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DNA degradation by ionizing radiation in Bacillus subtilis. Synergistic effect of actinomycin D
837
PRELIMINARY NOTES
BBA 91186 D N A degradation by ionizing radiation in Bacillus subtilis. Synergistic effect of actinomycin D In bacteria a striking effect of 7 radiation is the development of DNA degradation 1-5. The degradation is enzymatically mediated after an initial primary radiation effect and the question as to the nature of the enzyme is still open. Bacillus subtilis is of interest because it is permeable to actinomycin D which interferes with RNA synthesis 6 and has been used to study, for example, the decay of messenger RNA (ref. 6). In addition, attention has recently been focused on an enhancement by actinomycin D of the lethal effect of ionizing radiation on mammalian cellsV,s. The effect has been measured in terms of cell survival. It would be desirable if some relation between the effect of actinomycin D and ionizing radiation could be studied in a model system and related to a specific aspect of radiation damage. In our laboratory we have recently investigated DNA degradation produced by y irradiation of B. subtilis. We find that it occurs in a manner similar to that in Escherichia coli, for example, although at relatively higher doses relative to the IO % survival dose of 7000 R reported by KAPLAN AND ZAVARINE9. Our present data show a considerable increase in the amount of DNA degraded in the presence of actinomycin D and are in agreement with the conclusion s that the effects of actinomycin D andionizing radiation are not simply additive. Our experiments further sugge.st that the two-component feature of DNA degradation found by HUSTON AND POLLARD4 is also seen in B. subtilis, for the fraction of DNA degradation which is influenced by actinomycin D is only a part of the whole amount of degradation. B. subtilis 168 T - I n d - was obtained originally from Dr. JAMES L. FARMER, Department of Biophysics, University of Colorado Medical Center 1°. The glucose minimal medium of SPIZIZENn, supplemented with 5/,g/ml thymine and 2opg/ml L-tryptophan, was used in all experiments. Cultures were grown at 37 ° with aeration. A 50/~g/ml solution of actinomycin D (Merck, Sharp and Dohme Research Laboratory, Rahway, N. J.) in 8 % ethanol was prepared and stored at 4 ° protected from light. The exterior of all glassware used in conjunction with actinomycin D was painted flat black. Sterile aqueous solutions of E2J4Clthymine and E2-z4C]uracil (New England Nuclear) were used. Cultures for degradation experiments were grown in medium supplemented with [2-14C]thymine. At a titer of approx, lOs cells/ml, the culture was tiltered (142 mm diameter, 0.22 # pore-size Millipore), rinsed and resuspended at the original titer in fresh, warm, isotope-free medium. The culture was then divided into three equal portions, which were treated as follows: i. Actinomycin D was added to a final concn, of I.O #g/mh After IO min at 37 ° with aeration, the sample was oxygenated for I.O min and then exposed to the desired dose in a 6°Co source (Gammacell 2oo, Atomic Energy of Canada, Ltd.). i-ml samples were removed and added to I.O ml of cold IO % trichloroacetic acid at intervals up to and including 12o min. After I h in an ice bath, they were filtered through Schleicher and Schuell type B-6 filters, rinsed with io ml of cold 5 % trichloroacetic Biochim. Biophys. Acta, 145 (1967) 837-839
838
PRELIMINARY NOTES
acid, glued to planchets, dried and counted. The loss of radioactivity from the trichloroacetic acid-precipitable fraction as a function of time after irradiation was taken as a measure of DNA degradation. 2. A volume of 8 % ethanol equivalent to that used above, but without actinomycin D, was added. The sample was otherwise treated exactly the same as above. 3. Actinomycin D was added to a final concn, of I.O #g/ml. After io min at 37 ° with aeration, the sample was oxygenated for I.o rain, but was not irradiated. Samples were removed at intervals and analyzed as previously described. The effect of actinomycin D on DNA degradation at two different doses is shown in Fig. I. At low doses degradation proceeds rapidly to a definite end. The amount degraded depends on dose. When actinomycin D was present, a sharp increase in the extent of degradation was observed at lower doses. The amount degraded approached 42-45 % and did not change significantly over the dose-range investigated. No degradation was observed in the unirladiated control, regardless of whether actinomycin D was present or absent. It should be noted that, beginning about 60-75 min after irradiation, a partial lysis of the cells was observed in samples irradiated without actinomycin D. No lysis was observed when actinomycin D was present. . z
.
.
.
.
.
.
.
.
,
CONTROL NO ACT D
.
.
.
.
.
--T
CONTROL,ACT D 1.0 pg/ml
IO0 E~< 9O m
"
, NO
ACT.O
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.
.
.
.
.
.
.
.
.
.
