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Variations in the mitotic cycle in vitro
Experimental
Cell Reseurch 22. 521~525 (1961)
VARIATIONS
IN THE MITOTIC J. E. SISKEN
Department
of Experimen.tal
521
Pathology,
City
CYCLE
II\; T’ITRO1
and R. KINOSITA
of Hope
Medical
Center, Duarte,
Culifornia,
Li.S..41
Received May 5, 1960
in the length of the mitotic cycle [l , 4 j and the occurrence rif abnormal divisions have been reported for cells in tissue culture [l, li, 5 1, Swh phenomena must be taken into account in the interpretation of data derived from in uitro studies at the cellular level. Since we are now studying the timing of the syntheses of the nucleic acids in relation to the mitotic cycle in such cells, we have undertaken to assess the degree of variation in the length of the mitotic cycle in two tissue culture lines under our culture
\T~~~~.~A~~~~~~
conditions. Some of this work has been published addition, we hare made a number of observations whicl-n are included in this report.
MATERIALS
-4ND
in abstract form [9!. In on multipolar divisions
METHODS
Cells derived from Nakanishi’s kitten lung strain [G] and from Fernandes’ human amnion strain [2] were cultured in a medium made with a modified Gey’s balanced salt solution [3] (modified by doubling the glucose concentration and by increasing the magnesium chloride concentration 2.1 times), Eagle’s amino acids and vitamins (Microbiological -4ssociates), 10 per cent horse serum, 100 unit.s/ml penicillin, 70 mg/liter neomycin, 0.292 g/liter glutamine, and 0.0125 g/liter phenol red, and adjusted to pH 7.8. Stock cultures were grown in rubber-stoppered bottles. Sub-culturing was carried out every week by scrapin g the cells off the glass surface with a rubber policeman and separating them from each other by passage through a syringe. The medium was exchanged for fresh medium two or three times each week. Cultures used for experimental work were grown in Rose chambers [S]. For these cultures, cells were not completely separated from each other but left in small clumps, and the volume of cell suspension added to the chamber was so adjusted that there were only a few clumps in each chamber. All experiments were begun when Rose chamber cultures were 18 to 2-2 hours old. Cellular events were recorded by time-lapse cinematography which was carried out at 37.5 k 0.5”C, usually at. the rate of one frame per minute for periods up to 7T hours. It was determined that under these conditions the pH of the medium in the chamber dropped only 0.1 pH unit. 1 This investigation was supported ‘LT.S. Public Health Service.
by research grant 4526 from the National
Experimenfai
Cancer Institute.
Cell Reseurrh 22
J. E. Sisken and R. Kinosita
522
The length of the mitotic cycle was determined by following individual cells from the beginning of one anaphase to the beginning of the next on a motion picture editor while the number of frames was being recorded by a film-measuring machine (Neumade Products, N.Y.). RESULTS
In Figs. 1 a, 1 b, and 1 c, the time after the beginning of the motion picture recording at which a cell was first seen to divide is plotted against the time which elapsed between that division and the division of its daughter cells. It was often possible to follow both daughter cells, and these are represented by vertically paired points on the graphs. TABLE I. Length O-20 hours
Aa A” KL
24.5 21.0 22.5
of mitotic
S.D.
N
k1.7 _f2.7S ll.62
10 16 13
cycles.
20-40-t
hours
22.9 25.5
S.D.
N
k3.2 f4.8
9 14
’ From film kindly loaned to us by Dr. C. RI. Pomerat. * One cell showing an extreme.ly high value was excluded from these calculations.
