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Pleomycin-induced mitotic inhibition and chromosomal abnormalities in cultured human leucocytes
SHORT COMMUNICATIONS
251
9 WITKIN, E. M., Mutation-proof and mutation-prone modes of survival in derivatives of E. coli B differing in sensitivity to ultraviolet light, Recovery and repair mechanisms in radiobiology, Brookhaven Symp. Biol., 20 (1967) 17.
Received December I3th, 1968 Mutation Res., 7 (1969) 248-251 Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
Phleomycin-induced mitotic inhibition and chromosomal abnormalities in cultured human leucocytes* MAEDA et al. 5 identified phleomycin in a culture of Streptomyces in 1966. This water-soluble antibiotic inhibited the growth of mycobacteria, had no delayed toxicity, and had an LDs0 in the mouse of 250-500 mg/kg. Phleomycin was found to have antitumor activity against mouse sarcoma 18o, adenocarcinoma 755, and Ehrlich ascites carcinoma; it was ineffective against L-I2IO leukemia 1. FALASCHI AND KORNBERG2 studied the effects of phleomycin on DNA polymerase in vitro. At low concentrations, inhibition of DNA polymerase, but not of RNA polymerase, was demonstrated. The mechanism of action of phleomycin appeared to be a binding of the DNA primer and consequent inhibition of DNA polymerase. The sites of preferential binding are areas high in adenine-thymine base pairs. The first investigation of the effects of phleomycin on chromosome structure was described by MATTINGLY~. Mitotic inhibition, decrease in DNA synthesis, and chromosome breakage occurred in Vicia faba exposed to phleomycin. The chromosomal abnormalities were present only in cells that had been exposed to phleomycin during the G2 period. KIHLMAN et al? reported obtaining chromosomal damage in cells that had been exposed to phleomycin during DNA synthesis (S period), as well as in those exposed following DNA synthesis (G2 period). They also found, contrary to reports of TANAKA et al.L that RNA synthesis in Viciafaba is inhibited almost as much as DNA synthesis. This report concerns the effects of this drug on cultured human leucocytes. Phleomycin (Lot 64LI238) was obtained as a sterile preparation from Bristol Laboratories (courtesy of Dr. WILLIAM BRADNER). Solutions of varying strengths were made using Chromosome Medium IA (Grand Island Biological Co.) as the solvent. Since phleomycin has been reported to be unstable in solution at room temperature 8, the solutions were stored at --12 °. Samples of peripheral blood were obtained from 4 adult volunteers with normal karyotypes and cultured in Chromosome Medium IA. The total incubation period was 72 h. 24 h before harvesting concentrations of o, o.I, I.O, IO, 25 and 5 ° / , g / m l of phleomycin were added to each donor's leucocytes. 50 metaphases were analyzed at each concentration, except as noted in Table I. The mitotic index of each culture was determined by counting the number of metaphases in IOOO cells. The results show clearly that phleomycin produces a marked * This study was aided by grants AM-o25o 4, TI-AM-5277 and 5-TO-2-CH-Io8I from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. Mutation Res., 7 (1969) 251-253
25 2 TABLE
SHORT COMMUNICATIONS I
THE PER CENT METAPHASES
WITH
ABNORMALITIES
AT V A R I O U S
CONCENTRATIONS
OF
PHLEOMYCIN
Donor
Phleomycin concentration (#g/ml) o o.± ±.o ±o 25
JM RN NJ JB
o 2 4 o
4 2 6 4
14 16 14 18
a Less than 5° metaphases TABLE
II
THE
32 32 3° 24
50
68 64 72a 84a
74 7° Ioo a __a
available for analysis.
