Carcinogens induce genetic tandem duplications in Salmonella

Mutation Research, 152 (1985) 131-145 Elsevier

131

MTR 04106

Carcinogens induce genetic tandem duplications in Salmonella M a r t i n L. P a l l a n d B e v e r l y

J. H u n t e r

Program in Genetics and Cell Biology, and Program in Biochemistry/Biophysics, Washington State University, Pullman, WA 99164-4350

(U.S.A.) (Received 23 April 1984) (Revision received 11 April 1985) (Accepted 25 April 1985)

Summary Extensive studies have shown that chemical carcinogenesis involves an initiation-promotion pattern. A gene amplification model of carcinogenesis predicts that initiation involves induction of a genetic tandem duplication. We use a system developed by Anderson and Roth to select for tandem duplication of the histidine operon of Salmonella typhimurium by selection for resistance to 3-amino-l,2,4-triazole. Evidence reported here shows that, consistent with prediction, 10 carcinogens are all active in inducing tandem duplications. Two toxic noncarcinogens show little or no activity under the conditions used in inducing tandem duplication but azide, a mutagenic noncarcinogen, did show some activity. 9 types of evidence now support the gene amplification initiation-promotion model of carcinogenesis.

Many studies have shown that chemical carcin o g e n e s i s o f t e n involves a s e q u e n c e of i n i t i a t i o n - p r o m o t i o n - p r o g r e s s i o n (Berenblum, 1941, 1974; Slaga et al., 1978). From these studies, it appears that carcinogenesis ofter requires a single initiation event produced by a single exposure to any of a large number of carcinogens. Subsequent to initiation, repeated exposure to a tumor promoter such as 12-O-tetradecanoyl 13-phorbol acetate (TPA) can then lead to the induction of tumors. To be effective in producing tumors, exposure to promoters must follow exposure to initiators and must be repeated over an extended period of time. The temporal sequence of initiation-promotion is difficult to explain based on the simple model that chemical carcinogenesis involves multiple mutations of a cell genome. This difficulty arises in part, because tumor promoters are reported not to be mutagenic in standard mutagen tester systems (Trosko et al., 1977; McCann et al., 1975). In addition, repeated promotion events must follow

initiation in order to be effective in inducing tumors, whereas mutational events should be able to occur in any order. Consequently, barring selection of intermediate mutants, no ordering of mutational effectiveness should be seen. The tumors induced are often benign but they may progress to malignancy and to increased growth rates and metastatic activity, presumably by subsequent genetic events. One of us has proposed a model which accounts for the initiation-promotion pattern and is in agreement with many other observations on carcinogenesis and cancer cell properties (Pall, 1981). The model was stimulated by the finding of oncogenes and their normal cellular homologues known as proto- or cellular oncogenes. Such proto-oncogenes must be activated in order to be involved in carcinogenesis. The model proposes that the activation may involve a high level of gene amplification of the proto-oncogene, producing many extra copies of the gene involved. According

002%5107/85/$03.30 sO 1985 Elsevier Science Publishers B.V. (Biomedical Division)

132 to the model (Pall, 1981), the initiation evqat in the process of carcinogenesis (and gene amplification) is the induction of a tandem duplication of a proto-oncogene. T u m o r promoters then act by stimulating a series of unequal sister-chromatid exchanges in subsequent cell cycles which results in high levels of gene amplification. One of the predictions of this gene amplification model is that carcinogens are active in initiation because they can induce genetic tandem duplications. In this paper, we use a test system to show that 10 different carcinogens are active in inducing tandem duplications. The system used is one developed by Anderson and Roth (1977, 1978) in which one can select for tandem duplication of the histidine operon of Salmonella by selecting for resistance to 3-amino-l,2,4-triazole (AT) in a strain carrying the his02355 mutation which destroys the regulation of the histidine operon. Because AT inhibits one of the histidine biosynthetic enzymes, the original strain grows poorly on AT-containing medium but strains duplicated for the histidine operon produce increased levels of the biosynthetic enzymes and grow quite well. Materials and methods Bacterial strains and bacteriophage The Salmonella typhimurium strains TR4179 (srl-201, h i s 0 2 3 5 5 ) and TT1984 (srl-201, his02355, hisG8575::TnlO) (Anderson and Roth, 1978) and bacteriophage P22HT, a nonlysogenizing derivative of the high-transducing phage of Schmeiger, P22HT105/1 int-201 were provided by J.R. Roth (University of Utah, Salt Lake City, UT). Media Minimal media (ME) is Vogel and Bonner (1956) E medium containing 2% glucose. AT medium is ME medium supplemented with 3amino-l,2,4-triazole (1.4 raM), adenine (0.4 mM) and thiamine (0.05 mM). For the tranduction experiment, tetracycline (10 ffg/ml) and histidine (50 # m / m t ) were added to ME as required. Difco nutrient broth (NB) was used as a nonselective growth medium. Solid media contained 1.5% Difco agar.

