMENT

ON

CELL

SURVIVAL

AND

MUTATION

TPA concentration (#g/m/)

Survival (%)

Mean number of mutants/plate b

Mutants/106 survivors

0 10 -3 10 -2 10 -1 1.0 2.0 5.0

34 33 32 34 34 36 25

21.6 26.1 29.0 32.0 32.8 39.1 17.7

318 395 453 470 482 544 354

± 2.3 ± 3.3 ± 2.8 -+ 2.4 ± 3.1 ± 3.0 ± 2.2

FREQUENCY

c d d d

a All plates treated w i t h 24 ~ g / m l MAM for 24 h prior to T P A t r e a t m e n t , w h i c h w a s c o n t i n u e d for the r e m a i n d e r o f the e x p e r i m e n t . b 9 5 % c o n f i d e n c e interval ( S t u d e n t ' s t test). c p ~ 0.05 compared to no TPA treatment. d p <: 0 . 0 1 .

exposed to MAM, nor that of untreated cells. Furthermore, the degree of enhancement of MAM-induced mutagenesis increased with increasing TPA concentration up to a maximum occurring in the region of 2.0 #g/ml TPA. The subsequent decrease in enhancement was associated with toxicity observed at higher concentrations of the promoter. A primary factor in promotion of tumorigenesis in vivo is the length of time of exposure o f the tissue to the promoting agent. To determine the importance of this factor for this system, cells were treated with an optimally active, nontoxic dose of TPA after treatment with MAM, and TPA treatment discontinued at various times thereafter. Selection of mutants resistant to ouabain was then carried out with TPA present in the selection medium for the 5 and 14 day

TABLE 3 E F F E C T OF V A R Y I N G T H E D U R A T I O N OF E X P O S U R E TO TPA ON T H E MAM-INDUCED MUTATION FREQUENCY TPA exposure time a

Survival (%)

Mean number of mutants/plate b

Mutants/106 survivors

0 2 6 27 5 2

21 21 20 21 22 21

14.6 16.9 15.3 21.2 36.1 24.3

347 402 383 504 c 820 d 578 d

h h h days weeks

-+ 2.3 + 4.9 -+ 2.9 + 2.8 +- 4.5 + 1.8

a All plates treated w i t h 2 4 # g l m / M A M f o r 2 4 h prior t o e x p o s u r e to 2 # g l m l , w h i c h w a s d i s c o n t i n u e d after t i m e s stated. b 9 5 % c o n f i d e n c e interval ( S t u d e n t ' s t test). c p <: 0 . 0 2 c o m p a r e d to n o T P A t r e a t m e n t . d p <~ 0 . 0 0 1 .

324 exposure groups. Table 3 shows that if promoter treatment is discontinued at a time too far in advance of ouabain selection, its effects are lost, and no significant enhancement of m u t a t i o n frequency is observed. When added right up to or during the selection period, increasing degrees of enhancement are observed. The reduction in effect after 2 weeks may be due to reduced m u t a n t survival in the medium which is clearly observable to be low in pH and probably depleted in nutrients following the first few days of selection. This medium depletion probably arises from the fact that ouabain cytolethality is not instantaneous, and the large preponderance of wild-type cells are still able to survive and perform certain metabolic functions for a short time. In the 5-day group the mutants are placed in fresh ouabain~containing medium with a full complement o f nutrients, and most wild-type cells have ceased function by that time. Alternatively, toxic effects resulting from extended exposure of mutants to TPA or its metabolites or decomposition products could explain the observed decrease. Also intrinsic to 2-stage carcinogenesis is the ubiquitous observation that promoting agents induce or enhance tumorigenesis only when applied after initiation, and not preceding. To further validate the in vitro system described herein as a model for in vivo response, the effect of promoter pretreatment on carcinogen-induced m u t a t i o n frequencies was determined. Since the previous experiment indicated that a 5-day post-treatment gave the highest recovery of mutants (see Table 3), this time period was chosen for the pretreatment experiment. To insure as closely identical cell populations as possible, a single group of cells was equally divided, and one-half exposed to promoter while the other received no treatment. Each group was then subcultured and plated in the same medium and treated with MAM as initiator, or left untreated as controls. After an expression time of 70 h, m u t a t i o n frequencies were determined as described previously. Table 4 shows that TPA pretreatment did n o t affect cell survival or m u t a t i o n frequencies relative to controls containing no promoter, and confirms the lack of toxicity and mutagenicity of this agent at a concentration of 2/ag/ ml. Experiments in mouse skin have shown that, within an extensive period of time, the magnitude of response to t r e a t m e n t with promoting agents does not change significantly when the interval between initiation and promotion is increased. In order to determine whether parallel behavior is observed in the

