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Estradiol-induced mitotic delay in hela cells: Reversal by calcium chloride and putrescine
Experimental
ESTRADIOL-INDUCED REVERSAL
Cell Research 57 (1969) 230-234
MITOTIC
BY CALCIUM
DELAY
CHLORIDE
IN HELA AND
CELLS:
PUTRESCINE
P. N. RAO Department of Biophysics, University of Colorado Medical Center, Denver, Colo. 80220, USA
SUMMARY Estradiol-17p induced reversible mitotic delay in HeLa cells which was localized at the beginning of mitosis. The duration of the mitotic lag was dose dependent. Only those cells that were in S or G2 periods at the time of estradiol treatment suffered the delay but not those in Cl period. A synergism between estradiol and colcemide was also observed. Calcium chloride or putrescine successfully reversed the mitotic delay induced by estradiol.
While studying the structural specificy of estrogens in inducing chromosomal non-disjunction in HeLa cells it was observed that HeLa cells suffered mitotic delay immediately following a treatment with estradiol-17fi [12]. Subsequently, a detailed study was taken up to gain insight into the nature of this mitotic delay and the results from this study form the subject of this report. The mitotic delay induced by estradiol in HeLa cells was manifested as a prolongation of G2 + early prophase and was reversed by the addition of calcium chloride or putrescine to the medium. MATERIALS
AND METHODS
HeLa cells grown as suspension cultures were used in the present study. The cultures were always maintained in exponential growth at 37°C in Eagle’s MEM for sninner cultures without calcium, supplemented with non-essential amino acids, sodium pyruvate and 5 % calf serum. They were gassed with a mixture of carbon dioxide (2.7 X) and air. Estradiol-17b (Calbiochem Co.) was dissolved in 95 % ethyl alcohol and then added to the cell suspensions. Final concentration of alcohol did not exceed 0.5 % at any time. Preparation of cells for autoradiography and for scoring mitotic index were described in a previous publication [lo].
Scoring for mitotic index The cells scored as mitoses included cells in all phases of mitosis except those in very early prophase. Because of the uncertainty involved in distinguishing some of the very early prophases from interphase nuclei they were grouped along with the latter. The prophases in which the chromosomal fibers were detectable were classified as mitoses for the determination of the mitotic index. Exptl Cell Res 57
RESULTS Effect of continuous exposure to estradiol on the life cycle of HeLa Cells
Some insight into the effect of estradiol on the life cycle of HeLa cells was obtained by the simultaneous addition of colcemide (6.8 x 1O-7 M) and varying concentrations of estradiol to a culture in exponential growth (fig. 1). Estradiol induced an immediate lag in the collection function for mitotic cells. The greater the concentration of estradiol, the greater was the lag. Subsequent to the initial lag in the collection function the rate of increase in the mitotic index for the treatment (2 x 1OP M of estradiol + cc!cemide) was the same as for the control (colcemide only). There was no delay or decrease in the rate of flow of cells from Gl into S-period as measured by the increase in the percentage of cells labeled with 3H-thymidine following the addition of estradiol and colcemide to a random culture. At concentrations of 2 x 10-j M or below, estradiol had no significant effect on the rate of movement of cells through interphase. At higher doses of estradiol the curves for mitotic accumulation function significantly diverged from that of the control indicating that the cells were progressively slowed down in their movement towards mitosis. With 8 X 1O-5M of estradiol in the medium, a major portion of the cells failed to reach mitosis. For this reason a
Estradiol-induced mitotic delay in HeLa cells 231
Abscissa: Time, h; ordinate: mitotic index.
Fig. 1. Estradiol dose-effect relationships: Estradiol and colcemide (6.8 x lo-’ M) were added simultaneously at zero time. A, Control with colcemide alone; B, 2 x lo-& M; C, 4 x 1O-5M; D, 8 x 1O-5M of estradiol plus colcemide.
