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Induction of chromosomal aberrations and SCE by camptothecin, and inhibitor of mammalian topoisomerase I
Mutation Research, 211 (1989) 125-130 Elsevier
125
MTR 04721
Induction of c h r o m o s o m a l aberrations and S C E by c a m p t o t h e c i n , an inhibitor of m a m m a l i a n t o p o i s o m e r a s e I * Francesca Degrassi 1, Rosella De Salvia 1, Caterina Tanzarella 1,2 and Fabrizio Palitti 1,3 l Centro di Genetica Eooluzionistica del C.N.R., c / o 2 Dipartimento di Genetica e Biologia Molecolare, Universitgt "La Sapienza', 00185 Rome (Italy) and 3 Dipartimento di Agrobiologia, Universitgt della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo (Italy)
(Received 28 September 1988) (Accepted 7 October 1988)
Keywords: Camptothecin; Chromosome aberrations; Sister-chromatid exchange
Summary The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with camptothecin (CM), an inhibitor of mammalian topoisomerase I. While no chromosome-type aberrations were found in Gl-treated cells, instead there was a dose-dependent induction of chromatid-type aberrations. These types of chromosomal alteration were not induced during the treatment itself but during the S phase, as CM is not efficiently removed with the normal washing procedure after treatment.
DNA topoisomerases are nuclear enzymes that control and modify the topological state of DNA and thereby participate in several genetic processes (for recent reviews see Liu, 1984; Wang, 1985). In particular, type I topoisomerases break and reseal one DNA strand at a time, changing linking numbers by steps of one. This enzyme has been found to be associated with transcriptionally active chromatin in mammalian cells (Weisbrod, 1982) and Drosophila polytene chromosomes (Fleischmann et al., 1984). Camptothecin (CM), an alkaloid derived from Camptotheca acuminata, has been shown to be effective against several experimental neoplasms
Correspondence: Dr. Francesca Degrassi, Centro di Genetica Evoluziortistica, c / o Dipartimento di Genetica e Biologia Molecolare, Universith "La Sapienza', Piazzale Aldo Moro 5, 00185 Rome (Italy). * This paper is dedicated to Prof. A.T. Natarajan on the occasion his 60th birthday.
(Gallo et al., 1971). Existing evidence indicates that the primary intracellular target of this compound is the mammalian enzyme topoisomerase I. Camptothecin has been shown to inhibit topoisomerase I in vitro by stabilizing tlie intermediate enzyme-associated DNA single-strand breaks (Hsiang et al., 1985). In L1210 cells and isolated nuclei CM has been reported to induce proteinconcealed DNA single-strand breaks and DNAprotein cross-links in a ratio close to 1 : 1 (Mattern et al., 1987). This suggests that CM acts by inhibiting the rejoining activity of topoisomerase I and therefore, stabilizing a 'cleavable DNA-topoisomerase complex' (Hsiang et al., 1985). Protein-concealed DNA double-strand breaks are characteristic of several antitumor drugs that have been recognized as topoisomerase II inhibitors (Zwelling et al., 1981; Pommier et al., 1982). It is suggested that these protein-concealed DNA breaks are involved in the production of chromosomal aberrations and sister-chromatid exchanges (SCE) by topoisomerase II inhibitors
0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
126
(Pommier et al., 1985; Dillehay et al., 1987; Kohn et al., 1987). Since very few data are available on the production of chromosomal alterations by CM we studied the induction of chromosomal aberrations and SCE by this chemical in different stages of the cell cycle of human lymphocytes grown in vitro. This has allowed us to investigate (i) the types of aberrations induced at the various stages of the cell cycle and the mechanism involved in the production of chromosomal alterations by CM (whether S-phase-independent or S-phase-dependent) (Kihlman and Natarajan, 1984) and (ii) the relationships between induction of protein-concealed DNA single-strand breaks and production of chromosomal aberrations and SCE. Materials
Ham's F-10 medium (Flow) supplemented with 20% fetal calf serum (Flow), antibiotics, 0.5% Lglutamine and 0.05 m g / m l phytohemagglutinin (PHA, Wellcome). Cultures were incubated at 3 7 ° C in a 5% CO 2 atmosphere and harvested at 52 h or 72 h after a 3-h pretreatment with Colcemid (0.15 #g/ml). For SCE analysis the medium contained 9 /~g/ml 5-bromodeoxyuridine (BrdUrd). Treatments were performed according to the experimental schedule in Fig. 1. In G 1 experiments cultures were incubated with CM for 1 h, starting 8 h from PHA addition. At the end of this treatment cultures were washed twice with F-10 medium and reincubated in complete medium. Half an hour later, the medium was discarded in half of the cultures, which were subsequently incubated for another half hour in fresh medium. At the end of this interval the medium was replaced again with fresh medium. Chromosome preparations were obtained by the air-drying technique. Slides from cultures grown in presence of BrdUrd were differentially stained as previously described (Palitti et al., 1982). For each experimental point, 1000 cells were scored for mitotic index, 100 cells were analyzed for M1, M2, M3+ determination and chromosomal aberrations, 20 differentially stained metaphases were scored for SCE.
