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Actinomycin D: Inhibition of chromosomal repair in human lymphocytes
126
Mutation Research Elsevier Publishing Company, A m s t e r d a m Printed in The Netherlands
Actinomycin D: Inhibition of chromosomal repair in human lymphocytes Actinomycin D is described as an inhibitor of DNA-dependent RNA synthesis and subsequent protein synthesis15,1". More recently other effects on biological systems have been reportedS, TM.The effect of actinomycin D has been attributed to its complex formation with the guanine bases in nucleic acid which would additionally create a block to the synthesis of DNA~," (when the complexed G-base is in DNA) and also the inhibition of RNA replication (when the complexed G-base is in RNA) in poliovirusq influenza virus I and RNA bacteriophage MS 21°. One of the more interesting consequences of actinomycin D treatments is the enhanced response of biological systems to subsequent treatments with other agents especially X-ray. The simplest explanation of this observation is that the effect of actinomycin D on X-ray responses is the result of its ability to inhibit repair of X-ray induced damage either directly or indirectly. In a previous study actinomycin D was placed in the culture medium between 2 doses of X-ray for various periods of time. The measurement of cell survival indicated that there was little repair between the doses 6. In fact what was observed was a diminution of the increase in survival normally seen between doses of X-ray. As the time between doses was increased (the time of actinomycin D exposure necessarily increased) the increase in survival decreased. At the higher concentration of actinomycin D, survival decreased even in the absence ot X-ray. These effects have led to the belief that actinomycin D may exert its influence by mechanisms other than repair inhibition 7. The mechanism for the effects of actinomycin D on "transformed" or neoplastic cells such as HeLa or the ubiquitous chinese hamster cell lines used so frequently remains questionable. The theory that actinomycin D can or cannot inhibit repair and enhance the response of cells to the effects of X-ray treatment, on the other hand, can be tested. This report concerns the results of those tests which indicate that at concentrations which do not induce chromosomal aberrations, actinomycin D inhibits the repair of chromatid strands broken by X-ray. Human lymphocytes were obtained from the peripheral blood of two normal male individuals and cultured using a modification of the method first reported by MOORHEAD et al. 11. Treatments were initiated at the time when most of the cells were in S stage 3,8 (48 h after incubation). Actinomycin D was added to the culture medium for the duration of the interval between two equal fractions of X-irradiation and that interval was made equal to the known repair time of chromosomal breaks in the S stage 14. The optimal concentration of actinomycin D was o.o5 ¢/g/ml for 1. 5 h. When the concentration was equal to or in excess of o. 5/~g/ml for the 1.5-h treatment time, there was a gross inhibition of mitosis. The results of the actinomycin D treatments over the entire range of concentrations are given in Table I. Both effects on mitotic index and chromosomal aberrations were observed. As shown (Table I) the aberration frequency in the treated cells over the entire range was no different than the control rate. All aberrations were single chromatid gaps. We conclude that at the concentrations used, and the treatment durations, actinomycin D does not induce chromosome breaks which can be seen in the first post~Vlutation Res., 7 (1969) 126--128
SHORT COMMUNICATIONS TABLE
127
I
THE EFFECT OF VARYING CONCENTRATIONS OF ACTINOMYCIN D ON MITOTIC ACTIVITY AND CHROMOSOME ABERRATION RATES IN HUMAN LYMPHOCYTIgS
Final concentration (izg/ml in L 5 h)
Number of metaphases scored
E~ect on mitosis (mitotic index)
% Chromatid aberration Gaps Deletion Exchange
5.o o. 5
None None
0.05 O.OI None
IOO 5° 2oo
No mitotic figure Mitotic inhibition was extreme M.I. less t h a n o . 1 % M.1. 4,o °/o M . I . 5.00,0 M.I. 4.5%
IO I2 9
N.B.M.I. TABLE
o o o
o o o
= Mitotic index. II
THE EFFECT OF ACTINOMYCIN D (0.05
X - R a y treatment 15o R 15o R - - - a c t i n o m y c i n D f o r 90 m i n 300 1R 15o R - - t i m e i n t e r v a l 60 m i n - - i 5 o R 15o R - - t i m e i n t e r v a l 9 ° m i n - - i 5 o R 15o R - - t i m e i n t e r v a l 90 r a i n w i t h actinomycin D--iSo R
