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Mitotic non-disjunction of sister chromatids and anomalous mitosis induced by low temperatures in HeLa cells
332
Experimental
Cell Research
43, 332-342
MITOTIC NON-DISJUNCTION OF SISTER CHROMATIDS AND ANOMALOUS MITOSIS INDUCED LOW TEMPERATURES IN HeLa CELLS P. N. RAO Department
(1966)
BY
and J. ENGELBERG
of Physiology and Biophysics, Lexington, KY., 40506,
University U.S.A.
of Kentucky,
Received January 21, 1966l
MITOSIS follows the classical pattern in the normal HeLa cell. At metaphase the chromosomes align in the equatorial region of the cell to form the “metaphase plate”. Anaphase begins when the sister chromatids of each chromosome separate and continues as the sister chromosomes move in synchrony towards opposite poles. These events may be observed in HeLa cells grown at temperatures from 33°C through 39°C. When the temperature of HeLa cells grown at 37°C was reduced to 29”C, however, the pattern of mitosis changed with time [S, 91. Initially the mitoses were normal in appearance though the duration of mitosis was increased; for about 12 hr the mitotic index remained at about 0.03 indicating that cells were entering mitosis at roughly the same rate at which cells in mitosis were entering interphase. The mitotic index then increased in linear fashion toward a mitotic index of about 0.36 which was reached about 60 hr after the temperature shift. During the period of mitotic index increase cells entered mitosis at a rate that exceeded the rate with which they completed mitosis. The mitosis in question was normal in some respects but abnormal in many others. (1) A mitotic spindle was present. (2) A metaphase plate of normal appearance formed. (3) The plate often subsequently became disorganized with chromosomes scattered throughout the cell. (4) Following such a diffusion of chromosomes the metaphase plate often reformed. (5) During this period there appeared to be a progressive drift of chromosomes away from the plate and an accumulation of these chromosomes at the poles. (6) In cells which divided there was finally a concerted movement of chromosomes from the equatorial region towards the poles. The number of chromosomes moving to each pole was, however, unequal. (7) Sister chromatids in a given chromosome remained attached to one another in the kinetochore region. Thus,
the chromosomes
arriving
at the poles
rather than anaphase chromosomes. 1 Revised Experimental
version Cell
received Research
May 43
16, 1966.
were
metaphase
chromosomes,
Mitotic
nondisjunction
MATERIALS
and anomalous
AND
333
mitosis
METHODS
The HeLa cell line studied was one originally isolated for suspensiongrowth by Dr Norman Salzman of the National Institute of Health. These cells were regularly grown as suspensioncultures in Eagle’s medium at 37°C. Some of the experimental techniques used in this study were described in previous publications 18, 91. In the present study direct phase-contrast microscope observations were made on living cells growing in plastic Cooper Dishes(Falcon Plastics, Inc.). To make possible the use of a high power objective (Zeiss “Neofluar” objective, 43 x , NA 0.75) the rim of the bottom dish was slightly trimmed so as to bring the bottom and the cover closer together. The ambient temperature of the culture dish on the microscope stage was maintained at constant temperature in a chamber designedby Dr Ralph Hinegardner of Columbia University, New York. This chamber consisted of two transparent plastic bags-a smaller bag enclosedwithin a larger one. The microscopewas placed within the small bag and the bag sealed around the binocular head of the microscope by a rubber band. This whole set up was placed within a bigger bag and similarly sealedleaving a small air vent to a side. Warm air was blown into the outer plastic bag from a hair drier. The inner bag was areated with a humidified mixture of carbon dioxide (4 per cent) in air. The temperature of the culture dish was monitored by means of a thermistor probe. One of the objects in the present study was to locate the relative positions of chromosomeson the mitotic spindle. Unfortunately, a HeLa cell is not a good system for the study of chromosome movements during mitosis. We were able to overcome someof the difficulties to a certain extent by making detailed observations on living mitotic cells first and then comparing these cells before and after they were fixed and stained. For this purpose the following procedure was adopted. The bottom half of the Cooper Dish was attached with Duco cement to a thin rectangular lucite plate having a circular opening in the center. The lucite plate fitted the slide holder on the microscope stage. The covered Cooper Dish was placed on the microscope stage and a field with a number of cells in mitosis was picked. The general outline of the field and the relative positions of groups of chromosomeswithin mitotic cells were sketched. After recording the position of the field, medium was removed from the dish by gentle suction and the cells were fixed in Bouin’s fluid for 2 min. Two washings with 50 per cent ethanol and one washing with absolute ethanol followed taking care that no cells were dislodged. A drop of acetoorcein was placed on the cells and they were covered with a cover glass. This method was quite satisfactory in retaining the same microscopic field with all the cells intact, fixed and nicely stained.