and methods
Chemical Camptothecin (CM) (NSC 94600) was obtained from the National Cancer Institute, Bethesda, MD. The drug was dissolved at appropriate stock concentrations in dimethyl sulfoxide and stored at - 20 o C before use. Lymphocyte cultures and treatments Lymphocyte cultures were established from 2 healthy donors using 0.5 ml whole blood in 5 ml
CM CONTINUOUS
PHA + BrdUrd
EXPERIMENTS
..............................
Colc
0
Fix
// . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
69
72h
CM G
I
EXPERIMENTS
PHA
2W
Colc
............................ 0
8
9
51
W W ............... 9.30
10
Fix
// . . . . . . . . . . . . . . . . . 54h
C o l c Fix // . . . . . . . . . ~ . . . . . . . . 51
54h
CM + G 2
PHA
EXPERIMENTS
........................... 0
Fig. l. Expefimentalschedule.
Colc
Fix
// . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
72h
127 TABLE 1 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN H U M A N LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR THE LAST 24 h BEFORE HARVESTING Concentration
Abnormal
Aberrations per 100 cells
of camptothecin (10 -9 M)
cells (%)
Gaps
Chromatid breaks
Isochromatid breaks
Chromatid exchanges
Chromosome exchanges
Total ( - gaps)
0 1 5 10
1 1 4 8
0 1 0 1
1 0 2 4
0 0 3 0
0 0 0 4
0 0 0 0
1 0 5 8
The proliferation index (PI) values were obtained by subtracting the fraction of cells in first mitosis from the fraction of cells in third mitosis +1. Results
Fig. 2 shows the frequencies of SCE obtained following exposure of whole-blood cultures to various concentrations of CM (1-10 × 10 -9 M) for the last 24 h before harvesting. Camptothecin induced a significant increase of SCE over the control values at all tested doses in a concentration-dependent manner. At the highest doses chro-
mosomal aberrations of the chromatid type (chromatid and isochromatid breaks, chromatid exchanges) were also observed (Table 1). In the same cultures the compound also induced a cell-cycle delay as can be seen by the decrease in the proliferation index at the highest doses (Table 2). Table 3 shows that in CM treatments (1-100 × 10 -7 M) in the G~ phase of the cell cycle, only aberrations of the chromatid type are induced with a high induction of exchanges. In a previous work (De Salvia et al., 1987) we showed that a great part of the aberrations induced in G~ treatments by a topoisomerase II inhibitor (etoposide) were caused by traces of the substance remaining in the cells after the standard washing procedure, exerting their action during the S and G 2 phases. Similar results have been obtained by Andersson and Kihlman (1988) with another topoisomerase II inhibitor (m-AMSA). To explore whether this was also true for CM, a topoisomerase I inhibitor, we studied the effects of 2 further changes of medium at 30-rain interTABLE 2 MITOTIC INDEX A N D PERCENTAGE OF M1, M2, M3+ CELLS IN H U M A N LYMPHOCYTES EXPOSED TO C A M P T O T H E C I N F O R T H E LAST 24 h BEFORE HARVESTING
1
5
10
1~9MCAMPTOTHECIN
Fig. 2. Frequencies of sister-chromatid exchanges (SCE) induced in human lymphoeytes exposed to camptotheein for the last 24 h before harvesting. Bars represent standard errors.