final concentration of 2 x 1O-5 M of estradiol, at which the toxicity was minimal, was chosen to be the optimal dose and had been used uniformly throughout this study unless otherwise stated. The dosage effect of estradiol on mitotic delay was also studied in synchronized cultures. The procedure for synchronization of HeLa cells in suspension by excess thymidine double block technique had been previously described [ll]. To such synchronized population of HeLa cells various concentrations of estradiol and 6.8 x lo-’ M of colcemide were simultaneously added at 6 h after the reversal of the second thymidine block and the collection functions for mitosis were obtained at regular intervals. The mitotic
Abscissa: Hours after releaseof thymidine block; ordinate:
mitotic index. Fig. 2. Mitotic delay in synchronized cultures: (A), Synergism between estradiol and colcemide. Various concentrations of estradiol and colcemide (6.8 x lo-’ M) were added at 6 h after the releaseof the secondthymidin block. P, Control; colcemide only; Q, 2 x 1O-5 M; R, 4 x 1O-5 M; S, 8 x 1O-5M of estradiol plus colcemide. (B) Synchronized cultures treated with estradiol alone. The treatments represented by the curves Q’, R’ and S’ are the same as in fig. 2 a except that no colcemide was added in this case, The decreasein mitotic index following the peak was due to the completion of mitosis and re-entry of cells into interphase.
index in the control reached 50 % level at t ~9.0 h, where as it was delayed by 0.5 to 2 h in cultures treated with colcemide plus estradiol, depending on the dose of the estrogen (fig. 24. When synchronized cultures were similarly treated with estradiol alone (without colcemide) the durations of the mitotic lag were relatively shorter (ranging from 0 to 1 h) than those when colcemide was also present in the medium along with estradiol. In other words the presence of colcemide enhanced the effect of estradiol in inducing mitotic delay indicating some degree of synergism between the two drugs (fig. 2A, B). The gradual decrease of the mitotic index following the peak (curves Q’, R’ and S’ of fig. 211) was due to the completion of mitosis and re-entry of cells into interphase. At lower concentrations of estradiol the cells completed mitosis at a faster rate. Increased concentrations of estradiol prolonged the duration of mitosis in general and the metaphase in particular [12]. The effectiveness of estradiol in inducing the mitotic delay was tested in another experiment in which the estrogen was added to a synchronized and colcemide-treated culture when the wave of mitotic activity was in progress. The addition of the hormone resulted in an immediate cessation in the increase of mitotic index which remained constant for an hour before resuming the normal rate of increase (fig. 3). Effect of pulse treatment with estradiol To answer the question, whether continuous presence of estradiol was necessary for the induction of mitotic delay, the mitotic accumulation function was studied in a random culture following a pulse treatment with estradiol. Estradiol dissociates from the cells rather easily [6, 71. A change of medium would remove almost all the estradiol from the cells. HeLa cells were exposed ro estradiol (2 x 1O-5 M) for 15 min, centrifuged and resuspended in fresh medium containing colcemide. The increase in mitotic index was plotted as a function of time (fig. 4). The mitotic delay was exhibited by cells that entered mitosis during the estradiol treatment and also during a period of 10.5 to 11.0 h imExptl Cell Res 57
232
P. N. Rao
Abscissa: Hours after release of TdR block; ordinate: mitntic index. Fig. 3. The effect of estradiol on the entry of G2 + early prophase cells into mitosis. Colcemide (6.8 x 1O-7 M) was added to a synchronized culture at 6 h after the release of the thymidine block. Estradiol (2 x IO-’ MI was added 2 h later as indicated by the arrow. O-O, control with colcemide alone; x - x , cells were treated with colcemidefirst, to which estradiol was added2 h later.
Abscissa: Time, h; ordinate: log [l + N(M)]. Fig. 4. Effect of pulse lrealment of HeLa cells with estradiol on mitotic delay: Colcemide (6.8 x lo-’ M) was present in the control and the treatments throughout the course of the experiment. O-O, 15 min; and A-A, 5 h of estradiol treatment; O-O , control with colcemide alone. The arrows a and b indicate the termination of estradiol treatment and the arrows u’ and b’ indicate the reversal of the mitotic delay. N(M)-Mitotic index.
the treatment. For this cell mediately following line the combined duration of G2 and S-periods was equal to 10.5 to 11.0 h. In other words the cells that were in S- or G2 periods at the time of estradiol treatment suffered the mitotic delay. If cell division was not blocked by adding colcemide following a 15 min exposure to estradiol the mitotic index curve showed a Ushaped depression at its origin (fig. 5). This depression was the result of an immediate decrease in the rate of arrival of G2 + early prophase cells into mitosis, but not in the departure of cells from mitosis into Gl. At the end of the lag period (about 2 h) the mitotic index gradually rose from about 0.01 to 0.037 which was normal for a culture in exponential growth.