.
B 0 ~-
~ ~o ~_o BO
60
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90+
A C T . O LO V g / m l
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201
DOSE
i 25
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ACT. 0 I.O ! J g / m l
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DOSE : 6 5 K I L O R A D z0
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Io : ~ ~_
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I I I 15 3 0 4 5 ',L AFTER ---BEFORE
I 60
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I 90
I I lOS 120 ',', 15 3 0 4 5 6 0 7 5 ,L_ AFTER L__ BEFORE TiME IN MINUTESAFTER iRRADIATION
90
lOB 120
0
I
0
20
40
60
I
I
I
80 Joo 120 140 DOSE IN K I L O R A O
I
I
r
I
160
180
200
220
240
Fig. I. The effect of actinomycin D on DNA degradation at two different doses. Curves have been normalized by setting the trichloroacetic acid (TCA)-precipitable radioactivity in the sample removed before irradiation equal to ioo %. Fig. 2. Dose-response of DNA degradation in B. subtilis i68 T-Ind-.
A dose-response curve for DNA degradation is shown in Fig. 2. The enhancement by actinomycin D can be seen to diminish as the dose increases. This suggests that at Mast two processes are involved in DNA degradation and that only one is affected by actinomycin D. As previously mentioned, evidence for two components in DNA degradation has been obtained also by HUSTON AND POLLARD4 on the basis of high lethal radiation studies with F.. coli. The effect of this concentration of actinomycin D on the synthesis of DNA and RNA in unirradiated cells was studied separately. The data are in good agreement with those of ROLFE12 using similar concentrations on B. subtilis S. Thus, the concentration of actinomycin D was used in the range where RNA synthesis is drastically reduced while DNA synthesis is not greatly affected. Biochim. Biophys. dcta, 145 (1967) 837-839
PRELIMINARY NOTES
839
Without claiming that the mechanism is explained, the effect of actinomycin D on DNA degradation suggests that some relationship between it and the synthesis of RNA should be sought. At the present time there appear to be at least three interpretations of the actinomycin pocentiation which are consistent with the data presented. In all of these it is assumed that the increase in degradation obtained at high doses is attributable to a second process which is unaffected by actinomycin D and is dependent only on dose. I. The effect of actinomycin D could be due to inhibition either of existing DNA repair systems or of the synthesis of any repair enzymes which would otherwise be induced by radiation action. 2. The binding of actinomycin D to DNA might function to put the DNA in a condition which renders degradation more rapid and effective. 3. The strain of B. subtilis used in these expeliments has been shown to be inducible for a defective prophage 1~. Should ionizing radiation give rise to induction, then development of the prophage could result in interference with various cell processes, including DNA degradation. In the presence of actinomycin D, the prophage would be unable to develop and, hence, would not interfere in the degradation process. Whatever the explanation, these results suggest that the action of actinomycin D in potentiating radiation effects in mammalian cells may well be concerned with the potentiation of radiation-induced DNA degradation. This work was supported by the National Aeronautics and Space Administration (NsG-324).
Biophysics Department The Pennsylvania State University University Park, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13
LEO J . GRADY ERNEST C. POLLARD
J. H. STUY, J. Bacteriol., 79 (196o) 707. B. MIL:ETIC, Z. KUCAN AND DJ. NOVAK, Nature, 202 (1964) lO6. ~E. C. POLLARD AND P. M. ACHEY, Radiation Res., 27 (1966) 419. D. C. HUSTON AND E. C. POLLARD, Biophys. J., in the press. E. C. POLLARD, J. SwEz AND L. GRADY, Radiation Res., 28 (1966) 585 . C. LEVINTHAL, A. KEYNAN AND A. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. W. ZEMAN, T. SAMORAJSKI AND M. ORDY, Abstr. z5th Ann. Meeting Radiation Res. Soc., x967, p. 3 o. i . M. ELKIND, W. B. MOSES, H. SUTTON-GILBERT AND C. KAMPER, Abstr. z5th Ann. Meeting Radiation Res. Soc., x967, p. 5 o. H. S. KAPLAN AND t{. ZAVARINE, Biochem. Biophys. Res. Commun., 8 (1962) 432. J. L. FARMER AND F. ROTHMAN, J. Bacteriol., 89 (1965) 262. J. SPIZlZEN, Proc. Natl. Acad. Sci. U.S., 44 (1958) lO72. R. ROLFE, Proc. Natl. Acad. Sci. U.S., 57 (1967) 114. E. SEAMAN, E. TARMY AND J. MARMUR, Biochemistry, 3 (1964) 6o7-
Received August Ioth, 1967 Biochim. Biophys. Acta, 145 (I967) 837-839
PRELIMINARY NOTES
