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Inducible repair of ionizing radiation damage in higher eukaryotic cells
Mutation Research, 173 (1986) 291-293 Elsevier
291
MRLett 0834
Inducible repair of ionizing radiation damage in higher eukaryotic cells Thomas M. Koval Department o f Radiology, George Washington University School o f Medicine and Health Sciences, Washington, DC 20037 (U.S.A.) and National Council on Radiation Protection and Measurements, Bethesda, MD 20814 (U.S.A.) (Accepted 9 December 1985)
Although there is widespread acceptance of inducible repair pathways in prokaryotes (Witkin, 1976; Peters and Jagger, 1981) and lower eukaryotes (Ruby and Szostak, 1985), the existence of such inducible systems in higher eukaryotes has remained a controversial issue (Radman, 1980). The presence of such systems in higher eukaryotic organisms could significantly alter current estimations of the human risks of radiation and chemicals in the environment. This study establishes the existence of an inducible process in cultured TN-368 lepidopteran insect cells that is responsible for increased cellular survival in the presence of otherwise lethal ionizing radiation damage. The resulting increase in cellular survival is completely inhibited by cycloheximide or actinomycin D, indicating that protein and RNA synthesis are necessary for its induction. This inducible repair process is apparently not present in unirradiated cells or in cells receiving less than some minimal dose. The TN-368 leipidopteran cell line exhibits a pronounced resistance to the lethal effects of ionizing radiation (Koval, 1983). The molecular rationale for this radioresistance is believed to involve very efficient DNA-repair processes (Koval, 1980). Initial inferences of an inducible repair proCorrespondence to: Thomas M. Koval, Ph.D., National Council on Radiation Protection and Measurements, 7910 Woodmont Avenue, Suite 1016, Bethesda MD 20814 (U.S.A.)
cess emanated from efforts to characterize the multiphasic survival response of the TN-368 line (Koval, 1984). These inferences were strengthened and supported by experiments intended to assess potentially lethal damage (PLD) and sublethal damage (SLD) repair capabilities. Methods of cell cultivation and colony formation have been described in detail (Koval, 1980, 1983). When a TN-368 cell population entering stationary growth phase is irradiated on ice with ~37Cs ~,-rays and then incubated from 0.25 to several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation (Figs. 1 and 2, open symbols). The increase in survival is maximal by 6 h of incubation. Similarly, the survival of cells plated immediately following the second of two equivalent radiation doses separated by 0.25 to several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses (Fig. 3, open symbols), the increase in survival again reaching its peak by 6 h. These phenomena appeared initially to be analogous to PLD (Phillips and Tolmach, 1966) and SLD (Elkind and Sutton, 1959) repair as described for mammalian cells. However, when the cells are incubated in 10-2 or 10-3 M cycloheximide (Fig. 1) o r 10 - 3 M actinomycin D (Fig. 2) following irradiation, but prior to cell dilution and plating (PLD experiments), no increase in survival is observed.
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
292 10 0
10 0
J
-
~
~ . ~ . ~ _ ~
10-I
_J o ¢0
alO c
== > > 10 - 2
O 350 Gy o n l y
10-3
•
3 5 0 Gy, 10 - 3 M cycloheximide
•
350 Gy, 10 - 2 M cycloheximide
I
I
I
I
I
I
1
2
3
4
5
6
Time between irradiation and plating, hr
O • Z~ • 10- 2
I 1
200 200 350 350 I 2
Gy Gy, Gy Gy, I 3
only 10 - 3 M a c t i n o m y c i n D only 10 - 3 M a c t i n o m y c i n D
4
I 5
I 6
Time between irradiation and plating, hr
Fig. I. Cell monolayers entering plateau growth were irradiated on ice with J37Cs, incubated at 28°C in medium without (O) or with l0 -3 M (e) or l0 -2 M (A) cycloheximide, and rinsed immediately prior to plating cells for colony formation in cycloheximide-free medium. Each point represents the mean _+ S.E. of 3 Expts. with 5 replicates per Expt.
Fig. 2. Cell monolayersentering plateau growth were irradiated on ice with '37Cs, incubated at 28°C in medium without (©, A) or with 10-~ M actinomycin D (e,&) and rinsed immediately prior to plating cells for colony formation in actinomycin D-free medium. Each point represents the mean of 5 replicates from a single experiment.