cells in spinner-B were exposed to estradiol for only 15 min. Colcemide was added to both the spinners to block cell division and samples taken at regular intervals for autoradiography. The mitotic index in the non-labeled segment of the population reached its maximum in two steps (fig. 6). The first step represented the arrival of G2 cells into mitosis while the second step represented that of Gl cells. Except for the initial lag of 2.5 h the durations of G2, S and Gl in continuous estradiol treatment were identical with those in the control. The difference between the two estradiol treatments (continuous and pulse) lie in the rate of Gl cells reaching mitosis. The slope of mitotic accumulation function of Gl cells in continuous treatment was parallel to those of G2 and S cells (fig. 6).
Specificity of estradiol effect to S and G2 periods The following experiment was designed to confirm the observation that the induction of mitotic delay by a pulse treatment with estradiol was specific to cells in S and G2 periods. A random culture was treated with 3H-thymidine (1 &ml; 0.36 c/mM, New England Nuclear Corp.) for 15 min to label the cells in DNA synthetic period and then divided into two spinner flasks-A and B. The cells in spinner-A were treated with estradiol continuously whereas the Erptl Cell Res 57
Abscissa: After pulse treatment with estradiol; ordinate: mitotic index. Fig. 5. A plot of mitotic index in a random culture of HeLa cells following 15 min pulse treatment with 2 x 1O-5 M of estradiol. In this culture cell division was not blocked by colcemide.
Estradiol-induced
0
4
6
12
16
20
24
Abscissa: Time, h; ordinate: log [l I N(M)]. Fig. 6. Effect of estradiol on cells in S and G2 periods at the time of the treatment. The solid symbols represent continuous presence of estradiol in the medium; whereas, the open symbols represent a 15-min pulse treatment with estradiol. 0.-.O,G2; A---A,S; q --n,Gl+Mcells at the time of estradiol treatment. N(M), Mitotic index.
In the pulse treatment the slope was greater than normal indicating that the Gl cells did not suffer the mitotic delay. It is evident from fig. 6 that the lag period is located at the end of G2 or at the beginning of mitosis. The cells which were in S or G2 periods at the time of estradiol treatment were delayed in the beginning of mitosis. Reversal of the mitotic delay by CaClz and putrescine
As mentioned earlier estradiol binding to HeLa cells was completely and quickly reversible.
Abscissa: Hours after estradiol treatment; ordinate: log
11+ WWI. Fig. 7. Reversal of mitotic delay. Mitotic collection functions in random cultures of HeLa cells after: ----, addition of colcemide (6.8 x lo-’ M) at t=O h; O-U, 15min pulse treatment with estradiol followed by addition of colcemide; 0 * * * C , 15-min pulse treatment with estradiol immediately followed by the addition of CaCl, (8 x 1OP M) and colcemide; and n---A, 15-min pulse treatment with estradiol followed by addition of putrestine (8 x 1O-L M) and colcemide.
