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Strand scission of DNA in KB cells by macromomycin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 193, No. 2, April 1, pp. 560-563, 1979
COMMUNICATION Strand
Scission
of DNA in KB Cells by Macromomycin
Macromomycin, an antitumor protein, produces strand scission of DNA in KB cells, which might appear to correlate with its concommitant inhibition of thymidine and uridine incorporation into nucleic acids. However, an acetyl derivative of macromomycin does not cause the same extent of strand breakage, yet it shows inhibitory and cytotoxic properties similar to the native protein. This is also seen in a derivative of macromomycin resulting from its reaction with the Bolton-Hunter reagent. Cesalin, another antitumor protein, does not possess DNA cleavage activity. Macromomycin is a protein, isolated from the culture filtrates of Streptomyces macromomyceticus (l), that is cytotoxic to several tumor cell lines, such as P388 and L1210 leukemias, B16 melanoma, and Lewis lung carcinoma (2). The protein has recently been obtained homogeneous (3, 4) and been shown to inhibit in viva the incorporation of thymidine into the DNA of P388 cells to less than 10% of controls within 30 min of their release &om G,/S phase synchronization (3). A similar antitumor protein, neocarzinostatin (5, S), isolated from S. carzinosfaticus (‘7) inhibits thymidine incorporation into treated cells, and the process has been correlated with strand scission of cellular DNA (8). The present studies demonstrate that the DNA in KB cells treated with macromomycin is cleaved. Modification of macromomycin by acetylation or reaction with BoltonHunter reagent yields derivatives that are cytotoxic and inhibit thymidine incorporation into cellular DNA, but the degree of strand breakage does not correlate with the eytotoxic characteristics. Crude macromomycin in dried culture filtrates of S. macromomyceticus, obtained from Dr. John D. Douros, Natural Products Branch, DTP, DCT, NCI, was purified as described previously (3). DE-52,’ a DEAE-cellulose anion exchanger, was purchased from Whatman Laboratory Products, [methyZ-3H]thymidine was from Amersham Corp., and Bolton-Hunter reagent, N-succinimidyl 3-(4hydroxyphenyl)propionate, was from Pierce Chemical Co. All other materials were of reagent grade quality and obtained from standard sources. Macromomycin was acetylated by the procedure of Montelaro and Rueckert (9) with only slight modification. The reaction products were chromatographed on a column (29 x 1.4 cm) of DE-52 equilibrated with 0.01 M Tris buffer, pH 8.0, using a gradient of O-O.2 M NaCl in the Tris buffer. No unreacted macromomycin was found, and the acetyl derivatives 1 Abbreviations diethylaminoethyl
used: MCR, macromomycin; cellulose.
0003~9861/79/040560-04$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
DE-52,
were fractionated into four peaks. The principal product (peak 2) was used in these studies. The reaction of macromomycin with the BoltonHunter reagent (10) was carried out in 0.1 M borate buffer, pH 8.5. To 10 mg of macromomycin dissolved in 2 ml of borate buffer was added 10 ~1 of methanol containing 0.46 mg of Bolton-Hunter reagent. The reaction proceeded at 4°C for 45 min, followed by dialysis overnight and chromatography on DE-52 as described for the acetylated mixture. Only two peaks were identified; most of the macromomycin was recovered unchanged. Gel electrophoresis of the acetyl and Bolton-Hunter derivatives is illustrated in Fig. 1. Kl3 cells were grown as monolayer cultures in 25cm2 Falcon flasks in Eagle’s minimum essential medium supplemented with 10% heat-inactivated fetal calf serum and Pen-strep. The cells were labeled overnight by adding 10 PCi [methyl-3H]thymidine to the culture medium, which was replaced by fresh media without labeled thymidine 15 min prior to the addition of macromomycin. The cells were harvested after appropriate times, and 20-~1 aliquots containing approximately 5 x lo4 cells placed on top of a 5-20% sucrose gradient (11) above which had been layered 