Correspondingly, when the cells are incubated in 10- 2 M cycloheximide during the interval between split radiation doses (SLD experiments), no increase in survival occurs (Fig. 3). These concentrations of cycloheximide and actinomycin D inhibit protein and R N A synthesis, respectively, and cause little or no toxicity to control cells as determined by lack of effect on colony forming ability (e.g., • symbol in Fig. 3) and absence of microscopicallydetermined cytopathic effects (morphological alterations). In addition, when cycloheximide is added prior to the first irradiation, but removed during the interval between split doses, survival increases as for cells treated with radiation only, indicating that the lack of survival increase for the reciprocal treatment is not due to synergistic ef-
fects between the radiation and cycloheximide (v symbol, Fig. 3). The absence o f an increase in survival in the presence of either cycloheximide or actinomycin D therefore serves as evidence that protein and RNA synthesis are essential for the development of the enhanced survival observed in the P L D and SLD repair studies (Figs. 1-3). Interestingly enough, the inhibition of colony formation, is one of the treatments that permits the expression of and enhances P L D repair (Phillips and Tolmach, 1966). The inhibition of cellular survival in the presence of cycloheximide and actinomycin D indicates the induction of a radiation-induced repair system that is dependent upon transcriptional and translational activities which are not present in unit-
293
crease in survival, inducible repair in prokaryotes is usually associated with an increase in mutagenesis (Witkin, 1976). Mindful of this, work is currently underway in this laboratory to develop a suitable mutation assay for the TN-368 cells.
10 0
~ 10 o
-I
Acknowledgements I thank M.L. Flippin and M.C. Augustyn for excellent technical assistance. This work was supported by USPHS grant RO1-CA34158, awarded by the National Cancer Institute, DHHS.
== >
10- 2
References •
3 0 0 Gy, 1 0 - 2
• •
10 - 2 M c y c l o h e x i m i d e only 10 - 2 M c y c l o h e x i m i d e , 3 0 0 Gy
M cycloheximide
1( 3 1 2 3 4 ]~me b e t w e e n i r r a d i a t i o n s ,
5 hr
6
Fig. 3. Exponentially growing cell monolayers were irradiated on ice with J3~Cs and plated for colony formation immediately following a single dose or the second of two equivalent split doses. Between split doses, the cells were incubated at 28°C in medium without (O) or with l0 -2 M cycloheximide (o). An additional monolayer ( , ) was incubated at 28°C in medium containing l0 -2 M cycloheximide for 3 h, rinsed and reincubated in cycloheximide-free medium for l h before receiving the same treatment as (~). A final monolayer was incubated in medium containing 10- 2 M cycloheximide for 3 h, rinsed, reincubated for 4 h and plated (,,). Each point represents the mean +_ S.E. of 2 Expts. with 5 replicates per Expt.
radiated cells or cells receiving less than some minimal amount of radiation essential for activation of the process. In addition, recent studies examining the functional significance of heat stress proteins in Drosophila cells have suggested the possibility that an inducible repair system is activated in these cells in response to heat damage (Tomasovic and Koval, 1985). Along with an in-
Elkind, M.M., and H.A. Sutton (1959) X-Ray damage and recovery in mammalian cells in culture, Nature (London), 184, 1293-1295. Koval, T.M. (1980) Relative responses of mammalian and insect cells, in: R.E. Meyn and H.R. Withers (Eds.), Radiation Biology in Cancer Research, Raven, New York, pp. 169-184. Koval, T.M. (1983) Intrinsic resistance to the lethal effects of X-irradiation in insect and arachnid cells, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4"/52-4755. Koval, T.M. (1984) Multiphasic survival response of a radioresistant lepidopteran insect cell line, Radiat. Res., 98, 642-648. Peters, J., and J. Jagger (1981) Inducible repair of near-UV radiation lethal damage in E. coil, Nature (London), 289, 194-195. Phillips, R.A., and L.J. Tolmach (1966) Repair of potentially lethal damage in X-irradiated HeLa cells, Radiat. Res., 29, 413-432. Radman, M. (1980) Is there SOS induction in mammalian cells?, Photochem. Photobiol., 32, 823-830. Ruby, S.W., and J.W. Szostak (1985) Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents, Mol. Cell. Biol., 5, 75-84. Tomasovic, S.P., and T.M. Koval 0985) Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line, Int. J. Radiat. Biol., 48, 535-650. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in E. coli, Bacteriol. Rev., 40, 869-907. Communicated by R.J. Preston