RESULTS
Cytological evidence for anomalous mitosis in HeLa cells grown at 29°C Evidence for an anomalous mitosis became manifest in the period between 12 and 60 hr after the temperature was shifted from 37°C to 29°C. Fixed and stained preparations of cells obtained during this period characteristically Experimenlal
Cell Research
43
P. N. Rae and J. Engelberg
exhibited mitoses in which most chromosomes were at or near the equatorial plane while some were at the poles (Fig. 1 A and B). The number of chromosomes at each of the two poles in a given cell were generally not the same. The chromosomes at the poles consisted of two sister chromatids joined in the kinetochore region, a point to which we will return later in our discussion of mitotic chromosomal non-disjunction. In most cells there were many Experimental
Cell
Research
43
Mitotic
nondisjunction
and anomalous
335
mitosis
chromosomes at the plate but few at the poles. Further observations suggested that the chromosomes moved from the plate to the poles in an asynchronous manner. However, other interpretations were possible and so the matter was investigated along other lines. Studies on synchronized cells.-A series of studies were made on cells synchronized by the double thymidine block technique [7, 91. When cells in the post-DNA synthesis period (G2) were transferred from 37” to 29°C the appearance of the mitoses which followed was normal and the cell division regular. Rut for a few exceptions (1 or 2 per cent) normal mitoses were obtained when the temperature shift took place during the middle or late part of the DNA synthesis period (S). However, when the temperature shift took place in the pre-DNA synthetic period (Gl) or in early S, the cells entered a mitosis which followed an anomalous course. Originally we attributed these results to an event in Gl or early S which was necessary for a normal mitosis but which was inhibited at 29°C. Dr L. J. Tolmach suggested to us that, alternatively, the effect on the Gl cells could be due merely to the period of time which the cells spend at the lower temperature. Subsequent experiments favored his contention. Synchronized cells in late S were blocked by the addition of thymidine and were then maintained at 29°C for 17 hr. Upon release of the block the cells manifested the same anomalous pattern as had been observed with cells which had been transferred to 29°C during Gl. This suggests that it is the time that the interphase cell spends at 29”C, rather than the specific phase of the mitotic cycle during
Fig. 2.-Kinetics of a synchronized HeLa cell culture. The temperature of the culture was shifted from 37°C to 29°C at a time (t = 0) when the cells were in GI. A, Mitotic index ( x 100) versus time for a portion of the above culture to which colcemide was added at t = 0; B, control. Same as (A) but no colcemide added; C, average number of chromosomes per pole among the mitotic cells in the “control”.
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Cell
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P. N. Rao and J. Engelberg
which the temperature shift occurs, which correlates with the appearance of abnormal mitoses. Another study on synchronized cells was carried out to determine the average chromosome number per pole as a function of time after synchronized cells in the Gl phase of the cell cycle were shifted from 37°C to 29°C. The results are shown in Fig. 2. From t =O, the time of the temperature shift, to t =20 hr, normal prophases and metaphases were observed in fixed preparations and no chromosomes were found at the poles. The average number of chromosomes at the poles of mitotic cells, nominally in metaphase, began to increase continually with time about 20 hr after the temperature shift. In Fig. 2 the divergence of the two mitotic index curves, one of them representing a culture containing colcemide, indicates that some of the cells were able to pass through mitosis. By means of a formula [9] one can estimate that by 60 hr about 13 per cent of the cells initially present had divided. Direct
microscopic
observations
of living cells in mitosis
Mitotic events in HeLa cells growing in monolayers in Cooper Dishes were followed under phase-contrast microscopy. In control cultures at 37°C HeLa cells proceeded normally from prophase to metaphase with the alignment of chromosomes at the equatorial region. The metaphase plate remained sharp and clear for about 15 to 30 min after which it started to widen, following which the broadened metaphase plate split into two sharp bands signaling the initiation of anaphase. The anaphase bands moved away from each other towards the opposite poles. This was followed by cell elongation and cytokinesis. About 8 min elapsed from the beginning of anaphase to the completion of cytokinesis. Mitosis at 29°C was studied in a similar fashion. For approximately the first 12 hr after the temperature shift from 37°C to 29°C mitosis followed the normal course, though the duration of mitosis at 29°C was greater, of course, than it would have been at 37°C. Thereafter, anomalous mitoses were observed. Observations on a typical anomalous mitosis follow. The progression through prophase to the formation of the “metaphase plate” appeared to be normal. The metaphase plate was often sharp and clear. Occasionally one had the impression of seeing dark masses detach themselves from the metaphase plate and migrate to the poles. When fixed and stained such a cell might look like Fig. 1 A or 1 B. About 1 hr after the formation of the plate, the plate might start to broaden and become diffuse within 2 or 3 min: the whole cell looked grey. (See Fig. 1 C for an example of such a cell after fixation and staining.) The duration of this diffuse state was highly Experimental
Cell Research
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Mitotic
nondisjunction
and
anomalous
337
mitosis