itg/ml, 1,5 h) ON CHROMOSOMAL REPAIR TIME Cells scored
Observed
°/o Chromatid exchange ( i f additive)
Calculated for interaction
200 ioo 25o 200 163
8 7 3I 26 15
16 16
32 32
ioo
29
16
32
treatment division. To test the effect of actinomycin D on the ability of ceils to repair chromosomal breaks, X-ray treatments were divided into 2 equal fractions with a time interval between the two doses during which repair could take place. The known interval between dosesi3, I4 which is equal to or exceeds the repair time for chromosomal breaks in the S stage in human lymphocytes is 9 ° min. The chromatid exchange frequency was used as an assay for determination of repair. A full discussion of this technique has appeared previously13,IL The results of this experiment are listed in Table II. All exchange values represent the peak frequency response after treatment. These peaks occurred at the same time in both the actinomycin D treated and the untreated populations. Mitotic delay was, therefore, not considered a factor. Further evidence that delay was not a factor comes from concurrent labelling (E3HJthymidine) tests. The time of appearance of the labelled S cells were also found to be the same in treated and untreated cells. It should be clear from the data (Table II) that the repair time of chromatid breaks is longer than 6o min and equal to or less than 9 ° min after I5o R since the observed rate differs from the calculated interaction figure and the rate observed after a single 3oo-R dose, only after a 9o-min interval between fractions. At that time (9° min) the observed rate is approximately what is expected if the two treatments were additive and repair of the chromosomal breaks induced by the first dose had occurred. But when the 9o-min interval includes the presence of actinomycin D (o.o5 #g/ml following the first dose) in the culture medium the yield of chromatid exchanges was 290/o. This figure is approximately the same as that calculated for interaction and the frequency observed after a single 3oo-R dose (no interval). It clearly indicates Mutation Res., 7 (1969) I 2 6 - I 2 S
128
SHORT COMMUNICATIONS
no repair during that interval. This is more certainly true since actinomycin D at these concentrations neither inhibits division nor induces aberrations (Table I). In addition we have observed in simultaneous experiments that actinomycin D (o.o5 ~,g/ml) given to cells right after a I5o-R dose of X-ray for 1.5 h does not change tile frequency of chromatid exchanges observed after I5o R alone. What ever else may be assumed about the action of actinomycin D on cells that are or have been irradiated, one is forced to consider the effect of actinomycin D on the repair of X-ray induced chromosomal breaks in any discussion of the cidal effects of X-ray in the presence of actinomycin D. This work was partially supported by grants GM lO8-O2-O5 and GRS 46-68.
The Johns Hopkins University, School of Hygiene and Public Health, Department of RadioIogical Science, Baltimore, Md. (U.S.A.)
T. PREMPREE T. MERZ
I BARRY, R. D., D. R. IRES AND J. G. GRUICKSHANK, Nature, 194 (1962) II39. BASERGA, R., AND S. HEFFLER, Exptl. Cell. Res., 46 (1967) 571. BENDER, M. A., AND D. M. PRESCOTT, Exptl. Cell Res., 27 (1962) 221. COOPER, P. D., Virology, 28 (1966) 663. DEITCH, A. D., AND G. C. GODMAN, ]grOc. Natl. Acad. Sci. (U.S.), 57 (1967) 16o7ELKIND, M. M., G. F. WttlTMORE AND T. ALESClO, Science, 143 (1964) 1454. ELKIND, M. M., C. IKAMPER, W. B. MOSES AND H. SUTTON-GILBERT, Recovery and Repair Mechanisms in Radiobiology, Brookhaven Syrup. Biol., No. 2 ( J u n e 5-7, 1967). 8 GERMAN, J. L., Trans. N . Y . Acad. Sci., 24 (1962) 395. 9 KORN, D., J. Biol. Chem., 242 (1966) 16o. io LUNT, M. R., AND R. L. SINSHEIMER, J, 21/Iol. Biol., 18 (1966) 541. 2 3 4 5 o 7
II MOORHEAD, P. S., P. C. NO'WELL, W. J. ~IELLMAN, D. M. BATTIPS AND D. A. HUNGERFORD, Exptl. Cell Res., 20 (196o) 613. 12 PASTAN, I., AND R. M. FRIEDMAN, Science, 16o (1968) 316. 13 PREMPREE, T., Chromosomal repair time of X-ray induced breaks in human cells, Thesis, Johns Hopkins University, Baltimore, Md., I968. 14 PREMPREE, T., AND T. MERZ, Abstract of 5th Annual Meeting of the American Society for Cell Biology, J. Cell Biol., 27 (1965) 78A, 15 REICH, E., I. H. GOLDBERG AND M. I~ABINOXVITZ, Nature, 196 (1962) 743. 16 REICH, E., R. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Proc. Nail. 3cad. Sci. (U.S.), 48 (1962) 1238. 17 WOLVP, S., AND K. C. ATWOOO, Proc. Natl. Aead. Sci. (U.S.), 4 ° (I954) 187.
