153
Mutation Research, 45 (1977) 153--156
© Elsevier/North-Holland Biomedical Press
E F F E C T OF TUMOR PROMOTING AGENTS ON MUTATION F R E Q U E N C I E S IN C U L T U R E D V79 CHINESE HAMSTER CELLS *
GEORGE R. LANKAS Jr., C. STUART BAXTER and ROBERT T, CHRISTIAN Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267 (U.S.A.)
(Received 1 March, 1977) (Revision received 31 May, 1977) (Accepted 8 June, 1977)
The two-stage model of carcinogenesis, first demonstrated in skin by Berenblum [5], has n o w been extended to other organs [2,3]. In this model initiation, which is conceived as an irreversible change in DNA, is followed by promotion, which enhances the expression of the transformed phenotype. All carcinogens are considered to be initiators [7] and most are also mutagenic [1]. The observed promotion of carcinogenesis in other organs and transformation in vitro [9] suggests that a two-stage process may be fundamental to carcinogenesis and is n o t a unique property of skin, as many have claimed. If initiation is a mutagenic change then it seems reasonable to deduce that known promoting agents, such as phorbol and TPA will enhance the chemically induced mutation frequency in Chinese hamster cells in culture. Recently, Trosko et al. [12] have demonstrated that the ultra-violet light-induced mutation frequency in Chinese hamster cells is enhanced by these promoters. Here we report evidence that the frequency of chemically-induced ouabain-resistant mutations is increased by tumor-promoters. Chinese hamster V79 cells (kindly provided by Dr. R. Hart, Ohio State University) were grown in 100 mm plastic petri dishes in a modified minimum essential medium (Eagle's) supplemented with 1.5X vitamins and amino acids and 10% fetal calf serum. Cells were seeded at a density of 4 × l 0 s cells/plate in 10 ml medium and 3 h allowed for cell attachment. Plates were then dosed with either MAM dissolved in medium without serum or MNNG dissolved in acetone. MAM exposure was for 48 h and MNNG exposure for 2 h, at which time medium was poured from the plates and replaced with fresh medium. TPA and phorbol were dissolved in acetone at equimolar concentrations (1 mg/ml * This w o r k w a s s u p p o r t e d b y N I H g r a n t s ES 0 0 1 5 9 , ES 0 0 1 2 7 , a n d 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 i e s Institute of Toxicology.
Abbreviations: T P A , 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e , MAM, m e t h y l a z o x y m e t h a n o l M N N G , N-methyl-N'-nitro-N-nitrosoguanidine.
a c e t a t e , and
154 TPA, 0.59 mg/ml phorbol). Forty-two hours after dosing with the mutagen, 10 gl of the TPA or phorbol solutions were added to the appropriate plates. After an additional 6 h, medium was poured off all plates and replaced with medium containing 1 mM ouabain, and promoter where appropriate. The promoter was thus present throughout the selection period. After 10 days incubation, the plates were then fixed, stained and scored for ouabain-resistant mutants. Concurrently, toxicity assays were done to determine cell survival after the various treatments. Cells were seeded at a density of 2 × 102/plate in 60 mm dishes and treated as above, except that medium did not contain ouabain. Plates were fixed and stained after one week and the number of colonies determined. Preliminary experiments indicated that addition of phorbol compounds at 42 h after treatment, and continued exposure during selection with ouabain, gave the highest recovery of mutants. A non-toxic dose of both phorbol and TPA was used, since this concentration has been shown not to inhibit DNA repair synthesis in Chinese hamster cells [13]. Therefore, inhibition of repair does n o t appear to be the mechanism of action of phorbol compounds. Results shown in Table 1 indicate that both TPA and phorbol increase the recovery of ouabain-resistant mutants at all 3 doses of MNNG tested w i t h o u t enhancing the cytotoxic effects of the agent. Phorbol was n o t as effective in increasing the recovery of mutants as was TPA at equimolar concentrations. Table 2 shows the results of a similar experiment with MAM as the mutagen. Again, phorbol and TPA increased the recovery of ouabain-resistant mutants w i t h o u t enhancing the cytotoxicity of the agent. Furthermore, the effect of TPA was greatest at the highest mutagen dose. Phorbol was again not as effective as TPA in enhancing m u t a n t recovery. Preliminary results (Lankas, unpublished data) indicate that TPA can enhance m u t a n t recovery 4 weeks after treatment with MAM, though to a somewhat lesser extent compared to treatment 42 h after addition of the muta-
TABLE
1
EFFECT
OF PHORBOL
AND TPA ON MNNG-INDUCED
OUABAIN-RESISTANT
Treatment
No. of plates
% survival
Control Acetone (0.2%) T P A (1 p g / m l ) A c e t o n e + T P A (1 I J g / m l ) Acetone + phorbol (0.59 Hg/ml) MNNG (pg/ml) 0.5 0.5 + TPA 1.0 1.0 + TPA 1.5 1.5 + phorbol 1.5 + TPA
10 10 10 5 5
85 87 78 83 85
20 25 19 5 0
10 8 9 8 9 5 9
70 72 56 54 46 44 42
81 125 138 158 279 168 349
a p values compare b p < 0.05.