Concentration of camptothecin (10 -9 M)
Mitotic index (%)
M~ (%)
M2 (%)
M 3+ (%)
PI
0 1 5 10
5.3 5.9 6.5 5.0
33 31 42 47
61 62 57 53
6 7 2 0
0.73 0.76 0.60 0.53
128 TABLE 3 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR 1 h IN G 1 PHASE: EFFECT OF DIFFERENT WASHING PROCEDURES Concentration of campto-
Abnormal cells
thecin (10- 7 M)
( %)
Aberrations per 100 cells Gaps
Chromatid breaks
lsochromatid breaks
Chromatid exchanges
Chromosome exchanges
Total ( - gaps)
Standard washing procedure 0 0
0
0
0
0
0
0
5 10 50 100
5 7 4 19 No mitoses No mitoses
5 2
11 16
0 0
23 37
Standard washing procedure + 1 / 2 h F-IO + washing + 1 / 2 h F-IO + washing 0 1 1 0 0 5 1 1 0 0
0 0
0 0
0 0
10 50 100
0 4 9
0 0 0
2 8 17
23 34
4 8 19
2 0 3
1 4 3
1 0 5
For experimental details see Materials and methods.
vals, after the s t a n d a r d w a s h i n g procedure. T a b l e 3 shows that the use o f n u m e r o u s changes o f m e d i u m results in a d r a m a t i c r e d u c t i o n in the frequencies o f all types of aberrations. F u r t h e r more, a m u c h w e a k e r c y t o t o x i c effect was d e t e c t e d in the cultures subjected to several changes of m e d i u m (Table 3). T h e effect of a 2oh t r e a t m e n t w i t h C M in the G2 phase o f the cell cycle is r e p o r t e d in T a b l e 4. A d o s e - d e p e n d e n t i n d u c t i o n of c h r o m o s o m a l aberrations was found. T h e a b e r r a t i o n s o b s e r v e d were
m a i n l y c h r o m a t i d breaks in c o n t r a s t to the G t t r e a t m e n t s w h e r e m o s t l y c h r o m a t i d exchanges were found. Discussion
O n the basis of the results p r e s e n t e d in this p a p e r it can be c o n c l u d e d that C M induces chrom o s o m a l al t er at i o n s n e i t h e r simply as an agent acting b y an S - p h a s e - d e p e n d e n t n o r as an S-phaseindependent mechanism. Camptothecin Gl-phase
TABLE 4 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR THE LAST 2 h BEFORE HARVESTING Concentration of camptothecin (10 7 M)
Abnormal cells (%)
Aberrations per 100 cells Gaps Chromatid breaks
Isochromatid breaks
Chromatid exchanges
Total ( - gaps)
0 10 50 100
2 8 10 19
1 1 2 5
0 2 1 4
0 2 1 1
1 8 13 22
1 4 11 17
129 t r e a t m e n t o f h u m a n l y m p h o c y t e s induces c h r o m a t i d - t y p e a b e r r a t i o n s b u t no c h r o m o s o m e - t y p e aberrations; SCE and chromosomal aberrations are d r a s t i c a l l y r e d u c e d w h e n a d d i t i o n a l c h a n g e s o f m e d i u m are p e r f o r m e d after the C M t r e a t m e n t . T h e s e d a t a show that a G t - p h a s e t r e a t m e n t d o e s n o t give rise to lesions in D N A which will persist until S phase and then produce SCE or c h r o m a t i d - t y p e a b e r r a t i o n s , b u t that, in o r d e r to i n d u c e c h r o m o s o m a l d a m a g e , C M has to b e p r e sent d u r i n g the S phase. F u r t h e r m o r e , while G l - p h a s e t r e a t m e n t does n o t give rise to c h r o m o s o m e - t y p e a b e r r a t i o n s , G2-phase t r e a t m e n t , o n the c o n t r a r y , i n d u c e s chromatid-type aberrations. The single-strand b r e a k s i n d u c e d in G 1 b y C M seem to r e s t i t u t e w i t h o u t giving rise to c h r o m o s o m a l d a m a g e if the i n h i b i t o r is efficiently r e m o v e d ; whenever, instead, the d r u g is n o t efficiently r e m o v e d , the singles t r a n d b r e a k s will persist until the S phase, giving rise to c h r o m a t i d - t