Chemicals 3-Amino-l,2,4-triazole, tetracycline, histidineHC1, adenine, thiamine. HC1 and all chemicals tested for tandem duplication induction were purchased from Sigma Chemical Co. A T resistance assay For the AT resistance assay 50 ffl of a stationary phase, overnight (16-18 h) NB culture of TR4179 was added to 940-948 ixl of ME in 16 m m × 120 m m test tubes. The compounds to be tested were dissolved in DMSO, ethanol or ME and delivered in 2-10-/~1 volumes to give a final volume of 1 ml. Controls received 10/~1 of solvent in place of the test chemical. The tubes were vigorously shaken in rotary water bath shaker at 37°C for 4 h. After incubation, 0.1-ml aliquots of a 10 2 dilution (in ME) were plated onto each of four 100-mm AT plates. Viable cell counts were obtained by plating 0.1-ml aliquots of 10 -6 dilution on to each of two nutrient agar plates. The plates were incubated 48 h at 37°C prior to counting. For UV treatment, 250 /~1 NB culture of TR4179 was mixed with 4750 /11 ME in a sterile petri dish. The mixture was exposed to UV light at 1000 e r g / s e c / c m 2 for 1-10 sec while being swirled. After exposure to UV light, 1 ml of the mixture was pipetted into 16 m m x 120 mm test tubes and treated as described above. AT-resistant colonies induced were calculated as follows: AT-resistant viable count~ (AT[ - ATe) colonies inviable count t duced/10 s = nmoles of agent used bacteria/ nmole/ml X

1 ×10 w viable count ~.

AT-resistant colonies induced by UV light were calculated as A T - r e s i s t a n t c o l o n i e s / 1 0 w bacteria/100 erg/cm2; where t stands for culture treated with test chemical, c stands for control (or not treated with test chemical), and AT r stands for number of AT-resistant colonies/ml of culture plated. The increase in AT-resistant colonies due to induction was calculated as follows:

133

Ratio of AT-resistant colonies/105 bacteria plated (treated/uninduced control)

viable count~ ~ coun~i (ATtr) AT,.~

Tandem duplication assays Individual AT-resistant colonies were cloned by streaking onto AT medium. After 48 h incubation, isolated colonies were selected and grown in nutrient broth overnight (16-18 h). For test A (Fig. 1),

0.1 ml of a 1 0 - 6 dilution (in ME) was plated onto AT medium. For test B (Fig. 1) the NB cultures were centrifuged at 1450 × g for 30 rain and the pellets resuspended in ME to an approximate concentration of 1 x 10 l° bacteria/ml. P22HT lysate of TT1984 was mixed with 0.1 ml of the bacterial suspension in a phage : bacteria ratio of 5 : 1. After 30-min incubation at room temperature, 50 ~1 of the mixture was plated onto one plate each of tetracycline and tetracycline + histidine ME medium. All plates were incubated 48 h at 37°C.

T W O T E S T S FOR T A N D E M DUPLICATION

A. Reversion of Duplication by Unequal Crossing Over. his+

his+

| unequal crossover

: his-ll- X i

!

I

his+

I

his'}"

his+

his Jr

1

r a v a r t e n t (AT-sensitive)

t

|

his+

I

triplication

B. Duplication Demonstration by H e t e r o z y g o s i t y of Transductents.

Tandem duplication

I his+

I hill+

TnlO • v " his-

Nortdupllcstion

I

I his+ I trensduction of

:

his-::Tn 10 ( c a r r i e s tat r)

TnlO | his'

1 | his'l" •

I

1 TnlO '

• h,,-

i

TnlO

I

' his-

I

Trensductent le both his-}- (left copy)

Trensductent is t e t r a c y c l i n e

end t e t r a c y c l i n e resistent(right copy).

resistent but hie-.

Fig. 1. Two tests were used to distinguish tandem duplications form other genetic changes producing AT resistance as discussed in Anderson and Roth (1978), see Materials and Methods for details. (A) tandem duplications revert at high frequencies by unequal crossing over. High frequency reversion will lead to substantial numbers of small colonies on 1.4 m M AT medium. (B) Duplication was also tested by tranducing AT-resistant clones using a his ::Tnl0 strain (TT1984) as donor. Duplicants should produce similar numbers of colonies minimal-tetracycline and histidine+ tetracycline media but nonduplicants (B, right) will produce many colonies only on histidine + tetracycline medium.

134 A T plates were examined under 10 × magnification for the presence of large and small colonies. Tetracycline-containing plates were examined for growth.

Results and discussion A large fraction of spontaneously occurring AT-resistant colonies are due to tandem duplication of the histidine operon as shown by two tests (Fig. 1) previously described by A n d e r s o n and Roth (1978). Duplicants revert at high frequency because recombination between the duplications can restore the original single copy n u m b e r (Fig. 1A). Because duplicants have two copies of the histidine operon, they can be made heterozygous for it by transducing from a his : : T n l 0 strain (See Fig. 1B). In duplicants, one of the two his regions is replaced on transduction by his-::TnlO but the second his region remains

functional, causing the transductant

to remain

his +. Such his +, tet-resistant transductants are found in high frequency only when the recipient strain is duplicated for the his region and can be used, therefore, to test for possible duplications (for further discussion see Fig. 1, Anderson and Roth, 1978). Anderson and Roth (1978) showed that 77 out of 77 spontaneously occurring AT-resistant colonies were tandem duplicants and in our studies, 48 out of 48 such colonies were tandem duplicants. It may be inferred that all or almost all such spontaneously occurring colonies are due to tandem duplication. In the experiment discussed below, when ATresistant colonies induced by various agents were tested using the above two tests, individual colonies were either positive in both tests, showing that they are tandem duplicants or negative in both tests, showing that they are not. The studies described below are divided into

TABLE 1 AZASERINE AND INDUCTION OF AT RESISTANCE IN SALMONELLA (a) Concentration of azaserine (b) Viable count/ml of culture (×10 7) (c) AT-resistant colonies/ml of culture plated

Concurrent control (0)

0.116 ~M

139.6_+13.5

184.3_+2.1

12.0_+1.5

44.1_+8.1

0.289 ,ttM

0.405 #M

0.58 taM

1.16 taM

170.6_+5.0

149.5_+16.1

149.9-+14.1

99.0 _+20.1

73.6_+9.9

99.9+10.0

60.0_+10.8

87.3±9.9

( × 1 0 -3 )

(d) Number of ATresistant colonies counted (e) Number of AT plates counted (f) AT-resistant colonies/10 5 bacteria plated (c/b) (g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

432

485

660

1177

1598

36

11

11

16

16

0.86

2.39

3.52

4.92

6.66

2.78

4.09

5.73

7.75

Rows b and c are expressed as mean_+standard error.