mitotic delay in HeLa cells
233
However, removal of the estrogen from the medium did not result in an immediate recovery from the lag (fig. 4). Assuming that the mitotic delay was due to the negative charge of the e&radio1 molecule, attempts were made to reverse this effect by cationic substances like calcium chloride or putrescine. The addition of CaCl, (8 x 1O-5 M) or putrescine (8 x 1O-5 M) to a culture exposed to estradiol for only 1.5min facilitated the recovery of cells from the mitotic lag (fig. 7). The cells to which CaCl, or putrescine was added following a pulse treatment with estradiol suffered a smaller delay and their rate of mitotic accumulation reached the level of the untreated control faster than those cells that were not rescued (fig. 7). DISCUSSION The data presented in this paper indicate that estradiol induces a reversible mitotic delay in HeLa cells. Simultaneous addition of estradiol and colcemide to a suspension culture of HeLa cells caused an immediate lag in the mitotic accumulation function (figs 1, 3). The duration of the lag period was directly proportional to the concentration of estradiol. The lag was shorter in cultures synchronized by excess thymidine method than in random cultures. The unbalanced growth that is associated with the inhibition of DNA synthesis while the synthesis of RNA and protein was still in progress, seem to reduce the impact of estradiol as manifested by the reduction in the lag period by about 50 % of that of the exponential culture. The effect of estradiol in inducing the mitotic delay was enhanced by the presence of colcemide in the medium. Estradiol (2 X 1O-5M) plus colcemide (6.8 x lo-’ M) induced a lag of 0.5 h in a synchronized culture while estradiol alone did not cause any significant delay (compare curves Q and Q’ with P of fig. 2A, B). Similarly, estradiol at a final dose of 8 x 1O-5M induced a 2 h lag in the presence of colcemide and only of 1 h lag in its absence (fig. 2; curves S and S’). In a previous study it was shown that some of the effects of estradiol on HeLa cells were similar to those of colcemide [12]. As a mitotic inhibitor Exptl Cell Res 57
234 P. N. Rao estradiol was less specific and far less effective than colcemide. Mitotic delay was induced by e&radio1 and not by colcemide even at higher concentrations [13]. Becauseof the fact that both the drugs are mitotic inhibitors they could have some common sites of binding within the cell, such as micro-tubular proteins. Colcemide is highly specific in binding to micro-tubular protein [3] whereas estradiol is not. It is possible that the saturation of these common binding sites by colcemide would relieve more estradiol molecules and thus enhance the effect of e&radio1 in causing mitotic delay. The mitotic lag was seen in all the cells if they were exposed to estradiol continuously. A brief exposure of a random population of HeLa cells to estradiol for 15 min induced a lag period that extended over 10.5 h, after which the curve for mitotic index rose sharply and joined the curve for the control (fig. 4). For this cell line the duration of G2 plus S is equal to 10.5 to 11.0 h. The steep rise of the mitotic index curve to the level of the control was due to the arrival of Gl cells which did not suffer the mitotic delay. This fact was confirmed by the data in fig. 6 which indicate that only those cells that have completed DNA synthesis (in G2) or those that have commenced to do so (in S period) at the time of the estradiol treatment were delayed at the beginning of mitosis. One of the events that distinguishes the interphase nuclei from the prophase nuclei is the condensation of chromatin. The fluctuations in the ionic concentrations in the cells seem to have a great influence on this event [17]. Ionizing radiations are equally effective in inducing mitotic delay [4, 81.The mitotic delay induced by estradiol in HeLa cells is similar to the division delay reported in HeLa S, and hamster cells following X-irradiation [5, 9, 141and in both the cases it is reversed by positively charged compounds like polyamines or CaCl, [15-171. Anderson [l, 21 proposed that the condensation of chromatin which marks the initiation of mitosis is the result of an interaction between positively charged substances like polyamines and the negatively charged DNA strands of the Exptl Cell Res 57
chromosomes, According to this hypothesis one would expect the cationic compounds to enhance the entry of interphase (G2) cells into prophase while anionic substances delay this event. This has been confirmed earlier by the studies on the reversal of radiation-induced mitotic delay in mouse cells and rat thymocytes by calcium salts and agmatine [15-171. Likewise, in the present study CaCl, and putrescine successfully reversed the mitotic delay in HeLa cells induced by estradiol-17p (fig. 7). This fact lends support to Anderson’s hypothesis and would suggest that the ionic equilibrium within the cell plays an important role in the initiation of mitosis. A major portion of this work was done while 1 was with the Department of Physiology and Biophysics, University of Kentucky, Lexington, Ky. I thank Dr James Flesher for performing a pilot experiment to study the extent of reversibility of tritium labeled estradiol binding in HeLa cells. My thanks are also due to Mrs Nora Mitchell and Mrs Mattie Mitchell for their technical assistance. This investigation was aided by PHS grant NIH-CA06835 from the National Cancer Institute. Contribution no. 321, Department of Biophysics, University of Colorado Medical Center, Denver, Colo. 80220. USA.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Anderson, N G, Quart rev biol 31 (1965) 169. - Ibid 31 (1956) 243. Borisv. G G & Tavlor. E W. J cell bio134 (1967) 525. Carl&n, J G & Gaulden,’ M E, Methods in cell physiology (ed D M Prescott). Academic Press, New York (1964). Froese. G. Int i rad biol 10 (1966) 353. Kuchler, R J $ Grauer, R C; Proc sot exptl biol med 110 (1962) 287. Lester, G, Stone, D & Hechter, 0, Arch biochem biophys 75 (1958) 196. McGrath, R A, Rad res 19 (1963) 526. Puck, T T & Steffen, J, Biophys j 3 (1963) 379. Rao, P N & Engelberg, J, Science 148 (1965) 1092. - Cell svnchronv: Studies in biosvnthetic reeulation (ed I L Cameron & G M Padilla). Academyc Press New York ( 1966). - Exptl cell res 48 (1967) 71. Tavlor. E W. J cell bio125 (No. 1. nart 2) (1965) 145. Teiasima, T ‘& Tolmach, L J, Biophys j ‘3‘(1963) 11. Whitfield, J F & Rixon, R H, Exptl cell res 27 (1962) 154. Whitfield, J F, Rixon, R H & Youdale, T, Exptl cell res 27 (1962) 143. Whitfield, J F & Youdale, T, Exptl cell res 43 (1966) 602.
