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Induction of prematurely condensed chromosomes by mitoplasts
Cell Biology
international
Reports,
Vol. 4, No. 7 1, November
1980
INDUCTION OF PREMATURELY CONDENSED CHROMOSOMES BY MITOPLASTS Prasad S. Sunkara*, Abdullatif A. Al-Bader*, Mark A. Rikerl and Potu N. Raol 1Department of Developmental Therapeutics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 (USA), and *Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait ABSTRACT The objective of this study was to obtain pure prematurely condensed chromosomes (PCC) by a fusion between mitoplasts and The stabilization of mitoplasts with interphase Gl cells. spermine (25 ~.IM)and a modification of the Sendai virus-mediated fusion enabled us to obtain pure PCC without contaminating mitotic chromosomes. This study clearly suggests that the factors for premature chromosome condensation induction are present in the cytoplasm of the mitotic cells. Pure PCC obtained by this method may help us to understand the nature of the factors initiating chromosome condensation and cell division. INTRODUCTION The chromosomes of eukaryotic cells are usually visualized only during mitosis. However, it is possible to induce premature chromosome condensation of interphase cells by fusion with mitotic cells with the help of inactivated Sendai virus (Johnson and Rao, 1970). The factors present in mitotic cells condense the interphase nucleus into discrete chromosomes. The morphology of the PCC depends upon the position of the cell in the cell cycle at the time of fusion (Rao -et -9 al 1977). During the induction of premature chromosome condensation, mitotic proteins have been shown to become associated with the PCC (Rao and Johnson, 1974). The * Present address:
0309-1651/80/l
Merrell Research Center 2110 East Galbraith Road Cincinnati, Ohio 45215 U.S.A.
11025-05/$02.00/0
0 1980 Academic
Press Inc. (London)
Ltd
1026
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1980
nature of these factors is not yet clearly understood because of the difficulties in getting pure preparations of PCC without the presence of contaminating mitotic chromosomes that is essential for the characterization of mitotic factors responsible for the induction of PCC. Sunkara --et al (1977) have successfully extruded the chromosomes from mitotic cells leaving the cytoplasts of mitotic cells (mitoplasts) intact by using cytochalasin B. However, pure PCC could not be obtained by a fusion between mitoplasts and interphase cells, because the mitoplasts were easily disrupted during the fusion procedure. In the present the mitoplasts with spermine, a naturally study, we stabilized occurring polyamine known to strengthen bacterial membranes (Tabor, 1962), and successfully induced premature chromosome condensation by mitoplasts. MATERIAL AND METHODS Chemicals. A stock solution of cytochalasin B (CB) (Aldrtch Chemical Co Inc. Milwaukee, Wisconsin) was prepared by dissolving it in dim;?;hyl sulfoxide (4.0 mg/ml), and stored at 4%. Ficoll (Pharmacia Laboratories, Inc., Piscataway, N.J.) was dissolved by stirring in Eagle's minimal essential medium (MEM) with serum to final concentration of 25%, 17X, 16%, 15% and 12.5% and then stored at 4OC. Spermine was obtained from Cafbiochem (San Diego, California). B. Cells and Cell Synchrony. HeLa cells were routinely grown in a suspension culture at 37OC in MEM supplemented with nonessential amino acids, sodium pyruvate, glutamine (1% each), and 10% heat-inactivated fetal calf serum. Mitotic HeLa cells were obtained by partially synchronizing an exponentially growing culture with a single excess thymidine block, which was followed by a nitrous oxide block (Rao, 1968). The cells blocked in mitosis were harvested by selective detachment. Cells thus obtained had a mitotic index of about 98%. Gl cells were obtained by the reversal of nitrous oxide blocked mitotic cells. of Chromosomes from Mitotic Cells. C. Extrusion The density gradients were prepared as follows: polyallomer tubes were carefully filled with the following layers of Ficoll: 4 ml of 25%, 5 ml of 17X, 1 ml of 16X, 1 ml of 15%, and 5 ml of 12.52, all in complete MEM plus CB (10 Ug/ml). Mitotic cells were collected, centrifuged at 1500 rpm for 5 minutes, and resuspended in MEM complete to a final concentration of 1 x 10' cells. This cell suspension, essentially free of clumps, was applied on the already prepared gradients and overlayed with 20 ml of MEM containing CB. The gradients were then centrifuged in a Beckman (Fullerton, California) SW27 swinging bucket rotor in an L5 ultracentrifuge for 1 hour at 25,000 and 35OC. After centrifugation, cell fractions at appropriate banding interfaces were carefully aspirated with a Pasteur pipette from the top of the tube. The
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1980
1027
LEGEND Fig. 1: (A.) Gl PCC induced by a fusion between a mitotic cell and a Gl cell. The darkly stained chromosomes with two chromatids are mitotic chromosomes. The Gl PCC are thin long chromosomes with one chromatid. .(C) mid and (D) late Cl PCC were (B) early, obtained by a fusion between ReLa mitoplasts and HeLa Gl cells.