Fig. 1 a and Table I contain data derived from a motion picture of Fernandes’s human amnion, a copy of which was loaned to us by Dr. C. AI. Pomerat who made the film in his laboratory when the strain was fairly young. In this culture, cells which had their first division during the first 20 hours of the motion picture recording were able to carry out their next division, on the average, in 24.5 (S.D. i 1.7) hours. After this period, an increasing amount of time was apparently required for the cells to complete their c.ycles. Similar motion pictures were made in our laboratory of the same amnion kitten lung strain (Fig. 1 c), but they line (Fig. 1 b), as well as of Nakanishi’s did not offer such clear-cut results. Although the differenc.es are probably not statistically significant, cycle time in our cultures appeared to be a little shorter at the beginning of the experiment (Table I), particularly if one very high point in each of Figs. 1 b and 1 c is disregarded. Although we did not observe any trend toward an increase in the mitotic cycle time in our cultures as they aged, in one case there was an increase in variability as shown by a three-fold increase in the standard deviation. The increase in time of the cell cycle which parallels the increase in age of Ezperimenfal
Cell Research 22
Vuriations
l6l
0
161 0
5
’
5
10
’
10
in mitotic cycle in vitro
15
’
15
20
25
30
’
’
’
25 30 20 Hours under comero
35
’
35
40
’
40
L
45
’
50
Fig. 1.
the culture
as observed in Fig. 1 (I might have been caused by the eshaustion of the medium in the chamber. B somewhat higher initial co~ce~t~atio~ of cells and therefore a more rapid exhaustion would explain why this phcnomenon was observed in Fig. 1 CLbut not in Figs. 1 b or 1 c, T’ariability between individual cells and even between two daughter cells of the same division is noticeable in all three figures. We have observed such sister cells diriding xithin as little as three minutes of each other and as far apart as 13.4 hours. Hsu’s recent report [4] shows a similar variation. In the course of our studies, we collected information on a total of 29 multipolar divisions. 1Ye were able to observe the deri~alion of such dirisions and/or the c.apabilities of the resultant daughter cells. Seven representative cases haw been listed in Table II. These observations parallel and extend the
524
J. E. Sisken and R. Kinosita
findings of Moorhead and Hsu [5]. We may summarize our findings in the following way: (1) Cells which are apparently separate can fuse to form cells with two or more nuclei which eventually result in multipolar spindles (Cell 6). (2) The nuclei involved may be from sister cells of a previous division either normal or multipolar, and the multipolar division-fusion-multipolar division cycle can be repeated by the same cells (Cells 2, 1, and perhaps 6). (3) The c.ells which result from multipolar divisions range from those that are viable and can undergo apparently normal (as well as abnormal) clivisions after a normal interval, to those which may die in a short time. Both possibilities map occur among the daughters of the same division (Cells 1, 7). TABLE
Cell
Culture
Premitotic nucleation
II.
Xnaphase polarity
Fate of daughter
cells
die shortly
division.
KL-29
Bi- or trinucleate
Tri- or tetrapolar
All daughters
IiL33
Binucleate
Tripolar
Daughters fuse to form multinucleate cell which undergoes a second multipolar division 23.9 hours later. Again daughter cells fuse to form a cell with 4 or 5 nuclei which remains this way until end of sequence.
A152
Binucleate
Tripolar
One daughter divides in 15.9 hours. Second daughter divides in 16.9 hours. Third retracts in 18.4 hours as if to divide but remains this way until sequence ends 8.2 hours later.
A152
Binucleate
Tripolar
Daughters fuse and undergo a second multipolar division 17.2 hours later. hgain fusion of daughter cells with no abnormal results up to end of sequence 5.3 hours later.
A152
l\Iononuclear
Tripolar
Two daughter cells appear as normal interphase cells until end of sequence 22.2 hours later. Third daughter divides in 17.9 hours following violent bubbling.
A152
Mononuclear
Tripolar
Two daughters fuse and retract in 19.3 hours. Third daughter remains in interphase until end of sequence 23 hours later.
X147
Bi- or trinucleate
Tripolar
First daughter divides in 22 hours. Second daughter divides in 24 hours. Third retracts at 28 hours, dies 7 hours later.
