61
Mutation Research, 60 (1979) 61--72
© Elsevier/North-Holland Biomedical Press
E F F E C T OF DNA REPAIR INHIBITORS ON THE INDUCTION AND REPAIR OF BLEOMYCIN-INDUCED CHROMOSOME DAMAGE
MARGUERITE A. SOGNIER *, WALTER N. HITTELMAN and POTU N. RAO Department of Deve!opmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 (U.S.A.)
(Received 30 August 1978) (Revision received 17 October 1978) (Accepted 30 October 1978)
Summary The phenomenon of premature chromosome condensation (PCC) was used to study the effects of 4 purported DNA repair inhibitors (hydroxyurea, hycanthone, cycloheximide, and streptovitacin A) on the induction and repair of chromosome aberrations after bleomycin treatment of Chinese hamster ovary cells. To test whether these repair inhibitors influenced the initial develo p m e n t of aberrations, exponentially growing populations of cells were treated with 25 gg/ml bleomycin for 30 min with or without the simultaneous presence of each inhibitor. Chromatid aberrations (gaps, breaks and exchanges) were then scored in the G2 PCC-induced immediately after treatment. None of the purported DNA-repair inhibitors tested were found to alter the initial frequency of bleomycin-induced chromatid aberrations. To determine whether these same agents had an effect on the repair of chromosome damage, cells were first treated with bleomycin in the presence of inhibitor and then incubated for 1 h in bleomycin-free media with or without the presence of inhibitor prior to cell fusion. In all cases, exchanges, once formed, were n o t repaired. While h y d r o x y u r e a and h y c a n t h o n e had no effect on the repair of chromosome damage, the protein synthesis inhibitors, cycloheximide and streptovitacin A, blocked the repair of bleomycin-induced chromatid breaks. These results suggest that protein synthesis inhibitors can prevent chromosome repair, however, the exact role of protein synthesis in the repair process remains to be elucidated. Since cycloheximide and streptovitacin A were found to inhibit the repair of chromatid breaks without altering the exchange frequency, these
* Please s e n d r e p r i n t r e q u e s t to this a u t h o r . A b b r e v i a t i o n s : BLM, b l e o m y c i n ; C H O , Chinese h a m s t e r o v a r y ; H a n k s ' BSS, H a n k s ' b a l a n c e d salt solution~ PCC, p r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n , or p r e m a t u r e l y c o n d e n s e d c h r o m o s o m e s ; PIPES~ p i p e r a z i n e - N , N - b i s ( 2 = e t h a n e s u l f o n i e acid).
62 results suggest that chromatid exchanges are not simply the product of misrepair or recombinational repair of chromatid breaks.
Chromosome damage in mammalian cells can be induced by a wide variety of agents, including X-rays and UV-irradiation [35], chemicals [31], viruses [ 26], and temperature changes [13 ]. However, little is known about the molecular basis of chromosome damage and repair. Most clastogens are known either to interact with cellular chromatin or to interfere with DNA synthesis, yet it remains uncertain how chromatin lesions are translated into chromosome aberrations. To have a better understanding of this process, we decided to study the effects of the purported inhibitors of DNA-repair synthesis on the induction and repair of bleomycin-induced chromosome damage. Until recently, chromosome aberrations could only be visualized when the cell reaches mitosis. The level of aberrations observed in mitosis, however, reflects that a m o u n t of damage initially induced in the interphase cell which has not been repaired by the time the cell reaches mitosis. For this reason, it has been difficult to measure chromosome repair by the conventional method of scoring metaphase chromosomes since the exact extent of the initial damage suffered by the cell is n o t known. Further, most DNA-repair inhibitors also block the progression of cells into mitosis. The phenomenon of premature chromosome condensation has been shown to be useful in the direct measurement of chromosome during and its repair after treatment of cells with a variety of agents, including X-rays [19,34], bleomycin [20] and adriamycin [21]. With this technique, clastogen-treated interphase cells can be fused with mitotic cells immediately after treatment (to determine the initial level of damage) or after a post-treatment incubation period (to determine the amount of chromosome repair). The objective of the present study was to employ the PCC technique to assay the effects of 4 purported inhibitors of DNA repair, viz., hydroxyurea, hycanthone, cycloheximide, and streptovitacin A, on the production and repair of bleomycin-induced chromosome aberrations in cells. Bleomycin is a mixture of glycopeptides that has been reported to bind to DNA, cause the release of free bases, and produce breaks in DNA [11,25]. Previous studies also have shown that bleomycin-induced damage is rapidly repaired at both DNA [10,23] and chromosomal levels [20], which is reflected in an increased cell survival [3]. Our results indicate that of the 4 purported DNA repair inhibitors studied, none decreased the initial frequency of bleomycin-induced chromatid aberrations. However, the inhibitors of protein synthesis, cycloheximide and streptovitacin A, blocked the repair of bleomycin-induced chromosome damage while the other two inhibitors had little or no effect on the repair process. Materials and Methods Cells Chinese hamster ovary (CHO) cells were routinely grown as monolayer cultures in Lux plastic dishes in McCoy's 5A medium (Gibco) supplemented with 16% heat-inactivated fetal calf serum (Gibco) and a 1% penicillin-streptomycin
63 mixture. Exponentially growing cells, subcultured the day prior to the experiment, were used in all of the experiments.
