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3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells
Mutation Research, 143 (1985) 113-115 Elsevier
113
MRLett. 0715
3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells Robert B. Painter Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (US.A.) (Accepted 25 March 1985)
Summary 3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADPribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.
Fibroblasts from patients with the autosomal recessive genetic disease, ataxia-telangiectasia (AT), are uniformly hypersensitive to ionizing radiation (Paterson and Smith, 1979) and to chemical agents that directly produce DNA-strand breaks (Shiloh et al., 1983). These cells also exhibit radioresistant DNA synthesis, i.e., their rate of DNA synthesis is inhibited to a lesser degree than that of normal cells after treatment with ionizing radiation (Painter and Young, 1980; DeWit et al., 1981; Painter, 1981) or radiomimetic chemicals (Cramer and Painter, 1981; Shiloh et al., 1983). Because poly(ADP-ribose) synthesis is stimulated by strand breaks (Benjamin and Gill, 1980) and appears to be sluggish in irradiated A-T lymphoblasts (Edwards and Taylor, 1980), it has been suggested that A-T cells fail to inhibit DNA synthesis because they synthesize poly(ADP-ribose) more slowly after irradiation than do normal cells (Edwards and Taylor, 1980). However, this concept was challenged by Zwelling et al. (1983), who reported that synthesis of poly(ADP-ribose) is not reduced in irradiated A-T fibroblasts.
Poly(ADP-ribose) is inhibited by a number of chemicals; among them, 3-aminobenzamide (3AB) is one of the most powerful (Purnell and Whish, 1980). I report here that 3AB fails completely to affect the inhibition of DNA synthesis by Xradiation in normal human fibroblasts, and infer therefore that poly(ADP-ribose) synthesis has no role in the radioresistant DNA synthesis observed in A-T cells. Materials and methods A-T fibroblasts BMA, KIM, and BRO were gifts of Dr. Richard Gatti, Department of Pathology, UCLA School of Medicine. HS-27 is a human fibroblast cell line of normal origin, supplied by the Cell Culture Facility, University of California, San Francisco. All cells were grown in Eagle's minimal essential medium supplemented with 15070 fetal bovine serum. A-T and normal cells were inoculated into 4 35-mm petri dishes at about 5 x 10 cells per dish. After 1 day of growth, medium containing 0.01
0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
114
{tCi/ml [t4C]thymidine (spec. act. 50 mCi/mmole) was added to all cultures and incubation was continued for 24 h. For dose-response experiments, the medium was removed and two dishes containing A-T cells and two containing HS-27 cells were simultaneously exposed to 0-20 Gy of X-rays (250 kVp max, about 4 Gy/min). Immediately after irradiation, nonradioactive medium was placed in all dishes and the cells were returned to the incubator for 30 min. This medium was then removed and all cultures were incubated with medium containing 20 {tCi/ml [3H]thymidine (50 Ci/mmole) for 10 min. This medium was then rapidly removed and the cells were washed with ice-cold SSC (0.15 M sodium chloride, 0.015 M sodium citrate) and scraped into SSC; ice-cold 4lJ1o perchloric acid was added and the cell suspension was filtered through GF/C (Whatman) filters. The filters were rinsed sequentially with 4lJ1o perchloric acid, 70lJlo alcohol and 100lJlo alcohol, dried, and assayed for radioactivity in a liquid scintillation spectrometer. The resulting 3H/ t4C ratios were measures of the specific activity of DNA and therefore of the rate of DNA synthesis. In experiments with 3AB, at 1 h before irradiation the medium on all cells was changed to one containing either 3AB (5 or 10 mM) or no 3AB, and incubation continued for 30 min after irradiation.
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40
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30
15
20
Fig. I. Effect of X-radiation on the rate of DNA synthesis in normal HS-27 fibroblasts (~) and in A-T fibroblasts BMA (l>.), KIM (e). and BRO (0). Error bars represent the range of duplicate samples.