TABLE 4 EFFECT OF TPA PRETREATMENT

Treatment

ON MAM-INDUCED OUABAIN-RESISTANT

Survival (%)

Mean number of

MUTATIONS

Mutants/106 survivors

mutants/plate a Control TPA pretreatment b MAM (24/~g/mD TPA preWeatment b + MAM (24/~g/ml)

72 80 10

0 . 4 -+ 0 . 7 0 1 2 . 9 -+ 1.6

2.8 0 620

9

1 1 . 7 -+ 2.2

650

a 9 5 % c o n f i d e n c e i n t e r v a l ( S t u d e n t ' s t test). b C e n s t r e a t e d w i t h 2 / ~ g / m l T P A f o r 5 d a y s p r i o r t o p l a t i n g for mutagenesis a s s a y .

325 TABLE 5 PERSISTENCE OF S E N S I T I V I T Y OF M A M - T R E A T E D CELLS TO T P A - I N D U C E D P R O M O T I O N OF MUTAGENSIS

Time of addition of TPA a

Mean number of mutants/plate

0 TPA Control ( no TPA) 1 week TPA Control 2 weeks TPA Control 3 weeks TPA Control 4 weeks TPA Control 6 weeks TPA Control

17.6 10.4 44.0 37.6 38.6 32.5 30.4 26.2 52.4 48.0 48.0 42.0

MAM + T P A b

Ratio MAM 1 7 . 4 -+ 0 . 1 8 c 1 . 2 0 -+ 0.21 1.21 -+ 0 . 1 0 c 1.20 + 0.20 1.11 -+ 0 . 0 8 c 1 . 1 5 -+ 0 . 1 1 c

a M A M ( 2 5 / ~ g / m l ) r e m o v e d at t i m e 0. T P A ( 2 / ~ g / m l ) added at various t i m e s thereafter. b 95% c o n f i d e n c e interval (t test) calculated b y r a n d o m pairing o f treated and c o n t r o l plates ( 1 0 plates per p o i n t ) . c Significant differences (P ~ 0 . 0 5 ) . N o significant differences w e r e f o u n d in cell survival rates b e t w e e n a n y groups.

present mutagenesis system, carcinogen-induced mutation frequencies were measured when various time periods were allowed to elapse prior to promoter treatment. Following treatment with the carcinogen, MAM, ceUs were treated with TPA, and ouabain-selection medium containing TPA was added subsequently. A second group was subcultured every 3 days following the same carcinogen treatment. At intervals following mutagen treatment cells were replated in normal medium or medium containing TPA(2 #g/ml) and 24 h later the selection medium containing ouabain and TPA where appropriate, added. The addition of TPA did not affect cell survival. Therefore, results are compared on a per mutant colony basis. Results presented in Table 5 suggest that the greatest response to the promoting agent occurred when the latter was applied shortly after the carcinogen, in analogy with another report (Trosko et al., 1977). Within 7 days the degree of enhancement of mutagenesis due to TPA apparently declined markedly, but thereafter remained constant for 6 weeks. The recurrent question of whether cells showing resistance to ouabain represent true mutants appears to be answered in the positive by experiments whereby we have replated and grown numerous carcinogen-induced colonies, following ouabain selection, in normal medium and subsequently found a high plating efficiency on replating into ouabain-containing medium (data not shown). In agreement with others we have also found the ouabain-resistant phenotype to be stable over the periods required for our experiments (Mankowitz et al., 1974).