variable-ranging from 20 min to 5 or 6 hr, at the end of which the metaphase plate reformed again as sharp and clear as before. The cycle of diffusion and reformation of the metaphase plate might be repeated a number of times-in some cells as many as six times. (No such diffusion of the metaphase chromosomes was observed in the control culture at 37°C.) Ultimately some of these cells entered “anaphase”. Prior to the initiation of anaphase the metaphase plate was reformed though often small dark patches were present in the polar regions. These patches probably represented the undivided chromosomes that migrated earlier to the poles. Finally the metaphase plate once again broadened and became diffuse. In 7 or 8 min a line of clearance appeared in the middle demarcating the cell into two diffused zones. These diffused areas gradually narrowed down into clear and sharp anaphase bands. Upon fixation and staining the anaphase bands appeared to consist of undivided chromosomes (Fig. 3 B). At times some lagging chromosomes were observed between the anaphase plates. After the formation of the anaphase plates, the cell started elongating. This was followed by cytokinesis and the formation of two daughter cells. At 29°C anaphase and telophase together lasted for about 30 min whereas the duration of the metaphase-like period ranged from 2 to 20 hr. The duration of mitosis at 29°C showed a great deal of variability. Some cells divided within 1 or 2 hr after observations were begun while others did not divide even 24 hr after entering mitosis. The mitotic apparatus was present in HeLa cells in mitosis even after prolonged exposures to 29°C. As indirect evidence for this we cite the presence of well formed “metaphase plates” at 29°C. Confirmation by microscopy of the presence of a spindle was obtained in cells fixed with Bouin’s fluid and stained with hematoxylin. In another experiment an exponential (non-synchronized) suspension culture of HeLa cells was transferred to 29°C and samples were collected at 2-hr intervals. Fixed and stained cells were scored for anomalies during mitosis. Mitotic phases appeared to be normal during the first 10 or 12 hr at 29°C. No chromosome scatter was observed while the metaphase plate was intact. During this period anaphases were perfectly normal with sister chromatids being pulled to opposite poles (Fig. 3 A). About 12 to 14 hr following the decrease in temperature anomalies started appearing. There was a metaphase-like period with a good metaphase plate with one or two undivided chromosomes at the poles. The number of chromosomes accumulating at the poles gradually increased with time. Subsequently anomalous anaphases were observed in these cells at 29’C (Fig. 3 B and 3C). When such a culture after 48 hr exposure to 29°C was returned to 37°C the mitoses continued to Experimental
Cell Research
43
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P. N. Rue and J. Engelberg
b
Fig. 3.PAnomalous “Anaphase” in HeLa cells at 29°C. A, Normal anaphase: synchronous movement of sister chromatids to opposite poles. Normal anaphases were observed until about 12 hr after reducing the temperature from 37°C to 29°C; B, anomalous anaphase: undivided chromosomes were randomly distributed between poles; C, anomalous anaphase: random distribution of undivided chromosomes at anaphase often results in unequal number of chromosomes going to opposite poles.
be anomalous. No normal mitoses were observed even 24 hr after returning the culture to 37°C. Mifofic non-disjunction of sister chromafids.-Our attention was drawn to the phenomenon of mitotic chromosomal non-disjunction by three observations. First, the number of chromosomes per pole was generally not the same for two poles of a given cell. Had sister chromosomes separated from Experimental
Cell Research
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Mitotic
nondisjunction
and anomalous
mitosis
339
one another before traversing to the poles one might have expected in a given cell, an equal number of chromosomes at the two poles. Second, under direct observation by phase contrast microscopy of dividing cells at 29°C it was found that occasionally the two anaphase chromosome groups at the opposite poles differed greatly in size. Such unequal distribution of chromosomes yielded daughter cells of unequal size. Third, the chromosomes that moved to the poles appeared to have not divided and to consist of a pair of sister chromatids (Fig. 1 B and 3 B).
DISCUSSION
Terms like metaphase and anaphase have precise meanings when applied to a normal mitosis. In the case of anomalous mitoses these terms can only be used as rough parallels to the normal case. From the observations which we have described here the following picture emerges. (1) As previously reported [9] HeLa cells at 29°C are able to progress through interphase and to enter mitosis. The mitotic cells are able to form a spindle and to arrive at a “metaphase” in which the chromosomes are aligned in the equatorial plane. (2) The mitoses are anomalous, however, in that the aligned chromosomes at the equatorial plane may become disorganized and scattered. Furthermore, after the chromosomes have been scattered for periods of time up to several hours, they often again become aligned in the equatorial plane. (3) During the period of metaphase plate formation and scattering there appears to be a gradual drift of some of the chromosomes to the poles. This is an interpretation of the result that in synchronized cultures the chromosome number per pole increases with time (Fig. 2). Other interpretations are possible. For example, one could interpret the presence of chromosomes at the poles of cells exhibiting a metaphase plate as representing chromosomes which were not incorporated into the plate at the time the plate was formed. George ef al. [2] who found chromosomes at the poles in metaphase figures of cells treated with vincristine interpreted the results this way. The HeLa cell appears to be a poor material for settling this question by direct observation in living cells because of the large number chromosomes and their poor visibility. We should like to attempt to study this phenomenon in a more favorable material. (4) A fraction of HeLa cells passing through an anomalous mitosis at 29°C are able to divide. The division is preceded by a phenomenon, which though dissimilar in some respects, bears some similarity to anaphase in Experimental
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P. N. Rao and J. Engelberg
normal cells. It is similar in the sense that the chromosomes aggregate into two groups following which cell cleavage is initiated and completed. It is dissimilar in that (a) the chromosomes in the two groups appear to be undivided-the sister chromatids are joined at the kinetochore region, and (b) the two groups do not necessarily have an equal number of chromosomes, We have not been able to ascertain by direct observation how the chromosomes are sorted into two groups. The two groups emerge and become progressively more well defined from amidst a diffuse, cloud of chromosomes.
TABLE
I. Non-classical mitosis. Anaphase chromosome movement
Metaphase plate
Kinetochores Spindle separate?