BBA 91186 D N A degradation by ionizing radiation in Bacillus subtilis. Synergistic effect of actinomycin D In bacteria a striking effect of 7 radiation is the development of DNA degradation 1-5. The degradation is enzymatically mediated after an initial primary radiation effect and the question as to the nature of the enzyme is still open. Bacillus subtilis is of interest because it is permeable to actinomycin D which interferes with RNA synthesis 6 and has been used to study, for example, the decay of messenger RNA (ref. 6). In addition, attention has recently been focused on an enhancement by actinomycin D of the lethal effect of ionizing radiation on mammalian cellsV,s. The effect has been measured in terms of cell survival. It would be desirable if some relation between the effect of actinomycin D and ionizing radiation could be studied in a model system and related to a specific aspect of radiation damage. In our laboratory we have recently investigated DNA degradation produced by y irradiation of B. subtilis. We find that it occurs in a manner similar to that in Escherichia coli, for example, although at relatively higher doses relative to the IO % survival dose of 7000 R reported by KAPLAN AND ZAVARINE9. Our present data show a considerable increase in the amount of DNA degraded in the presence of actinomycin D and are in agreement with the conclusion s that the effects of actinomycin D andionizing radiation are not simply additive. Our experiments further sugge.st that the two-component feature of DNA degradation found by HUSTON AND POLLARD4 is also seen in B. subtilis, for the fraction of DNA degradation which is influenced by actinomycin D is only a part of the whole amount of degradation. B. subtilis 168 T - I n d - was obtained originally from Dr. JAMES L. FARMER, Department of Biophysics, University of Colorado Medical Center 1°. The glucose minimal medium of SPIZIZENn, supplemented with 5/,g/ml thymine and 2opg/ml L-tryptophan, was used in all experiments. Cultures were grown at 37 ° with aeration. A 50/~g/ml solution of actinomycin D (Merck, Sharp and Dohme Research Laboratory, Rahway, N. J.) in 8 % ethanol was prepared and stored at 4 ° protected from light. The exterior of all glassware used in conjunction with actinomycin D was painted flat black. Sterile aqueous solutions of E2J4Clthymine and E2-z4C]uracil (New England Nuclear) were used. Cultures for degradation experiments were grown in medium supplemented with [2-14C]thymine. At a titer of approx, lOs cells/ml, the culture was tiltered (142 mm diameter, 0.22 # pore-size Millipore), rinsed and resuspended at the original titer in fresh, warm, isotope-free medium. The culture was then divided into three equal portions, which were treated as follows: i. Actinomycin D was added to a final concn, of I.O #g/mh After IO min at 37 ° with aeration, the sample was oxygenated for I.O min and then exposed to the desired dose in a 6°Co source (Gammacell 2oo, Atomic Energy of Canada, Ltd.). i-ml samples were removed and added to I.O ml of cold IO % trichloroacetic acid at intervals up to and including 12o min. After I h in an ice bath, they were filtered through Schleicher and Schuell type B-6 filters, rinsed with io ml of cold 5 % trichloroacetic Biochim. Biophys. Acta, 145 (1967) 837-839
838
PRELIMINARY NOTES
acid, glued to planchets, dried and counted. The loss of radioactivity from the trichloroacetic acid-precipitable fraction as a function of time after irradiation was taken as a measure of DNA degradation. 2. A volume of 8 % ethanol equivalent to that used above, but without actinomycin D, was added. The sample was otherwise treated exactly the same as above. 3. Actinomycin D was added to a final concn, of I.O #g/ml. After io min at 37 ° with aeration, the sample was oxygenated for I.o rain, but was not irradiated. Samples were removed at intervals and analyzed as previously described. The effect of actinomycin D on DNA degradation at two different doses is shown in Fig. I. At low doses degradation proceeds rapidly to a definite end. The amount degraded depends on dose. When actinomycin D was present, a sharp increase in the extent of degradation was observed at lower doses. The amount degraded approached 42-45 % and did not change significantly over the dose-range investigated. No degradation was observed in the unirladiated control, regardless of whether actinomycin D was present or absent. It should be noted that, beginning about 60-75 min after irradiation, a partial lysis of the cells was observed in samples irradiated without actinomycin D. No lysis was observed when actinomycin D was present. . z
.
.
.
.
.
.
.
.
,
CONTROL NO ACT D
.
.
.
.
.
--T
CONTROL,ACT D 1.0 pg/ml
IO0 E~< 9O m
"
, NO
ACT.O
~BO
.
.
.
.
.
.
.
.
.
.
.
B 0 ~-
~ ~o ~_o BO
60
i BO
x
.