400 ~1 of a solution containing 0.2% sarkosyl and 2.5% sucrose in which the cells rapidly lysed. The tubes were centrifuged at 27,000 rpm in a Beckman SW-SOL rotor for 45 min at 20°C. The gradient contents were fractionated by piercing the bottom of the plastic centrifuge tubes. The fractions (200 ~1) were collected and counted for radioactivity. The inhibition of thymidine incorporation was determined after treatment of 5 x lo5 KB cells for 30 min with macromomycin followed by a IO-min pulse of 1.0 &i of [methyl-3H]thymidine. The cells were washed three times with ice-cold isotonic phosphatebuffered saline, pH 7.5, then dissolved in 1 N NaOH followed by precipitation of macromolecules with 15% trichloroacetate. The precipitate was counted for 3H. By this method the IDso of macromomycin was 0.1 pglml; the acetyl and Bolton-Hunter derivatives 560
561
STRAND SCISSION OF DNA BY MACROMOMYCIN
FIG. 1. Gel electrophoresis of macromomycin and its derivatives on 7.5% polyacrylamide in barbital buffer, pH 7.2. a and g, macromomycin; b-e, acetyl macromomycin (Peaks l-4, respectively); f, mixture of a-e; h, macromomycin treated with Bolton-Hunter reagent; i, mixture of g and h. were approximately 10 and 190 times less inhibitory, respectively (See Table I). The results from the alkaline sucrose gradient sedimentation of cellular DNA indicate that single strand cleavage commences within 1 min and is complete by 30 min of exposure to macromomycin (Fig. 2). Little further change occurs from 15 min to 9 h of treatment, but in the neutral sucrose gradient (Fig. 3) the double strand cleavage is only apparent when single strand scission is approaching its maximum. It TABLE I EFFECTOF MACROMOMYCIN AND ITSACETYLAND BOLTON-HUNTERDERIVATIVESONTHE INCORPORATION OF(WZ&Z@~H)!~IYMIDINE INTO KB CELLS
Concentration of protein b.dnl) 25
2.5 0.25 0.925
(methyVH)Thymidine incorporation (S of control) MCR
Acetyl-MCR
BoltonHunter-MCR
4
8
37
4 19 111
44 100 103
77 102 102
would appear that the double strand fragments represent the statistical chance of two single strand breaks occurring in the same area of the helix rather 2( Alkaline
‘D 5 2 k! cc 2 5 2
Sucrose Gradient 0 Control q
1 Mmute
A 5 Minutes p
l
15 Minutes
MCR
(5pg/ml)
MCR MCR
1(
f z z c
z s
(
5
10 Fraction
15 Number
20
FIG. 2. Alkaline sucrose gradient of macromomycintreated cells. Cells treated for 1 and 5 mm were released from the culture flask prior to the addition of macromomycin to a final concentration of 5 pg/ml.
562
VANDREANDMONTGOMERY
than highly specific nucleotide sequence breaks. The extent of strand scission is also dependent upon the concentration of the macromomycin. The extent of inhibition produced by macromomycin, at a concentration of 5 pglml, increased with time; aRer 5 min, 30 min, 60 min, and 5 h of exposure to macromomytin the incorporation of thymidine into KB cells was 43, 10, 4, and 61, respectively, of the control values. The time courses of strand scission of DNA (Figs. 2 and 3) follow that for the inhibition of thymidine incoporation, but the correlation, if any, between these two events, remains to be established. It is clear that the macromomycin derivatives, when used at concentrations which produce similar effects on thymidine incorporation to native macromomycin, result in much reduced, nearly insignificant, cleavage of the single strands of DNA (Fig. 4). It is interesting to note that tallysomycin, an analog of bleomycin, shows greater antitumor activity than bleomycin but less cleavage of cellular DNA (12). Also, a different antitumor protein, cesalin (13, 14), does not produce strand scission of DNA but is very active in inhibiting the incorporation of thymidine in viwo; ID,,, 1 x 1Om6 pglml (unpublished results). Single strand cleavage of DNA by macromomycin in HeLa S3(15) and Novikoff hepatoma cells (16) has been noted. Some discrepancies exist in the published results relating to the ability of macromomycin to cleave cell free DNA and probably arise due to its interaction with the plasma membrane through which its action is effected by as yet unknown mechanisms.