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Radioresistant DNA synthesis was observed in all three A-T fibroblast strains (Fig. 1), in accordance with previous reports (Painter and Young, 1980; DeWit et al., 1981; Painter, 1981), but not in HS-27 cells. When HS-27 cells were incubated with 5 mM 3AB for 1 h before irradiation and 30 min after irradiation, there was no significant effect on the inhibition of DNA synthesis induced by Xradiation (Fig. 2). 3AB also had no significant effect on DNA synthesis in unirradiated cells. At 10 mM 3AB, DNA synthesis in normal cells was inhibited by 1O-50lJlo, but in no case was there a change in the dose-response curve for radiationinduced inhibition. Similarly, 3AB had no effect
10 Gy
Vl
Results and discussion
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Fig. 2. Effect of 5 mM 3AB on X-radiation-induced inhibition of DNA synthesis in HS-27 fibroblasts. Cells not incubated with 3AB (e); cells incubated with 5 mM 3AB (0). The dashed line indicates the response expected if inhibition of poly(ADP· ribose) caused normal cells to respond as A-T cells do. Error bars represent the range of duplicate samples.
115
on the Xvradiarion-induced inhibition of DNA synthesis in A- T cells (data not shown). Although 3AB is one o f th e mo st po werful inhibitors o f poly(ADP-ribose) synt hesis, it ha s been show n to inhibit other metabolic processes as well (Milan and Clea ver, 1984). Therefore, if 3AB is found to affect some biological endpoint , one cannot un equivocally state that thi s result is caused by inhi bitio n of poly(ADP-ribo se) synt hesis. However , if 3AB fails to affect a biological endpoint, and 3AB is known to inhibit poly(ADP-ribose) synthesis in the system under study, then one can state cat egorically that inh ibition of poly(ADP-ribose) synthesis plays no role in the biological effect; th is is th e case here Jacobson et al. (1983) have sho wn th at concent rations of ;:::: 3 mM 3AB completely sup press t he synt hes is of pol y(ADP-ribose) fro m its precursor, NAD, in human fib ro blas ts. Ther efore , th e failure of 5 and lO mM 3AB to a ffec t t he radiationinduced inhibition of DN A synthesis in normal human fibrobl asts, let alone establi sh an A-T-like radi oresistant DNA synthesis , shows that in hibited pol y(ADP-ribose) synthesis is not so lely resp on sible for th e radioresistant DNA synt hesis obse rved in A-T cells. Even though th ere is strong evidence that an intermediary factor act s as a signal in normal cells to blo ck chain elongation - because the sa me damage that causes a blo ck to chai n elongation in normal human cells does not do so in A- T cell s (Painter, 1983; Ockey, 1983) - the results reported here indicate that poly(ADP-ribose) is not that fa ctor. Acknowledgements Thi s wo rk was supported b y th e U .S . Department of Energy (DE-AC03-76-SFOlOI2) .
References Benjam in, R.C. , and D.M . Gill (1980) Poly(ADP-ribose) synthe sis in vitro program med by damaged DNA . A comparison of DNA molecules containing different type s of strand breaks, J . BioI. C hern., 255, 10502- 10508. Cramer, 1'., a nd R.B . Paint er (1981) Bleom ycin-resista nt DNA synt hesis in ataxia tela ngiectasia cells, Nature (Lo ndon) , 291, 67 1-672. DeWit, 1., N.G .J . Jaspers and D. Bootsma (1981) T he rate o f DNA synthesis in normal h uman and ataxia telang iectasia cells afte r expos ure