MITOTIC INDEX
AT V A R I O U S C O N C E N T R A T I O N S
(THE
PERCENTAGE
OF METAPHASES
OBSERVED
IN
IOOO
CELLS)
OF P H L E O M Y C I N
Donor
Phleomycin concentration (~g /ml) 0
0.i
I.o
I0
2,5
50
JM RN NJ JB
9.9 4.1 8.6 3.0
6.9 2.2 7.2 1. 4
4.5 2.9 4.6 i.i
3 .8 0.8 3.5 0. 5
3 .6 1. 3 1.6 0. 7
2.2 0. 5 0.6 o
inhibition of mitosis and also causes severe chromosomal damage (Tables I and II). Both the mitotic inhibition and the induction of abnormalities increased as the phleomycin concentration increased. At low concentrations (o.I and I.O pg/ml) of phleomycin, almost all of the abnormal metaphases had only one aberration. The incidence of multiple aberrations within a single metaphase was generally higher at higher phleomycin concentrations; at 25 ¢~g/ml and 5o t~g/ml in some cells the chromosomes were so badly fragmented that the number of abnormalities could not be determined. In the controls the only abnormalities that were observed were gaps, and no metaphase exhibited more than one of these. The term "gap" refers to a non-staining segment of a chromatid with no visible connection between the proximal and distal portions of the chromatid and with no loss of alignment of the chromatid segments. If there is nonalignment, then the aberration is scored as a "break". In the treated cultures gaps, breaks, and acentric fragments occurred at all concentrations; dicentrics were observed at concentrations of IO/,g/inl, 25/~g/ml, and 50 Fg/ml. A number of metaphases with triradial or quadriradial formations were observed at concentrations of 25/:g/ml and 5 ° f,g/ml (Fig. I). One metaphase with a possible ring chromosome was observed at 25 F*g/ml. The marked inhibition of mitosis seen in the treated cultures may represent, at least in part, the formation of nonviable daughter cells due to extensive chromosomal damage caused by the drug. Thus, the actual capability of this substance to damage chromosomes may be greater than the percent of abnormal metaphases recovered from the culture would indicate. In addition, the in vivo production of extensively damaged but still relatively viable lymphocytes, would be of concern for two reasons. First, if the drug damages in vivo a large proportion of the cells, a difficulty might arise in maintaining a sufficient number of normally functioning lymphocytes. Also, the abnormal lymphocytes might play some role in tile genesis of disease. GERMAN3 has suggested that some quadriradial formations may represent somatic crossing-over; as a result of this process, lymphocytes with a genetic complement different from that of the host can be produced. Part of the host genome would be "foreign" to these lymphocytes, and the result could be an autoimmune disease. BIutation Res., 7 (1969) 2 5 1 - 2 5 3
253
SHORT COMMUNICATIONS
I b
,i A
! II
L iJ, j g
C
q
D
Fig. i. Examples of chromosome aberrations induced by phleomycin. A, gap. B, break. C, dicentric. D, a possible ring. E, radial configurations (triradials and quadriradials). The present investigation has shown that phleomycin damages human chromos o m e s in vitro. S i m i l a r a g e n t s w h i c h a r e i s o l a t e d i n t h e f u t u r e s h o u l d b e i n v e s t i g a t e d w i t h r e s p e c t t o t h e i r effect o n c h r o m o s o m a l s t r u c t u r e , a n d t h e r e s u l t s s h o u l d b e t a k e n i n t o c o n s i d e r a t i o n b e f o r e t h e d r u g s a r e r e l e a s e d for c l i n i c a l use. F u r t h e r e f f o r t s t o d e t e r m i n e if in vitro d a m a g e is i n d i c a t i v e of in vivo d a m a g e s h o u l d b e u n d e r t a k e n for d r u g s s u c h as p h l e o m y c i n w h i c h p r o d u c e c h r o m o s o m a l a b n o r m a l i t i e s in vitro. T h e a u t h o r s w i s h t o a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e of Miss SADDIE KING.
Genetic and Endocrine Unit, Department of Pediatrics, State University of New York, Upstate Medical Center, Syracuse, N . Y . (U.S.A.)
NORMAN F. JACOBS* RICHARD L. NEU LYTT I. GARDNER
I ]~RADNER, W., AND M. PINDELL, Antitumour properties of phleomycin, Nature, 196 (1962) 682. 2 FALASCHI, A., AND A. KORNBERG, Phleomycin, an inhibitor of DNA polymerase, Federation Proc., 23 (1964) 94 o. 3 GERMAN, J., Cytological evidence for crossing-over in vitro in h u m a n lymphoid cells, Science, 144 (1964) 298. 4 KIHLMAN, B. A., G. ODMARK AND B. HARTLEY, Studies on the effects of phleomycin on chromosome structure and nucleic acid synthesis in Viciafaba, Mutation Res., 4 (1967) 783-79 °. 5 MAZDA, K., H. I~OSAKA, K. YAGISHITA AND l~I. UMEZAWA, A new antibiotic, phleomycin, J. Antibiot. (A), 9 (1956) 82. 6 MATTINGLY, E., Induction of chromosome- and chromatid-type aberrations by phleomycin, Mutation Res., 4 (1967) 51-57 • 7 TANAKA, N., H. YAMAGUCHI AND H. UMEZAVv'A,Mechanism of action of angustmycins, mikamycins, and phleomycin, Japan. J. Med. Sci. Biol., 16 (1963) 240. 8 UMEZAWA, H., I<[. ?¢~AEDA, T. TAKEUCHI AND Y. OKAMI, New antibiotics, phleomycin A and B, J. Antibiot. (A), 19 (1966) 200.
Received January
6 t h , 1969
* Predoctoral trainee, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health.