E.zperimental
Cell Research 22
after
\‘ariations
in mitotic cycle in vitro
(~4) The majority of the multipolar spindles resultecl from multinucleated ceUs (Cells 1-4, 7). Tbe mitotic capabilities of cells resulting from multipolar divisions appear most easily explainable on the basis of variant chromosome complements. For example, man>- of the tripolar divisions observed were derived from cells which contained more than a single nucleus so that chromosome numbers different from those of the parent nuclei would be expected in the resultant daughter cells. In addition, we hare observed chromosome abnormalities in stained preparations of our cultures, and rariations in chromosome numbers hare been reported for many lines of cells in tissue culture, including the lines used in the present studies [6, 71. Variations in chromosome complements due, perhaps, to non disjunction could even account for differences observed in the mitotic cycle times between the two daughter cells of a bipolar division. However, this is not the only possible explanation. For example, a slightly unequal distribution of cytoplasm during division might give one of the daughter cells a larger share of one or more essential cptoplasmic components which xi11 enable that cell to diride sooner. SUMMARY
1. Under our conditions, wide variations are apparent in mitotic cycle times of cells within a single culture and e\-en between the two daughter cells of a single division. 2. hlultipolar divisions often result from cells with more than one nucleus and the fate of the daughters of these divisions may range from immediate death to a repeated multipolar division-fusion-multipolar division cycle. 4. The observed variabilities must be taken into account in the interpretation of data derived from in ~tif~o studies at the cellular level. REFERENCES 1. FELL, H. B. and HUGHES, A. F., Quurl. J. .Uicroscop. Sci. 90: 355 (1949). 2. FERNASDES, M. V., Texas Repfs. Biol. Med. 16, 48 (1958). 3. GEP, G. 0. and GEY, hl. K., Am. J. Cancer 27, 45 (193F). -1. Hsu, T. C., Texus Repts. Biol. Med. 18, 31 (1960). 5. MOOHHEAD, P. S. and Hsu, T. C., J. Matl. Cuncer Inst. 16, 1047 (1956). 6. NAKAPI’ISHI, Y. H., Z. Zellforsch. 51. 138 (1960). 7. NAUNISHI. Y. H., FERNANDES, 31. V., MIZUTAKI, &I. and POSIERAT, C. N., Med. 17, 345 (1959). 8. ROSE, G. G., Texus Repts. BioZ. Med. 12, 1074 (1954). 9. SISICE~L, J. E.. (Xbstract) Genetics 44, 536 (1959).
Experimentul
Terns
Repts. BioZ,
Gel! Research 22
Cell Reseurch 22. 521~525 (1961)
VARIATIONS
IN THE MITOTIC J. E. SISKEN
Department
of Experimen.tal
521
Pathology,
City
CYCLE
II\; T’ITRO1
and R. KINOSITA
of Hope
Medical
Center, Duarte,
Culifornia,
Li.S..41
Received May 5, 1960
in the length of the mitotic cycle [l , 4 j and the occurrence rif abnormal divisions have been reported for cells in tissue culture [l, li, 5 1, Swh phenomena must be taken into account in the interpretation of data derived from in uitro studies at the cellular level. Since we are now studying the timing of the syntheses of the nucleic acids in relation to the mitotic cycle in such cells, we have undertaken to assess the degree of variation in the length of the mitotic cycle in two tissue culture lines under our culture
\T~~~~.~A~~~~~~
conditions. Some of this work has been published addition, we hare made a number of observations whicl-n are included in this report.
MATERIALS
-4ND
in abstract form [9!. In on multipolar divisions
METHODS
Cells derived from Nakanishi’s kitten lung strain [G] and from Fernandes’ human amnion strain [2] were cultured in a medium made with a modified Gey’s balanced salt solution [3] (modified by doubling the glucose concentration and by increasing the magnesium chloride concentration 2.1 times), Eagle’s amino acids and vitamins (Microbiological -4ssociates), 10 per cent horse serum, 100 unit.s/ml penicillin, 70 mg/liter neomycin, 0.292 g/liter glutamine, and 0.0125 g/liter phenol red, and adjusted to pH 7.8. Stock cultures were grown in rubber-stoppered bottles. Sub-culturing was carried out every week by scrapin g the cells off the glass surface with a rubber policeman and separating them from each other by passage through a syringe. The medium was exchanged for fresh medium two or three times each week. Cultures used for experimental work were grown in Rose chambers [S]. For these cultures, cells were not completely separated from each other but left in small clumps, and the volume of cell suspension added to the chamber was so adjusted that there were only a few clumps in each chamber. All experiments were begun when Rose chamber cultures were 18 to 2-2 hours old. Cellular events were recorded by time-lapse cinematography which was carried out at 37.5 k 0.5”C, usually at. the rate of one frame per minute for periods up to 7T hours. It was determined that under these conditions the pH of the medium in the chamber dropped only 0.1 pH unit. 1 This investigation was supported ‘LT.S. Public Health Service.
by research grant 4526 from the National
Experimenfai
Cancer Institute.