to X-ir rad ia tion , Mu tat ion Res., 80, 221-2 26. Ed war ds, M .J ., and A.M.R. Tayl or (1980) Unusual levels of (A DP-ribose)n and DNA synthesi s in ataxia telangiectasia cells following -y-ray irradiation, Natu re (Lo ndon), 287, 745- 747. Jacobson, E .L., K.M. Antol , H. J uare z-Salinas and M.K. Jacobso n (1983) P oly(ADP -ribose) metab olism in ultraviolet irradiated huma n fibro blasts , J . BioI. Chern. , 258, 103-107 . Milam, K.M ., and J. E . Cleaver (1984) Inh ibitors of poIy(adenosine dip hosp ha te-r ibose) synt hesis: effe ct on ot her metabolic processes, Science, 223, 589-591. Ockey, C. H . (1983) Differences in replico n behavior between X-irr adiatio n-sensitive L5178Y mo use lymph oma cells and A-T fibrob lasts using DNA fibe r autorad iography, Rad ial. Res., 94, 427-438. Pa inter , R.B. (1981) Radi or esistant DNA synt hesis: an intrinsic featu re of ataxia telan giecta sia, Mutatio n Res., 84, 183-1 90. Painter, R.B. (1983) Are lesions induced by ionizing radia tion direct blocks to DNA cha in elongation" , Rad ial. Res., 95, 42 1-426. Painter , R.B., an d B.R . Young (1980) Radio sensitivity in ataxia telangi ectas ia: A new explanat ion, Proc. Nat l. Acad. Sci. (U.S.A.) , 77, 7315- 7317. Paterson , M.C., and P.J. Smith (1979) At axia telang iectasia: an inh erited human disorder invol ving hyp ersensitivity to ion izing rad iation and related DN A-d am aging chemicals, Ann u. Rev. Genet. , 13, 291-31 8. Pu rnell, M.R., an d W.J.D . Whish (1980) Novel inhibito rs of po ly(ADP -ribose) synthetase, Biochem . J ., 185,775-777. Shiloh , Y., E. Ta bo r a nd Y. Becker (1983) A bn ormal respo nse of ataxia-tela ngiectasia cells 10 agent s tha t break the deox yribose moiety of DNA via a targeted free rad ical mechani sm, Ca rcinog enesis, 4, 1317- 1322. Zwelling , L.A ., D. Ker rigan a nd M. R. Mattern (1983) Ataxiatelang iecta sia cells are not uniformly deficient in poly(ADPribose) synthesis follo wing X-irr adiation, Mutation Res. , 120, 69- 78.
113
MRLett. 0715
3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells Robert B. Painter Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (US.A.) (Accepted 25 March 1985)
Summary 3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADPribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.
Fibroblasts from patients with the autosomal recessive genetic disease, ataxia-telangiectasia (AT), are uniformly hypersensitive to ionizing radiation (Paterson and Smith, 1979) and to chemical agents that directly produce DNA-strand breaks (Shiloh et al., 1983). These cells also exhibit radioresistant DNA synthesis, i.e., their rate of DNA synthesis is inhibited to a lesser degree than that of normal cells after treatment with ionizing radiation (Painter and Young, 1980; DeWit et al., 1981; Painter, 1981) or radiomimetic chemicals (Cramer and Painter, 1981; Shiloh et al., 1983). Because poly(ADP-ribose) synthesis is stimulated by strand breaks (Benjamin and Gill, 1980) and appears to be sluggish in irradiated A-T lymphoblasts (Edwards and Taylor, 1980), it has been suggested that A-T cells fail to inhibit DNA synthesis because they synthesize poly(ADP-ribose) more slowly after irradiation than do normal cells (Edwards and Taylor, 1980). However, this concept was challenged by Zwelling et al. (1983), who reported that synthesis of poly(ADP-ribose) is not reduced in irradiated A-T fibroblasts.