(mutagen
and promoter)
to (mutagen
No. of mutants
only) treatment.
MUTATIONS Mutants/ 106 survivors a 5.8 7.2 6.1 2.9 0 28.9 54.2 b 68.3 91.7 168.5 190.9 230.4 b
155 TABLE 2 EFFECT OF PHORBOL AND TPA ON MAM-INDUCED OUABAIN-RESISTANT
Treatment
Control T P A (1 p g / m l ) Phorbol (0.59 pg/ml) MAM (pg/ml) 12 12 + T P A 18 18 + T P A 24 24 + p h o r b o l 24 + T P A a p bP c P d p
No. of plates
% survival
N o . of mutants
MUTATIONS Mutants/ 10 6 s u r v i v o r s a
6 6 6
96 89 95
4 7 8
1.7 3.2 3.5
10 9 8 8 10 10 10
62 65 15 15 7 7 7
57 67 64 90 73 109 123
22.9 28.6 133.3 187.5 260.7 390.0 439.3
v a l u e s c o m p a x e ( m u t a g e n and p r o m o t e r ) t o ( m u t a g e n o n l y ) t r e a t m e n t . < 0.10. < 0.05. < 0.02.
gen. Thus, effects of TPA on expression time cannot solely explain its enhancement of mutagenesis. Studies are currently being performed to determine the mechanism of p r o m o t e r enhancement of mutagenesis. Since TPA or phorbol are not mutagenic per se, either in our system or in the 6-thioguanine mutation system [13], b u t are able to increase the induced mutation frequency by two chemical mutagens, we interpret these results as indicating that phorbol c o m p o u n d s can modify gene expression. In the ouabain mutation system, changes in membrane b o u n d Na÷/K + ATPase sensitivity to ouabain are determined [4]. If a cell has an altered but repressed gene, it will behave as a ouabain-sensitive cell and be eliminated from the population under conditions selecting for ouabain-resistant mutants. Apparently, both TPA and phorbol can convert these "latent" mutants into "expressed" mutants, allowing their recovery in the ouabain medium. Our conclusions are supported by several other reports. It has been shown that TPA can induce expression of normally repressed proteins in several cell lines [14] and in mouse skin [7]. It has also been shown that TPA increases the phosphorylation of histones in mouse skin [11]. Histone phosphorylation has been correlated with increased transcription and protein syntl?esis [10]. Phorbol was inactive in these systems and was thought not to be a promoter. However, recently it has been shown that phorbol is a promoter of chemicallyinduced lung and liver tumors in mice [2] and mammary tumors in rats [3]. Our results are consistent with the in vivo experiments showing phorbol to be a promoter. Also, preliminary experiments indicate that phenyldodecane, a p o t e n t promoter of tumors in mouse skin experiments [6], enhances the recovery of ouabain-resistant mutants in this system [8]. Finally, this data supports the role of mutagenesis as the initial event in carcinogenesis because promoting agents which enhance tumorigenesis in vivo also enhance mutagenesis in vitro. We recognize that mutagenesis may not be the only prerequisite for malignancy. Our results also demonstrate the potential of
156