Mutation Res., 7 (1969) 251-253
251
9 WITKIN, E. M., Mutation-proof and mutation-prone modes of survival in derivatives of E. coli B differing in sensitivity to ultraviolet light, Recovery and repair mechanisms in radiobiology, Brookhaven Symp. Biol., 20 (1967) 17.
Received December I3th, 1968 Mutation Res., 7 (1969) 248-251 Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
Phleomycin-induced mitotic inhibition and chromosomal abnormalities in cultured human leucocytes* MAEDA et al. 5 identified phleomycin in a culture of Streptomyces in 1966. This water-soluble antibiotic inhibited the growth of mycobacteria, had no delayed toxicity, and had an LDs0 in the mouse of 250-500 mg/kg. Phleomycin was found to have antitumor activity against mouse sarcoma 18o, adenocarcinoma 755, and Ehrlich ascites carcinoma; it was ineffective against L-I2IO leukemia 1. FALASCHI AND KORNBERG2 studied the effects of phleomycin on DNA polymerase in vitro. At low concentrations, inhibition of DNA polymerase, but not of RNA polymerase, was demonstrated. The mechanism of action of phleomycin appeared to be a binding of the DNA primer and consequent inhibition of DNA polymerase. The sites of preferential binding are areas high in adenine-thymine base pairs. The first investigation of the effects of phleomycin on chromosome structure was described by MATTINGLY~. Mitotic inhibition, decrease in DNA synthesis, and chromosome breakage occurred in Vicia faba exposed to phleomycin. The chromosomal abnormalities were present only in cells that had been exposed to phleomycin during the G2 period. KIHLMAN et al? reported obtaining chromosomal damage in cells that had been exposed to phleomycin during DNA synthesis (S period), as well as in those exposed following DNA synthesis (G2 period). They also found, contrary to reports of TANAKA et al.L that RNA synthesis in Viciafaba is inhibited almost as much as DNA synthesis. This report concerns the effects of this drug on cultured human leucocytes. Phleomycin (Lot 64LI238) was obtained as a sterile preparation from Bristol Laboratories (courtesy of Dr. WILLIAM BRADNER). Solutions of varying strengths were made using Chromosome Medium IA (Grand Island Biological Co.) as the solvent. Since phleomycin has been reported to be unstable in solution at room temperature 8, the solutions were stored at --12 °. Samples of peripheral blood were obtained from 4 adult volunteers with normal karyotypes and cultured in Chromosome Medium IA. The total incubation period was 72 h. 24 h before harvesting concentrations of o, o.I, I.O, IO, 25 and 5 ° / , g / m l of phleomycin were added to each donor's leucocytes. 50 metaphases were analyzed at each concentration, except as noted in Table I. The mitotic index of each culture was determined by counting the number of metaphases in IOOO cells. The results show clearly that phleomycin produces a marked * This study was aided by grants AM-o25o 4, TI-AM-5277 and 5-TO-2-CH-Io8I from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. Mutation Res., 7 (1969) 251-253
25 2 TABLE
SHORT COMMUNICATIONS I
THE PER CENT METAPHASES
WITH
ABNORMALITIES
AT V A R I O U S
CONCENTRATIONS
OF
PHLEOMYCIN
Donor
Phleomycin concentration (#g/ml) o o.± ±.o ±o 25
JM RN NJ JB
o 2 4 o
4 2 6 4
14 16 14 18
a Less than 5° metaphases TABLE
II
THE
32 32 3° 24
50
68 64 72a 84a
74 7° Ioo a __a
available for analysis.