Poly(ADP-ribose) is inhibited by a number of chemicals; among them, 3-aminobenzamide (3AB) is one of the most powerful (Purnell and Whish, 1980). I report here that 3AB fails completely to affect the inhibition of DNA synthesis by Xradiation in normal human fibroblasts, and infer therefore that poly(ADP-ribose) synthesis has no role in the radioresistant DNA synthesis observed in A-T cells. Materials and methods A-T fibroblasts BMA, KIM, and BRO were gifts of Dr. Richard Gatti, Department of Pathology, UCLA School of Medicine. HS-27 is a human fibroblast cell line of normal origin, supplied by the Cell Culture Facility, University of California, San Francisco. All cells were grown in Eagle's minimal essential medium supplemented with 15070 fetal bovine serum. A-T and normal cells were inoculated into 4 35-mm petri dishes at about 5 x 10 cells per dish. After 1 day of growth, medium containing 0.01
0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
114
{tCi/ml [t4C]thymidine (spec. act. 50 mCi/mmole) was added to all cultures and incubation was continued for 24 h. For dose-response experiments, the medium was removed and two dishes containing A-T cells and two containing HS-27 cells were simultaneously exposed to 0-20 Gy of X-rays (250 kVp max, about 4 Gy/min). Immediately after irradiation, nonradioactive medium was placed in all dishes and the cells were returned to the incubator for 30 min. This medium was then removed and all cultures were incubated with medium containing 20 {tCi/ml [3H]thymidine (50 Ci/mmole) for 10 min. This medium was then rapidly removed and the cells were washed with ice-cold SSC (0.15 M sodium chloride, 0.015 M sodium citrate) and scraped into SSC; ice-cold 4lJ1o perchloric acid was added and the cell suspension was filtered through GF/C (Whatman) filters. The filters were rinsed sequentially with 4lJ1o perchloric acid, 70lJlo alcohol and 100lJlo alcohol, dried, and assayed for radioactivity in a liquid scintillation spectrometer. The resulting 3H/ t4C ratios were measures of the specific activity of DNA and therefore of the rate of DNA synthesis. In experiments with 3AB, at 1 h before irradiation the medium on all cells was changed to one containing either 3AB (5 or 10 mM) or no 3AB, and incubation continued for 30 min after irradiation.
w
100
I-
-c
'"
Vl
iii w
80
I-
z
70
-c z c
60
'" Z
50
J:
>Vl ..... 0
I-
0
U
u.
0
40
0~
30
15
20
Fig. I. Effect of X-radiation on the rate of DNA synthesis in normal HS-27 fibroblasts (~) and in A-T fibroblasts BMA (l>.), KIM (e). and BRO (0). Error bars represent the range of duplicate samples.
w
................
I-
-c
""" .......... ....................
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Vl
iii w J:
I-
Z
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70
-cz
60
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.....
Radioresistant DNA synthesis was observed in all three A-T fibroblast strains (Fig. 1), in accordance with previous reports (Painter and Young, 1980; DeWit et al., 1981; Painter, 1981), but not in HS-27 cells. When HS-27 cells were incubated with 5 mM 3AB for 1 h before irradiation and 30 min after irradiation, there was no significant effect on the inhibition of DNA synthesis induced by Xradiation (Fig. 2). 3AB also had no significant effect on DNA synthesis in unirradiated cells. At 10 mM 3AB, DNA synthesis in normal cells was inhibited by 1O-50lJlo, but in no case was there a change in the dose-response curve for radiationinduced inhibition. Similarly, 3AB had no effect
10 Gy
Vl
Results and discussion
5
0
'" Z I-
50
•~::::::::!
1~!
0
U
u.
0
0~
is
40
30
""" .........................
5
10
15
20
Gy
Fig. 2. Effect of 5 mM 3AB on X-radiation-induced inhibition of DNA synthesis in HS-27 fibroblasts. Cells not incubated with 3AB (e); cells incubated with 5 mM 3AB (0). The dashed line indicates the response expected if inhibition of poly(ADP· ribose) caused normal cells to respond as A-T cells do. Error bars represent the range of duplicate samples.
115
on the Xvradiarion-induced inhibition of DNA synthesis in A- T cells (data not shown). Although 3AB is one o f th e mo st po werful inhibitors o f poly(ADP-ribose) synt hesis, it ha s been show n to inhibit other metabolic processes as well (Milan and Clea ver, 1984). Therefore, if 3AB is found to affect some biological endpoint , one cannot un equivocally state that thi s result is caused by inhi bitio n of poly(ADP-ribo se) synt hesis. However , if 3AB fails to affect a biological endpoint, and 3AB is known to inhibit poly(ADP-ribose) synthesis in the system under study, then one can state cat egorically that inh ibition of poly(ADP-ribose) synthesis plays no role in the biological effect; th is is th e case here Jacobson et al. (1983) have sho wn th at concent rations of ;:::: 3 mM 3AB completely sup press t he synt hes is of pol y(ADP-ribose) fro m its precursor, NAD, in human fib ro blas ts. Ther efore , th e failure of 5 and lO mM 3AB to a ffec t t he radiationinduced inhibition of DN A synthesis in normal human fibrobl asts, let alone establi sh an A-T-like radi oresistant DNA synthesis , shows that in hibited pol y(ADP-ribose) synthesis is not so lely resp on sible for th e radioresistant DNA synt hesis obse rved in A-T cells. Even though th ere is strong evidence that an intermediary factor act s as a signal in normal cells to blo ck chain elongation - because the sa me damage that causes a blo ck to chai n elongation in normal human cells does not do so in A- T cell s (Painter, 1983; Ockey, 1983) - the results reported here indicate that poly(ADP-ribose) is not that fa ctor. Acknowledgements Thi s wo rk was supported b y th e U .S . Department of Energy (DE-AC03-76-SFOlOI2) .