Alkaline
Sucrose
Gradient
0Control
0 Bolton-Hunter MCR (50pg/ml) A Acetyl MCR (50pg/ml)
OL 0 Fraction
Number
FIG. 4. Alkaline sucrose gradient of cells treated with macromomycin derivatives. Cells were exposed to the derivatives for 30 min before being applied to the gradient. The acetyl-derivative used in all experiments corresponds to gel c, Fig. 1. The control refers to untreated KB cells centrifuged simultaneously. Cells treated with 50 pg of MCR for 30 min had a pattern similar to that seen in Fig. 2 at 15 min. ACKNOWLEDGMENT
80 Neutral
Sucrose
Gradient
~Control
Minutes MCR (5pg/ml A30 Minutes MCR 060 Mmutes MCR
The research was supported in part by Grant GM-14013 from the National Institutes of Health.
015
i /
REFERENCES 1. CHIMURA, H., ISHIZUKA, M., HAMADA, M., HORI, S., KIMURA, K., IWANAGA,J., TAKEUCHI, T., AND UMEZAWA, H. (1968) J. Antibiot. 21, 44-49. 2. LIPPMAN, M. M., LASTER, W. R., ABBOTT, B. J., VENDITTI, J., AND BARATTA, M. (1975) Cancer Res. 35, 939-945.
Fraction
Number
FIG. 3. Neutral sucrose gradient of macromomycintreated cells. No apparent change was observed before 15 min. The concentration of MCR was 5 pg/ml.
3. IM, W. B., CHIANG, C. K., AND MONTGOMERY,R. (1978) J. Biol. Chem. 253, 3259-3264. 4. YAMASHITA, T., NAOI, N., WATANABE, K., TAKEUCHI, T., AND UMEZAWA, H. (1976) J. Antibiot. 29, 415-423. 5. MAEDA, H., KUMAGAI, K., AND ISHIDA, N. (1366) J. Antibiot. Ser. A 19, 253-259. 6. MAEDA, H., GLASER, C. B., CZOMBOS,J., AND MEIENHOFER, J. (19’74) Arch. Biochem. Biophys. 164,369-378. 7. ISHIDA, N., MIYAZAKI, K., KUMAGAI, K., AND RIKIMARU, M. (1365) J. Antibiot. Ser. A 18, 68-76.
STRAND
SCISSION
8. BEERMAN, T. A., AND GOLDBERG, I. H. Biochim. Biophys. Acta. 475, 281-293. 9. MONTELARO, R. C., AND RUECKERT, R. R. J. Biol. Chem. 250, 1413-1421. 10. BOLTON, A. E., AND HUNTER, W. M. Biochem. J. 133, 529-539. 11. BEERMAN, T. A., AND GOLDBERG, I. H. Biochem. Biophys. Res. Commun. 59, 1261. 12. STRONG, J. E., AND CROOKE, S. T. Cancer Res. 38, 3322-3326.
OF DNA BY MACROMOMYCIN (1977) (1975) (1973) (1974) 1254(1978)