Cell Reseurrh 22
J. E. Sisken and R. Kinosita
522
The length of the mitotic cycle was determined by following individual cells from the beginning of one anaphase to the beginning of the next on a motion picture editor while the number of frames was being recorded by a film-measuring machine (Neumade Products, N.Y.). RESULTS
In Figs. 1 a, 1 b, and 1 c, the time after the beginning of the motion picture recording at which a cell was first seen to divide is plotted against the time which elapsed between that division and the division of its daughter cells. It was often possible to follow both daughter cells, and these are represented by vertically paired points on the graphs. TABLE I. Length O-20 hours
Aa A” KL
24.5 21.0 22.5
of mitotic
S.D.
N
k1.7 _f2.7S ll.62
10 16 13
cycles.
20-40-t
hours
22.9 25.5
S.D.
N
k3.2 f4.8
9 14
’ From film kindly loaned to us by Dr. C. RI. Pomerat. * One cell showing an extreme.ly high value was excluded from these calculations.
Fig. 1 a and Table I contain data derived from a motion picture of Fernandes’s human amnion, a copy of which was loaned to us by Dr. C. AI. Pomerat who made the film in his laboratory when the strain was fairly young. In this culture, cells which had their first division during the first 20 hours of the motion picture recording were able to carry out their next division, on the average, in 24.5 (S.D. i 1.7) hours. After this period, an increasing amount of time was apparently required for the cells to complete their c.ycles. Similar motion pictures were made in our laboratory of the same amnion kitten lung strain (Fig. 1 c), but they line (Fig. 1 b), as well as of Nakanishi’s did not offer such clear-cut results. Although the differenc.es are probably not statistically significant, cycle time in our cultures appeared to be a little shorter at the beginning of the experiment (Table I), particularly if one very high point in each of Figs. 1 b and 1 c is disregarded. Although we did not observe any trend toward an increase in the mitotic cycle time in our cultures as they aged, in one case there was an increase in variability as shown by a three-fold increase in the standard deviation. The increase in time of the cell cycle which parallels the increase in age of Ezperimenfal
Cell Research 22
Vuriations
l6l
0
161 0
5
’
5
10
’
10
in mitotic cycle in vitro
15
’
15
20
25
30
’
’
’
25 30 20 Hours under comero
35
’
35
40
’
40
L
45
’
50
Fig. 1.
the culture
as observed in Fig. 1 (I might have been caused by the eshaustion of the medium in the chamber. B somewhat higher initial co~ce~t~atio~ of cells and therefore a more rapid exhaustion would explain why this phcnomenon was observed in Fig. 1 CLbut not in Figs. 1 b or 1 c, T’ariability between individual cells and even between two daughter cells of the same division is noticeable in all three figures. We have observed such sister cells diriding xithin as little as three minutes of each other and as far apart as 13.4 hours. Hsu’s recent report [4] shows a similar variation. In the course of our studies, we collected information on a total of 29 multipolar divisions. 1Ye were able to observe the deri~alion of such dirisions and/or the c.apabilities of the resultant daughter cells. Seven representative cases haw been listed in Table II. These observations parallel and extend the
524
J. E. Sisken and R. Kinosita
findings of Moorhead and Hsu [5]. We may summarize our findings in the following way: (1) Cells which are apparently separate can fuse to form cells with two or more nuclei which eventually result in multipolar spindles (Cell 6). (2) The nuclei involved may be from sister cells of a previous division either normal or multipolar, and the multipolar division-fusion-multipolar division cycle can be repeated by the same cells (Cells 2, 1, and perhaps 6). (3) The c.ells which result from multipolar divisions range from those that are viable and can undergo apparently normal (as well as abnormal) clivisions after a normal interval, to those which may die in a short time. Both possibilities map occur among the daughters of the same division (Cells 1, 7). TABLE
Cell
Culture
Premitotic nucleation
II.