326 Discussion The observations reported above demonstrate that the responses to tumorpromoting agents of both chemically-induced mutagenesis in V79 cell culture, and chemical tumorgenesis in vivo show remarkably similar characteristics, considering the biological diversity of the 2 systems. The mechanisms of action of tumor-promoting agents in the 2 systems may thus share c o m m o n features, and the V79 mutagenesis assay may prove valuable in providing information on the mechanism of t u m o r p r o m o t i o n and chemical carcinogenesis in vivo. At least one major difference persists, however, between the 2 systems, that being the magnitude of the response to the promoting agent. In cell-transformation experiments the effects of the p r o m o t e r are approximately an order of magnitude greater than those reported here (Mondal et al., 1976). This difference may be due to differences in cell types being investigated or in the type of initiator used prior to addition of the promoting agent or both. But it may be more than fortuitous that the difference in response mentioned above is strinkingly similar to the differences in response between mutation and transformation frequencies measured in the same cells with the same chemical carcinogens (Huberman et al., 1976). Therefore, the lower response of mutagenesis to promoting agents may be due to intrinsic differences in the reactivity of genes controlling cell transformation and ouabain resistance or in their relative target sizes. The lack of intrinsic mutagenicity of TPA, as described above, or other t u m o r promoters (Lankas et al., 1978} parallels the lack of carcinogenicity of these agents, a fact which has been well established previously. This finding is consistent with proposals that promoters owe their activity in both systems not to direct interaction with DNA. TPA has been shown to modulate several factors implicated in regulation of cell growth, gene activity and also neoplasia. These include stimulation of mouse epidermal histone kinase activity (Allfrey, 1970; Raineri et al., 1973), and ornithine decarboxylase and resulting polyamine synthesis in vivo (O'Brien et al., 1975) and in vitro (Yuspa et al., 1976). The p o t e n t phorbol diester promoters have also been shown to inhibit differentiation in vitro (Lowe et al., 1978) and to induce proteolytic enzyme activity (Troll et al., 1978). These findings have led to the proposal that promoters enhance carcinogenesis b y epigenetic mechanisms. Although the above considerations suggest a mechanism of promotion involving effects on cell growth or gene regulatory processes, which is considered by ourselves and others to be the most appealing, others have been proposed. The possibility that inhibition of DNA-repair processes is responsible for enhancement of mutation frequencies (Gaudin et al., 1972) has been cast into d o u b t by our observation that TPA is able to elicit additional increases in mutation frequency in cells long after the mutation expression time following carcinogen treatment. DNA-repair replication in vitro has been reported to be essentially complete within a period of 24 h following DNA damage (Teebor et al., 1973). A similar conclusion to ours has been reached b y other authors (Trosko and Yager, 1974), who have observed firstly that V79 cells do not do much excision repair, and secondly that TPA causes a small nonspecific inhibition of both normal and repair replication DNA synthesis in treated cells.