“C-Mitosis”
Absent
Absent
No
Absent
UV microbeam irradiation of cytoplasm
“False anaphase”
“Quasirosette”
Absent
NO
Rosettes (whole chromosome clusters) move apart
Recovery from cold
“Incompact spindles”
“Star”
Present
Yes
Asynchronous
None
“Asynchronous division of kinetochores”
Normal
Present
Yes
Asynchronous
Mota, 1952 Onion [41 Hyppio et Onion; al., 1955 [3] Pisum
Seed extracts
“Diffuse anaphase”
Normal
Present
Yes
Asynchronous
Cyclochlorohexane
“Scattered anaphase”
“Scattered”
Present
Yes
Multipolar migration
Ostergren and Bajer, 1961 [6]
Haemanthus
Methanol
Present
No
Irregular and incomplete
Rao and Engelberg, 1965 [9]
HeLa cells
Cold (29°C)
Author
Material
-
Name given to phenomenon
Treatment
Plant and Colchicine animal cells
Zirkle, 1957 Newt
1111
Barber and Callan, 1943 [l]
Newt
Upcott, 1939 Tulipa [lOI
Experimental
Sp.
Cell Research
43
(?) “Mitosis with Forms with undivided difficulty chromosomes” “Anomalous mitosis”
Normal but undergoes a cycle of diffusion and reformation
Present
A few chromosomes move tc$ poles individul ally while thq rest go throug@ anomalous anaphase
Mitotic
nondisjunction
and anomalous
341
mitosis
The most economical working hypothesis we can put forth to explain all of these phenomena is this: The anomalous mitosis is the consequence of the inability of the sister chromatids to separate in the region of the kinetochore. A lesion of this kind would not prevent the chromosomes from If there were an event which initiated a aligning in the equatorial plane. normal anaphase, however, the chromosomes would not be able to respond normally because the sister chromatids would not be able to separate. It is possible that a scattering of the chromosomes would result. Various departures from the “classical mitosis” have been described in the literature in cells grown under normal conditions as well as in cells grown under abnormal conditions (Table I). Among the different kinds of abnormal mitosis chromosomal non-disjunction has been observed usually in cells in which intense radiation [ 111 or colchicine has obliterated the visible achromatic spindle. After radiation, however, the migration of the chromosomes during the “false anaphase” is in terms of a whole group of chromosomes arranged in the form of a rosette. By contrast, a spindle is present in the course of the anomalous mitosis described here. The phenomenon described in this report has a striking similarity to the mitosis with single chromatids observed by Ostergren and Bajer [6] in living endosperm cells of Haemanthus katharinae in the presence of methanol. Yet the following differences between the two phenomena are significant. They are: (1) In Haemanthus the chromosomes consisted of a single chromatid. In HeLa cells the chromosomes had two sister chromatids connected at the kinetochore region. (2) The metaphase plate in Haemanthus endosperm was formed with great difficulty and many chromosomes were left scattered along the spindle. In HeLa cells at 29°C a good metaphase plate was formed without any difficulty and remained stable for varying lengths of time. The diffusion and reformation of the metaphase plate over a number of times prior to the anaphase which we observed was not observed by Ostergren and Bajer. Ostergren [5] discussed a number of reports dealing with mitosis with undivided chromosomes including that in Haemanthus. In all of these cases either the metaphase plate formed more or less normally or not at all. But once the chromosomes were stabilized at the equatorial plate no reversal of this structure was reported until the cell entered some sort of anaphase. We are in the process of preparing a report on chemical means of inducing the anomalous mitosis phenomenon. This phenomenon has been induced by treating HeLa cells with estradiol-17B, diethylstilbesterol and low concentrations of colcemide. Experimental
Cell Research
43
342
P. N. Rao and J. Engelberg
SUMMARY
An anomalous mitosis was observed in HeLa cells within 24 hr after a shift in growth temperature from 37% to 29°C. The anomalous mitosis was characterized by (a) the presence of a mitotic spindle, (b) the formation of a “metaphase plate” which passed through a number of diffusions and reformations prior to the onset of “anaphase”, (c) the failure of the separation of sister chromatids in the kinetochore region, (d) the asynchronous movement and random distribution of (undivided) chromosomes towards the poles. The induction of the anomalous mitosis appeared to be dependent upon the time spent by the interphase cell at 29°C rather than on the specific phase of the cell cycle during which the temperature shift occurred. We thank Mrs Nora Mitchell and Mrs Mattie Mitchell for their valuable technical assistance. This investigation was aided by PHS grant NIH-CA-06835 from the National Cancer Institute and by research grant E-303 from the American Cancer Society. REFERENCES BARBER, H. N. and CALLAN, H. G., Proc. Roy. SW London, Ser. B. 131, 258 (1943). GEORGE, P., JOURNEY, L. J. and GOLDSTEIN, M. N., J. N~tl Cnncer Inst. 35, 355 (1965). HYPPIO, P. A., Tsou, T. M. and WILSON, J. B., Cytologia (Tokyo) 20, 166 (1955). MOTA, M., Arquiu. Patol. (Lisbon) 24, 335 (1952). ~STERGREN, G., Chromosoma (Bed.) 12, 80 (1961). OSTERGREN, G. and BAJER, A., Chromosoma (Berl.) 12, 72 (1961). PUCK, T. T., Science 144, 566 (1964). RAO, P. N. and ENGELBERG, J., Science 148, 1092 (1965). -in I. L. CAMERON and G. M. PADILLA (eds.), Cell Synchrony-Studies in Biosynthetic Regulation. Academic Press, New York, 1966. LO. UPCOTT, M., Chromosoma (Berl.) 1, 178 (1939). 11. ZIRKLE, FL E., Advan. Biol. Med. Phys. 5, 103 (1957). 1. 2. 3. 4. 5. 6. 7. 8. 9.