1397 16

8.82

10.3

135 TABLE 2 f l - P R O P I O L A C T O N E A N D I N D U C T I O N OF A T R E S I S T A N C E IN S A L M O N E L L A (a) Concentration of /3-propiolactone

Concurrent control

(0) (b) Viable c o u n t / m l of culture ( × 1 0 7) (c) AT-resistant colonies/ml of culture plated

0.069 m M

0.138 m M

0.277 m M

0.554 m M

0.69 m M

1.1 m M

198.3 +_23.5

169.2+_13.7

199.2+_23.2

99.7_+7.5

119.0+-21.1

94.5+_5.9

46.7+_10.2

22.7 _+2.3

31.2_+5.3

35.7+3.2

54.3+4.0

75.7_+4.2

71.3+_7.7

57.2+_6.5

(xlO 3) (d) Number of ATresistant colonies counted (e) Number of AT plates counted

680

404

464

707

984

927

744

30

13

13

13

13

13

13

(f) AT-resistant colonies/10 ~ bacteria plated (c/b) (g) Ratio of ATresistant colonies/10 s bacteria plated (treated/uninduced control)

1.14

1.84

1.79

5.45

6.36

7.54

12.2

-

1.61

1.56

4.76

5.56

6.59

10.7

Rows b and c are expressed as mean_+ standard error.

two different parts. Firstly, 13 different agents were tested to determine if t h e y are capable of inducing A T resistance in our standard strain (TR4179) of Salmonella typhimurium. Secondly, individual induced colonies were tested to determine whether they were tandem duplicants by the two tests described above. Based on the frequency of duplicants and the induction level, the activity of the agents in inducing tandem duplications was calculated. 10 different carcinogens and 3 noncarcinogens were tested as inducers of AT resistance in Salmonella (see McCann et al., 1975 for refs. on carcinogenicity). All 10 carcinogens appear to be active in inducing A T resistance whereas 2 out of 3 noncarcinogens showed little or no inducing activity (Tables 1-13). For example, when cultures are grown in the presence of azaserine (Table 1), levels of AT-resistant colonies increase with increasing concentra-

tions of azaserine (row c). When the number of AT-resistant colonies are corrected for the viable bacterial count for each culture (row f), a still more consistent increase in AT resistance is seen with increasing dose of mutagen/carcinogen used. The results with 10 different carcinogens are shown in Tables 1-10. Each table represents the pooled averages from 3 or more separate experiments. In each case, an increasing level of AT resistance is seen with increasing dose of agent (rows f and g, Tables 1-10). In each case, induction appears to occur at levels of agent which produce little or no toxicity. The uninduced level of A T resistance was fairly consistent in these experiments, averaging about 1 per 105 bacteria plated. As indicated above, all or almost all of these spontaneous, AT-resistant colonies contain tandem duplications of the histidine operon. M o s t - o f the mutagen/carcinogens appear to produce a roughly linear dose-response curve of

136 TABLE 3 D I E T H Y L S U L F A T E (DES) A N D I N D U C T I O N OF AT RESISTANCE IN S A L M O N E L L A (a) Concentration of DES

Concurrent control

(0) (b) Viable c o u n t / m l of culture ( × 1 0 7) (c) AT-resistant colonies/ml of culture plated ( × 1 0 ~) (d) N u m b e r of ATresistant colonies counted (e) Number of AT plates counted

1.3 m M

1.95 mM

3.25 mM

5.2 m M

6.5 mM

165.8_+13.8

190.6_+17.0

141.8_+12.4

148.1_+12.8

99.8+9.5

75.4_+6.5

22.2 ± 1.5

61.5 _+7.7

76.1+6.3

99.1_+8.0

132.5_+11.8

163.5_+19.7

1 377

1 292

1 902

2378

2518

3270

62

21

25

24

19

20

13.3

21.7

(f) AT-resistant colonies/105 bacteria plated (c/b)

1.34

3.23

5.37

6.69

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

-

2.41

4.01

5.00

9.92

16.2

Rows b and c are expressed as mean -+ standard error.

