EXPERIMENTALCELL RESEARCH 2 0 0 , 215-217 (1992)
SHORT NOTE Okadaic Acid Inhibits Sister Chromatid Separation in Mammalian Cells SIBDAS G H O S H * ' t AND N E I D H A R D P A W E L E T Z *'1
*Institute of Cell and Tumor Biology, German Cancer Research Centre, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany; and tCentre of Advanced Study in Botany, University of Calcutta, Calcutta 700019, India
Mitotic HeLa cells were treated with different concentrations of okadaic acid inhibiting phosphatase 2A a c t i v i t y a l o n e or i n a d d i t i o n to p h o s p h a t a s e 1 a c t i v i t y . Phosphatase 2A inhibition alone had no visible effect on mitosis, but inhibition of both phosphatase 1 and 2A produced mitotic abnormalities, including inhibition of anaphase mimicking the effect of colchicine. Recovery e x p e r i m e n t s i n o k a d a i c a c i d - f r e e m e d i u m s h o w e d formation of diplochromosomes, indicating a failure of sister chromatid separation in the treated mitotic cells. The universality of the phosphatase 1 requirement in s i s t e r c h r o m a t i d s e p a r a t i o n is d i s c u s s e d . 9 1992 Academic Press, Inc.
INTRODUCTION Earlier we have shown that several mitotic events are dissociable from each other [1-4] and they may be controlled by independent factors. Genetic experiments [5, 6] indicate that sister chromatid separation is a distinct mitotic event, dissociable from other events such as chromosome condensation, nuclear envelope breakdown, spindle formation, etc. In ts-Bim G mutants in Aspergillus nidulans [5] and also in cs-dis mutants in fission yeast [6] sister chromatids fail to separate and anaphase separation is incomplete. It is of interest to note that both these mutants fail to encode phosphoprotein phosphatase I, and yeast and mammalian phosphatases are remarkably similar [7]. The present investigations were designed to see the effect of phosphatase 1 inhibition on mitosis, specially in relation to sister chromatid separation in mammalian cells. In recent years okadaic acid has been reported to be a specific inhibitor of phosphatase 2A and phosphatase 1 [8]. If sister chromatid separation is really a dis-
1 To whom correspondence and reprint requests should be addressed. Fax: 49-6221-402 598.
tinct mitotic event and is controlled by phosphatase 1, mitosis is expected to proceed in okadaic acid-treated cells, but the sister chromatids would fail to separate. Following a DNA-replication cycle the chromosomes should appear with four chromatids in the next mitosis. Part of our work, indicating the requirement of phosphatase 1 in sister chromatid separation in mammalian cells, is presented in this short note. MATERIALS AND METHODS Okadaic acid (a potent tumor promoter) was obtained from Boehringer (Mannheim). Different concentrations of okadaic acid (OA) were used: 1 n M (for phosphatase 2A inhibition), 12 n M (IC~o for phosphatase 1), and 100 n M (for almost total inhibition of phosphatase 1 and phosphatase 2A). As material HeLa cells were used. They were grown on tube slips and were synchronized by adding 10 m M deoxythymidine for 24 h to the medium. Eleven hours from the release from the thymidine block, when the cells started to enter mitosis, they were treated with OA. Cells were fixed at hourly intervals up to 3 h and stained with hemalum. Others were transferred to OA-free medium and were left for 24 h. Cells were treated with 100 n M OA detached from the glass surface and were further handled as cell suspension. Chromosome preparations were made from cells after 24 h recovery, following usual colcemid (2 h) and hypotonic (0.075 M KC1) treatment. The chromosomes were stained with Giemsa.