y p e a b e r r a t i o n s a n d SCE. T h e effect in G 2 is u n e x p e c t e d as s i n g l e - s t r a n d b r e a k s s h o u l d n o t give rise to c h r o m a t i d - t y p e aberrations. The production of chromatid-type a b e r r a t i o n s b y C M in G2 c o u l d b e a t t r i b u t e d to the p e c u l i a r t y p e of single-strand b r e a k s i n d u c e d b y this substance, i.e., p r o t e i n - c o n c e a l e d b r e a k s , a n d to the fact that the i n h i b i t o r is n o t r e m o v e d until the cells e n t e r mitosis a n d are fixed. T h e s e p e c u l i a r lesions c o u l d interfere w i t h the v a r i o u s processes o c c u r r i n g in G 2 involved in the coiling o f the c h r o m o s o m e a n d therefore i n d u c e chrom o s o m a l d a m a g e . I t is well k n o w n t h a t the G2 p h a s e is a crucial p e r i o d in the p r o d u c t i o n o f c h r o m o s o m a l a b e r r a t i o n s (for review see K i h l m a n a n d N a t a r a j a n , 1984). Similar d a t a have b e e n o b t a i n e d in G 2 e x p e r i m e n t s with agents k n o w n to interfere with D N A synthesis a n d r e p a i r a n d w i t h l o n g - w a v e U V i r r a d i a t i o n of calls with c h r o m o s o m e - m o n o s u b s t i t u t e d with B r d U r d ( K i h l m a n et al., 1978).
Acknowledgements T h e s t u d y was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l R e s e a r c h C o u n c i l (Prog. Fin. O n c o l o g i a ) a n d from M i n i s t e r o della P u b b l i c a I s t r u z i o n e
(40~). W e wish to t h a n k Mr. M a r i o F i o r e for his excellent technical assistance.
References Andersson, H.C., and B.A. Kihlman (1988) The production of chromosomal alterations in human lymphocytes by drugs known to interfere with the activity of DNA topoisomerase II, I. m-AMSA, submitted for publication. De Salvia, R., F. Degrassi, C. Tanzarella, M. Fiore, F. Palitti, H.C. Andersson and B.A. Kihlman (1987) Induction of chromosomal aberrations and SCE by VP-16 in human lymphocytes, in: International Congress on DNA Damage and Repair, Rome, July 1987, p. 98. Dillehay, L.E., S.C. Denstman and J.R. Williams (1987) Cell cycle dependence of sister chromatid exchange induction by DNA topoisomerase II inhibitors in Chinese hamster V79 cells, Cancer Res., 47, 206-209. Fleischmann, G., G. Pflugfelder, E.K. Steiner, K. Javaherian, G.C. Howard, J.C. Wang and S.C.R. Elgin (1984) Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome, Proc. Natl. Acad. Sci. (U.S.A.), 41, 541-551. Gallo, R.C., J. Whang-Peng and R.H. Adamson (1971) Studies on the antitumor activity, mechanism of action and the cell cycle effects of camptothecin, J. Natl. Cancer Inst., 46, 789-795. Hsiang, Y., R. Hertzberg, S. Hecht and L.F. Liu (1985) Camptothecin induces protein-linked DNA-breaks via mammalian DNA topoisomerase I, J. Biol. Chem., 260, 14873-14878. Kihlman, B.A., and A.T. Natarajan (1984) Potentiation of chromosomal alterations by inhibitors of DNA repair, in: A. Collins et al. (Eds.), DNA Repair and Its Inhibition, IRL Press, Oxford, pp. 319-339. Kihlman, B.A., A.T. Natarajan and H.C. Andersson (1978) Use of the 5-bromodeoxyuridine-labelling technique for exploring mechanisms involved in the formation of chromosomal aberrations, Mutation Res., 52, 199-206. Kohn, K.W., Y. Pommier, D. Kerrigan, J. Markovits and J.M. Covey (1987) in: NCI Monographs, First Conference on DNA Topoisomerases in Cancer Chemotherapy, New York, November 1986, Vol. 4, pp. 61-71. Liu, L. (1984) DNA