Cell Biology
International
Reports,
Vol. 4, No. 7 1, November
1980
banding fractions were diluted with 15 ml of MPMcomplete and harvested by centrifugation at 1000 for 6 minutes. The cells were washed once, then resuspended in MHMplus spermine (25 pM), plated in 35-mm Petri dishes, and incubated at 37OC in a humidified CO2 incubator for 1 hour. The purity of the mitoplasts was determined by cytocentrifuge preparations. The cytocentrifuge preparations were fixed in methanolacetic acid (3:1), air dried, and stained with aceto-orcein. D. Chromosome Preparations. The chromosome preparations were made as follows: The fusion mixture was suspended in 0.075 M potassium chloride and left at room temperature for 10 minutes. At the end of incubation, it was in Carnoy's solution (3 parts absolute methanol and 1 part glacial acetic acid) and incubated for 5 minutes at room temperature. It was resuspended in 5 ml of Carnoy's solution and spun down immediately. To the pellet, 2 to 3 drops of Carnoy's solution was added and then dropped on wet slides. The chromosome preparations were stained with Giemsa and scored for the PCC without any contaminating mitotic chromosomes. RESULTSAND DISCUSSION The purity of mitoplasts obtained ranged from 95% to 98%. A 25 pM concentration of spermine was found to be the optimum for stabilizing the membranes of the mitoplasts. Hence, the mitoplasts were incubated for 1 hour with 25 pM spermine at 37OC before fusion. Conventional fusion procedure involving 15 minutes incubation at 4'C and 45 minutes at 37OC tend to break the mitoplasts. Hence, we have standardized the fusion procedure to get better yields of PCC with mitoplasts. This involves the following: The mitoplasts and the Gl cells (2 x 106 each) were spun at 800 for 5 minutes and washed in MEMwithout serum. The supernatant was removed and 0.5 ml of ultraviolet-inactivated Sendai virus was added. It was kept at 4OC for 5 minutes, followed by a lominute incubation at 37'C. After this, 2 drops of heatinactivated fetal calf serum was added to each fusion. We were able to obtain pure PCC by fusion between mitoplasts and Gl cells (Fig. 1). About 5% of the fusion products of the mitoplasts and Gl cells exhibited PCC. The results of this study clearly indicate that it is possible to induce premature chromosome condensation by the use of mitoplasts. This study also suggests that the inducers are present in the cytoplasm of mitotic cells and are not associated with the chromosomes. The availability of large quantities of pure PCC by this procedure would enable us to isolate and characterize the factors necessary for chromosome condensation and cell division.
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1029
1980
ACKNOWLEDGEMENTS This study was supported in part by grants CA-16480, CA-19856 and CA-23878 from National Cancer Institute.
CA-11520,
REFERENCES Johnson,
R. T. and Rao, P. N.
Rao, P. N.
(1968)
Science
Rao, P. NT. and Johnson, tion in animal cells,
l&,
(1970)
Nature
226,
714-722.
774-776.
R. T. (1974) Control of cell proliferaCold Spring Harbor Laboratory Symposium,
785-800.
Rao, P. N., Wilson, 91, 131-141.
B. and Puck,
Sunkara, P. S., Al-Bader, Res., 107, 445-448. Tabor,
C. W.
Received:
(1962)
T. T.
(1979)
J. Cell
Physiol.
(1977)
Exp.
A. A. and Rao, P. N.
J. Bact.,
18th March 1980
83,
Cell
1101-1111.