13. ELTING, J., AND MONTGOMERY, R. (1979) J. Biol. Chem. 254,453-458.
563
14. MONTGOMERY, R., YAMAUCHI, F., AND BRADNER, W. T. (1977) Lloydia 40, 269-274. 15. BEERMAN, T. A. (1978) Biochem. Biophys. Res. Commun. 83, 908-914. 16. SAWYER, T. H., CROOKE, S. T., AND PRESTAYKO, A. W. (1978) American Association of Cancer Research, Abstract #151, April, 1978. DALE VANDR$ REX MONTGOMERY Department of Biochemistry University of Iowa Iowa City, Iowa 52240 Received November 21,1978; revised January
2,1%+9
COMMUNICATION Strand
Scission
of DNA in KB Cells by Macromomycin
Macromomycin, an antitumor protein, produces strand scission of DNA in KB cells, which might appear to correlate with its concommitant inhibition of thymidine and uridine incorporation into nucleic acids. However, an acetyl derivative of macromomycin does not cause the same extent of strand breakage, yet it shows inhibitory and cytotoxic properties similar to the native protein. This is also seen in a derivative of macromomycin resulting from its reaction with the Bolton-Hunter reagent. Cesalin, another antitumor protein, does not possess DNA cleavage activity. Macromomycin is a protein, isolated from the culture filtrates of Streptomyces macromomyceticus (l), that is cytotoxic to several tumor cell lines, such as P388 and L1210 leukemias, B16 melanoma, and Lewis lung carcinoma (2). The protein has recently been obtained homogeneous (3, 4) and been shown to inhibit in viva the incorporation of thymidine into the DNA of P388 cells to less than 10% of controls within 30 min of their release &om G,/S phase synchronization (3). A similar antitumor protein, neocarzinostatin (5, S), isolated from S. carzinosfaticus (‘7) inhibits thymidine incorporation into treated cells, and the process has been correlated with strand scission of cellular DNA (8). The present studies demonstrate that the DNA in KB cells treated with macromomycin is cleaved. Modification of macromomycin by acetylation or reaction with BoltonHunter reagent yields derivatives that are cytotoxic and inhibit thymidine incorporation into cellular DNA, but the degree of strand breakage does not correlate with the eytotoxic characteristics. Crude macromomycin in dried culture filtrates of S. macromomyceticus, obtained from Dr. John D. Douros, Natural Products Branch, DTP, DCT, NCI, was purified as described previously (3). DE-52,’ a DEAE-cellulose anion exchanger, was purchased from Whatman Laboratory Products, [methyZ-3H]thymidine was from Amersham Corp., and Bolton-Hunter reagent, N-succinimidyl 3-(4hydroxyphenyl)propionate, was from Pierce Chemical Co. All other materials were of reagent grade quality and obtained from standard sources. Macromomycin was acetylated by the procedure of Montelaro and Rueckert (9) with only slight modification. The reaction products were chromatographed on a column (29 x 1.4 cm) of DE-52 equilibrated with 0.01 M Tris buffer, pH 8.0, using a gradient of O-O.2 M NaCl in the Tris buffer. No unreacted macromomycin was found, and the acetyl derivatives 1 Abbreviations diethylaminoethyl
used: MCR, macromomycin; cellulose.
0003~9861/79/040560-04$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
DE-52,
were fractionated into four peaks. The principal product (peak 2) was used in these studies. The reaction of macromomycin with the BoltonHunter reagent (10) was carried out in 0.1 M borate buffer, pH 8.5. To 10 mg of macromomycin dissolved in 2 ml of borate buffer was added 10 ~1 of methanol containing 0.46 mg of Bolton-Hunter reagent. The reaction proceeded at 4°C for 45 min, followed by dialysis overnight and chromatography on DE-52 as described for the acetylated mixture. Only two peaks were identified; most of the macromomycin was recovered unchanged. Gel electrophoresis of the acetyl and Bolton-Hunter derivatives is illustrated in Fig. 1. Kl3 cells were grown as monolayer cultures in 25cm2 Falcon flasks in Eagle’s minimum essential medium supplemented with 10% heat-inactivated fetal calf serum and Pen-strep. The cells were labeled overnight by adding 10 PCi [methyl-3H]thymidine to the culture medium, which was replaced by fresh media without labeled thymidine 15 min prior to the addition of macromomycin. The cells were harvested after