tandem duplication induction as a function of dose of agent. Two possible exceptions to this pattern are M N N G and EMS (Tables 7 and 3). M N N G shows more than a 10-fold increase in AT resistance with a doubling of concentration (from 0.68 mM to 1.36 mM, Table 7, rows f and g). Ethyl methanesulfonate (EMS) shows a smaller deviation from linearity. EMS resembles M N N G by having an increased yield of resistant colonies per millimole of agent at higher dose. Both of these compounds are reported to give nonlinear dose-response curves in the induction of point mutations (McCann et al., 1975). Two noncarcinogens, chloramphenicol and potassium cyanide were chosen as toxic agents not known to induce genetic damage. They were tested at levels producing partial growth inhibition similar to growth inhibition produced by the carcinogens (Tables 11 and 12). They fail to induce substantial levels of AT resistance, showing less than

a doubling of AT-resistant colonies/105 cells, and we conclude that toxicity is not sufficient to effectively induce tandem duplications, The last noncarcinogen, sodium azide, has been shown to induce point mutations in Salmonella (Nilan et al., 1973) and is shown here to have a low activity in inducing A T resistance (Table 13). We present evidence below that the azide-induced AT-resistant colonies are largely tandem duplications of the histidine operon. The point mutations previously studied are actually induced by a metabolite of azide (Owais et al., 1981) which is probably not synthesized in mammalian cells and this metabolite may also be the active agent for tandem duplication induction. The data presented in Tables 1-13 provide evidence that 10 carcinogens are active in inducing AT resistance in a strain of Salmonella in which AT resistance can be produced by tandem duplication of the histidine operon. Are these agents

137 TABLE 4 ETHYL METHANESULFONATE (EMS) AND INDUCTION OF AT RESISTANCE IN SALMONELLA (a) Concentration of EMS (b) Viable count/ml of culture ()<10 -7) (c) AT-resistant colonies/ml of culture plated ( x l 0 -3) (d) Number of ATresistant colonies counted (e) Number of AT plates counted (f) AT-resistant colonies/105 bacteria plated (c/b)

Concurrent control {0)

4.02mM

5.63mM

8.05mM

13.1 mM

151.4 ± 10.6

165.8±17.6

191.8±18.1

180.6±11.4

114.4±6.5

116.8±7.5

19.1 ± 1.6

49.8±6.4

60.3±6.6

77.8±7.9

120.5±12.0

227.8±30.7

916

868

723

48

20

12

1.26

(g) Ratio of ATresistant colonies/10 ~ bacteria plated (treated/uninduced control)

1 479

19

3.00

3.14

4.31

2.38

2.50

3.41

1 446

21 mM

3644

12

16

10.5

19.5

8.35

15.5

Rows b and c expressed as mean ± standard error. actually active in inducing such tandem duplications? As shown in Table 14, column d, a series of AT-resistant colonies isolated after induction with each agent were cloned and tested for tandem duplication of the histidine operon, using the two tests outlined above in Fig. 1. Because essentially all spontaneous AT-resistant colonies are tandem duplicants, the expected fraction of AT-resistant colonies if an agent does not induce duplications, can be readily calculated. For example, an agent that does not induce duplications and shows a ratio of t r e a t e d / u n i n d u c e d A T resistance of 10 would be expected to show only 10% tandem duplications. Five of the m u t a g e n / c a r c i n o g e n s (azaserine, /3-propiolactone, epichlorohydrin, MMS, and UV light) show both a substantial induction of A T resistance (ratios of t r e a t e d / c o n t r o l of 4.06 to 10.7, column b) and high percentages of tandem duplicants (82-100%, column d). F o u r others (DES, EMS, M N N G and streptozotocin) show

lower levels of tandem duplicants (22-44%, colu m n d) but exhibit a high induction of A T resistance (ratio of t r e a t e d / u n i n d u c e d A T resistance of 16.2 to 82.7, column b). These results make it clear that these four are also active in inducing tandem duplications. N M U has the highest level of induction of A T resistance (191-fold) but only about 5% (3 out of 60) of them are tandem duplicants (Table 14, column d). If N M U is n o t inducing tandem duplication, the probability of finding 3 spontaneous duplicants with 191-fold induction is less than 1%. We infer, consequently, that N M U p r o b a b l y induces tandem duplication and conclude that all 10 tested carcinogens are active in such induction. Sodium azide also shows a low level of apparent induction of tandem duplication (Table 14, bottom). The other two noncarcinogens show less than a 2-fold change in A T resistance and were not investigated further. The mechanism of tandem duplication induc-

TABLE 5 E P I C H L O R O H Y D R I N A N D I N D U C T I O N OF A T R E S I S T A N C E IN S A L M O N E L L A (a) Concentration of epichlorohydrin

Concurrent control (0)

(b) Viable c o u n t / m l of culture ( × 1 0 -7) (c) AT-resistant colonies/ml of culture plated ( × 1 0 3) (d) N u m b e r of ATresistant colonies counted (e) N u m b e r of AT plates counted (f) AT-resistant colonies/105 bacteria plated (c/b)

0.54 m M

0.86 m M

1.08 m M

2.16 m M

163.5 ± 10.2

172.2 ± 11.5

196.7 ± 13.8

152.8 ± 18.1

73.7 ± 13.3

12.7 ± 1.8

31.6 ± 5.4

34.7 ± 5.0

44.1 ± 6.1

39.6 ± 4.6

356

253

271

485

634

28

8

11

11

16

0.78

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

1.84

1.76

2.89

5.37

2.36

2.26

3.72

6.92

Rows b and c are expressed as mean ± standard error. TABLE 6 M E T H Y L M E T H A N E S U L F O N A T E (MMS) A N D I N D U C T I O N OF AT RESISTANCE IN S A L M O N E L L A (a) Concentration of MMS (b) Viable c o u n t / m l of culture ( × 1 0 7) (c) AT-resistant colonies/ml of culture plated ( x l 0 -3) (d) N u m b e r of ATresistant colonies counted (e) N u m b e r of AT plates counted (f) AT-resistant colonies/105 bacteria plated (c/b) (g) Ratio of ATresistant colonies/l 05 bacteria plated (treated/uninduced control)

Concurrent control (0)

0.045mM

0.09mM

0.18mM

150.1 ± 12.9

167.7±22.9

197.0±18.5

160.3±10.2

132.2±8.7

80.6±14.9

17.3±1.7

31.5±5.9

31.4±5.3

94.1±8.0

117.0±10.9

100.4±15.9

0.455mM

0.91mM

692

378

377

1 505

2 032

1 607

40

12

12

16

16

16

1.18

1.88

1.59

5.87

8.85

12.46

1.60

1.36

4.99

7.50

10.6

Rows b and c are expressed as mean_+ standard error.