Drugs Blenoxane (Bristol Laboratories), a mixture of A and B group bleomycins, was dissolved just before use in 0.9% NaC1 solution to give a stock solution of 1000 pg/ml. The stock solution was then diluted to a final concetration of 25 pg/ml in the treatment medium. The treatment medium consisted of McCoy's 5A medium, with 1% heat-inactivated fetal calf serum, 1% penicillin and streptomycin, and 15 mM PIPES (piperazine.N,N-bis(2-ethanesulfonic acid), Calbiochem). Since many studies have indicated that DNA damage and cell killing by bleomycin are dependent on the pH of the treatment medium [23,24,33], PIPES buffer was used to minimize pH changes. Although bleomycin has little binding to serum components [18], low serum levels were used in an effort to minimize the effects of different serum batches. Hycanthone (Etrenol, Winthrop Products, Inc.) h y d r o x y u r e a (Sigma Chemical Corp.), streptovitacin A (Upjohn Co.), and cycloheximide (Sigma Chemical Corp.) were dissolved just before use in sterile water to make stock solutions which were then diluted to the final concentration in the treatment media. The following final concentrations were employed: 10 mM hydroxyurea, 25 #g/ml hycanthone, 25 pg/ml streptovitacin A, and 25 ug/ml cycloheximide. Experimental pro tocol The effects of the DNA-repair inhibitors on the initial frequency of bleomycin-induced chromatid aberrations were determined by treating cells with 25 #g/ml bleomycin for 30 rain with or without the simultaneous presence of the particular repair inhibitor {Fig. 1). The drugs were then washed from the RTLBX.A]lENT T PROTOCOL
ABERRATION
ABERRATION
INDUCTION
REPAIR
PERIOD
EERIOD
BLEOMYCIN + REPAIR
INHIBITOR
(A) < 30 MIN, (B)
Mutation Research, 60 (1979) 61--72
© Elsevier/North-Holland Biomedical Press
E F F E C T OF DNA REPAIR INHIBITORS ON THE INDUCTION AND REPAIR OF BLEOMYCIN-INDUCED CHROMOSOME DAMAGE
MARGUERITE A. SOGNIER *, WALTER N. HITTELMAN and POTU N. RAO Department of Deve!opmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 (U.S.A.)
(Received 30 August 1978) (Revision received 17 October 1978) (Accepted 30 October 1978)
Summary The phenomenon of premature chromosome condensation (PCC) was used to study the effects of 4 purported DNA repair inhibitors (hydroxyurea, hycanthone, cycloheximide, and streptovitacin A) on the induction and repair of chromosome aberrations after bleomycin treatment of Chinese hamster ovary cells. To test whether these repair inhibitors influenced the initial develo p m e n t of aberrations, exponentially growing populations of cells were treated with 25 gg/ml bleomycin for 30 min with or without the simultaneous presence of each inhibitor. Chromatid aberrations (gaps, breaks and exchanges) were then scored in the G2 PCC-induced immediately after treatment. None of the purported DNA-repair inhibitors tested were found to alter the initial frequency of bleomycin-induced chromatid aberrations. To determine whether these same agents had an effect on the repair of chromosome damage, cells were first treated with bleomycin in the presence of inhibitor and then incubated for 1 h in bleomycin-free media with or without the presence of inhibitor prior to cell fusion. In all cases, exchanges, once formed, were n o t repaired. While h y d r o x y u r e a and h y c a n t h o n e had no effect on the repair of chromosome damage, the protein synthesis inhibitors, cycloheximide and streptovitacin A, blocked the repair of bleomycin-induced chromatid breaks. These results suggest that protein synthesis inhibitors can prevent chromosome repair, however, the exact role of protein synthesis in the repair process remains to be elucidated. Since cycloheximide and streptovitacin A were found to inhibit the repair of chromatid breaks without altering the exchange frequency, these
* Please s e n d r e p r i n t r e q u e s t to this a u t h o r . A b b r e v i a t i o n s : BLM, b l e o m y c i n ; C H O , Chinese h a m s t e r o v a r y ; H a n k s ' BSS, H a n k s ' b a l a n c e d salt solution~ PCC, p r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n , or p r e m a t u r e l y c o n d e n s e d c h r o m o s o m e s ; PIPES~ p i p e r a z i n e - N , N - b i s ( 2 = e t h a n e s u l f o n i e acid).