RESULTS AND DISCUSSION The sensitivity to OA is identical in almost all the species tested so far from mammals to higher plants [8]. As such HeLa cells are also expected to have identical sensitivity to OA. At a concentration of 1 n M OA had no visible effect on the mitotic process. In the following text the effect of OA at 12 n M concentration is presented and discussed. OA did not show any immediate effect on mitosis. However, within 1 h abnormal mitoses appeared. Chromosomes became highly condensed and scattered in the cytoplasm mimicking the effect of colchicine (Figs. 1, 2). Clumping of chromosomes was also evident in many cells (Fig. 3). Some interphase cells started to round up 215
0014-4827/92 $3.00 Copyright 9 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
216
SHORT NOTE and d e t a c h at about 2 h at this c o n c e n t r a t i o n . At higher c o n c e n t r a t i o n s all these events s t a r t e d m u c h earlier. Vacuolation was evident in these cells. T h i s p h e n o m e n o n has also b e e n n o t e d in O A - t r e a t e d h u m a n neurob l a s t o m a cells [9]. Mitotic cells b e c a m e a r r e s t e d i n metaphases. After 2 h no a n a p h a s e cells were observed. On recovery, the a r r e s t e d c h r o m o s o m e groups p r o d u c e d a large n u m b e r o f micronuclei forming m u l t i n u c l e a r ceils (Fig. 4). About 15% multinucleate cells was observed in p r e p a r a t i o n s t r e a t e d with 100 n M OA for 3 h followed by 24 h recovery. T h i s f r e q u e n c y is m u c h higher t h a n t h a t in control cells (less t h a n 1%). Moreover, the multinucleate cells in these p r e p a r a t i o n s h a d a large n u m b e r of small nuclei, in c o n t r a s t to a small n u m b e r of large nuclei in m u l t i n u c l e a t e cells in control p r e p a r a t i o n s . All these effects can be c o m p a r e d to those of colchicine. T h e main effect of colchicine is on the microtubules. W e have u n d e r t a k e n detailed u l t r a s t r u c t u r a l a n d i m m u n o cytochemical studies on O A - t r e a t e d mitotic cells. In these cells, unlike in colchicine-treated cells, microtubules are formed, b u t t h e y b e c o m e d e r a n g e d (details with o t h e r observations on p o s t m e t a p h a s e events will be published elsewhere). Moreover, c h r o m o s o m e p r e p a rations f r o m O A - t r e a t e d cells after 24 h recovery showed the f o r m a t i o n of d i p l o c h r o m o s o m e s in the seco n d cell generation (Fig. 5). Cell t r e a t e d with 12 n M OA showed a b o u t 20% of the mitotic cells c o n t a i n e d dip l o c h r o m o s o m e s . T r e a t m e n t with 100 n M OA revealed a m u c h higher p e r c e n t a g e (over 50%). In our control p r e p a r a t i o n s no cell with d i p l o c h r o m o s o m e s could be observed. Evidently, the sister c h r o m a t i d s e p a r a t i o n was affected in O A - t r e a t e d mitotic cells. Colchicine, in contrast, c a n n o t inhibit c h r o m a t i d s e p a r a t i o n [10]. T h e results clearly indicate t h a t OA inhibits sister c h r o m a tid s e p a r a t i o n at c o n c e n t r a t i o n s inhibiting b o t h phosp h a t a s e 1 a n d 2A. P h o s p h a t a s e 2A inhibition alone has no effect on mitosis. As such the effect m a y be conside r e d either due to inhibition of p h o s p h a t a s e 1 alone or due to inhibition of b o t h p h o s p h a t a s e 1 a n d 2A. However, genetic e x p e r i m e n t s indicate the i n v o l v e m e n t only o f p h o s p h a t a s e 1 in sister c h r o m a t i d separation. In tsB i m G m u t a n t s of AspergiUus [5] a n d cs-dis m u t a n t s of fission y e a s t [6] failure of sister c h r o m a t i d s e p a r a t i o n is associated with the lack of synthesis of p h o s p h a t a s e 1 alone. A Drosophila m u t a n t at locus 87 B 6-12 of chrom o s o m e 3 is also deficient in p h o s p h a t a s e 1 activity a n d dies at the larval stage [8, 11]. T h e mitotic cells were f o u n d to be defective during a n a p h a s e separation. T h u s
FIGS. 1 and 2. HeLa cells treated with okadaic acid (12 nM) for 1 h (Fig. 1) and 2 h (Fig. 2) showing condensed and scattered chromosomes in metaphase. FIG. 3. Metaphase cells with clumped chromosomes. Vacuola-
tion in these cells is evident. Note rounding up of the interphase cells accompanied by blebbing. FIG. 4. A multinucleate cell with a number of micronuclei formed after okadaic acid treatment. FIG. 5. Spread preparation from cells 24 h after recovery shows ing formation of diplochromosomes.