327 A further mechanism of tumor promotion, based on expression of genetic damage by mitotic recombination, has been proposed (Kinsella and Radman, 1978). However the significance of the phenomenon described, sister-chromatid exchange, in carcinogenesis, is still highly debated. The effects of TPA reported, furthermore, have not been reproduced by other authors (Loveday and Latt, 1979). The question of whether the effects seen with TPA are due to selection of otherwise nonviable mutants is answered clearly in the negative, firstly by our findings that increases in mutation frequency are observed even if promoter treatment is terminated before selection with ouabain (see Table 3 and Wey and Baxter, manuscript in preparation) and secondly by experiments showing that TPA h a s no effect on the recovery of ouabain-resistant mutant cells replated together with varying numbers of wild-type cells in reconstruction experiments (J.E. Trosko, personal communication). The finding that the majority of chemical carcinogens are mutagenic, and that mutagenicity in mammalian cells and initiating capability are well correlated (Huberman, 1975) has led to the conclusion that somatic mutation is a required early step in carcinogenesis and its equation with initiation (Boutwell, 1974). The observed ability of promoters to enhance frequencies of mutation by chemical initiators, without mutagenic activity per se, is thus consistent with this conclusion. Furthermore, a report has recently appeared demonstrating that TPA alone can induce neoplastic transformation of mutant human fibroblasts from patients genetically predisposed to cancer (Kopelovich et at., 1979). This mutation apparently results in the cells existing in an "initiated state" such that TPA treatment alone results in expression of the malignant phenotype. These findings are in agreement with our data demonstrating that TPA can enhance mutagenesis in cell cultures. Comparison of the effects of TPA in V79 cell culture, mouse skin, and rodent tissues in vivo shows that the former system shows a qualitatively parallel response to that shown in the in vivo situations, in that the promoter is effective when applied a considerable time after carcinogen administration. Further common features are the lack of effect when the promoter is added prior to administration of carcinogen (BoutweU, 1974), and apparent reversil~ ility of promoter action. These observations agree with previous findings that in both systems the changes resulting from interaction of normal cells and prorooter are reversible. This has also been found to be true for most biochemical promoter effects such as enhancement of cyclic GMP levels (Trosko et al., 1975) and stimulation of ornithirre decarboxylase, DNA synthesis (Yuspa et al., 1976) and histone phosphorylation activity (Raineri et al., 1973). TPA is also able to elicit mutations or tumors (Berenblum, 1954) when applied after extended periods following carcinogen treatment, and is inactive when applied beforehand. Our experiments demonstrate that the effect of TPA subsequently decreases somewhat from that observed for the first few days after mutagen treatment, but thereafter remains constant. It would appear, therefore, that a certain population of ouabain-resistant mutants is slowly reversible or nonviable. This appears to be less true for mutants resistant to 6thioguanine, since the enhancing effects of TPA in this system persist for much longer (Trosko et al., 1977; Wey and Baxter, 1980). Whether a similar decrease

328

to that in the ouabain system occurs on mouse skin is difficult to judge, since in the corresponding experiments in the in vivo system, a period of 1 week is also allowed between initiator and promoter treatment (Boutwell, 1964). Thus it is difficult to say on this basis which mutation assay more closely parallels the in vivo situation. We have also observed that a range of linear alkanes shows the same relative activities as promoters of carcinogenesis and mutagenesis, resp., in the skin (Sick, 1966) and culture systems (Lankas et al., 1978), a very strong indication of similar biochemical mechanisms of action of promoters in both systems. E~periments are at present in progress in our laboratory to explore this possibility. Human populations are invariably exposed simultaneously both to tumor initiating and p r o m o t i n g agents. While exposure to the latter may enhance cancer incidence, promotion is both reversible for a considerable period, and inhibitable by several types of agents (Belman and Troll, 1972). Thus recognition of compounds with promoting activity may lead to measures for effectively reducing cancer risk. The system we describe herein has been shown to be capable of correctly identifying several classes of tumor-promoting agent and to behave toward TPA in a qualitatively similar fashion in several respects compared to mouse epidermis. Furthermore, it shares several biochemical responses to Tr'A with mouse epidermis (Trosko et al., 1975; Wey and Baxter, manuscript in preparation). Thus it shows promise for identification of tumor-promoting agents, and for study of biochemical mechanisms involved in their action. References Allfrey, V.G. ( 1 9 7 0 ) Changes in c h r o m o s o m a l p r o t e i n s a t times o f gene a c t i v a t i o n , F e d e r a t i o n Proc., 29, 1447--1460. Ames, B.N., J. 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