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Mechanisms Ofcarcinogenesis Proc. Natl. Acad. Sci. USA Vol. 75, No. 12, pp. 6149-6153, December 1978 Genetics Tumor promoter induces sister chromatid exchanges: Relevance to mechanisms of carcinogenesis (12-0-tetradecanoylphorbol 13-acetate/bromodeoxyuridine labeling/recombination/mitotic segregation/recessive mutations) ANNE R. KINSELLA AND MIROSLAV RADMAN D6partement de Biologie Moleculaire, Universite Libre de Bruxelles, B 1640 Rhode-St-Genese, Belgium Communicated by J. D. Watson, August 31, 1978 ABSTRACT 12-0-Tetradecanoylphorbol 13-acetate (TPA), Six possible mechanisms for the segregation of a recessive a powerful tumor promoter, is shown to induce sister chromatid mutation from a heterozygous cell are presented in 1. exchanges (SCEs), whereas the nonpromoting derivative 4-0- Fig. (i) methyl-TPA does not. Inhibitors of tumor promotion-antipain, Chromosomal rearrangement or deletion and (ii) one-step leupeptin, and fluocinolone acetonide-inhibit formation of nondisjunction both lead to hemizygosity, while (iii) two-step such TPA-induced SCEs. TPA is a unique agent in its induction nondisjunction, (iv) aberrant mitotic segregation (in the absence of SCEs in the absence of DNA damage, chromosome aberra- of nondisjunction or mitotic recombination), (v) increase in tions, mutagenesis, or significant toxicity. Because TPA is ploidy plus chromosome loss, and (vi) mitotic recombination known to induce several gene functions, we speculate that it all lead to might also induce enzymes involved in genetic recombination. homozygosity. Only the events leading to homozy- Thus, the irreversible step in tumor promotion might be the re- gosity (iii-vi) are consistent with the observation that initiation sult of an aberrant mitotic segregation event leading to the ex- must precede promotion in order to produce an enhanced pression of carcinogen/mutagen-induced recessive genetic or carcinogenic effect. Furthermore, mechanisms i and ii are epigenetic chromosomal changes. made unlikely by the fact that chromosome loss or deletion is usually lethal to normal diploid cells (11). Carcinogenesis is usually a multistep process. Epidemiological We present here a cytological study of the effects of TPA on studies of human carcinomas indicate that several distinct Chinese hamster cells in culture to test the hypothesis of tumor heritable changes are necessary to change a normal cell into a promotion outlined in Fig. 1. This study was encouraged by the malignant cell (1). This stepwise process is best illustrated in the demonstration of promotion processes in cell culture (ref. 12 two-stage mouse skin carcinogenesis system, which is composed and references therein) and by the observation that griseofulvin, of "initiation" and "promotion" stages (2, 3). Initiators, such a known promoter of dimethylbenzanthracene-initiated mouse as radiation or chemical carcinogens, cause rapid irreversible skin tumors (13), induces high ploidy and subsequent chro- changes, mutagenic or epigenetic in origin, which result in a mosome loss in cell cultures (14), which would be consistent with "premalignant" state that is inherited by both daughters when mechanism v listed above. "initiated" cells divide. Promoters are neither significantly carcinogenic (3) or mutagenic (4, 5) when tested alone, but MATERIALS AND METHODS greatly increase tumor frequency and shorten the lag time for tumor appearance when added after an initiator. Thus, tumor V79-4 Chinese hamster lung fibroblasts were maintained in promoters complete a process begun by initiators. Dulbecco's modification of Eagle's minimal essential medium These facts prompted us to consider the possibility that ini- supplemented with 10% fetal bovine serum in a humidified tiated cells contain specific recessive, autosomal, somatic incubator at 37°C in the presence of 5% CO2. For analysis of mutations and that promotion causes the expression of these sister chromatid exchange (SCE), exponentially growing cells mutations by inducing an aberrant mitotic segregation event. were exposed simultaneously to 30 ,M bromodeoxyuridine The initiated heterozygous cell is thus converted into a homo- (BrdUrd) and the tumor promoter TPA (diluted from a 10 zygous or hemizygous cell (see Fig. 1 and legend). This would mg/ml stock solution in acetone) for 28 hr (i.e., two genera- be consistent with the finding that the nonviral malignant (or tions). TPA and its derivative 4-O-methyl-TPA (4-O-Me-TPA) transformed) phenotype is usually suppressed on hybridization were obtained from the Consolidated Midland Chemical with nontransformed cells and that malignant segregants can Corporation, Brewster, NY. Similar incubations were carried reappear as a result of chromosome loss (7, 8). Although the out in the presence of inhibitors of tumor promotion such as term "recessive mutation" is used throughout this paper, our antipain and leupeptin or fluocinolone acetonide (FA). These hypothesis is compatible with any model of