SHORT NOTE
the requirement of phosphatase 1 activity for sister chromatid separation in mitosis seems to be universal. Recently a number of proteins has been located at the centromeric regions of the chromosomes as CENPs, INCENPs, and CLiPs [12-14]. It is possible that some of them are the substrate of phosphatase 1 and are involved in sister chromatid separation. However, it is not known whether they remain phosphorylated at metaphase or whether they become dephosphorylated at the onset of anaphase. The involvement of phosphatase 1 in sister chromatid separation suggests a dephosphorylation process during this event. The failure of sister chromatid separation may also be caused by OA indirectly. An overcondensation caused by OA may render the substrate inaccessible. In fission yeast phosphatase 2A inhibition has been reported to induce premature mitosis [15]. From observations in Xenopus, Lee et al. [16] infer that phosphatase 2A might be required to reverse a phosphorylation needed for M P F activation. It may influence the initiation of mitosis, but unlike phosphatase 1, may not affect the mitotic progression. Our detailed studies into these aspects are in progress. This work is part of an Indo-German science collaboration program between GSF (Munich) and ICMR (New Delhi). The support is gratefully acknowledged. We thank Mr. G. Withers for the photographic work and Ms. C. Kamp and Ms. I. Purkert for typing the manuscript. Received October 28, 1991 Revised version received January 13, 1992
217 REFERENCES
1. Ghosh, S., and Paweletz, N. (1984) Chromosoma 90, 57-67. 2. Ghosh, S., and Paweletz, N. (1987) Chromosoma 95, 136-143. 3. Ghosh, S., and Paweletz, N. (1987) Exp. Cell Res. 171,243-249. 4. Ghosh, S., Paweletz, N., and Armas-Portela, R. (1988) CellBiol. Int. Rep. 12, 443-447. 5. Doonan, J. H., and Morris, R. (1989) Cell 57, 987-996. 6. Ohkura, H., Kinosita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007. 7. Cohen, P., Schelling, D. L., and Stark, M. J. R. (1989) F E B S Lett. 250, 601-606. 8. Cohen, P., and Cohen, P. T. W. (1989) J. Biol. Chem. 264, 21,435-21,438. 9. Boe, R., Gjertsen, B. T., Vintermyr, O. K., Houge, G., Lanotte, M., and Doskeland, S. O. (1991) Exp. Cell Res. 195, 237-246. 10. Lambert, A. (1980) Chromosoma 76, 295-308. 11. Dombradi, V., Axton, J. M., Glover, D. M., and Cohen, P. T. W. (1989) Eur. J. Biochem. 183,603-610. 12. Earnshaw, W. C., and Rattner, J. B. (1989) in Mechanism of Chromosome Distribution and Aneuploidy (Resnick, M. A., and Vig, B. K., Eds), pp. 33-42, A. R. Liss, New York. 13. Cooke, C. A., Heck, M. M. S., and Earnshaw, W. C. (1987) J. Cell Biol. 105, 2053-2067. 14. Rattner, J. B., Kingwell, B. G., and Fritzler, M. G. (1988) Chromosoma 96, 360-367. 15. Kinoshita, N., Ohkura, H., and Yanagida, M. (1990) Cell 63, 405-415. 16. Lee, T. H., Solomon, M. J., Mumby, M. C., and Kirschner, M. W. (1991) Cell 64, 415-423.