Experimental
Cell Research
43
Experimental
Cell Research
43, 332-342
MITOTIC NON-DISJUNCTION OF SISTER CHROMATIDS AND ANOMALOUS MITOSIS INDUCED LOW TEMPERATURES IN HeLa CELLS P. N. RAO Department
(1966)
BY
and J. ENGELBERG
of Physiology and Biophysics, Lexington, KY., 40506,
University U.S.A.
of Kentucky,
Received January 21, 1966l
MITOSIS follows the classical pattern in the normal HeLa cell. At metaphase the chromosomes align in the equatorial region of the cell to form the “metaphase plate”. Anaphase begins when the sister chromatids of each chromosome separate and continues as the sister chromosomes move in synchrony towards opposite poles. These events may be observed in HeLa cells grown at temperatures from 33°C through 39°C. When the temperature of HeLa cells grown at 37°C was reduced to 29”C, however, the pattern of mitosis changed with time [S, 91. Initially the mitoses were normal in appearance though the duration of mitosis was increased; for about 12 hr the mitotic index remained at about 0.03 indicating that cells were entering mitosis at roughly the same rate at which cells in mitosis were entering interphase. The mitotic index then increased in linear fashion toward a mitotic index of about 0.36 which was reached about 60 hr after the temperature shift. During the period of mitotic index increase cells entered mitosis at a rate that exceeded the rate with which they completed mitosis. The mitosis in question was normal in some respects but abnormal in many others. (1) A mitotic spindle was present. (2) A metaphase plate of normal appearance formed. (3) The plate often subsequently became disorganized with chromosomes scattered throughout the cell. (4) Following such a diffusion of chromosomes the metaphase plate often reformed. (5) During this period there appeared to be a progressive drift of chromosomes away from the plate and an accumulation of these chromosomes at the poles. (6) In cells which divided there was finally a concerted movement of chromosomes from the equatorial region towards the poles. The number of chromosomes moving to each pole was, however, unequal. (7) Sister chromatids in a given chromosome remained attached to one another in the kinetochore region. Thus,
the chromosomes
arriving
at the poles
rather than anaphase chromosomes. 1 Revised Experimental
version Cell
received Research
May 43
16, 1966.
were
metaphase
chromosomes,
Mitotic
nondisjunction
MATERIALS
and anomalous
AND
333
mitosis
METHODS
The HeLa cell line studied was one originally isolated for suspensiongrowth by Dr Norman Salzman of the National Institute of Health. These cells were regularly grown as suspensioncultures in Eagle’s medium at 37°C. Some of the experimental techniques used in this study were described in previous publications 18, 91. In the present study direct phase-contrast microscope observations were made on living cells growing in plastic Cooper Dishes(Falcon Plastics, Inc.). To make possible the use of a high power objective (Zeiss “Neofluar” objective, 43 x , NA 0.75) the rim of the bottom dish was slightly trimmed so as to bring the bottom and the cover closer together. The ambient temperature of the culture dish on the microscope stage was maintained at constant temperature in a chamber designedby Dr Ralph Hinegardner of Columbia University, New York. This chamber consisted of two transparent plastic bags-a smaller bag enclosedwithin a larger one. The microscopewas placed within the small bag and the bag sealed around the binocular head of the microscope by a rubber band. This whole set up was placed within a bigger bag and similarly sealedleaving a small air vent to a side. Warm air was blown into the outer plastic bag from a hair drier. The inner bag was areated with a humidified mixture of carbon dioxide (4 per cent) in air. The temperature of the culture dish was monitored by means of a thermistor probe. One of the objects in the present study was to locate the relative positions of chromosomeson the mitotic spindle. Unfortunately, a HeLa cell is not a good system for the study of chromosome movements during mitosis. We were able to overcome someof the difficulties to a certain extent by making detailed observations on living mitotic cells first and then comparing these cells before and after they were fixed and stained. For this purpose the following procedure was adopted. The bottom half of the Cooper Dish was attached with Duco cement to a thin rectangular lucite plate having a circular opening in the center. The lucite plate fitted the slide holder on the microscope stage. The covered Cooper Dish was placed on the microscope stage and a field with a number of cells in mitosis was picked. The general outline of the field and the relative positions of groups of chromosomeswithin mitotic cells were sketched. After recording the position of the field, medium was removed from the dish by gentle suction and the cells were fixed in Bouin’s fluid for 2 min. Two washings with 50 per cent ethanol and one washing with absolute ethanol followed taking care that no cells were dislodged. A drop of acetoorcein was placed on the cells and they were covered with a cover glass. This method was quite satisfactory in retaining the same microscopic field with all the cells intact, fixed and nicely stained.