initiation based inhibitors were the gifts of W. Troll (New York University) and upon stable, recessive, genetic, or epigenetic chromosomal S. Yuspa (National Institutes of Health, Bethesda, MD), re- changes. spectively. Control cultures were treated with the same volume Aneuploidy and aberrant mitotic segregations have already of acetone as the TPA-treated cultures (10 ,ul per 10 ml of me- been postulated as a prerequisite for malignant transformation dium). Until chromosome fixation, all cultures were kept in the (1, 9). This speculation was applied specifically to tumor pro- dark. Twenty-six to 28 hr after TPA treatment, Colcemid (0.2 motion (6), and a preliminary study has shown that a nontoxic ,uM) was added to the cells for 2 hr, and the mitotic cells were dose of the tumor promoter 12-0-tetradecanoylphorbol 13- isolated by "mitotic shake-off." Metaphase preparations and acetate (TPA) induces the segregation of two recessive traits, differential staining were essentially as described by Perry and 6-thioguanine resistance and growth on agar, from doubly Wolff (15). All visible metaphases were analyzed for SCEs. heterozygous cell hybrids (10). Because promoting activity of TPA is correlated with skin ir- ritation and inflammation (3), the activity of the agents used The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: SCE, sister chromatid exchange; TPA, 12-0-tetra- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate decanoylphorbol 13-acetate; 4-O-Me-TPA, 4-O-methyl-TPA; BrdUrd, this fact. 5-bromodeoxyuridine; FA, fluocinolone acetonide. 6149 6150 Genetics: Kinsella and Radman Proc. Nati. Acad. Sci. USA 75 (1978) A MUTAGENIC__| -|- 2n .- AGENT | somatic cell z cK to Induction Direct 0 of cellular mutagenic mutagenesis .2 processes <Px .9 Heterozygous mutant G_ (wild-type phenotype) .c % Ploidy phase % |S p sk4~~~~~~~~~~~~~~~~~~~~~~~. I *-FI Conserv-JE Aberrant oI- ative chromosomal / / segre- segregations /._. gation Two-step Non- Chromosomal Mitotic # it nondisjunction disjunction rearrangement recombination 6 B W4 Fi < \ >~~~~~~~~Oa % 0 M BM M 3 M l 0 E 0 0L Vo M@ Ml \ M \[E[a~~~~~ AD t a" [3 Oco Dilution of the wild-type protein 11Mao - x Expressed mutant phenotype FIG. 1. Fixation, segregation, and expression ofrecessive mutations in somatic cells as a model in the study of carcinogenesis. For sim- C plicity, only one homologous chromosome pair of a diploid somatic ds- cell is shown. The homozygous (+/+), wild-type constitution for one NNW- gene on this chromosome pair can be changed to the heterozygous A&- state (m/+) by exposure to mutagenic agents. Depending on the na- 19M ture of the mutagen, the mutation fixation either can occur directly 4w during DNA replication or may require induction of cellular muta- genic processes (6). Circles symbolize centromeres. Broken lines with .mr.,W. -W -.IRA open circles represent the newly synthesized chromatids. M, homo- 0 t zygous (/rm) or hemizygous (inO) mutant cells in which phenotype *k.*- will be expressed. See text for other comments. T v ol in this study was tested by spotting 10 ,l of 10 Ag/ml solutions 41 in acetone onto the ears of BALB/c mice. TPA spots caused 1 inflammation within 12 hr, while 4-0-Me-TPA and acetone V 40, alone did not. laid 0 RESULTS AddL--- 00 lb To test the hypothesis presented in Fig. 1, the effects of TPA Ah on ploidy, chromosome loss, chromosomal aberrations, and SCEs have been studied. Only an increase in SCE frequency was observed (Fig. 2 A, B, and C). In addition to the approxi- mately 2-fold increase in countable SCEs seen in the majority D e N_ of the cells,* up to 25% of the TPA-treated cells showed SCEs that were "too many to count" (more than 100 SCEs per me- ".~ ~~~~ taphase) (Fig. 2C). This "two-population effect" was also ob- served with two independent V79 clones derived from our cultures, thus eliminating the involvement of stable mixed cell populations (results not shown). TPA toxicity was seen to vary between batches, but in general 4e4 the maximum induction of SCEs was found in the absence of major toxicity as measured by cloning efficiency (Table 1) and growth curves (Fig. 3). The optimum SCE induction occurred at 1.6 ,gM (1 ,g/ml) TPA (Table 2); TPA at 5 ,gg/ml was lethal without causing further increase in SCEs (Table 2). In an attempt to correlate SCE induction with mouse skin FIG. 2. Effects of TPA on chromosomes from V79 cells differ- tumor promotion, 4-0-Me-TPA, a nonpromoting derivative entially stained for detection of SCEs. SCEs are shown in (16), was found to have no effect on SCEs (Fig. 4), although its metaphase chromosomes from untreated control cells (A) and cells treated with TPA at 1 ,tg/ml (B and C). A and B show 8 and 34 SCEs, * Because TPA induction of SCEs is independent of BrdUrd concen- respectively, while C is an example of metaphases that were classified tration (see Table 2), we assume that BrdUrd is not a requirement as having "too many SCEs to count" (see Table 2).
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