Received February 18, 1969 Revised version received April 14, 1969
ESTRADIOL-INDUCED REVERSAL
Cell Research 57 (1969) 230-234
MITOTIC
BY CALCIUM
DELAY
CHLORIDE
IN HELA AND
CELLS:
PUTRESCINE
P. N. RAO Department of Biophysics, University of Colorado Medical Center, Denver, Colo. 80220, USA
SUMMARY Estradiol-17p induced reversible mitotic delay in HeLa cells which was localized at the beginning of mitosis. The duration of the mitotic lag was dose dependent. Only those cells that were in S or G2 periods at the time of estradiol treatment suffered the delay but not those in Cl period. A synergism between estradiol and colcemide was also observed. Calcium chloride or putrescine successfully reversed the mitotic delay induced by estradiol.
While studying the structural specificy of estrogens in inducing chromosomal non-disjunction in HeLa cells it was observed that HeLa cells suffered mitotic delay immediately following a treatment with estradiol-17fi [12]. Subsequently, a detailed study was taken up to gain insight into the nature of this mitotic delay and the results from this study form the subject of this report. The mitotic delay induced by estradiol in HeLa cells was manifested as a prolongation of G2 + early prophase and was reversed by the addition of calcium chloride or putrescine to the medium. MATERIALS
AND METHODS
HeLa cells grown as suspension cultures were used in the present study. The cultures were always maintained in exponential growth at 37°C in Eagle’s MEM for sninner cultures without calcium, supplemented with non-essential amino acids, sodium pyruvate and 5 % calf serum. They were gassed with a mixture of carbon dioxide (2.7 X) and air. Estradiol-17b (Calbiochem Co.) was dissolved in 95 % ethyl alcohol and then added to the cell suspensions. Final concentration of alcohol did not exceed 0.5 % at any time. Preparation of cells for autoradiography and for scoring mitotic index were described in a previous publication [lo].
Scoring for mitotic index The cells scored as mitoses included cells in all phases of mitosis except those in very early prophase. Because of the uncertainty involved in distinguishing some of the very early prophases from interphase nuclei they were grouped along with the latter. The prophases in which the chromosomal fibers were detectable were classified as mitoses for the determination of the mitotic index. Exptl Cell Res 57
RESULTS Effect of continuous exposure to estradiol on the life cycle of HeLa Cells
Some insight into the effect of estradiol on the life cycle of HeLa cells was obtained by the simultaneous addition of colcemide (6.8 x 1O-7 M) and varying concentrations of estradiol to a culture in exponential growth (fig. 1). Estradiol induced an immediate lag in the collection function for mitotic cells. The greater the concentration of estradiol, the greater was the lag. Subsequent to the initial lag in the collection function the rate of increase in the mitotic index for the treatment (2 x 1OP M of estradiol + cc!cemide) was the same as for the control (colcemide only). There was no delay or decrease in the rate of flow of cells from Gl into S-period as measured by the increase in the percentage of cells labeled with 3H-thymidine following the addition of estradiol and colcemide to a random culture. At concentrations of 2 x 10-j M or below, estradiol had no significant effect on the rate of movement of cells through interphase. At higher doses of estradiol the curves for mitotic accumulation function significantly diverged from that of the control indicating that the cells were progressively slowed down in their movement towards mitosis. With 8 X 1O-5M of estradiol in the medium, a major portion of the cells failed to reach mitosis. For this reason a
Estradiol-induced mitotic delay in HeLa cells 231
Abscissa: Time, h; ordinate: mitotic index.