RESULTS
Cytological evidence for anomalous mitosis in HeLa cells grown at 29°C Evidence for an anomalous mitosis became manifest in the period between 12 and 60 hr after the temperature was shifted from 37°C to 29°C. Fixed and stained preparations of cells obtained during this period characteristically Experimenlal
Cell Research
43
P. N. Rae and J. Engelberg
exhibited mitoses in which most chromosomes were at or near the equatorial plane while some were at the poles (Fig. 1 A and B). The number of chromosomes at each of the two poles in a given cell were generally not the same. The chromosomes at the poles consisted of two sister chromatids joined in the kinetochore region, a point to which we will return later in our discussion of mitotic chromosomal non-disjunction. In most cells there were many Experimental
Cell
Research
43
Mitotic
nondisjunction
and anomalous
335
mitosis
chromosomes at the plate but few at the poles. Further observations suggested that the chromosomes moved from the plate to the poles in an asynchronous manner. However, other interpretations were possible and so the matter was investigated along other lines. Studies on synchronized cells.-A series of studies were made on cells synchronized by the double thymidine block technique [7, 91. When cells in the post-DNA synthesis period (G2) were transferred from 37” to 29°C the appearance of the mitoses which followed was normal and the cell division regular. Rut for a few exceptions (1 or 2 per cent) normal mitoses were obtained when the temperature shift took place during the middle or late part of the DNA synthesis period (S). However, when the temperature shift took place in the pre-DNA synthetic period (Gl) or in early S, the cells entered a mitosis which followed an anomalous course. Originally we attributed these results to an event in Gl or early S which was necessary for a normal mitosis but which was inhibited at 29°C. Dr L. J. Tolmach suggested to us that, alternatively, the effect on the Gl cells could be due merely to the period of time which the cells spend at the lower temperature. Subsequent experiments favored his contention. Synchronized cells in late S were blocked by the addition of thymidine and were then maintained at 29°C for 17 hr. Upon release of the block the cells manifested the same anomalous pattern as had been observed with cells which had been transferred to 29°C during Gl. This suggests that it is the time that the interphase cell spends at 29”C, rather than the specific phase of the mitotic cycle during
Fig. 2.-Kinetics of a synchronized HeLa cell culture. The temperature of the culture was shifted from 37°C to 29°C at a time (t = 0) when the cells were in GI. A, Mitotic index ( x 100) versus time for a portion of the above culture to which colcemide was added at t = 0; B, control. Same as (A) but no colcemide added; C, average number of chromosomes per pole among the mitotic cells in the “control”.
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which the temperature shift occurs, which correlates with the appearance of abnormal mitoses. Another study on synchronized cells was carried out to determine the average chromosome number per pole as a function of time after synchronized cells in the Gl phase of the cell cycle were shifted from 37°C to 29°C. The results are shown in Fig. 2. From t =O, the time of the temperature shift, to t =20 hr, normal prophases and metaphases were observed in fixed preparations and no chromosomes were found at the poles. The average number of chromosomes at the poles of mitotic cells, nominally in metaphase, began to increase continually with time about 20 hr after the temperature shift. In Fig. 2 the divergence of the two mitotic index curves, one of them representing a culture containing colcemide, indicates that some of the cells were able to pass through mitosis. By means of a formula [9] one can estimate that by 60 hr about 13 per cent of the cells initially present had divided. Direct
microscopic
observations
of living cells in mitosis
Mitotic events in HeLa cells growing in monolayers in Cooper Dishes were followed under phase-contrast microscopy. In control cultures at 37°C HeLa cells proceeded normally from prophase to metaphase with the alignment of chromosomes at the equatorial region. The metaphase plate remained sharp and clear for about 15 to 30 min after which it started to widen, following which the broadened metaphase plate split into two sharp bands signaling the initiation of anaphase. The anaphase bands moved away from each other towards the opposite poles. This was followed by cell elongation and cytokinesis. About 8 min elapsed from the beginning of anaphase to the completion of cytokinesis. Mitosis at 29°C was studied in a similar fashion. For approximately the first 12 hr after the temperature shift from 37°C to 29°C mitosis followed the normal course, though the duration of mitosis at 29°C was greater, of course, than it would have been at 37°C. Thereafter, anomalous mitoses were observed. Observations on a typical anomalous mitosis follow. The progression through prophase to the formation of the “metaphase plate” appeared to be normal. The metaphase plate was often sharp and clear. Occasionally one had the impression of seeing dark masses detach themselves from the metaphase plate and migrate to the poles. When fixed and stained such a cell might look like Fig. 1 A or 1 B. About 1 hr after the formation of the plate, the plate might start to broaden and become diffuse within 2 or 3 min: the whole cell looked grey. (See Fig. 1 C for an example of such a cell after fixation and staining.) The duration of this diffuse state was highly Experimental
Cell Research
43
Mitotic
nondisjunction
and
anomalous
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mitosis