TABLE 7 N - M E T H Y L - N ' - N I T R O - N - N I T R O S O G U A N I D I N E ( M M N G ) A N D I N D U C T I O N OF AT RESISTANCE IN S A L M O N E L L A

(a) Concentration of M N N G

Concurrent control

(0)

0.136 ~ M

0.34 ~ M

0.467 p,M

0.68 p,M

1.36 p,M

3.4 ~M

(b) Viable c o u n t / m l of culture (>(10 -7 )

190.0 +15.2

174.3 +21.2

175.0 ±7.9

162.3 +14.8

159.5 ±13.1

58,1 _+7.0

23.8 ±3.2

(c) AT-resistant colonies/ml of culture plated ( >( 10 - 3 )

12.6 ± 1.6

22.4 __.6,0

46.8 + 10.3

52.9 ± 9.0

129.5 ± 23.1

317.0 + 45.8

334.3 ± 35.4

(d) N u m b e r of ATresistant colonies counted

554

314

1 217

741

2 720

6 657

7 020

44

14

26

14

21

21

21

(e) N u m b e r of AT plates counted (f) AT-resistant colonies/105 bacteria plated (c/b)

0.66

1.29

2,67

3.26

8.12

54.6

140.5

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)-

-

1.95

4.05

4.94

12.30

82.7

212.9

Rows b and c are expressed as mean +__standard error. TABLE 8 N I T R O S O M E T H Y L U R E A ( N M U ) A N D I N D U C T I O N OF AT R E S I S T A N C E IN S A L M O N E L L A (a)Concentration of N M U

Concurrent control

(0) (b) Viable c o u n t / m l of culture ( x l 0 -7) (c) AT-resistant colonies/ml of culture plated (xl0 -3) (d) N u m b e r of ATresistant colonies counted (e) N u m b e r of A T plates counted

0.145mM

0.36mM

0.51mM

0.725mM

196.4 ± 18.2

143.0± 14.0

60.5 ± 26.0

47.5 ± 14.7

16.3 ± 7.2

6.8 ± 1.4

286.7 + 45.4

359.9 + 49.2

314.5 ± 51.6

147.5 ± 20.7

163

3 440

4319

3774

1770

24

12

12

12

12

59.5

66.2

90.5

(Io AT-resistant colonies/10 ~ bacteria plated (c/b)

0.346

20.0

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

-

57.9

Rows b and c are expressed as mean 5: standard error.

172

191

261

TABLE 9 S T R E P T O Z O T O C I N A N D I N D U C T I O N OF AT RESISTANCE IN S A L M O N E L L A (a) Concentration of streptozotocin (b) Viable c o u n t / m l of culture ( × 1 0 7) (c) AT-resistant colonies/ml of culture plated (>(10 -3) (d) N u m b e r of ATresistant colonies counted (e) Number of AT plates counted

Concurrent control (0)

0.076/tM

139.8 ± 18.8

151.3±27.4

152.6±7.3

110.0±11,3

126.2±11.5

142.0±21.5

13.1 ± 1.8

188.7 ± 60.5

320.4 ± 69.4

425.0 ± 54.4

583.3±9.3

607.2 ± 90.2

0,113 ~M

0.19 ,uM

0.26 # M

0.38 ,aM

366

2 264

3 845

5100

6999

7286

28

12

12

12

12

12

12.5

21.0

38.6

46.2

42.8

13.3

22.4

41.2

49.3

45.6

5000 erg/cm 2

6000 erg/cm 2

7000 erg/cm ~

(f) AT-resistant colonies/10 s bacteria plated (c/b)

0.94

(g) Ratio of ATresistant colonies/10 s bacteria plated (treated/uninduced control)

Rows b and c are expressed as mean + standard error. T A B L E 10 UV A N D I N D U C T I O N OF A T RESISTANCE IN S A L M O N E L L A (a) Dose of UV light

(b) Viable c o u n t / m l of culture ( x l O -7) (c) AT-resistant colonies/ml of culture plated ( x 1 0 -3 ) (d) Number of ATresistant colonies counted

Concurrent Control (0)

3 000 erg/cm 2

4000 erg/cm 2

109.8 ± 9.2

100.5 _+9.1

89.7±9.5

87.7±9.0

73.3±6.9

64.5±3.8

25.8 ± 4.5

45.4 + 4.2

52.7±2.8

62.4±4.1

57.1±5.5

61.6±4.0

232

409

474

562

514

554

(e) N u m b e r of A T plates counted

9

9

9

9

9

9

(f) AT-resistant colonies/l 05 bacteria plated (c/b)

2.35

4.52

5.88

7.12

7.79

9.55

(g) Ratio of ATresistant colonies/10 s bacteria plated (treated/uninduced control)

-

1.92

2.50

3.03

3.31

4.06

Rows b and c are expressed as mean + standard error.