this system as a means of determining the tumor-promoting ability of environmental agents. Little is known about the significance of promoters in the human environment. The system described above may prove valuable in assessing this question. Acknowledgement We wish to express our thanks to Dr. R. Niemeier, National Institutes of Occupational Safety and Health, Cincinnati, Ohio, for valuable discussions. References 1 A m e s , B.N., J. M c C a n n , E. C h o i a n d E. Y a m a s a k i , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e B a / m i c r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. U.S., 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 8 . 2 A r m u t h , V. a n d I. B e r e n b l u m , S y s t e m i c p r o m o t i n g a c t i o n o f p h o r b o l in liver a n d l u n g c a r c i n o g e n e s i s in A K R m i c e , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 2 2 5 9 - - 2 2 6 2 . 3 A r m u t h , V. a n d I. B e r e n b l u m , P r o m o t i o n of m a m m a r y c a r c i n o g e n e s i s a n d l e u k e m o g e n i c a c t i o n of p h o r b o l in virgin f e m a l e Wistar r a t s , C a n c e r R e s . , 3 4 ( 1 9 7 4 ) 2 7 0 4 - - 2 7 0 7 . 4 B a k e r , R . M . , D.M. B r u n e t t e , R . M a n k o v i t z , L . H . T h o m p s o n , G . F . W h i t m o r e , L. S i m i n o v i t c h a n d J . E . Till, O u a b a l n - r e s i s t a n t m u t a n t s o f m o u s e a n d h a m s t e r cells in c u l t u r e , Cell, 1 ( 1 9 7 4 ) 9 - - 2 1 . 5 B e r e n b l u m , I., T h e c o c a r c i n o g e n i c a c t i o n o f c r o t o n resin, C a n c e r R e s . , 1 ( 1 9 4 1 ) 4 4 - - 4 7 . 6 B i n g h a m , E., Tb-resholds in c a n c e r i n d u c t i o n , A r c h . E n v i r o n . H e a l t h , 2 2 ( 1 9 7 1 ) 6 9 2 - - 6 9 5 . 7 Boutwell, R.K., The function and mechanism of promoters of carcinogenesis, CRC, Critical Reviews in T o x i c o l o g y , 2 ( 1 9 7 4 ) 4 1 9 - - 4 4 3 . 8 L a n k a s , G . R . , R . T . C h r i s t i a n a n d C.S. B a x t e r , m a n u s c r i p t in p r e p a r a t i o n . 9 M o n d a l , S., D.W. B r a n k o w a n d C. H e i d e l b e r g e r , T w o - s t a g e c h e m i c a l o n c o g e n e s i s in c u l t u r e s o f C 3 H / 1 0 T 1 / 2 cells, C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 2 2 5 4 - - 2 2 7 0 . 1 0 O r d , M . G . a n d L . A . S t o c k e n , P h o s p h a t e a n d t h i o l g r o u p s o n h i s t o n e f3 f r o m r a t liver a n d t h y m u s n u c l e i , B i o c h e m . J., 1 0 2 ( 1 9 6 7 ) 6 3 1 - - 6 3 6 . 11 R a i n e r i , R . , R . C . S i m s i m a n a n d R . K . B o u t w e U , S t i m u l a t i o n o f t h e p h o s p h o r y l a t i o n o f m o u s e e p i d e r mal histones by tumor promoting agents, Cancer Res., 33 (1973) 134--139. 1 2 T r o s k o , J . E . , C. C h a n g , L.P. Y o t t i a n d E . Y . H . C h u , E f f e c t o f p h o r b o l m y r i s t a t e a c e t a t e o n t h e r e c o v ery of spontaneous and ultraviolet-light induced 6-thioguanine and ouabainoresistant Chinese hamster cells, C a n c e r R e s . , 3 7 ( 1 9 7 7 ) 1 8 8 - - 1 9 3 . 1 3 T r o s k o , J . E . , J . D . Y a g e r , G . T . B o w d e n a n d F . R . B u t c h e r , T h e e f f e c t s o f several c r o t o n oil c o n s t i t u e n t s o n t w o t y p e s o f D N A r e p a i r a n d c y c l i c n u c l e o t i d e levels in m a m m a l i a n cells in v i t r o , C h e m . - B i o l . I n t e r a c t i o n s , 11 ( 1 9 7 5 ) 1 9 1 - - 2 0 5 . 1 4 Wigler, M. a n d I. W e i n s t e i n , T u m o r p r o m o t e r i n d u c e s p l a s m i n o g e n a c t i v a t o r , N a t u r e , 2 5 9 ( 1 9 7 6 ) 232--233.