MITOTIC INDEX
AT V A R I O U S C O N C E N T R A T I O N S
(THE
PERCENTAGE
OF METAPHASES
OBSERVED
IN
IOOO
CELLS)
OF P H L E O M Y C I N
Donor
Phleomycin concentration (~g /ml) 0
0.i
I.o
I0
2,5
50
JM RN NJ JB
9.9 4.1 8.6 3.0
6.9 2.2 7.2 1. 4
4.5 2.9 4.6 i.i
3 .8 0.8 3.5 0. 5
3 .6 1. 3 1.6 0. 7
2.2 0. 5 0.6 o
inhibition of mitosis and also causes severe chromosomal damage (Tables I and II). Both the mitotic inhibition and the induction of abnormalities increased as the phleomycin concentration increased. At low concentrations (o.I and I.O pg/ml) of phleomycin, almost all of the abnormal metaphases had only one aberration. The incidence of multiple aberrations within a single metaphase was generally higher at higher phleomycin concentrations; at 25 ¢~g/ml and 5o t~g/ml in some cells the chromosomes were so badly fragmented that the number of abnormalities could not be determined. In the controls the only abnormalities that were observed were gaps, and no metaphase exhibited more than one of these. The term "gap" refers to a non-staining segment of a chromatid with no visible connection between the proximal and distal portions of the chromatid and with no loss of alignment of the chromatid segments. If there is nonalignment, then the aberration is scored as a "break". In the treated cultures gaps, breaks, and acentric fragments occurred at all concentrations; dicentrics were observed at concentrations of IO/,g/inl, 25/~g/ml, and 50 Fg/ml. A number of metaphases with triradial or quadriradial formations were observed at concentrations of 25/:g/ml and 5 ° f,g/ml (Fig. I). One metaphase with a possible ring chromosome was observed at 25 F*g/ml. The marked inhibition of mitosis seen in the treated cultures may represent, at least in part, the formation of nonviable daughter cells due to extensive chromosomal damage caused by the drug. Thus, the actual capability of this substance to damage chromosomes may be greater than the percent of abnormal metaphases recovered from the culture would indicate. In addition, the in vivo production of extensively damaged but still relatively viable lymphocytes, would be of concern for two reasons. First, if the drug damages in vivo a large proportion of the cells, a difficulty might arise in maintaining a sufficient number of normally functioning lymphocytes. Also, the abnormal lymphocytes might play some role in tile genesis of disease. GERMAN3 has suggested that some quadriradial formations may represent somatic crossing-over; as a result of this process, lymphocytes with a genetic complement different from that of the host can be produced. Part of the host genome would be "foreign" to these lymphocytes, and the result could be an autoimmune disease. BIutation Res., 7 (1969) 2 5 1 - 2 5 3
253
SHORT COMMUNICATIONS
I b
,i A
! II
L iJ, j g
C
q
D
Fig. i. Examples of chromosome aberrations induced by phleomycin. A, gap. B, break. C, dicentric. D, a possible ring. E, radial configurations (triradials and quadriradials). The present investigation has shown that phleomycin damages human chromos o m e s in vitro. S i m i l a r a g e n t s w h i c h a r e i s o l a t e d i n t h e f u t u r e s h o u l d b e i n v e s t i g a t e d w i t h r e s p e c t t o t h e i r effect o n c h r o m o s o m a l s t r u c t u r e , a n d t h e r e s u l t s s h o u l d b e t a k e n i n t o c o n s i d e r a t i o n b e f o r e t h e d r u g s a r e r e l e a s e d for c l i n i c a l use. F u r t h e r e f f o r t s t o d e t e r m i n e if in vitro d a m a g e is i n d i c a t i v e of in vivo d a m a g e s h o u l d b e u n d e r t a k e n for d r u g s s u c h as p h l e o m y c i n w h i c h p r o d u c e c h r o m o s o m a l a b n o r m a l i t i e s in vitro. T h e a u t h o r s w i s h t o a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e of Miss SADDIE KING.
Genetic and Endocrine Unit, Department of Pediatrics, State University of New York, Upstate Medical Center, Syracuse, N . Y . (U.S.A.)
NORMAN F. JACOBS* RICHARD L. NEU LYTT I. GARDNER
I ]~RADNER, W., AND M. PINDELL, Antitumour properties of phleomycin, Nature, 196 (1962) 682. 2 FALASCHI, A., AND A. KORNBERG, Phleomycin, an inhibitor of DNA polymerase, Federation Proc., 23 (1964) 94 o. 3 GERMAN, J., Cytological evidence for crossing-over in vitro in h u m a n lymphoid cells, Science, 144 (1964) 298. 4 KIHLMAN, B. A., G. ODMARK AND B. HARTLEY, Studies on the effects of phleomycin on chromosome structure and nucleic acid synthesis in Viciafaba, Mutation Res., 4 (1967) 783-79 °. 5 MAZDA, K., H. I~OSAKA, K. YAGISHITA AND l~I. UMEZAWA, A new antibiotic, phleomycin, J. Antibiot. (A), 9 (1956) 82. 6 MATTINGLY, E., Induction of chromosome- and chromatid-type aberrations by phleomycin, Mutation Res., 4 (1967) 51-57 • 7 TANAKA, N., H. YAMAGUCHI AND H. UMEZAVv'A,Mechanism of action of angustmycins, mikamycins, and phleomycin, Japan. J. Med. Sci. Biol., 16 (1963) 240. 8 UMEZAWA, H., I<[. ?¢~AEDA, T. TAKEUCHI AND Y. OKAMI, New antibiotics, phleomycin A and B, J. Antibiot. (A), 19 (1966) 200.
Received January
6 t h , 1969
* Predoctoral trainee, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health.
Mutation Res., 7 (1969) 251-253