291
MRLett 0834
Inducible repair of ionizing radiation damage in higher eukaryotic cells Thomas M. Koval Department o f Radiology, George Washington University School o f Medicine and Health Sciences, Washington, DC 20037 (U.S.A.) and National Council on Radiation Protection and Measurements, Bethesda, MD 20814 (U.S.A.) (Accepted 9 December 1985)
Although there is widespread acceptance of inducible repair pathways in prokaryotes (Witkin, 1976; Peters and Jagger, 1981) and lower eukaryotes (Ruby and Szostak, 1985), the existence of such inducible systems in higher eukaryotes has remained a controversial issue (Radman, 1980). The presence of such systems in higher eukaryotic organisms could significantly alter current estimations of the human risks of radiation and chemicals in the environment. This study establishes the existence of an inducible process in cultured TN-368 lepidopteran insect cells that is responsible for increased cellular survival in the presence of otherwise lethal ionizing radiation damage. The resulting increase in cellular survival is completely inhibited by cycloheximide or actinomycin D, indicating that protein and RNA synthesis are necessary for its induction. This inducible repair process is apparently not present in unirradiated cells or in cells receiving less than some minimal dose. The TN-368 leipidopteran cell line exhibits a pronounced resistance to the lethal effects of ionizing radiation (Koval, 1983). The molecular rationale for this radioresistance is believed to involve very efficient DNA-repair processes (Koval, 1980). Initial inferences of an inducible repair proCorrespondence to: Thomas M. Koval, Ph.D., National Council on Radiation Protection and Measurements, 7910 Woodmont Avenue, Suite 1016, Bethesda MD 20814 (U.S.A.)
cess emanated from efforts to characterize the multiphasic survival response of the TN-368 line (Koval, 1984). These inferences were strengthened and supported by experiments intended to assess potentially lethal damage (PLD) and sublethal damage (SLD) repair capabilities. Methods of cell cultivation and colony formation have been described in detail (Koval, 1980, 1983). When a TN-368 cell population entering stationary growth phase is irradiated on ice with ~37Cs ~,-rays and then incubated from 0.25 to several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation (Figs. 1 and 2, open symbols). The increase in survival is maximal by 6 h of incubation. Similarly, the survival of cells plated immediately following the second of two equivalent radiation doses separated by 0.25 to several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses (Fig. 3, open symbols), the increase in survival again reaching its peak by 6 h. These phenomena appeared initially to be analogous to PLD (Phillips and Tolmach, 1966) and SLD (Elkind and Sutton, 1959) repair as described for mammalian cells. However, when the cells are incubated in 10-2 or 10-3 M cycloheximide (Fig. 1) o r 10 - 3 M actinomycin D (Fig. 2) following irradiation, but prior to cell dilution and plating (PLD experiments), no increase in survival is observed.
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
292 10 0
10 0
J
-
~
~ . ~ . ~ _ ~
10-I
_J o ¢0
alO c
== > > 10 - 2
O 350 Gy o n l y
10-3
•
3 5 0 Gy, 10 - 3 M cycloheximide
•
350 Gy, 10 - 2 M cycloheximide
I
I
I
I
I
I
1
2
3
4
5
6
Time between irradiation and plating, hr
O • Z~ • 10- 2
I 1
200 200 350 350 I 2
Gy Gy, Gy Gy, I 3
only 10 - 3 M a c t i n o m y c i n D only 10 - 3 M a c t i n o m y c i n D
4
I 5
I 6
Time between irradiation and plating, hr
Fig. I. Cell monolayers entering plateau growth were irradiated on ice with J37Cs, incubated at 28°C in medium without (O) or with l0 -3 M (e) or l0 -2 M (A) cycloheximide, and rinsed immediately prior to plating cells for colony formation in cycloheximide-free medium. Each point represents the mean _+ S.E. of 3 Expts. with 5 replicates per Expt.
Fig. 2. Cell monolayersentering plateau growth were irradiated on ice with '37Cs, incubated at 28°C in medium without (©, A) or with 10-~ M actinomycin D (e,&) and rinsed immediately prior to plating cells for colony formation in actinomycin D-free medium. Each point represents the mean of 5 replicates from a single experiment.