Fig. 1. Estradiol dose-effect relationships: Estradiol and colcemide (6.8 x lo-’ M) were added simultaneously at zero time. A, Control with colcemide alone; B, 2 x lo-& M; C, 4 x 1O-5M; D, 8 x 1O-5M of estradiol plus colcemide.
final concentration of 2 x 1O-5 M of estradiol, at which the toxicity was minimal, was chosen to be the optimal dose and had been used uniformly throughout this study unless otherwise stated. The dosage effect of estradiol on mitotic delay was also studied in synchronized cultures. The procedure for synchronization of HeLa cells in suspension by excess thymidine double block technique had been previously described [ll]. To such synchronized population of HeLa cells various concentrations of estradiol and 6.8 x lo-’ M of colcemide were simultaneously added at 6 h after the reversal of the second thymidine block and the collection functions for mitosis were obtained at regular intervals. The mitotic
Abscissa: Hours after releaseof thymidine block; ordinate:
mitotic index. Fig. 2. Mitotic delay in synchronized cultures: (A), Synergism between estradiol and colcemide. Various concentrations of estradiol and colcemide (6.8 x lo-’ M) were added at 6 h after the releaseof the secondthymidin block. P, Control; colcemide only; Q, 2 x 1O-5 M; R, 4 x 1O-5 M; S, 8 x 1O-5M of estradiol plus colcemide. (B) Synchronized cultures treated with estradiol alone. The treatments represented by the curves Q’, R’ and S’ are the same as in fig. 2 a except that no colcemide was added in this case, The decreasein mitotic index following the peak was due to the completion of mitosis and re-entry of cells into interphase.
index in the control reached 50 % level at t ~9.0 h, where as it was delayed by 0.5 to 2 h in cultures treated with colcemide plus estradiol, depending on the dose of the estrogen (fig. 24. When synchronized cultures were similarly treated with estradiol alone (without colcemide) the durations of the mitotic lag were relatively shorter (ranging from 0 to 1 h) than those when colcemide was also present in the medium along with estradiol. In other words the presence of colcemide enhanced the effect of estradiol in inducing mitotic delay indicating some degree of synergism between the two drugs (fig. 2A, B). The gradual decrease of the mitotic index following the peak (curves Q’, R’ and S’ of fig. 211) was due to the completion of mitosis and re-entry of cells into interphase. At lower concentrations of estradiol the cells completed mitosis at a faster rate. Increased concentrations of estradiol prolonged the duration of mitosis in general and the metaphase in particular [12]. The effectiveness of estradiol in inducing the mitotic delay was tested in another experiment in which the estrogen was added to a synchronized and colcemide-treated culture when the wave of mitotic activity was in progress. The addition of the hormone resulted in an immediate cessation in the increase of mitotic index which remained constant for an hour before resuming the normal rate of increase (fig. 3). Effect of pulse treatment with estradiol To answer the question, whether continuous presence of estradiol was necessary for the induction of mitotic delay, the mitotic accumulation function was studied in a random culture following a pulse treatment with estradiol. Estradiol dissociates from the cells rather easily [6, 71. A change of medium would remove almost all the estradiol from the cells. HeLa cells were exposed ro estradiol (2 x 1O-5 M) for 15 min, centrifuged and resuspended in fresh medium containing colcemide. The increase in mitotic index was plotted as a function of time (fig. 4). The mitotic delay was exhibited by cells that entered mitosis during the estradiol treatment and also during a period of 10.5 to 11.0 h imExptl Cell Res 57
232
P. N. Rao
Abscissa: Hours after release of TdR block; ordinate: mitntic index. Fig. 3. The effect of estradiol on the entry of G2 + early prophase cells into mitosis. Colcemide (6.8 x 1O-7 M) was added to a synchronized culture at 6 h after the release of the thymidine block. Estradiol (2 x IO-’ MI was added 2 h later as indicated by the arrow. O-O, control with colcemide alone; x - x , cells were treated with colcemidefirst, to which estradiol was added2 h later.