appropriate times, and 20-~1 aliquots containing approximately 5 x lo4 cells placed on top of a 5-20% sucrose gradient (11) above which had been layered 400 ~1 of a solution containing 0.2% sarkosyl and 2.5% sucrose in which the cells rapidly lysed. The tubes were centrifuged at 27,000 rpm in a Beckman SW-SOL rotor for 45 min at 20°C. The gradient contents were fractionated by piercing the bottom of the plastic centrifuge tubes. The fractions (200 ~1) were collected and counted for radioactivity. The inhibition of thymidine incorporation was determined after treatment of 5 x lo5 KB cells for 30 min with macromomycin followed by a IO-min pulse of 1.0 &i of [methyl-3H]thymidine. The cells were washed three times with ice-cold isotonic phosphatebuffered saline, pH 7.5, then dissolved in 1 N NaOH followed by precipitation of macromolecules with 15% trichloroacetate. The precipitate was counted for 3H. By this method the IDso of macromomycin was 0.1 pglml; the acetyl and Bolton-Hunter derivatives 560
561
STRAND SCISSION OF DNA BY MACROMOMYCIN
FIG. 1. Gel electrophoresis of macromomycin and its derivatives on 7.5% polyacrylamide in barbital buffer, pH 7.2. a and g, macromomycin; b-e, acetyl macromomycin (Peaks l-4, respectively); f, mixture of a-e; h, macromomycin treated with Bolton-Hunter reagent; i, mixture of g and h. were approximately 10 and 190 times less inhibitory, respectively (See Table I). The results from the alkaline sucrose gradient sedimentation of cellular DNA indicate that single strand cleavage commences within 1 min and is complete by 30 min of exposure to macromomycin (Fig. 2). Little further change occurs from 15 min to 9 h of treatment, but in the neutral sucrose gradient (Fig. 3) the double strand cleavage is only apparent when single strand scission is approaching its maximum. It TABLE I EFFECTOF MACROMOMYCIN AND ITSACETYLAND BOLTON-HUNTERDERIVATIVESONTHE INCORPORATION OF(WZ&Z@~H)!~IYMIDINE INTO KB CELLS
Concentration of protein b.dnl) 25
2.5 0.25 0.925
(methyVH)Thymidine incorporation (S of control) MCR
Acetyl-MCR
BoltonHunter-MCR
4
8
37
4 19 111
44 100 103
77 102 102
would appear that the double strand fragments represent the statistical chance of two single strand breaks occurring in the same area of the helix rather 2( Alkaline
‘D 5 2 k! cc 2 5 2
Sucrose Gradient 0 Control q
1 Mmute
A 5 Minutes p
l
15 Minutes
MCR
(5pg/ml)
MCR MCR
1(
f z z c
z s
(
5
10 Fraction
15 Number
20
FIG. 2. Alkaline sucrose gradient of macromomycintreated cells. Cells treated for 1 and 5 mm were released from the culture flask prior to the addition of macromomycin to a final concentration of 5 pg/ml.
562
VANDREANDMONTGOMERY
than highly specific nucleotide sequence breaks. The extent of strand scission is also dependent upon the concentration of the macromomycin. The extent of inhibition produced by macromomycin, at a concentration of 5 pglml, increased with time; aRer 5 min, 30 min, 60 min, and 5 h of exposure to macromomytin the incorporation of thymidine into KB cells was 43, 10, 4, and 61, respectively, of the control values. The time courses of strand scission of DNA (Figs. 2 and 3) follow that for the inhibition of thymidine incoporation, but the correlation, if any, between these two events, remains to be established. It is clear that the macromomycin derivatives, when used at concentrations which produce similar effects on thymidine incorporation to native macromomycin, result in much reduced, nearly insignificant, cleavage of the single strands of DNA (Fig. 4). It is interesting to note that tallysomycin, an analog of bleomycin, shows greater antitumor activity than bleomycin but less cleavage of cellular DNA (12). Also, a different antitumor protein, cesalin (13, 14), does not produce strand scission of DNA but is very active in inhibiting the incorporation of thymidine in viwo; ID,,, 1 x 1Om6 pglml (unpublished results). Single strand cleavage of DNA by macromomycin in HeLa S3(15) and Novikoff hepatoma cells (16) has been noted. Some discrepancies exist in the published results relating to the ability of macromomycin to cleave cell free DNA and probably arise due to its interaction with the plasma membrane through which its action is effected by as yet unknown mechanisms.