Mutation Research, 45 (1977) 153--156
© Elsevier/North-Holland Biomedical Press
E F F E C T OF TUMOR PROMOTING AGENTS ON MUTATION F R E Q U E N C I E S IN C U L T U R E D V79 CHINESE HAMSTER CELLS *
GEORGE R. LANKAS Jr., C. STUART BAXTER and ROBERT T, CHRISTIAN Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267 (U.S.A.)
(Received 1 March, 1977) (Revision received 31 May, 1977) (Accepted 8 June, 1977)
The two-stage model of carcinogenesis, first demonstrated in skin by Berenblum [5], has n o w been extended to other organs [2,3]. In this model initiation, which is conceived as an irreversible change in DNA, is followed by promotion, which enhances the expression of the transformed phenotype. All carcinogens are considered to be initiators [7] and most are also mutagenic [1]. The observed promotion of carcinogenesis in other organs and transformation in vitro [9] suggests that a two-stage process may be fundamental to carcinogenesis and is n o t a unique property of skin, as many have claimed. If initiation is a mutagenic change then it seems reasonable to deduce that known promoting agents, such as phorbol and TPA will enhance the chemically induced mutation frequency in Chinese hamster cells in culture. Recently, Trosko et al. [12] have demonstrated that the ultra-violet light-induced mutation frequency in Chinese hamster cells is enhanced by these promoters. Here we report evidence that the frequency of chemically-induced ouabain-resistant mutations is increased by tumor-promoters. Chinese hamster V79 cells (kindly provided by Dr. R. Hart, Ohio State University) were grown in 100 mm plastic petri dishes in a modified minimum essential medium (Eagle's) supplemented with 1.5X vitamins and amino acids and 10% fetal calf serum. Cells were seeded at a density of 4 × l 0 s cells/plate in 10 ml medium and 3 h allowed for cell attachment. Plates were then dosed with either MAM dissolved in medium without serum or MNNG dissolved in acetone. MAM exposure was for 48 h and MNNG exposure for 2 h, at which time medium was poured from the plates and replaced with fresh medium. TPA and phorbol were dissolved in acetone at equimolar concentrations (1 mg/ml * This w o r k w a s s u p p o r t e d b y N I H g r a n t s ES 0 0 1 5 9 , ES 0 0 1 2 7 , a n d 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 i e s Institute of Toxicology.
Abbreviations: T P A , 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e , MAM, m e t h y l a z o x y m e t h a n o l M N N G , N-methyl-N'-nitro-N-nitrosoguanidine.
a c e t a t e , and
154 TPA, 0.59 mg/ml phorbol). Forty-two hours after dosing with the mutagen, 10 gl of the TPA or phorbol solutions were added to the appropriate plates. After an additional 6 h, medium was poured off all plates and replaced with medium containing 1 mM ouabain, and promoter where appropriate. The promoter was thus present throughout the selection period. After 10 days incubation, the plates were then fixed, stained and scored for ouabain-resistant mutants. Concurrently, toxicity assays were done to determine cell survival after the various treatments. Cells were seeded at a density of 2 × 102/plate in 60 mm dishes and treated as above, except that medium did not contain ouabain. Plates were fixed and stained after one week and the number of colonies determined. Preliminary experiments indicated that addition of phorbol compounds at 42 h after treatment, and continued exposure during selection with ouabain, gave the highest recovery of mutants. A non-toxic dose of both phorbol and TPA was used, since this concentration has been shown not to inhibit DNA repair synthesis in Chinese hamster cells [13]. Therefore, inhibition of repair does n o t appear to be the mechanism of action of phorbol compounds. Results shown in Table 1 indicate that both TPA and phorbol increase the recovery of ouabain-resistant mutants at all 3 doses of MNNG tested w i t h o u t enhancing the cytotoxic effects of the agent. Phorbol was n o t as effective in increasing the recovery of mutants as was TPA at equimolar concentrations. Table 2 shows the results of a similar experiment with MAM as the mutagen. Again, phorbol and TPA increased the recovery of ouabain-resistant mutants w i t h o u t enhancing the cytotoxicity of the agent. Furthermore, the effect of TPA was greatest at the highest mutagen dose. Phorbol was again not as effective as TPA in enhancing m u t a n t recovery. Preliminary results (Lankas, unpublished data) indicate that TPA can enhance m u t a n t recovery 4 weeks after treatment with MAM, though to a somewhat lesser extent compared to treatment 42 h after addition of the muta-
TABLE
1
EFFECT
OF PHORBOL
AND TPA ON MNNG-INDUCED
OUABAIN-RESISTANT
Treatment
No. of plates
% survival
Control Acetone (0.2%) T P A (1 p g / m l ) A c e t o n e + T P A (1 I J g / m l ) Acetone + phorbol (0.59 Hg/ml) MNNG (pg/ml) 0.5 0.5 + TPA 1.0 1.0 + TPA 1.5 1.5 + phorbol 1.5 + TPA
10 10 10 5 5
85 87 78 83 85
20 25 19 5 0
10 8 9 8 9 5 9
70 72 56 54 46 44 42
81 125 138 158 279 168 349
a p values compare b p < 0.05.