SHORT NOTE Okadaic Acid Inhibits Sister Chromatid Separation in Mammalian Cells SIBDAS G H O S H * ' t AND N E I D H A R D P A W E L E T Z *'1
*Institute of Cell and Tumor Biology, German Cancer Research Centre, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany; and tCentre of Advanced Study in Botany, University of Calcutta, Calcutta 700019, India
Mitotic HeLa cells were treated with different concentrations of okadaic acid inhibiting phosphatase 2A a c t i v i t y a l o n e or i n a d d i t i o n to p h o s p h a t a s e 1 a c t i v i t y . Phosphatase 2A inhibition alone had no visible effect on mitosis, but inhibition of both phosphatase 1 and 2A produced mitotic abnormalities, including inhibition of anaphase mimicking the effect of colchicine. Recovery e x p e r i m e n t s i n o k a d a i c a c i d - f r e e m e d i u m s h o w e d formation of diplochromosomes, indicating a failure of sister chromatid separation in the treated mitotic cells. The universality of the phosphatase 1 requirement in s i s t e r c h r o m a t i d s e p a r a t i o n is d i s c u s s e d . 9 1992 Academic Press, Inc.
INTRODUCTION Earlier we have shown that several mitotic events are dissociable from each other [1-4] and they may be controlled by independent factors. Genetic experiments [5, 6] indicate that sister chromatid separation is a distinct mitotic event, dissociable from other events such as chromosome condensation, nuclear envelope breakdown, spindle formation, etc. In ts-Bim G mutants in Aspergillus nidulans [5] and also in cs-dis mutants in fission yeast [6] sister chromatids fail to separate and anaphase separation is incomplete. It is of interest to note that both these mutants fail to encode phosphoprotein phosphatase I, and yeast and mammalian phosphatases are remarkably similar [7]. The present investigations were designed to see the effect of phosphatase 1 inhibition on mitosis, specially in relation to sister chromatid separation in mammalian cells. In recent years okadaic acid has been reported to be a specific inhibitor of phosphatase 2A and phosphatase 1 [8]. If sister chromatid separation is really a dis-
1 To whom correspondence and reprint requests should be addressed. Fax: 49-6221-402 598.
tinct mitotic event and is controlled by phosphatase 1, mitosis is expected to proceed in okadaic acid-treated cells, but the sister chromatids would fail to separate. Following a DNA-replication cycle the chromosomes should appear with four chromatids in the next mitosis. Part of our work, indicating the requirement of phosphatase 1 in sister chromatid separation in mammalian cells, is presented in this short note. MATERIALS AND METHODS Okadaic acid (a potent tumor promoter) was obtained from Boehringer (Mannheim). Different concentrations of okadaic acid (OA) were used: 1 n M (for phosphatase 2A inhibition), 12 n M (IC~o for phosphatase 1), and 100 n M (for almost total inhibition of phosphatase 1 and phosphatase 2A). As material HeLa cells were used. They were grown on tube slips and were synchronized by adding 10 m M deoxythymidine for 24 h to the medium. Eleven hours from the release from the thymidine block, when the cells started to enter mitosis, they were treated with OA. Cells were fixed at hourly intervals up to 3 h and stained with hemalum. Others were transferred to OA-free medium and were left for 24 h. Cells were treated with 100 n M OA detached from the glass surface and were further handled as cell suspension. Chromosome preparations were made from cells after 24 h recovery, following usual colcemid (2 h) and hypotonic (0.075 M KC1) treatment. The chromosomes were stained with Giemsa.