Received October I4th, i968 7vlutation Res., 7 (1969) 126-128
Mutation Research Elsevier Publishing Company, A m s t e r d a m Printed in The Netherlands
Actinomycin D: Inhibition of chromosomal repair in human lymphocytes Actinomycin D is described as an inhibitor of DNA-dependent RNA synthesis and subsequent protein synthesis15,1". More recently other effects on biological systems have been reportedS, TM.The effect of actinomycin D has been attributed to its complex formation with the guanine bases in nucleic acid which would additionally create a block to the synthesis of DNA~," (when the complexed G-base is in DNA) and also the inhibition of RNA replication (when the complexed G-base is in RNA) in poliovirusq influenza virus I and RNA bacteriophage MS 21°. One of the more interesting consequences of actinomycin D treatments is the enhanced response of biological systems to subsequent treatments with other agents especially X-ray. The simplest explanation of this observation is that the effect of actinomycin D on X-ray responses is the result of its ability to inhibit repair of X-ray induced damage either directly or indirectly. In a previous study actinomycin D was placed in the culture medium between 2 doses of X-ray for various periods of time. The measurement of cell survival indicated that there was little repair between the doses 6. In fact what was observed was a diminution of the increase in survival normally seen between doses of X-ray. As the time between doses was increased (the time of actinomycin D exposure necessarily increased) the increase in survival decreased. At the higher concentration of actinomycin D, survival decreased even in the absence ot X-ray. These effects have led to the belief that actinomycin D may exert its influence by mechanisms other than repair inhibition 7. The mechanism for the effects of actinomycin D on "transformed" or neoplastic cells such as HeLa or the ubiquitous chinese hamster cell lines used so frequently remains questionable. The theory that actinomycin D can or cannot inhibit repair and enhance the response of cells to the effects of X-ray treatment, on the other hand, can be tested. This report concerns the results of those tests which indicate that at concentrations which do not induce chromosomal aberrations, actinomycin D inhibits the repair of chromatid strands broken by X-ray. Human lymphocytes were obtained from the peripheral blood of two normal male individuals and cultured using a modification of the method first reported by MOORHEAD et al. 11. Treatments were initiated at the time when most of the cells were in S stage 3,8 (48 h after incubation). Actinomycin D was added to the culture medium for the duration of the interval between two equal fractions of X-irradiation and that interval was made equal to the known repair time of chromosomal breaks in the S stage 14. The optimal concentration of actinomycin D was o.o5 ¢/g/ml for 1. 5 h. When the concentration was equal to or in excess of o. 5/~g/ml for the 1.5-h treatment time, there was a gross inhibition of mitosis. The results of the actinomycin D treatments over the entire range of concentrations are given in Table I. Both effects on mitotic index and chromosomal aberrations were observed. As shown (Table I) the aberration frequency in the treated cells over the entire range was no different than the control rate. All aberrations were single chromatid gaps. We conclude that at the concentrations used, and the treatment durations, actinomycin D does not induce chromosome breaks which can be seen in the first post~Vlutation Res., 7 (1969) 126--128
SHORT COMMUNICATIONS TABLE
127
I
THE EFFECT OF VARYING CONCENTRATIONS OF ACTINOMYCIN D ON MITOTIC ACTIVITY AND CHROMOSOME ABERRATION RATES IN HUMAN LYMPHOCYTIgS
Final concentration (izg/ml in L 5 h)
Number of metaphases scored
E~ect on mitosis (mitotic index)
% Chromatid aberration Gaps Deletion Exchange
5.o o. 5
None None
0.05 O.OI None
IOO 5° 2oo
No mitotic figure Mitotic inhibition was extreme M.I. less t h a n o . 1 % M.1. 4,o °/o M . I . 5.00,0 M.I. 4.5%
IO I2 9
N.B.M.I. TABLE
o o o
o o o
= Mitotic index. II
THE EFFECT OF ACTINOMYCIN D (0.05
X - R a y treatment 15o R 15o R - - - a c t i n o m y c i n D f o r 90 m i n 300 1R 15o R - - t i m e i n t e r v a l 60 m i n - - i 5 o R 15o R - - t i m e i n t e r v a l 9 ° m i n - - i 5 o R 15o R - - t i m e i n t e r v a l 90 r a i n w i t h actinomycin D--iSo R
itg/ml, 1,5 h) ON CHROMOSOMAL REPAIR TIME Cells scored
Observed