variable-ranging from 20 min to 5 or 6 hr, at the end of which the metaphase plate reformed again as sharp and clear as before. The cycle of diffusion and reformation of the metaphase plate might be repeated a number of times-in some cells as many as six times. (No such diffusion of the metaphase chromosomes was observed in the control culture at 37°C.) Ultimately some of these cells entered “anaphase”. Prior to the initiation of anaphase the metaphase plate was reformed though often small dark patches were present in the polar regions. These patches probably represented the undivided chromosomes that migrated earlier to the poles. Finally the metaphase plate once again broadened and became diffuse. In 7 or 8 min a line of clearance appeared in the middle demarcating the cell into two diffused zones. These diffused areas gradually narrowed down into clear and sharp anaphase bands. Upon fixation and staining the anaphase bands appeared to consist of undivided chromosomes (Fig. 3 B). At times some lagging chromosomes were observed between the anaphase plates. After the formation of the anaphase plates, the cell started elongating. This was followed by cytokinesis and the formation of two daughter cells. At 29°C anaphase and telophase together lasted for about 30 min whereas the duration of the metaphase-like period ranged from 2 to 20 hr. The duration of mitosis at 29°C showed a great deal of variability. Some cells divided within 1 or 2 hr after observations were begun while others did not divide even 24 hr after entering mitosis. The mitotic apparatus was present in HeLa cells in mitosis even after prolonged exposures to 29°C. As indirect evidence for this we cite the presence of well formed “metaphase plates” at 29°C. Confirmation by microscopy of the presence of a spindle was obtained in cells fixed with Bouin’s fluid and stained with hematoxylin. In another experiment an exponential (non-synchronized) suspension culture of HeLa cells was transferred to 29°C and samples were collected at 2-hr intervals. Fixed and stained cells were scored for anomalies during mitosis. Mitotic phases appeared to be normal during the first 10 or 12 hr at 29°C. No chromosome scatter was observed while the metaphase plate was intact. During this period anaphases were perfectly normal with sister chromatids being pulled to opposite poles (Fig. 3 A). About 12 to 14 hr following the decrease in temperature anomalies started appearing. There was a metaphase-like period with a good metaphase plate with one or two undivided chromosomes at the poles. The number of chromosomes accumulating at the poles gradually increased with time. Subsequently anomalous anaphases were observed in these cells at 29’C (Fig. 3 B and 3C). When such a culture after 48 hr exposure to 29°C was returned to 37°C the mitoses continued to Experimental
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b
Fig. 3.PAnomalous “Anaphase” in HeLa cells at 29°C. A, Normal anaphase: synchronous movement of sister chromatids to opposite poles. Normal anaphases were observed until about 12 hr after reducing the temperature from 37°C to 29°C; B, anomalous anaphase: undivided chromosomes were randomly distributed between poles; C, anomalous anaphase: random distribution of undivided chromosomes at anaphase often results in unequal number of chromosomes going to opposite poles.
be anomalous. No normal mitoses were observed even 24 hr after returning the culture to 37°C. Mifofic non-disjunction of sister chromafids.-Our attention was drawn to the phenomenon of mitotic chromosomal non-disjunction by three observations. First, the number of chromosomes per pole was generally not the same for two poles of a given cell. Had sister chromosomes separated from Experimental
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nondisjunction
and anomalous
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one another before traversing to the poles one might have expected in a given cell, an equal number of chromosomes at the two poles. Second, under direct observation by phase contrast microscopy of dividing cells at 29°C it was found that occasionally the two anaphase chromosome groups at the opposite poles differed greatly in size. Such unequal distribution of chromosomes yielded daughter cells of unequal size. Third, the chromosomes that moved to the poles appeared to have not divided and to consist of a pair of sister chromatids (Fig. 1 B and 3 B).
DISCUSSION
Terms like metaphase and anaphase have precise meanings when applied to a normal mitosis. In the case of anomalous mitoses these terms can only be used as rough parallels to the normal case. From the observations which we have described here the following picture emerges. (1) As previously reported [9] HeLa cells at 29°C are able to progress through interphase and to enter mitosis. The mitotic cells are able to form a spindle and to arrive at a “metaphase” in which the chromosomes are aligned in the equatorial plane. (2) The mitoses are anomalous, however, in that the aligned chromosomes at the equatorial plane may become disorganized and scattered. Furthermore, after the chromosomes have been scattered for periods of time up to several hours, they often again become aligned in the equatorial plane. (3) During the period of metaphase plate formation and scattering there appears to be a gradual drift of some of the chromosomes to the poles. This is an interpretation of the result that in synchronized cultures the chromosome number per pole increases with time (Fig. 2). Other interpretations are possible. For example, one could interpret the presence of chromosomes at the poles of cells exhibiting a metaphase plate as representing chromosomes which were not incorporated into the plate at the time the plate was formed. George ef al. [2] who found chromosomes at the poles in metaphase figures of cells treated with vincristine interpreted the results this way. The HeLa cell appears to be a poor material for settling this question by direct observation in living cells because of the large number chromosomes and their poor visibility. We should like to attempt to study this phenomenon in a more favorable material. (4) A fraction of HeLa cells passing through an anomalous mitosis at 29°C are able to divide. The division is preceded by a phenomenon, which though dissimilar in some respects, bears some similarity to anaphase in Experimental
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normal cells. It is similar in the sense that the chromosomes aggregate into two groups following which cell cleavage is initiated and completed. It is dissimilar in that (a) the chromosomes in the two groups appear to be undivided-the sister chromatids are joined at the kinetochore region, and (b) the two groups do not necessarily have an equal number of chromosomes, We have not been able to ascertain by direct observation how the chromosomes are sorted into two groups. The two groups emerge and become progressively more well defined from amidst a diffuse, cloud of chromosomes.
TABLE
I. Non-classical mitosis. Anaphase chromosome movement
Metaphase plate
Kinetochores Spindle separate?