RESULTS AND DISCUSSION The sensitivity to OA is identical in almost all the species tested so far from mammals to higher plants [8]. As such HeLa cells are also expected to have identical sensitivity to OA. At a concentration of 1 n M OA had no visible effect on the mitotic process. In the following text the effect of OA at 12 n M concentration is presented and discussed. OA did not show any immediate effect on mitosis. However, within 1 h abnormal mitoses appeared. Chromosomes became highly condensed and scattered in the cytoplasm mimicking the effect of colchicine (Figs. 1, 2). Clumping of chromosomes was also evident in many cells (Fig. 3). Some interphase cells started to round up 215
0014-4827/92 $3.00 Copyright 9 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
216
SHORT NOTE and d e t a c h at about 2 h at this c o n c e n t r a t i o n . At higher c o n c e n t r a t i o n s all these events s t a r t e d m u c h earlier. Vacuolation was evident in these cells. T h i s p h e n o m e n o n has also b e e n n o t e d in O A - t r e a t e d h u m a n neurob l a s t o m a cells [9]. Mitotic cells b e c a m e a r r e s t e d i n metaphases. After 2 h no a n a p h a s e cells were observed. On recovery, the a r r e s t e d c h r o m o s o m e groups p r o d u c e d a large n u m b e r o f micronuclei forming m u l t i n u c l e a r ceils (Fig. 4). About 15% multinucleate cells was observed in p r e p a r a t i o n s t r e a t e d with 100 n M OA for 3 h followed by 24 h recovery. T h i s f r e q u e n c y is m u c h higher t h a n t h a t in control cells (less t h a n 1%). Moreover, the multinucleate cells in these p r e p a r a t i o n s h a d a large n u m b e r of small nuclei, in c o n t r a s t to a small n u m b e r of large nuclei in m u l t i n u c l e a t e cells in control p r e p a r a t i o n s . All these effects can be c o m p a r e d to those of colchicine. T h e main effect of colchicine is on the microtubules. W e have u n d e r t a k e n detailed u l t r a s t r u c t u r a l a n d i m m u n o cytochemical studies on O A - t r e a t e d mitotic cells. In these cells, unlike in colchicine-treated cells, microtubules are formed, b u t t h e y b e c o m e d e r a n g e d (details with o t h e r observations on p o s t m e t a p h a s e events will be published elsewhere). Moreover, c h r o m o s o m e p r e p a rations f r o m O A - t r e a t e d cells after 24 h recovery showed the f o r m a t i o n of d i p l o c h r o m o s o m e s in the seco n d cell generation (Fig. 5). Cell t r e a t e d with 12 n M OA showed a b o u t 20% of the mitotic cells c o n t a i n e d dip l o c h r o m o s o m e s . T r e a t m e n t with 100 n M OA revealed a m u c h higher p e r c e n t a g e (over 50%). In our control p r e p a r a t i o n s no cell with d i p l o c h r o m o s o m e s could be observed. Evidently, the sister c h r o m a t i d s e p a r a t i o n was affected in O A - t r e a t e d mitotic cells. Colchicine, in contrast, c a n n o t inhibit c h r o m a t i d s e p a r a t i o n [10]. T h e results clearly indicate t h a t OA inhibits sister c h r o m a tid s e p a r a t i o n at c o n c e n t r a t i o n s inhibiting b o t h phosp h a t a s e 1 a n d 2A. P h o s p h a t a s e 2A inhibition alone has no effect on mitosis. As such the effect m a y be conside r e d either due to inhibition of p h o s p h a t a s e 1 alone or due to inhibition of b o t h p h o s p h a t a s e 1 a n d 2A. However, genetic e x p e r i m e n t s indicate the i n v o l v e m e n t only o f p h o s p h a t a s e 1 in sister c h r o m a t i d separation. In tsB i m G m u t a n t s of AspergiUus [5] a n d cs-dis m u t a n t s of fission y e a s t [6] failure of sister c h r o m a t i d s e p a r a t i o n is associated with the lack of synthesis of p h o s p h a t a s e 1 alone. A Drosophila m u t a n t at locus 87 B 6-12 of chrom o s o m e 3 is also deficient in p h o s p h a t a s e 1 activity a n d dies at the larval stage [8, 11]. T h e mitotic cells were f o u n d to be defective during a n a p h a s e separation. T h u s
FIGS. 1 and 2. HeLa cells treated with okadaic acid (12 nM) for 1 h (Fig. 1) and 2 h (Fig. 2) showing condensed and scattered chromosomes in metaphase. FIG. 3. Metaphase cells with clumped chromosomes. Vacuola-
tion in these cells is evident. Note rounding up of the interphase cells accompanied by blebbing. FIG. 4. A multinucleate cell with a number of micronuclei formed after okadaic acid treatment. FIG. 5. Spread preparation from cells 24 h after recovery shows ing formation of diplochromosomes.