TABLE 11 C H L O R A M P H E N I C O L AND INDUCTION OF AT RESISTANCE IN SALMONELLA (a) Concentration of chloramphenicol (b) Viable c o u n t / m l of culture ( x l 0 7) (c) AT-resistant colonies/ml of culture plated ( × 1 0 s) (d) Number of ATresistant colonies counted (e) Number of AT plates counted (f) AT-resistant colonies/10 s bacteria plated (c/b)

Concurrent control (0)

0.155 ~M

190.1 ± 19.9

213.5±35.8

191.1 ±11.0

183.3±5.9

164.6+_10.7

120.7+19.4

18.1 -+ 1.9

18.3±2.5

23.5±3.4

18.4_+2.0

23.8± 3.1

14.6±1.8

0.31 /zM

0.62 /IM

1.55 gM

3.1 gM

688

347

446

350

428

277

38

19

19

19

18

19

0.952

(g) Ratio of ATresistant colonies/10 s bacteria plated (treated/uninduced control)

0.857

1.23

1.00

1.44

1.21

0.90

1.29

1.05

1.52

1.27

Rows b and c are expressed as mean ± standard error. TABLE 12 KCN AND INDUCTION OF AT RESISTANCE 1N SALMONELLA (a) Concentration

of KCN (b) Viable c o u n t / m l of culture ( x l 0 7) (c) AT-resistant colonies/ml of culture plated ( x l 0 -3) (d) Number of ATresistant colonies counted (e) Number of AT plates counted (f) AT-resistant colonies/105 bacteria plated (c/b)

Concurrent control (0)

3.1gM

7.7gM

10.7gM

15.4gM

169.6 -+ 15.9

170.6-+17.8

173.4-+16.7

169.4±27.4

117.5±17.4

74.1 ± 15.1

18.9 -+ 1.9

17.3±3.2

20.8±3.7

23.2±3.9

13.5±3.1

12.0±1.6

30.7/t M

831

277

332

278

148

239

44

16

1'6

12

11

20

1.11

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control) Rows b and c are expressed as mean + standard error.

1.01

1.20

1.37

1.15

1.62

0.91

1.08

1.23

1.03

1.45

TABLE 13 A Z I D E A N D I N D U C T I O N OF AT RESISTANCE IN SALMONELLA (a) Concentration of sodium azide

(b) Viable c o u n t / m l of culture ( x l 0 v) (c) AT-resistant colonies/ml of culture plated ( × 1 0 -3 ) (d) Number of ATresistant colonies counted (e) Number of AT plates counted (f) AT-resistant colonies/l 05 bacteria plated (c/b)

Concurrent control C0)

169.6-+ 15.3

18.4_+1.9

0.16 mM

149.9-+7.6

22.9_+2.6

0.32 mM

0.48 mM

0.80 mM

1.29 mM

107.1_+12.5

80.3_+10.5

74.6_+13.9

56.9_+12.1

23.8"_+2.4

25.6_+4.8

20.9+_4.0

27.9_+1.8

811

458

558

476

409

335

44

20

20

20

16

16

1.08

(g) Ratio of ATresistant colonies/105 bacteria plated (treated/uninduced control)

1.53

2.60

2.96

3.43

3.67

1.41

2.40

2.73

3.16

3.39

Rows b and c are expressed as mean + standard error.

TABLE 14 I N D U C T I O N OF T A N D E M DUPLICATIONS BY C A R C I N O G E N S (a) Dosage used for data presented

(b) Ratio of ATresistant colonies/ 105 bacteria plated (treated/ uninduced control) ~

(c) AT-resistant colonies/10 s bacteria/nmole/ ml

(d) Fraction of AT-resistant colonies which were tandem duplicants

(e) Tandem duplicants/10 s bacteria/nmole/ ml

1.00 0.82 0.22 0.35 0.86 0.97

1.0 × 103 8.1 0.685 0.32 1.8 16.8

Chemical carcinogens Azaserine fl-Propiolactone Diethyl sulfate Ethyl methanesulfonate (EMS) Epichlorohydrin Methyl methanesulfonate (MMS)

0.58/zM 1.1 mM 6.5 mM 21 mM 2.16 mM 0.455 mM

7.75 10.7 16.2 15.5 6.92 7.69

1.0 x 10 ~ 9.9 3.1 0.9 2.1 17.3

(86/86) " (37/45) (10/46) (27/77) (70/81) (74/76)

N-Methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) Nitrosomethyl urea (NMU) Streptozotocin

1.36 #M 0.51 mM 0.26 #M

82.7 191 49.3

4.2 M 1 0 4 129.5 1.71 × 105

0.39(12/31) 0.05 (3/60) 0.44 (40/90)

1.6×104 6.5 7.5 × 104

102.8 h

0.95 (19/20)

97.6 b

4.9

0.82 (36/44)

4.0

Physical carcinogens UV light

7000 e r g / c m 2

4.06

Noncarcinogens Chloramphenicol KCN Sodium azide

0.155 3.1 p.M 3.1-30.7 I~M 0.32 mM

< 2 < 2 2.40

Number of tandem duplicants/number tested. h UV light is expressed as a function of 100 e r g / c m 2, not nmoles/ml. • Column b is derived from Tables 1-13.