topoisomerases: enzymes that catalyse the breaking and rejoining of DNA, CRC Crit. Rev. Biochem., 14, 1-24. Mattern, M.R., S.M. Mong, H.F. Bartus, C.K. Mirabelli, S.T. Crooke and R.K. Johnson (1987) Relationship between the intracellular effect of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells, Cancer Res., 47, 1793-1798. Palitti, F., C. Tanzarella, R. Cozzi, R. Ricordy, E. Vitagliano and M. Fiore (1982) Comparison of the frequencies of SCEs induced by chemical mutagens in bone marrow, spleen and spermatogonial cells, Mutation Res., 103, 191-195. Pommier, Y., D. Kerrigan, R. Schwartz and L.A. Zwelling (1982) The formation and resealing of intercalator-induced DNA strand breaks in isolated L1210 cell nuclei, Biochem. Biophys. Res. Commun., 107, 576-583. Pommier, Y., L.A. Zwelling, C.S. Kao-Shan, J. Whang-Peng
130 and M.O. Bradley (1985) Correlations between intercalatorinduced D N A strand breaks and sister chromatid exchanges, mutations and cytotoxicity in Chinese hamster cells, Cancer Res., 45, 3143-3149. Wang, J.C. (1985) DNA topoisomerases, Annu. Rev. Biochem., 54, 665-697. Weisbrod, S.T. (1982) Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography, Nucl. Acids Res., 10, 2017-2042.
Zwelling, L.A., S. Michaels, L.C. Erickson, R.S. Ungerleider, M. Nichols and K.W. Kohn (1981) Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with deoxyribonucleic acid intercalating agents 4'-(9acddinylamino)methanesulfon-m-anisidide and adriamycin, Biochemistry, 20, 6553-6563.
125
MTR 04721
Induction of c h r o m o s o m a l aberrations and S C E by c a m p t o t h e c i n , an inhibitor of m a m m a l i a n t o p o i s o m e r a s e I * Francesca Degrassi 1, Rosella De Salvia 1, Caterina Tanzarella 1,2 and Fabrizio Palitti 1,3 l Centro di Genetica Eooluzionistica del C.N.R., c / o 2 Dipartimento di Genetica e Biologia Molecolare, Universitgt "La Sapienza', 00185 Rome (Italy) and 3 Dipartimento di Agrobiologia, Universitgt della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo (Italy)
(Received 28 September 1988) (Accepted 7 October 1988)
Keywords: Camptothecin; Chromosome aberrations; Sister-chromatid exchange
Summary The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with camptothecin (CM), an inhibitor of mammalian topoisomerase I. While no chromosome-type aberrations were found in Gl-treated cells, instead there was a dose-dependent induction of chromatid-type aberrations. These types of chromosomal alteration were not induced during the treatment itself but during the S phase, as CM is not efficiently removed with the normal washing procedure after treatment.
DNA topoisomerases are nuclear enzymes that control and modify the topological state of DNA and thereby participate in several genetic processes (for recent reviews see Liu, 1984; Wang, 1985). In particular, type I topoisomerases break and reseal one DNA strand at a time, changing linking numbers by steps of one. This enzyme has been found to be associated with transcriptionally active chromatin in mammalian cells (Weisbrod, 1982) and Drosophila polytene chromosomes (Fleischmann et al., 1984). Camptothecin (CM), an alkaloid derived from Camptotheca acuminata, has been shown to be effective against several experimental neoplasms
Correspondence: Dr. Francesca Degrassi, Centro di Genetica Evoluziortistica, c / o Dipartimento di Genetica e Biologia Molecolare, Universith "La Sapienza', Piazzale Aldo Moro 5, 00185 Rome (Italy). * This paper is dedicated to Prof. A.T. Natarajan on the occasion his 60th birthday.