SHORT NOTE
the requirement of phosphatase 1 activity for sister chromatid separation in mitosis seems to be universal. Recently a number of proteins has been located at the centromeric regions of the chromosomes as CENPs, INCENPs, and CLiPs [12-14]. It is possible that some of them are the substrate of phosphatase 1 and are involved in sister chromatid separation. However, it is not known whether they remain phosphorylated at metaphase or whether they become dephosphorylated at the onset of anaphase. The involvement of phosphatase 1 in sister chromatid separation suggests a dephosphorylation process during this event. The failure of sister chromatid separation may also be caused by OA indirectly. An overcondensation caused by OA may render the substrate inaccessible. In fission yeast phosphatase 2A inhibition has been reported to induce premature mitosis [15]. From observations in Xenopus, Lee et al. [16] infer that phosphatase 2A might be required to reverse a phosphorylation needed for M P F activation. It may influence the initiation of mitosis, but unlike phosphatase 1, may not affect the mitotic progression. Our detailed studies into these aspects are in progress. This work is part of an Indo-German science collaboration program between GSF (Munich) and ICMR (New Delhi). The support is gratefully acknowledged. We thank Mr. G. Withers for the photographic work and Ms. C. Kamp and Ms. I. Purkert for typing the manuscript. Received October 28, 1991 Revised version received January 13, 1992
217 REFERENCES
1. Ghosh, S., and Paweletz, N. (1984) Chromosoma 90, 57-67. 2. Ghosh, S., and Paweletz, N. (1987) Chromosoma 95, 136-143. 3. Ghosh, S., and Paweletz, N. (1987) Exp. Cell Res. 171,243-249. 4. Ghosh, S., Paweletz, N., and Armas-Portela, R. (1988) CellBiol. Int. Rep. 12, 443-447. 5. Doonan, J. H., and Morris, R. (1989) Cell 57, 987-996. 6. Ohkura, H., Kinosita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007. 7. Cohen, P., Schelling, D. L., and Stark, M. J. R. (1989) F E B S Lett. 250, 601-606. 8. Cohen, P., and Cohen, P. T. W. (1989) J. Biol. Chem. 264, 21,435-21,438. 9. Boe, R., Gjertsen, B. T., Vintermyr, O. K., Houge, G., Lanotte, M., and Doskeland, S. O. (1991) Exp. Cell Res. 195, 237-246. 10. Lambert, A. (1980) Chromosoma 76, 295-308. 11. Dombradi, V., Axton, J. M., Glover, D. M., and Cohen, P. T. W. (1989) Eur. J. Biochem. 183,603-610. 12. Earnshaw, W. C., and Rattner, J. B. (1989) in Mechanism of Chromosome Distribution and Aneuploidy (Resnick, M. A., and Vig, B. K., Eds), pp. 33-42, A. R. Liss, New York. 13. Cooke, C. A., Heck, M. M. S., and Earnshaw, W. C. (1987) J. Cell Biol. 105, 2053-2067. 14. Rattner, J. B., Kingwell, B. G., and Fritzler, M. G. (1988) Chromosoma 96, 360-367. 15. Kinoshita, N., Ohkura, H., and Yanagida, M. (1990) Cell 63, 405-415. 16. Lee, T. H., Solomon, M. J., Mumby, M. C., and Kirschner, M. W. (1991) Cell 64, 415-423.