Accepted:
21s.t July
1980
international
Reports,
Vol. 4, No. 7 1, November
1980
INDUCTION OF PREMATURELY CONDENSED CHROMOSOMES BY MITOPLASTS Prasad S. Sunkara*, Abdullatif A. Al-Bader*, Mark A. Rikerl and Potu N. Raol 1Department of Developmental Therapeutics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 (USA), and *Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait ABSTRACT The objective of this study was to obtain pure prematurely condensed chromosomes (PCC) by a fusion between mitoplasts and The stabilization of mitoplasts with interphase Gl cells. spermine (25 ~.IM)and a modification of the Sendai virus-mediated fusion enabled us to obtain pure PCC without contaminating mitotic chromosomes. This study clearly suggests that the factors for premature chromosome condensation induction are present in the cytoplasm of the mitotic cells. Pure PCC obtained by this method may help us to understand the nature of the factors initiating chromosome condensation and cell division. INTRODUCTION The chromosomes of eukaryotic cells are usually visualized only during mitosis. However, it is possible to induce premature chromosome condensation of interphase cells by fusion with mitotic cells with the help of inactivated Sendai virus (Johnson and Rao, 1970). The factors present in mitotic cells condense the interphase nucleus into discrete chromosomes. The morphology of the PCC depends upon the position of the cell in the cell cycle at the time of fusion (Rao -et -9 al 1977). During the induction of premature chromosome condensation, mitotic proteins have been shown to become associated with the PCC (Rao and Johnson, 1974). The * Present address:
0309-1651/80/l
Merrell Research Center 2110 East Galbraith Road Cincinnati, Ohio 45215 U.S.A.
11025-05/$02.00/0
0 1980 Academic
Press Inc. (London)
Ltd
1026
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1980
nature of these factors is not yet clearly understood because of the difficulties in getting pure preparations of PCC without the presence of contaminating mitotic chromosomes that is essential for the characterization of mitotic factors responsible for the induction of PCC. Sunkara --et al (1977) have successfully extruded the chromosomes from mitotic cells leaving the cytoplasts of mitotic cells (mitoplasts) intact by using cytochalasin B. However, pure PCC could not be obtained by a fusion between mitoplasts and interphase cells, because the mitoplasts were easily disrupted during the fusion procedure. In the present the mitoplasts with spermine, a naturally study, we stabilized occurring polyamine known to strengthen bacterial membranes (Tabor, 1962), and successfully induced premature chromosome condensation by mitoplasts. MATERIAL AND METHODS Chemicals. A stock solution of cytochalasin B (CB) (Aldrtch Chemical Co Inc. Milwaukee, Wisconsin) was prepared by dissolving it in dim;?;hyl sulfoxide (4.0 mg/ml), and stored at 4%. Ficoll (Pharmacia Laboratories, Inc., Piscataway, N.J.) was dissolved by stirring in Eagle's minimal essential medium (MEM) with serum to final concentration of 25%, 17X, 16%, 15% and 12.5% and then stored at 4OC. Spermine was obtained from Cafbiochem (San Diego, California). B. Cells and Cell Synchrony. HeLa cells were routinely grown in a suspension culture at 37OC in MEM supplemented with nonessential amino acids, sodium pyruvate, glutamine (1% each), and 10% heat-inactivated fetal calf serum. Mitotic HeLa cells were obtained by partially synchronizing an exponentially growing culture with a single excess thymidine block, which was followed by a nitrous oxide block (Rao, 1968). The cells blocked in mitosis were harvested by selective detachment. Cells thus obtained had a mitotic index of about 98%. Gl cells were obtained by the reversal of nitrous oxide blocked mitotic cells. of Chromosomes from Mitotic Cells. C. Extrusion The density gradients were prepared as follows: polyallomer tubes were carefully filled with the following layers of Ficoll: 4 ml of 25%, 5 ml of 17X, 1 ml of 16X, 1 ml of 15%, and 5 ml of 12.52, all in complete MEM plus CB (10 Ug/ml). Mitotic cells were collected, centrifuged at 1500 rpm for 5 minutes, and resuspended in MEM complete to a final concentration of 1 x 10' cells. This cell suspension, essentially free of clumps, was applied on the already prepared gradients and overlayed with 20 ml of MEM containing CB. The gradients were then centrifuged in a Beckman (Fullerton, California) SW27 swinging bucket rotor in an L5 ultracentrifuge for 1 hour at 25,000 and 35OC. After centrifugation, cell fractions at appropriate banding interfaces were carefully aspirated with a Pasteur pipette from the top of the tube. The
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1980
1027
LEGEND Fig. 1: (A.) Gl PCC induced by a fusion between a mitotic cell and a Gl cell. The darkly stained chromosomes with two chromatids are mitotic chromosomes. The Gl PCC are thin long chromosomes with one chromatid. .(C) mid and (D) late Cl PCC were (B) early, obtained by a fusion between ReLa mitoplasts and HeLa Gl cells.