Xnaphase polarity
Fate of daughter
cells
die shortly
division.
KL-29
Bi- or trinucleate
Tri- or tetrapolar
All daughters
IiL33
Binucleate
Tripolar
Daughters fuse to form multinucleate cell which undergoes a second multipolar division 23.9 hours later. Again daughter cells fuse to form a cell with 4 or 5 nuclei which remains this way until end of sequence.
A152
Binucleate
Tripolar
One daughter divides in 15.9 hours. Second daughter divides in 16.9 hours. Third retracts in 18.4 hours as if to divide but remains this way until sequence ends 8.2 hours later.
A152
Binucleate
Tripolar
Daughters fuse and undergo a second multipolar division 17.2 hours later. hgain fusion of daughter cells with no abnormal results up to end of sequence 5.3 hours later.
A152
l\Iononuclear
Tripolar
Two daughter cells appear as normal interphase cells until end of sequence 22.2 hours later. Third daughter divides in 17.9 hours following violent bubbling.
A152
Mononuclear
Tripolar
Two daughters fuse and retract in 19.3 hours. Third daughter remains in interphase until end of sequence 23 hours later.
X147
Bi- or trinucleate
Tripolar
First daughter divides in 22 hours. Second daughter divides in 24 hours. Third retracts at 28 hours, dies 7 hours later.
E.zperimental
Cell Research 22
after
\‘ariations
in mitotic cycle in vitro
(~4) The majority of the multipolar spindles resultecl from multinucleated ceUs (Cells 1-4, 7). Tbe mitotic capabilities of cells resulting from multipolar divisions appear most easily explainable on the basis of variant chromosome complements. For example, man>- of the tripolar divisions observed were derived from cells which contained more than a single nucleus so that chromosome numbers different from those of the parent nuclei would be expected in the resultant daughter cells. In addition, we hare observed chromosome abnormalities in stained preparations of our cultures, and rariations in chromosome numbers hare been reported for many lines of cells in tissue culture, including the lines used in the present studies [6, 71. Variations in chromosome complements due, perhaps, to non disjunction could even account for differences observed in the mitotic cycle times between the two daughter cells of a bipolar division. However, this is not the only possible explanation. For example, a slightly unequal distribution of cytoplasm during division might give one of the daughter cells a larger share of one or more essential cptoplasmic components which xi11 enable that cell to diride sooner. SUMMARY
1. Under our conditions, wide variations are apparent in mitotic cycle times of cells within a single culture and e\-en between the two daughter cells of a single division. 2. hlultipolar divisions often result from cells with more than one nucleus and the fate of the daughters of these divisions may range from immediate death to a repeated multipolar division-fusion-multipolar division cycle. 4. The observed variabilities must be taken into account in the interpretation of data derived from in ~tif~o studies at the cellular level. REFERENCES 1. FELL, H. B. and HUGHES, A. F., Quurl. J. .Uicroscop. Sci. 90: 355 (1949). 2. FERNASDES, M. V., Texas Repfs. Biol. Med. 16, 48 (1958). 3. GEP, G. 0. and GEY, hl. K., Am. J. Cancer 27, 45 (193F). -1. Hsu, T. C., Texus Repts. Biol. Med. 18, 31 (1960). 5. MOOHHEAD, P. S. and Hsu, T. C., J. Matl. Cuncer Inst. 16, 1047 (1956). 6. NAKAPI’ISHI, Y. H., Z. Zellforsch. 51. 138 (1960). 7. NAUNISHI. Y. H., FERNANDES, 31. V., MIZUTAKI, &I. and POSIERAT, C. N., Med. 17, 345 (1959). 8. ROSE, G. G., Texus Repts. BioZ. Med. 12, 1074 (1954). 9. SISICE~L, J. E.. (Xbstract) Genetics 44, 536 (1959).
Experimentul
Terns
Repts. BioZ,
Gel! Research 22