“C-Mitosis”
Absent
Absent
No
Absent
UV microbeam irradiation of cytoplasm
“False anaphase”
“Quasirosette”
Absent
NO
Rosettes (whole chromosome clusters) move apart
Recovery from cold
“Incompact spindles”
“Star”
Present
Yes
Asynchronous
None
“Asynchronous division of kinetochores”
Normal
Present
Yes
Asynchronous
Mota, 1952 Onion [41 Hyppio et Onion; al., 1955 [3] Pisum
Seed extracts
“Diffuse anaphase”
Normal
Present
Yes
Asynchronous
Cyclochlorohexane
“Scattered anaphase”
“Scattered”
Present
Yes
Multipolar migration
Ostergren and Bajer, 1961 [6]
Haemanthus
Methanol
Present
No
Irregular and incomplete
Rao and Engelberg, 1965 [9]
HeLa cells
Cold (29°C)
Author
Material
-
Name given to phenomenon
Treatment
Plant and Colchicine animal cells
Zirkle, 1957 Newt
1111
Barber and Callan, 1943 [l]
Newt
Upcott, 1939 Tulipa [lOI
Experimental
Sp.
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(?) “Mitosis with Forms with undivided difficulty chromosomes” “Anomalous mitosis”
Normal but undergoes a cycle of diffusion and reformation
Present
A few chromosomes move tc$ poles individul ally while thq rest go throug@ anomalous anaphase
Mitotic
nondisjunction
and anomalous
341
mitosis
The most economical working hypothesis we can put forth to explain all of these phenomena is this: The anomalous mitosis is the consequence of the inability of the sister chromatids to separate in the region of the kinetochore. A lesion of this kind would not prevent the chromosomes from If there were an event which initiated a aligning in the equatorial plane. normal anaphase, however, the chromosomes would not be able to respond normally because the sister chromatids would not be able to separate. It is possible that a scattering of the chromosomes would result. Various departures from the “classical mitosis” have been described in the literature in cells grown under normal conditions as well as in cells grown under abnormal conditions (Table I). Among the different kinds of abnormal mitosis chromosomal non-disjunction has been observed usually in cells in which intense radiation [ 111 or colchicine has obliterated the visible achromatic spindle. After radiation, however, the migration of the chromosomes during the “false anaphase” is in terms of a whole group of chromosomes arranged in the form of a rosette. By contrast, a spindle is present in the course of the anomalous mitosis described here. The phenomenon described in this report has a striking similarity to the mitosis with single chromatids observed by Ostergren and Bajer [6] in living endosperm cells of Haemanthus katharinae in the presence of methanol. Yet the following differences between the two phenomena are significant. They are: (1) In Haemanthus the chromosomes consisted of a single chromatid. In HeLa cells the chromosomes had two sister chromatids connected at the kinetochore region. (2) The metaphase plate in Haemanthus endosperm was formed with great difficulty and many chromosomes were left scattered along the spindle. In HeLa cells at 29°C a good metaphase plate was formed without any difficulty and remained stable for varying lengths of time. The diffusion and reformation of the metaphase plate over a number of times prior to the anaphase which we observed was not observed by Ostergren and Bajer. Ostergren [5] discussed a number of reports dealing with mitosis with undivided chromosomes including that in Haemanthus. In all of these cases either the metaphase plate formed more or less normally or not at all. But once the chromosomes were stabilized at the equatorial plate no reversal of this structure was reported until the cell entered some sort of anaphase. We are in the process of preparing a report on chemical means of inducing the anomalous mitosis phenomenon. This phenomenon has been induced by treating HeLa cells with estradiol-17B, diethylstilbesterol and low concentrations of colcemide. Experimental
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SUMMARY
An anomalous mitosis was observed in HeLa cells within 24 hr after a shift in growth temperature from 37% to 29°C. The anomalous mitosis was characterized by (a) the presence of a mitotic spindle, (b) the formation of a “metaphase plate” which passed through a number of diffusions and reformations prior to the onset of “anaphase”, (c) the failure of the separation of sister chromatids in the kinetochore region, (d) the asynchronous movement and random distribution of (undivided) chromosomes towards the poles. The induction of the anomalous mitosis appeared to be dependent upon the time spent by the interphase cell at 29°C rather than on the specific phase of the cell cycle during which the temperature shift occurred. We thank Mrs Nora Mitchell and Mrs Mattie Mitchell for their valuable technical assistance. This investigation was aided by PHS grant NIH-CA-06835 from the National Cancer Institute and by research grant E-303 from the American Cancer Society. REFERENCES BARBER, H. N. and CALLAN, H. G., Proc. Roy. SW London, Ser. B. 131, 258 (1943). GEORGE, P., JOURNEY, L. J. and GOLDSTEIN, M. N., J. N~tl Cnncer Inst. 35, 355 (1965). HYPPIO, P. A., Tsou, T. M. and WILSON, J. B., Cytologia (Tokyo) 20, 166 (1955). MOTA, M., Arquiu. Patol. (Lisbon) 24, 335 (1952). ~STERGREN, G., Chromosoma (Bed.) 12, 80 (1961). OSTERGREN, G. and BAJER, A., Chromosoma (Berl.) 12, 72 (1961). PUCK, T. T., Science 144, 566 (1964). RAO, P. N. and ENGELBERG, J., Science 148, 1092 (1965). -in I. L. CAMERON and G. M. PADILLA (eds.), Cell Synchrony-Studies in Biosynthetic Regulation. Academic Press, New York, 1966. LO. UPCOTT, M., Chromosoma (Berl.) 1, 178 (1939). 11. ZIRKLE, FL E., Advan. Biol. Med. Phys. 5, 103 (1957). 1. 2. 3. 4. 5. 6. 7. 8. 9.
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