°/o Chromatid exchange ( i f additive)
Calculated for interaction
200 ioo 25o 200 163
8 7 3I 26 15
16 16
32 32
ioo
29
16
32
treatment division. To test the effect of actinomycin D on the ability of ceils to repair chromosomal breaks, X-ray treatments were divided into 2 equal fractions with a time interval between the two doses during which repair could take place. The known interval between dosesi3, I4 which is equal to or exceeds the repair time for chromosomal breaks in the S stage in human lymphocytes is 9 ° min. The chromatid exchange frequency was used as an assay for determination of repair. A full discussion of this technique has appeared previously13,IL The results of this experiment are listed in Table II. All exchange values represent the peak frequency response after treatment. These peaks occurred at the same time in both the actinomycin D treated and the untreated populations. Mitotic delay was, therefore, not considered a factor. Further evidence that delay was not a factor comes from concurrent labelling (E3HJthymidine) tests. The time of appearance of the labelled S cells were also found to be the same in treated and untreated cells. It should be clear from the data (Table II) that the repair time of chromatid breaks is longer than 6o min and equal to or less than 9 ° min after I5o R since the observed rate differs from the calculated interaction figure and the rate observed after a single 3oo-R dose, only after a 9o-min interval between fractions. At that time (9° min) the observed rate is approximately what is expected if the two treatments were additive and repair of the chromosomal breaks induced by the first dose had occurred. But when the 9o-min interval includes the presence of actinomycin D (o.o5 #g/ml following the first dose) in the culture medium the yield of chromatid exchanges was 290/o. This figure is approximately the same as that calculated for interaction and the frequency observed after a single 3oo-R dose (no interval). It clearly indicates Mutation Res., 7 (1969) I 2 6 - I 2 S
128
SHORT COMMUNICATIONS
no repair during that interval. This is more certainly true since actinomycin D at these concentrations neither inhibits division nor induces aberrations (Table I). In addition we have observed in simultaneous experiments that actinomycin D (o.o5 ~,g/ml) given to cells right after a I5o-R dose of X-ray for 1.5 h does not change tile frequency of chromatid exchanges observed after I5o R alone. What ever else may be assumed about the action of actinomycin D on cells that are or have been irradiated, one is forced to consider the effect of actinomycin D on the repair of X-ray induced chromosomal breaks in any discussion of the cidal effects of X-ray in the presence of actinomycin D. This work was partially supported by grants GM lO8-O2-O5 and GRS 46-68.
The Johns Hopkins University, School of Hygiene and Public Health, Department of RadioIogical Science, Baltimore, Md. (U.S.A.)
T. PREMPREE T. MERZ
I BARRY, R. D., D. R. IRES AND J. G. GRUICKSHANK, Nature, 194 (1962) II39. BASERGA, R., AND S. HEFFLER, Exptl. Cell. Res., 46 (1967) 571. BENDER, M. A., AND D. M. PRESCOTT, Exptl. Cell Res., 27 (1962) 221. COOPER, P. D., Virology, 28 (1966) 663. DEITCH, A. D., AND G. C. GODMAN, ]grOc. Natl. Acad. Sci. (U.S.), 57 (1967) 16o7ELKIND, M. M., G. F. WttlTMORE AND T. ALESClO, Science, 143 (1964) 1454. ELKIND, M. M., C. IKAMPER, W. B. MOSES AND H. SUTTON-GILBERT, Recovery and Repair Mechanisms in Radiobiology, Brookhaven Syrup. Biol., No. 2 ( J u n e 5-7, 1967). 8 GERMAN, J. L., Trans. N . Y . Acad. Sci., 24 (1962) 395. 9 KORN, D., J. Biol. Chem., 242 (1966) 16o. io LUNT, M. R., AND R. L. SINSHEIMER, J, 21/Iol. Biol., 18 (1966) 541. 2 3 4 5 o 7
II MOORHEAD, P. S., P. C. NO'WELL, W. J. ~IELLMAN, D. M. BATTIPS AND D. A. HUNGERFORD, Exptl. Cell Res., 20 (196o) 613. 12 PASTAN, I., AND R. M. FRIEDMAN, Science, 16o (1968) 316. 13 PREMPREE, T., Chromosomal repair time of X-ray induced breaks in human cells, Thesis, Johns Hopkins University, Baltimore, Md., I968. 14 PREMPREE, T., AND T. MERZ, Abstract of 5th Annual Meeting of the American Society for Cell Biology, J. Cell Biol., 27 (1965) 78A, 15 REICH, E., I. H. GOLDBERG AND M. I~ABINOXVITZ, Nature, 196 (1962) 743. 16 REICH, E., R. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Proc. Nail. 3cad. Sci. (U.S.), 48 (1962) 1238. 17 WOLVP, S., AND K. C. ATWOOO, Proc. Natl. Aead. Sci. (U.S.), 4 ° (I954) 187.
Received October I4th, i968 7vlutation Res., 7 (1969) 126-128