Cell Biology
International
Reports,
Vol. 4, No. 7 1, November
1980
banding fractions were diluted with 15 ml of MPMcomplete and harvested by centrifugation at 1000 for 6 minutes. The cells were washed once, then resuspended in MHMplus spermine (25 pM), plated in 35-mm Petri dishes, and incubated at 37OC in a humidified CO2 incubator for 1 hour. The purity of the mitoplasts was determined by cytocentrifuge preparations. The cytocentrifuge preparations were fixed in methanolacetic acid (3:1), air dried, and stained with aceto-orcein. D. Chromosome Preparations. The chromosome preparations were made as follows: The fusion mixture was suspended in 0.075 M potassium chloride and left at room temperature for 10 minutes. At the end of incubation, it was in Carnoy's solution (3 parts absolute methanol and 1 part glacial acetic acid) and incubated for 5 minutes at room temperature. It was resuspended in 5 ml of Carnoy's solution and spun down immediately. To the pellet, 2 to 3 drops of Carnoy's solution was added and then dropped on wet slides. The chromosome preparations were stained with Giemsa and scored for the PCC without any contaminating mitotic chromosomes. RESULTSAND DISCUSSION The purity of mitoplasts obtained ranged from 95% to 98%. A 25 pM concentration of spermine was found to be the optimum for stabilizing the membranes of the mitoplasts. Hence, the mitoplasts were incubated for 1 hour with 25 pM spermine at 37OC before fusion. Conventional fusion procedure involving 15 minutes incubation at 4'C and 45 minutes at 37OC tend to break the mitoplasts. Hence, we have standardized the fusion procedure to get better yields of PCC with mitoplasts. This involves the following: The mitoplasts and the Gl cells (2 x 106 each) were spun at 800 for 5 minutes and washed in MEMwithout serum. The supernatant was removed and 0.5 ml of ultraviolet-inactivated Sendai virus was added. It was kept at 4OC for 5 minutes, followed by a lominute incubation at 37'C. After this, 2 drops of heatinactivated fetal calf serum was added to each fusion. We were able to obtain pure PCC by fusion between mitoplasts and Gl cells (Fig. 1). About 5% of the fusion products of the mitoplasts and Gl cells exhibited PCC. The results of this study clearly indicate that it is possible to induce premature chromosome condensation by the use of mitoplasts. This study also suggests that the inducers are present in the cytoplasm of mitotic cells and are not associated with the chromosomes. The availability of large quantities of pure PCC by this procedure would enable us to isolate and characterize the factors necessary for chromosome condensation and cell division.
Cell Biology
International
Reports,
Vol. 4, No. 11, November
1029
1980
ACKNOWLEDGEMENTS This study was supported in part by grants CA-16480, CA-19856 and CA-23878 from National Cancer Institute.
CA-11520,
REFERENCES Johnson,
R. T. and Rao, P. N.
Rao, P. N.
(1968)
Science
Rao, P. NT. and Johnson, tion in animal cells,
l&,
(1970)
Nature
226,
714-722.
774-776.
R. T. (1974) Control of cell proliferaCold Spring Harbor Laboratory Symposium,
785-800.
Rao, P. N., Wilson, 91, 131-141.
B. and Puck,
Sunkara, P. S., Al-Bader, Res., 107, 445-448. Tabor,
C. W.
Received:
(1962)
T. T.
(1979)
J. Cell
Physiol.
(1977)
Exp.
A. A. and Rao, P. N.
J. Bact.,
18th March 1980
83,
Cell
1101-1111.
Accepted:
21s.t July
1980