(Gallo et al., 1971). Existing evidence indicates that the primary intracellular target of this compound is the mammalian enzyme topoisomerase I. Camptothecin has been shown to inhibit topoisomerase I in vitro by stabilizing tlie intermediate enzyme-associated DNA single-strand breaks (Hsiang et al., 1985). In L1210 cells and isolated nuclei CM has been reported to induce proteinconcealed DNA single-strand breaks and DNAprotein cross-links in a ratio close to 1 : 1 (Mattern et al., 1987). This suggests that CM acts by inhibiting the rejoining activity of topoisomerase I and therefore, stabilizing a 'cleavable DNA-topoisomerase complex' (Hsiang et al., 1985). Protein-concealed DNA double-strand breaks are characteristic of several antitumor drugs that have been recognized as topoisomerase II inhibitors (Zwelling et al., 1981; Pommier et al., 1982). It is suggested that these protein-concealed DNA breaks are involved in the production of chromosomal aberrations and sister-chromatid exchanges (SCE) by topoisomerase II inhibitors
0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
126
(Pommier et al., 1985; Dillehay et al., 1987; Kohn et al., 1987). Since very few data are available on the production of chromosomal alterations by CM we studied the induction of chromosomal aberrations and SCE by this chemical in different stages of the cell cycle of human lymphocytes grown in vitro. This has allowed us to investigate (i) the types of aberrations induced at the various stages of the cell cycle and the mechanism involved in the production of chromosomal alterations by CM (whether S-phase-independent or S-phase-dependent) (Kihlman and Natarajan, 1984) and (ii) the relationships between induction of protein-concealed DNA single-strand breaks and production of chromosomal aberrations and SCE. Materials
Ham's F-10 medium (Flow) supplemented with 20% fetal calf serum (Flow), antibiotics, 0.5% Lglutamine and 0.05 m g / m l phytohemagglutinin (PHA, Wellcome). Cultures were incubated at 3 7 ° C in a 5% CO 2 atmosphere and harvested at 52 h or 72 h after a 3-h pretreatment with Colcemid (0.15 #g/ml). For SCE analysis the medium contained 9 /~g/ml 5-bromodeoxyuridine (BrdUrd). Treatments were performed according to the experimental schedule in Fig. 1. In G 1 experiments cultures were incubated with CM for 1 h, starting 8 h from PHA addition. At the end of this treatment cultures were washed twice with F-10 medium and reincubated in complete medium. Half an hour later, the medium was discarded in half of the cultures, which were subsequently incubated for another half hour in fresh medium. At the end of this interval the medium was replaced again with fresh medium. Chromosome preparations were obtained by the air-drying technique. Slides from cultures grown in presence of BrdUrd were differentially stained as previously described (Palitti et al., 1982). For each experimental point, 1000 cells were scored for mitotic index, 100 cells were analyzed for M1, M2, M3+ determination and chromosomal aberrations, 20 differentially stained metaphases were scored for SCE.
and methods
Chemical Camptothecin (CM) (NSC 94600) was obtained from the National Cancer Institute, Bethesda, MD. The drug was dissolved at appropriate stock concentrations in dimethyl sulfoxide and stored at - 20 o C before use. Lymphocyte cultures and treatments Lymphocyte cultures were established from 2 healthy donors using 0.5 ml whole blood in 5 ml
CM CONTINUOUS
PHA + BrdUrd
EXPERIMENTS
..............................
Colc
0
Fix
// . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
69
72h
CM G
I
EXPERIMENTS
PHA
2W
Colc
............................ 0
8
9
51
W W ............... 9.30
10
Fix
// . . . . . . . . . . . . . . . . . 54h
C o l c Fix // . . . . . . . . . ~ . . . . . . . . 51
54h
CM + G 2
PHA
EXPERIMENTS
........................... 0
Fig. l. Expefimentalschedule.