143 tion (and also deletion) in bacteria is thought to involve recombination between short (i.e. 10-15 base pairs) or long identical DNA sequences and occurs particularly frequently where long sequences, such as ribosomal sequences, can undergo recombination (see for example Hill et al., 1977; Anderson and Roth, 1981; Edlund and Normark, 1981; Pribnow et al., 1981; Albertini et al., 1982; Marvo et al., 1983). There is some evidence for a similar duplication mechanism in animal cells (Ruley and Fried, 1983; Goldberg et al., 1983). The frequency of spontaneous AT resistance is much lower in r e c A - strains of Salmonella compared to r e c ÷ strains (Anderson and Roth, 1978), suggesting that recombination is involved in the duplicants selected in this study. Because the histidine operon is not near the ribosomal sequences in Salmonella and E. coli, any recombination events involved here almost certainly do not involve ribosomal sequences. Several studies have been made on the induction of tandem duplications in the bacterial genome by chemical or physical agents (Hill and Combriato, 1973; Straus, 1974; Hoffmann et al., 1978), some of which are demonstrated carcinogens and others of uncertain carcinogenicity. These studies have used different selection systems than that used here. In these studies, the carcinogens, UV light, X-rays, EMS, and possibly M N N G , were active in inducing genetic duplications. The results reported here extend the study of induction by carcinogens to a substantially larger group of agents using another selection system. For all of the data presented in Table 14, the two tests for tandem duplication outlined in Fig. 1 gave consistent scoring. Individual colonies were either scored as duplicants by both tests or as nonduplicants by both tests. However, in a series of 145 MNNG-induced AT-resistant colonies, we found 3 colonies which appeared to be duplicants by the reversion test but were nonduplicants by transduction test. These were the only colonies where the two tests did not agree out of over 1300 induced and spontaneous AT-resistant colonies tested. We are unsure how to interpret the results on these 3 colonies. In any case, it is clear that in almost all cases both tests give consistent scoring for individual colonies. The induction of tandem duplications by

carcinogens confirms a prediction of the gene amplification model of carcinogenesis discussed above. There are now 9 different types of observations supporting that model, 6 of which were known at the time the model was published and 3 of which are confirmations of predictions of the model. The 6 original supporting observations which were discussed earlier (Pall, 1981) are: (1) The model is excellent agreement with the initiation-promotion pattern of chemical carcinogenesis. (2) It predicts the long latencies found in carcinogenesis studies. (3 + 4) The two cytogenetic manifestations of gene amplification, homogeneously staining regions and double minutes, are often found in cancer cells. (5) Tumor promoters and complete carcinogens are reported to stimulate sister-chromatid exchange. (6) It is consistent with the positive role of genes in the neoplastic process found in the best documented genetic mechanisms of carcinogenesis. Three confirmations of predictions of the model are: (1) Proto-oncogene gene amplification has now been reported to occur in various types of cancer cells (Collins and Groudine, 1982, 1983; Favera et al., 1982; Schwab et al., 1983a,b; Murray et al., 1983; Montgomery et al., 1983; Alitalo et al., 1983; Kanda et al., 1983; Little et al., 1983). (2) Gene amplification in cell culture is stimulated by tumor promoters (Varshavsky, 1981; Hayashi et al., 1983; Barsoum and Varshavsky, 1983; Bojan et al., 1983). (3) We report here that 10 different diverse carcinogens all induce tandem genetic duplications and thus may initiate carcinogenesis by this mechanism. Our work extends earlier studies showing that 4 carcinogens (3 of which were also studied above) induce duplications in bacteria (Hill and Combriato, 1973; Straus, 1974; Hoffmann et al., 1978). Consequently, a total of 11 carcinogens have been shown to be active in such induction. The gene amplification model of chemical carcinogenesis is, therefore, consistent with diverse lines of evidence on tumor initiation and promotion.

144

Acknowledgements We t h a n k Drs. J o h n R o t h a n d Michael K a h n for helpful discussions.

This study was supported by PHS grant CA33503 awarded by the National Cancer Institute. References Albertini, A.M., M. Hofer, M.P. Calos and J.H. Miller (1982) On the formation of spontaneous deletions: The importance of short sequence homologies in the generation of large deletions, Cell, 29, 319-328. Alitalo, K., M. Schwab, C.C. Lin, H.E. Varmus and J.M. Bishop (1983) Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (e-myc) in malignant neuroendocrine cells from a human colon carcinoma, Proc. Natl. Acad. Sci. (U,S.A.), 80, 1707 1711. Anderson, R.P., and J.R. Roth (1977) Tandem genetic duplications in phage and bacteria, Annu, Rev. Microbiol., 31, 473-505. Anderson, R.P., and J.R. Roth (1978) Tandem genetic duplications in Salmonella typhimurium: Amplification of the histidine operon, J. Mol. Biol., 126, 53-71. Anderson, R.P., and J. Roth (1981) Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons, Proc. Natl. Acad. Sci. (U.S.A.), 78, 3113-3117. Barsoum, J., and A. Varshavsky (1983) Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dehydrofolate reductase genes, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5330-5334. Berenblum, I. (1941) The mechanism of carcinogenesis, A study of the significance of cocarcinogenic action and related phenomena, Cancer Res., 1,807-814. Berenblum, I. (1974) Carcinogenesis as a Biological Problem, Elsevier, New York. Bojan, F., A.R. Kinsella and M. Fox (1983) Effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate on recovery of methotrexate-, N-(phosphonacetyl)-L-aspartate-, and cadmium-resistant colony-forming mouse and hamster cells, Cancer Res., 43, 5217-5221. Collins, S., and M. Groudine (1982) Amplification of endogenous myc-related DNA sequences in a human myeloid leukemia cell line, Nature (London), 298, 679-681. Collins, S.J., and M.T. Groudine (1983) Rearrangement and amplification of c-abl sequences in the human chronic myelogenous leukemia cell line K-562, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4813-4817. Edlund, T., and S. Normark (1981) Recombination between short DNA homologies causes tandem duplication, Nature (London), 292, 269-271. Favera, R.D., F. Wong-Staal and R.C. Gallo (1982) onc gene amplification in promyelocytic leukaemia cell line HL-60