90+
A C T . O LO V g / m l
~o
40
z z
201
DOSE
i 25
KILORAO
t
-'"
-
ACT. 0 I.O ! J g / m l
50 40
~'& .....
30
, 1 ~
DOSE : 6 5 K I L O R A D z0
]
Io : ~ ~_
0
I I I 15 3 0 4 5 ',L AFTER ---BEFORE
I 60
I 7B
I 90
I I lOS 120 ',', 15 3 0 4 5 6 0 7 5 ,L_ AFTER L__ BEFORE TiME IN MINUTESAFTER iRRADIATION
90
lOB 120
0
I
0
20
40
60
I
I
I
80 Joo 120 140 DOSE IN K I L O R A O
I
I
r
I
160
180
200
220
240
Fig. I. The effect of actinomycin D on DNA degradation at two different doses. Curves have been normalized by setting the trichloroacetic acid (TCA)-precipitable radioactivity in the sample removed before irradiation equal to ioo %. Fig. 2. Dose-response of DNA degradation in B. subtilis i68 T-Ind-.
A dose-response curve for DNA degradation is shown in Fig. 2. The enhancement by actinomycin D can be seen to diminish as the dose increases. This suggests that at Mast two processes are involved in DNA degradation and that only one is affected by actinomycin D. As previously mentioned, evidence for two components in DNA degradation has been obtained also by HUSTON AND POLLARD4 on the basis of high lethal radiation studies with F.. coli. The effect of this concentration of actinomycin D on the synthesis of DNA and RNA in unirradiated cells was studied separately. The data are in good agreement with those of ROLFE12 using similar concentrations on B. subtilis S. Thus, the concentration of actinomycin D was used in the range where RNA synthesis is drastically reduced while DNA synthesis is not greatly affected. Biochim. Biophys. dcta, 145 (1967) 837-839
PRELIMINARY NOTES
839
Without claiming that the mechanism is explained, the effect of actinomycin D on DNA degradation suggests that some relationship between it and the synthesis of RNA should be sought. At the present time there appear to be at least three interpretations of the actinomycin pocentiation which are consistent with the data presented. In all of these it is assumed that the increase in degradation obtained at high doses is attributable to a second process which is unaffected by actinomycin D and is dependent only on dose. I. The effect of actinomycin D could be due to inhibition either of existing DNA repair systems or of the synthesis of any repair enzymes which would otherwise be induced by radiation action. 2. The binding of actinomycin D to DNA might function to put the DNA in a condition which renders degradation more rapid and effective. 3. The strain of B. subtilis used in these expeliments has been shown to be inducible for a defective prophage 1~. Should ionizing radiation give rise to induction, then development of the prophage could result in interference with various cell processes, including DNA degradation. In the presence of actinomycin D, the prophage would be unable to develop and, hence, would not interfere in the degradation process. Whatever the explanation, these results suggest that the action of actinomycin D in potentiating radiation effects in mammalian cells may well be concerned with the potentiation of radiation-induced DNA degradation. This work was supported by the National Aeronautics and Space Administration (NsG-324).
Biophysics Department The Pennsylvania State University University Park, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13
LEO J . GRADY ERNEST C. POLLARD
J. H. STUY, J. Bacteriol., 79 (196o) 707. B. MIL:ETIC, Z. KUCAN AND DJ. NOVAK, Nature, 202 (1964) lO6. ~E. C. POLLARD AND P. M. ACHEY, Radiation Res., 27 (1966) 419. D. C. HUSTON AND E. C. POLLARD, Biophys. J., in the press. E. C. POLLARD, J. SwEz AND L. GRADY, Radiation Res., 28 (1966) 585 . C. LEVINTHAL, A. KEYNAN AND A. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. W. ZEMAN, T. SAMORAJSKI AND M. ORDY, Abstr. z5th Ann. Meeting Radiation Res. Soc., x967, p. 3 o. i . M. ELKIND, W. B. MOSES, H. SUTTON-GILBERT AND C. KAMPER, Abstr. z5th Ann. Meeting Radiation Res. Soc., x967, p. 5 o. H. S. KAPLAN AND t{. ZAVARINE, Biochem. Biophys. Res. Commun., 8 (1962) 432. J. L. FARMER AND F. ROTHMAN, J. Bacteriol., 89 (1965) 262. J. SPIZlZEN, Proc. Natl. Acad. Sci. U.S., 44 (1958) lO72. R. ROLFE, Proc. Natl. Acad. Sci. U.S., 57 (1967) 114. E. SEAMAN, E. TARMY AND J. MARMUR, Biochemistry, 3 (1964) 6o7-
Received August Ioth, 1967 Biochim. Biophys. Acta, 145 (I967) 837-839