(mutagen
and promoter)
to (mutagen
No. of mutants
only) treatment.
MUTATIONS Mutants/ 106 survivors a 5.8 7.2 6.1 2.9 0 28.9 54.2 b 68.3 91.7 168.5 190.9 230.4 b
155 TABLE 2 EFFECT OF PHORBOL AND TPA ON MAM-INDUCED OUABAIN-RESISTANT
Treatment
Control T P A (1 p g / m l ) Phorbol (0.59 pg/ml) MAM (pg/ml) 12 12 + T P A 18 18 + T P A 24 24 + p h o r b o l 24 + T P A a p bP c P d p
No. of plates
% survival
N o . of mutants
MUTATIONS Mutants/ 10 6 s u r v i v o r s a
6 6 6
96 89 95
4 7 8
1.7 3.2 3.5
10 9 8 8 10 10 10
62 65 15 15 7 7 7
57 67 64 90 73 109 123
22.9 28.6 133.3 187.5 260.7 390.0 439.3
v a l u e s c o m p a x e ( m u t a g e n and p r o m o t e r ) t o ( m u t a g e n o n l y ) t r e a t m e n t . < 0.10. < 0.05. < 0.02.
gen. Thus, effects of TPA on expression time cannot solely explain its enhancement of mutagenesis. Studies are currently being performed to determine the mechanism of p r o m o t e r enhancement of mutagenesis. Since TPA or phorbol are not mutagenic per se, either in our system or in the 6-thioguanine mutation system [13], b u t are able to increase the induced mutation frequency by two chemical mutagens, we interpret these results as indicating that phorbol c o m p o u n d s can modify gene expression. In the ouabain mutation system, changes in membrane b o u n d Na÷/K + ATPase sensitivity to ouabain are determined [4]. If a cell has an altered but repressed gene, it will behave as a ouabain-sensitive cell and be eliminated from the population under conditions selecting for ouabain-resistant mutants. Apparently, both TPA and phorbol can convert these "latent" mutants into "expressed" mutants, allowing their recovery in the ouabain medium. Our conclusions are supported by several other reports. It has been shown that TPA can induce expression of normally repressed proteins in several cell lines [14] and in mouse skin [7]. It has also been shown that TPA increases the phosphorylation of histones in mouse skin [11]. Histone phosphorylation has been correlated with increased transcription and protein syntl?esis [10]. Phorbol was inactive in these systems and was thought not to be a promoter. However, recently it has been shown that phorbol is a promoter of chemicallyinduced lung and liver tumors in mice [2] and mammary tumors in rats [3]. Our results are consistent with the in vivo experiments showing phorbol to be a promoter. Also, preliminary experiments indicate that phenyldodecane, a p o t e n t promoter of tumors in mouse skin experiments [6], enhances the recovery of ouabain-resistant mutants in this system [8]. Finally, this data supports the role of mutagenesis as the initial event in carcinogenesis because promoting agents which enhance tumorigenesis in vivo also enhance mutagenesis in vitro. We recognize that mutagenesis may not be the only prerequisite for malignancy. Our results also demonstrate the potential of
156