Colc
Fix
// . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
72h
127 TABLE 1 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN H U M A N LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR THE LAST 24 h BEFORE HARVESTING Concentration
Abnormal
Aberrations per 100 cells
of camptothecin (10 -9 M)
cells (%)
Gaps
Chromatid breaks
Isochromatid breaks
Chromatid exchanges
Chromosome exchanges
Total ( - gaps)
0 1 5 10
1 1 4 8
0 1 0 1
1 0 2 4
0 0 3 0
0 0 0 4
0 0 0 0
1 0 5 8
The proliferation index (PI) values were obtained by subtracting the fraction of cells in first mitosis from the fraction of cells in third mitosis +1. Results
Fig. 2 shows the frequencies of SCE obtained following exposure of whole-blood cultures to various concentrations of CM (1-10 × 10 -9 M) for the last 24 h before harvesting. Camptothecin induced a significant increase of SCE over the control values at all tested doses in a concentration-dependent manner. At the highest doses chro-
mosomal aberrations of the chromatid type (chromatid and isochromatid breaks, chromatid exchanges) were also observed (Table 1). In the same cultures the compound also induced a cell-cycle delay as can be seen by the decrease in the proliferation index at the highest doses (Table 2). Table 3 shows that in CM treatments (1-100 × 10 -7 M) in the G~ phase of the cell cycle, only aberrations of the chromatid type are induced with a high induction of exchanges. In a previous work (De Salvia et al., 1987) we showed that a great part of the aberrations induced in G~ treatments by a topoisomerase II inhibitor (etoposide) were caused by traces of the substance remaining in the cells after the standard washing procedure, exerting their action during the S and G 2 phases. Similar results have been obtained by Andersson and Kihlman (1988) with another topoisomerase II inhibitor (m-AMSA). To explore whether this was also true for CM, a topoisomerase I inhibitor, we studied the effects of 2 further changes of medium at 30-rain interTABLE 2 MITOTIC INDEX A N D PERCENTAGE OF M1, M2, M3+ CELLS IN H U M A N LYMPHOCYTES EXPOSED TO C A M P T O T H E C I N F O R T H E LAST 24 h BEFORE HARVESTING
1
5
10
1~9MCAMPTOTHECIN
Fig. 2. Frequencies of sister-chromatid exchanges (SCE) induced in human lymphoeytes exposed to camptotheein for the last 24 h before harvesting. Bars represent standard errors.
Concentration of camptothecin (10 -9 M)
Mitotic index (%)
M~ (%)
M2 (%)
M 3+ (%)
PI
0 1 5 10
5.3 5.9 6.5 5.0
33 31 42 47
61 62 57 53
6 7 2 0
0.73 0.76 0.60 0.53
128 TABLE 3 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR 1 h IN G 1 PHASE: EFFECT OF DIFFERENT WASHING PROCEDURES Concentration of campto-
Abnormal cells
thecin (10- 7 M)
( %)
Aberrations per 100 cells Gaps
Chromatid breaks
lsochromatid breaks
Chromatid exchanges
Chromosome exchanges
Total ( - gaps)
Standard washing procedure 0 0
0
0
0
0
0
0
5 10 50 100
5 7 4 19 No mitoses No mitoses
5 2
11 16
0 0
23 37
Standard washing procedure + 1 / 2 h F-IO + washing + 1 / 2 h F-IO + washing 0 1 1 0 0 5 1 1 0 0
0 0
0 0
0 0
10 50 100
0 4 9
0 0 0
2 8 17
23 34
4 8 19
2 0 3
1 4 3
1 0 5
For experimental details see Materials and methods.