References Benjam in, R.C. , and D.M . Gill (1980) Poly(ADP-ribose) synthe sis in vitro program med by damaged DNA . A comparison of DNA molecules containing different type s of strand breaks, J . BioI. C hern., 255, 10502- 10508. Cramer, 1'., a nd R.B . Paint er (1981) Bleom ycin-resista nt DNA synt hesis in ataxia tela ngiectasia cells, Nature (Lo ndon) , 291, 67 1-672. DeWit, 1., N.G .J . Jaspers and D. Bootsma (1981) T he rate o f DNA synthesis in normal h uman and ataxia telang iectasia cells afte r expos ure to X-ir rad ia tion , Mu tat ion Res., 80, 221-2 26. Ed war ds, M .J ., and A.M.R. Tayl or (1980) Unusual levels of (A DP-ribose)n and DNA synthesi s in ataxia telangiectasia cells following -y-ray irradiation, Natu re (Lo ndon), 287, 745- 747. Jacobson, E .L., K.M. Antol , H. J uare z-Salinas and M.K. Jacobso n (1983) P oly(ADP -ribose) metab olism in ultraviolet irradiated huma n fibro blasts , J . BioI. Chern. , 258, 103-107 . Milam, K.M ., and J. E . Cleaver (1984) Inh ibitors of poIy(adenosine dip hosp ha te-r ibose) synt hesis: effe ct on ot her metabolic processes, Science, 223, 589-591. Ockey, C. H . (1983) Differences in replico n behavior between X-irr adiatio n-sensitive L5178Y mo use lymph oma cells and A-T fibrob lasts using DNA fibe r autorad iography, Rad ial. Res., 94, 427-438. Pa inter , R.B. (1981) Radi or esistant DNA synt hesis: an intrinsic featu re of ataxia telan giecta sia, Mutatio n Res., 84, 183-1 90. Painter, R.B. (1983) Are lesions induced by ionizing radia tion direct blocks to DNA cha in elongation" , Rad ial. Res., 95, 42 1-426. Painter , R.B., an d B.R . Young (1980) Radio sensitivity in ataxia telangi ectas ia: A new explanat ion, Proc. Nat l. Acad. Sci. (U.S.A.) , 77, 7315- 7317. Paterson , M.C., and P.J. Smith (1979) At axia telang iectasia: an inh erited human disorder invol ving hyp ersensitivity to ion izing rad iation and related DN A-d am aging chemicals, Ann u. Rev. Genet. , 13, 291-31 8. Pu rnell, M.R., an d W.J.D . Whish (1980) Novel inhibito rs of po ly(ADP -ribose) synthetase, Biochem . J ., 185,775-777. Shiloh , Y., E. Ta bo r a nd Y. Becker (1983) A bn ormal respo nse of ataxia-tela ngiectasia cells 10 agent s tha t break the deox yribose moiety of DNA via a targeted free rad ical mechani sm, Ca rcinog enesis, 4, 1317- 1322. Zwelling , L.A ., D. Ker rigan a nd M. R. Mattern (1983) Ataxiatelang iecta sia cells are not uniformly deficient in poly(ADPribose) synthesis follo wing X-irr adiation, Mutation Res. , 120, 69- 78.