and primary leukaemia cells of the same patient, Nature (London), 299, 61-63. Goldberg, M.L., J.-Y. Sheen, W.J. Gehring and M.M. Green (1983) Unequel crossing-over associated with asymmetrical synapsis between nomadic elements in Drosophila melanogaster genome, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5013 5021. Hayashi, K., H. Fujiki and T. Sugimura (1983) Effects of tumor promoters on the frequency of metallothionein I gene amplification in cells exposed to cadmium, Cancer Res., 43, 5433-5436. Hill, C.W., and G. Combriato (1973) Genetic duplications induced at very high frequency by ultraviolet irradiation in Escheriehia coil, Mol. Gen. Genet., 127, 197-214. Hill, C.W., R.H. Grafstrom and B.S. Hillman (1977) Chromosomal rearrangements resulting from recombination between ribosomal RNA genes, in: A.I. Bukari, J.A. Shapiro and S.L. Adhya (Eds.), DNA, Inserted Elements, Plasmids and Episomes, pp. 497-506. Hoffmann, G.R., R.W. Morgan and R.C. Harvey (1978) Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium, Mutation Res., 52, 73-80. Kanda, N., R. Schreck, F. Alt, G. Bruns, D. Baltimore and S. Latt (1983) Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: Facilitation by fluorescence-activated flow sorting of metaphase chromosomes, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4069-4073. Little, C.D., M.M. Nau, D.N. Carney, A.F. Gazdar and JD. Minna (1983) Amplification and expression of the c-m~,c oncogene in human lung cancer cell lines, Nature (London), 306, 194-196. Marvo, S.L., S.R. King and S.R. Jaskunas (1983) Role of short regions of homology in intermolecular illegitimate recombination events, Proc. Natl. Acad. Sci. (U.S.A.), 80, 2452-2456. MeCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Selection of carcinogens as mutagens in the Salmonella/ microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Montgomery, K.T., J.L. Biedler, B.A. Spengler and P.W. Melera (1983) Specific DNA sequence amplification in human neuroblastoma cells, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5724 5728. Murray, M.J., J.M. Cunningham, L.F. Parada, F. Dautry, P. Lebowitz and R.A. Weinberg (1983) The HL-60 transforming sequence: A ras oncogene coexisting with altered myc genes in hematopoietic tumors, Cell. 33, 749-757. Nilan, R.A., E.G. Sideris, A. Kleinhofs, C. Sander and C.F. Konzak (1973) Azide - - a potent mutagen, Mutation Res., 17, 142-144. Owais, W.M., A. Kleinhofs and R.A. Nilan (1981) Effects of L-cysteine and O-acetyl-L-serine in the synthesis and mutagenicity of azide metabolite, Mutation Res., 80, 99-104. Pall, M.L. (1981) Gene-amplification model of carcinogenesis, Proc. Natl. Acad. Sci. (U.S.A.), 78, 2465-2468. Pribnow, D., D.C. Sigurdson, L. Gold, B.S. Singer, C. Napoli, J. Brosing, T.J. Dull and H.F. Noller (1981) rll cistrons of bacteriophage T4, DNA sequence around the intercistronic

145 divide and positions of genetic landmarks, J. Mol. Biol., 149, 337-376. Ruley, H.E,, and M. Fried (1983) Clustered illegitimate recombination events in mammalian cells involve very short sequence homologies, Nature (London), 304, 181-184. Schwab, M., K. Alitalo, K.-H. Klempnauer, H.E. Varmus, J.M. Bishop, F. Gilbert, G. Brodeur, M. Goldstein and J. Trent (1983a) Amplified DNA with limited homology to rnyc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumor, Nature (London), 305, 245-248. Schwab, M., K. Alitalo, H.E. Varmus, J.M. Bishop and D. George (1983b) A cellular oncogene (c-Ki-ras) is amplified, over expressed and located within karyotypic abnormalities in mouse adrenocortical turnout cells, Nature (London), 303, 497-501. Slaga, T.J., A. Sivak and R.K. Boutwell (Eds.) (1978) Carcino-

genesis - - A Comprehensive Survey, Vol. 2, Mechanisms of Tumor Promotion and Cocarcinogenesis, Raven Press, New York. Straus, D.S. (1974) Induction by mutagens of tandem duplications in the glyS region of the Escherichia coli chromosome, Genetics, 78, 823 830. Trosko, J.E., Chang, L.P. Yotti and E.H.Y. Chu (1977) Effect of phorbol myristate acetate on the recovery of spontaneous and ultraviolet light induced 6-thioguanine and ouabain-resistant Chinese hamster cells, Cancer Res., 37, 188-193. Varshavsky, A. (1981) Phorbol esters dramatically increase incidence of methotrexate-resistant, colony-forming mouse cells: Possible mechanisms and relevance to tumor promoters, Cell, 25, 561-572. Vogel, H., and D. Bonner (1956) Acetylornithinase of Escherichia coli: Partial purification and some properties, J. Biol. Chem., 218, 97-106.