vals, after the s t a n d a r d w a s h i n g procedure. T a b l e 3 shows that the use o f n u m e r o u s changes o f m e d i u m results in a d r a m a t i c r e d u c t i o n in the frequencies o f all types of aberrations. F u r t h e r more, a m u c h w e a k e r c y t o t o x i c effect was d e t e c t e d in the cultures subjected to several changes of m e d i u m (Table 3). T h e effect of a 2oh t r e a t m e n t w i t h C M in the G2 phase o f the cell cycle is r e p o r t e d in T a b l e 4. A d o s e - d e p e n d e n t i n d u c t i o n of c h r o m o s o m a l aberrations was found. T h e a b e r r a t i o n s o b s e r v e d were
m a i n l y c h r o m a t i d breaks in c o n t r a s t to the G t t r e a t m e n t s w h e r e m o s t l y c h r o m a t i d exchanges were found. Discussion
O n the basis of the results p r e s e n t e d in this p a p e r it can be c o n c l u d e d that C M induces chrom o s o m a l al t er at i o n s n e i t h e r simply as an agent acting b y an S - p h a s e - d e p e n d e n t n o r as an S-phaseindependent mechanism. Camptothecin Gl-phase
TABLE 4 FREQUENCIES OF CHROMOSOMAL ABERRATIONS IN HUMAN LYMPHOCYTES EXPOSED TO CAMPTOTHECIN FOR THE LAST 2 h BEFORE HARVESTING Concentration of camptothecin (10 7 M)
Abnormal cells (%)
Aberrations per 100 cells Gaps Chromatid breaks
Isochromatid breaks
Chromatid exchanges
Total ( - gaps)
0 10 50 100
2 8 10 19
1 1 2 5
0 2 1 4
0 2 1 1
1 8 13 22
1 4 11 17
129 t r e a t m e n t o f h u m a n l y m p h o c y t e s induces c h r o m a t i d - t y p e a b e r r a t i o n s b u t no c h r o m o s o m e - t y p e aberrations; SCE and chromosomal aberrations are d r a s t i c a l l y r e d u c e d w h e n a d d i t i o n a l c h a n g e s o f m e d i u m are p e r f o r m e d after the C M t r e a t m e n t . T h e s e d a t a show that a G t - p h a s e t r e a t m e n t d o e s n o t give rise to lesions in D N A which will persist until S phase and then produce SCE or c h r o m a t i d - t y p e a b e r r a t i o n s , b u t that, in o r d e r to i n d u c e c h r o m o s o m a l d a m a g e , C M has to b e p r e sent d u r i n g the S phase. F u r t h e r m o r e , while G l - p h a s e t r e a t m e n t does n o t give rise to c h r o m o s o m e - t y p e a b e r r a t i o n s , G2-phase t r e a t m e n t , o n the c o n t r a r y , i n d u c e s chromatid-type aberrations. The single-strand b r e a k s i n d u c e d in G 1 b y C M seem to r e s t i t u t e w i t h o u t giving rise to c h r o m o s o m a l d a m a g e if the i n h i b i t o r is efficiently r e m o v e d ; whenever, instead, the d r u g is n o t efficiently r e m o v e d , the singles t r a n d b r e a k s will persist until the S phase, giving rise to c h r o m a t i d - t y p e a b e r r a t i o n s a n d SCE. T h e effect in G 2 is u n e x p e c t e d as s i n g l e - s t r a n d b r e a k s s h o u l d n o t give rise to c h r o m a t i d - t y p e aberrations. The production of chromatid-type a b e r r a t i o n s b y C M in G2 c o u l d b e a t t r i b u t e d to the p e c u l i a r t y p e of single-strand b r e a k s i n d u c e d b y this substance, i.e., p r o t e i n - c o n c e a l e d b r e a k s , a n d to the fact that the i n h i b i t o r is n o t r e m o v e d until the cells e n t e r mitosis a n d are fixed. T h e s e p e c u l i a r lesions c o u l d interfere w i t h the v a r i o u s processes o c c u r r i n g in G 2 involved in the coiling o f the c h r o m o s o m e a n d therefore i n d u c e chrom o s o m a l d a m a g e . I t is well k n o w n t h a t the G2 p h a s e is a crucial p e r i o d in the p r o d u c t i o n o f c h r o m o s o m a l a b e r r a t i o n s (for review see K i h l m a n a n d N a t a r a j a n , 1984). Similar d a t a have b e e n o b t a i n e d in G 2 e x p e r i m e n t s with agents k n o w n to interfere with D N A synthesis a n d r e p a i r a n d w i t h l o n g - w a v e U V i r r a d i a t i o n of calls with c h r o m o s o m e - m o n o s u b s t i t u t e d with B r d U r d ( K i h l m a n et al., 1978).
Acknowledgements T h e s t u d y was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l R e s e a r c h C o u n c i l (Prog. Fin. O n c o l o g i a ) a n d from M i n i s t e r o della P u b b l i c a I s t r u z i o n e
(40~). W e wish to t h a n k Mr. M a r i o F i o r e for his excellent technical assistance.
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