Alkaline
Sucrose
Gradient
0Control
0 Bolton-Hunter MCR (50pg/ml) A Acetyl MCR (50pg/ml)
OL 0 Fraction
Number
FIG. 4. Alkaline sucrose gradient of cells treated with macromomycin derivatives. Cells were exposed to the derivatives for 30 min before being applied to the gradient. The acetyl-derivative used in all experiments corresponds to gel c, Fig. 1. The control refers to untreated KB cells centrifuged simultaneously. Cells treated with 50 pg of MCR for 30 min had a pattern similar to that seen in Fig. 2 at 15 min. ACKNOWLEDGMENT
80 Neutral
Sucrose
Gradient
~Control
Minutes MCR (5pg/ml A30 Minutes MCR 060 Mmutes MCR
The research was supported in part by Grant GM-14013 from the National Institutes of Health.
015
i /
REFERENCES 1. CHIMURA, H., ISHIZUKA, M., HAMADA, M., HORI, S., KIMURA, K., IWANAGA,J., TAKEUCHI, T., AND UMEZAWA, H. (1968) J. Antibiot. 21, 44-49. 2. LIPPMAN, M. M., LASTER, W. R., ABBOTT, B. J., VENDITTI, J., AND BARATTA, M. (1975) Cancer Res. 35, 939-945.
Fraction
Number
FIG. 3. Neutral sucrose gradient of macromomycintreated cells. No apparent change was observed before 15 min. The concentration of MCR was 5 pg/ml.
3. IM, W. B., CHIANG, C. K., AND MONTGOMERY,R. (1978) J. Biol. Chem. 253, 3259-3264. 4. YAMASHITA, T., NAOI, N., WATANABE, K., TAKEUCHI, T., AND UMEZAWA, H. (1976) J. Antibiot. 29, 415-423. 5. MAEDA, H., KUMAGAI, K., AND ISHIDA, N. (1366) J. Antibiot. Ser. A 19, 253-259. 6. MAEDA, H., GLASER, C. B., CZOMBOS,J., AND MEIENHOFER, J. (19’74) Arch. Biochem. Biophys. 164,369-378. 7. ISHIDA, N., MIYAZAKI, K., KUMAGAI, K., AND RIKIMARU, M. (1365) J. Antibiot. Ser. A 18, 68-76.
STRAND
SCISSION
8. BEERMAN, T. A., AND GOLDBERG, I. H. Biochim. Biophys. Acta. 475, 281-293. 9. MONTELARO, R. C., AND RUECKERT, R. R. J. Biol. Chem. 250, 1413-1421. 10. BOLTON, A. E., AND HUNTER, W. M. Biochem. J. 133, 529-539. 11. BEERMAN, T. A., AND GOLDBERG, I. H. Biochem. Biophys. Res. Commun. 59, 1261. 12. STRONG, J. E., AND CROOKE, S. T. Cancer Res. 38, 3322-3326.
OF DNA BY MACROMOMYCIN (1977) (1975) (1973) (1974) 1254(1978)
13. ELTING, J., AND MONTGOMERY, R. (1979) J. Biol. Chem. 254,453-458.
563
14. MONTGOMERY, R., YAMAUCHI, F., AND BRADNER, W. T. (1977) Lloydia 40, 269-274. 15. BEERMAN, T. A. (1978) Biochem. Biophys. Res. Commun. 83, 908-914. 16. SAWYER, T. H., CROOKE, S. T., AND PRESTAYKO, A. W. (1978) American Association of Cancer Research, Abstract #151, April, 1978. DALE VANDR$ REX MONTGOMERY Department of Biochemistry University of Iowa Iowa City, Iowa 52240 Received November 21,1978; revised January
2,1%+9