Correspondingly, when the cells are incubated in 10- 2 M cycloheximide during the interval between split radiation doses (SLD experiments), no increase in survival occurs (Fig. 3). These concentrations of cycloheximide and actinomycin D inhibit protein and R N A synthesis, respectively, and cause little or no toxicity to control cells as determined by lack of effect on colony forming ability (e.g., • symbol in Fig. 3) and absence of microscopicallydetermined cytopathic effects (morphological alterations). In addition, when cycloheximide is added prior to the first irradiation, but removed during the interval between split doses, survival increases as for cells treated with radiation only, indicating that the lack of survival increase for the reciprocal treatment is not due to synergistic ef-
fects between the radiation and cycloheximide (v symbol, Fig. 3). The absence o f an increase in survival in the presence of either cycloheximide or actinomycin D therefore serves as evidence that protein and RNA synthesis are essential for the development of the enhanced survival observed in the P L D and SLD repair studies (Figs. 1-3). Interestingly enough, the inhibition of colony formation, is one of the treatments that permits the expression of and enhances P L D repair (Phillips and Tolmach, 1966). The inhibition of cellular survival in the presence of cycloheximide and actinomycin D indicates the induction of a radiation-induced repair system that is dependent upon transcriptional and translational activities which are not present in unit-
293
crease in survival, inducible repair in prokaryotes is usually associated with an increase in mutagenesis (Witkin, 1976). Mindful of this, work is currently underway in this laboratory to develop a suitable mutation assay for the TN-368 cells.
10 0
~ 10 o
-I
Acknowledgements I thank M.L. Flippin and M.C. Augustyn for excellent technical assistance. This work was supported by USPHS grant RO1-CA34158, awarded by the National Cancer Institute, DHHS.
== >
10- 2
References •
3 0 0 Gy, 1 0 - 2
• •
10 - 2 M c y c l o h e x i m i d e only 10 - 2 M c y c l o h e x i m i d e , 3 0 0 Gy
M cycloheximide
1( 3 1 2 3 4 ]~me b e t w e e n i r r a d i a t i o n s ,
5 hr
6
Fig. 3. Exponentially growing cell monolayers were irradiated on ice with J3~Cs and plated for colony formation immediately following a single dose or the second of two equivalent split doses. Between split doses, the cells were incubated at 28°C in medium without (O) or with l0 -2 M cycloheximide (o). An additional monolayer ( , ) was incubated at 28°C in medium containing l0 -2 M cycloheximide for 3 h, rinsed and reincubated in cycloheximide-free medium for l h before receiving the same treatment as (~). A final monolayer was incubated in medium containing 10- 2 M cycloheximide for 3 h, rinsed, reincubated for 4 h and plated (,,). Each point represents the mean +_ S.E. of 2 Expts. with 5 replicates per Expt.
radiated cells or cells receiving less than some minimal amount of radiation essential for activation of the process. In addition, recent studies examining the functional significance of heat stress proteins in Drosophila cells have suggested the possibility that an inducible repair system is activated in these cells in response to heat damage (Tomasovic and Koval, 1985). Along with an in-
Elkind, M.M., and H.A. Sutton (1959) X-Ray damage and recovery in mammalian cells in culture, Nature (London), 184, 1293-1295. Koval, T.M. (1980) Relative responses of mammalian and insect cells, in: R.E. Meyn and H.R. Withers (Eds.), Radiation Biology in Cancer Research, Raven, New York, pp. 169-184. Koval, T.M. (1983) Intrinsic resistance to the lethal effects of X-irradiation in insect and arachnid cells, Proc. Natl. Acad. Sci. (U.S.A.), 80, 4"/52-4755. Koval, T.M. (1984) Multiphasic survival response of a radioresistant lepidopteran insect cell line, Radiat. Res., 98, 642-648. Peters, J., and J. Jagger (1981) Inducible repair of near-UV radiation lethal damage in E. coil, Nature (London), 289, 194-195. Phillips, R.A., and L.J. Tolmach (1966) Repair of potentially lethal damage in X-irradiated HeLa cells, Radiat. Res., 29, 413-432. Radman, M. (1980) Is there SOS induction in mammalian cells?, Photochem. Photobiol., 32, 823-830. Ruby, S.W., and J.W. Szostak (1985) Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents, Mol. Cell. Biol., 5, 75-84. Tomasovic, S.P., and T.M. Koval 0985) Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line, Int. J. Radiat. Biol., 48, 535-650. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in E. coli, Bacteriol. Rev., 40, 869-907. Communicated by R.J. Preston