this system as a means of determining the tumor-promoting ability of environmental agents. Little is known about the significance of promoters in the human environment. The system described above may prove valuable in assessing this question. Acknowledgement We wish to express our thanks to Dr. R. Niemeier, National Institutes of Occupational Safety and Health, Cincinnati, Ohio, for valuable discussions. References 1 A m e s , B.N., J. M c C a n n , E. C h o i a n d E. Y a m a s a k i , D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in t h e S a l m o n e B a / m i c r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. U.S., 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 8 . 2 A r m u t h , V. a n d I. B e r e n b l u m , S y s t e m i c p r o m o t i n g a c t i o n o f p h o r b o l in liver a n d l u n g c a r c i n o g e n e s i s in A K R m i c e , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 2 2 5 9 - - 2 2 6 2 . 3 A r m u t h , V. a n d I. B e r e n b l u m , P r o m o t i o n of m a m m a r y c a r c i n o g e n e s i s a n d l e u k e m o g e n i c a c t i o n of p h o r b o l in virgin f e m a l e Wistar r a t s , C a n c e r R e s . , 3 4 ( 1 9 7 4 ) 2 7 0 4 - - 2 7 0 7 . 4 B a k e r , R . M . , D.M. B r u n e t t e , R . M a n k o v i t z , L . H . T h o m p s o n , G . F . W h i t m o r e , L. S i m i n o v i t c h a n d J . E . Till, O u a b a l n - r e s i s t a n t m u t a n t s o f m o u s e a n d h a m s t e r cells in c u l t u r e , Cell, 1 ( 1 9 7 4 ) 9 - - 2 1 . 5 B e r e n b l u m , I., T h e c o c a r c i n o g e n i c a c t i o n o f c r o t o n resin, C a n c e r R e s . , 1 ( 1 9 4 1 ) 4 4 - - 4 7 . 6 B i n g h a m , E., Tb-resholds in c a n c e r i n d u c t i o n , A r c h . E n v i r o n . H e a l t h , 2 2 ( 1 9 7 1 ) 6 9 2 - - 6 9 5 . 7 Boutwell, R.K., The function and mechanism of promoters of carcinogenesis, CRC, Critical Reviews in T o x i c o l o g y , 2 ( 1 9 7 4 ) 4 1 9 - - 4 4 3 . 8 L a n k a s , G . R . , R . T . C h r i s t i a n a n d C.S. B a x t e r , m a n u s c r i p t in p r e p a r a t i o n . 9 M o n d a l , S., D.W. B r a n k o w a n d C. H e i d e l b e r g e r , T w o - s t a g e c h e m i c a l o n c o g e n e s i s in c u l t u r e s o f C 3 H / 1 0 T 1 / 2 cells, C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 2 2 5 4 - - 2 2 7 0 . 1 0 O r d , M . G . a n d L . A . S t o c k e n , P h o s p h a t e a n d t h i o l g r o u p s o n h i s t o n e f3 f r o m r a t liver a n d t h y m u s n u c l e i , B i o c h e m . J., 1 0 2 ( 1 9 6 7 ) 6 3 1 - - 6 3 6 . 11 R a i n e r i , R . , R . C . S i m s i m a n a n d R . K . B o u t w e U , S t i m u l a t i o n o f t h e p h o s p h o r y l a t i o n o f m o u s e e p i d e r mal histones by tumor promoting agents, Cancer Res., 33 (1973) 134--139. 1 2 T r o s k o , J . E . , C. C h a n g , L.P. Y o t t i a n d E . Y . H . C h u , E f f e c t o f p h o r b o l m y r i s t a t e a c e t a t e o n t h e r e c o v ery of spontaneous and ultraviolet-light induced 6-thioguanine and ouabainoresistant Chinese hamster cells, C a n c e r R e s . , 3 7 ( 1 9 7 7 ) 1 8 8 - - 1 9 3 . 1 3 T r o s k o , J . E . , J . D . Y a g e r , G . T . B o w d e n a n d F . R . B u t c h e r , T h e e f f e c t s o f several c r o t o n oil c o n s t i t u e n t s o n t w o t y p e s o f D N A r e p a i r a n d c y c l i c n u c l e o t i d e levels in m a m m a l i a n cells in v i t r o , C h e m . - B i o l . I n t e r a c t i o n s , 11 ( 1 9 7 5 ) 1 9 1 - - 2 0 5 . 1 4 Wigler, M. a n d I. W e i n s t e i n , T u m o r p r o m o t e r i n d u c e s p l a s m i n o g e n a c t i v a t o r , N a t u r e , 2 5 9 ( 1 9 7 6 ) 232--233.