62 results suggest that chromatid exchanges are not simply the product of misrepair or recombinational repair of chromatid breaks.
Chromosome damage in mammalian cells can be induced by a wide variety of agents, including X-rays and UV-irradiation [35], chemicals [31], viruses [ 26], and temperature changes [13 ]. However, little is known about the molecular basis of chromosome damage and repair. Most clastogens are known either to interact with cellular chromatin or to interfere with DNA synthesis, yet it remains uncertain how chromatin lesions are translated into chromosome aberrations. To have a better understanding of this process, we decided to study the effects of the purported inhibitors of DNA-repair synthesis on the induction and repair of bleomycin-induced chromosome damage. Until recently, chromosome aberrations could only be visualized when the cell reaches mitosis. The level of aberrations observed in mitosis, however, reflects that a m o u n t of damage initially induced in the interphase cell which has not been repaired by the time the cell reaches mitosis. For this reason, it has been difficult to measure chromosome repair by the conventional method of scoring metaphase chromosomes since the exact extent of the initial damage suffered by the cell is n o t known. Further, most DNA-repair inhibitors also block the progression of cells into mitosis. The phenomenon of premature chromosome condensation has been shown to be useful in the direct measurement of chromosome during and its repair after treatment of cells with a variety of agents, including X-rays [19,34], bleomycin [20] and adriamycin [21]. With this technique, clastogen-treated interphase cells can be fused with mitotic cells immediately after treatment (to determine the initial level of damage) or after a post-treatment incubation period (to determine the amount of chromosome repair). The objective of the present study was to employ the PCC technique to assay the effects of 4 purported inhibitors of DNA repair, viz., hydroxyurea, hycanthone, cycloheximide, and streptovitacin A, on the production and repair of bleomycin-induced chromosome aberrations in cells. Bleomycin is a mixture of glycopeptides that has been reported to bind to DNA, cause the release of free bases, and produce breaks in DNA [11,25]. Previous studies also have shown that bleomycin-induced damage is rapidly repaired at both DNA [10,23] and chromosomal levels [20], which is reflected in an increased cell survival [3]. Our results indicate that of the 4 purported DNA repair inhibitors studied, none decreased the initial frequency of bleomycin-induced chromatid aberrations. However, the inhibitors of protein synthesis, cycloheximide and streptovitacin A, blocked the repair of bleomycin-induced chromosome damage while the other two inhibitors had little or no effect on the repair process. Materials and Methods Cells Chinese hamster ovary (CHO) cells were routinely grown as monolayer cultures in Lux plastic dishes in McCoy's 5A medium (Gibco) supplemented with 16% heat-inactivated fetal calf serum (Gibco) and a 1% penicillin-streptomycin
63 mixture. Exponentially growing cells, subcultured the day prior to the experiment, were used in all of the experiments.
Drugs Blenoxane (Bristol Laboratories), a mixture of A and B group bleomycins, was dissolved just before use in 0.9% NaC1 solution to give a stock solution of 1000 pg/ml. The stock solution was then diluted to a final concetration of 25 pg/ml in the treatment medium. The treatment medium consisted of McCoy's 5A medium, with 1% heat-inactivated fetal calf serum, 1% penicillin and streptomycin, and 15 mM PIPES (piperazine.N,N-bis(2-ethanesulfonic acid), Calbiochem). Since many studies have indicated that DNA damage and cell killing by bleomycin are dependent on the pH of the treatment medium [23,24,33], PIPES buffer was used to minimize pH changes. Although bleomycin has little binding to serum components [18], low serum levels were used in an effort to minimize the effects of different serum batches. Hycanthone (Etrenol, Winthrop Products, Inc.) h y d r o x y u r e a (Sigma Chemical Corp.), streptovitacin A (Upjohn Co.), and cycloheximide (Sigma Chemical Corp.) were dissolved just before use in sterile water to make stock solutions which were then diluted to the final concentration in the treatment media. The following final concentrations were employed: 10 mM hydroxyurea, 25 #g/ml hycanthone, 25 pg/ml streptovitacin A, and 25 ug/ml cycloheximide. Experimental pro tocol The effects of the DNA-repair inhibitors on the initial frequency of bleomycin-induced chromatid aberrations were determined by treating cells with 25 #g/ml bleomycin for 30 rain with or without the simultaneous presence of the particular repair inhibitor {Fig. 1). The drugs were then washed from the RTLBX.A]lENT T PROTOCOL
ABERRATION
ABERRATION
INDUCTION
REPAIR
PERIOD
EERIOD
BLEOMYCIN + REPAIR
INHIBITOR
(A) < 30 MIN, (B)