Abscissa: Time, h; ordinate: log [l + N(M)]. Fig. 4. Effect of pulse lrealment of HeLa cells with estradiol on mitotic delay: Colcemide (6.8 x lo-’ M) was present in the control and the treatments throughout the course of the experiment. O-O, 15 min; and A-A, 5 h of estradiol treatment; O-O , control with colcemide alone. The arrows a and b indicate the termination of estradiol treatment and the arrows u’ and b’ indicate the reversal of the mitotic delay. N(M)-Mitotic index.
the treatment. For this cell mediately following line the combined duration of G2 and S-periods was equal to 10.5 to 11.0 h. In other words the cells that were in S- or G2 periods at the time of estradiol treatment suffered the mitotic delay. If cell division was not blocked by adding colcemide following a 15 min exposure to estradiol the mitotic index curve showed a Ushaped depression at its origin (fig. 5). This depression was the result of an immediate decrease in the rate of arrival of G2 + early prophase cells into mitosis, but not in the departure of cells from mitosis into Gl. At the end of the lag period (about 2 h) the mitotic index gradually rose from about 0.01 to 0.037 which was normal for a culture in exponential growth.
cells in spinner-B were exposed to estradiol for only 15 min. Colcemide was added to both the spinners to block cell division and samples taken at regular intervals for autoradiography. The mitotic index in the non-labeled segment of the population reached its maximum in two steps (fig. 6). The first step represented the arrival of G2 cells into mitosis while the second step represented that of Gl cells. Except for the initial lag of 2.5 h the durations of G2, S and Gl in continuous estradiol treatment were identical with those in the control. The difference between the two estradiol treatments (continuous and pulse) lie in the rate of Gl cells reaching mitosis. The slope of mitotic accumulation function of Gl cells in continuous treatment was parallel to those of G2 and S cells (fig. 6).
Specificity of estradiol effect to S and G2 periods The following experiment was designed to confirm the observation that the induction of mitotic delay by a pulse treatment with estradiol was specific to cells in S and G2 periods. A random culture was treated with 3H-thymidine (1 &ml; 0.36 c/mM, New England Nuclear Corp.) for 15 min to label the cells in DNA synthetic period and then divided into two spinner flasks-A and B. The cells in spinner-A were treated with estradiol continuously whereas the Erptl Cell Res 57
Abscissa: After pulse treatment with estradiol; ordinate: mitotic index. Fig. 5. A plot of mitotic index in a random culture of HeLa cells following 15 min pulse treatment with 2 x 1O-5 M of estradiol. In this culture cell division was not blocked by colcemide.
Estradiol-induced
0
4
6
12
16
20
24
Abscissa: Time, h; ordinate: log [l I N(M)]. Fig. 6. Effect of estradiol on cells in S and G2 periods at the time of the treatment. The solid symbols represent continuous presence of estradiol in the medium; whereas, the open symbols represent a 15-min pulse treatment with estradiol. 0.-.O,G2; A---A,S; q --n,Gl+Mcells at the time of estradiol treatment. N(M), Mitotic index.
In the pulse treatment the slope was greater than normal indicating that the Gl cells did not suffer the mitotic delay. It is evident from fig. 6 that the lag period is located at the end of G2 or at the beginning of mitosis. The cells which were in S or G2 periods at the time of estradiol treatment were delayed in the beginning of mitosis. Reversal of the mitotic delay by CaClz and putrescine
As mentioned earlier estradiol binding to HeLa cells was completely and quickly reversible.
Abscissa: Hours after estradiol treatment; ordinate: log
11+ WWI. Fig. 7. Reversal of mitotic delay. Mitotic collection functions in random cultures of HeLa cells after: ----, addition of colcemide (6.8 x lo-’ M) at t=O h; O-U, 15min pulse treatment with estradiol followed by addition of colcemide; 0 * * * C , 15-min pulse treatment with estradiol immediately followed by the addition of CaCl, (8 x 1OP M) and colcemide; and n---A, 15-min pulse treatment with estradiol followed by addition of putrestine (8 x 1O-L M) and colcemide.
mitotic delay in HeLa cells
233
However, removal of the estrogen from the medium did not result in an immediate recovery from the lag (fig. 4). Assuming that the mitotic delay was due to the negative charge of the e&radio1 molecule, attempts were made to reverse this effect by cationic substances like calcium chloride or putrescine. The addition of CaCl, (8 x 1O-5 M) or putrescine (8 x 1O-5 M) to a culture exposed to estradiol for only 1.5min facilitated the recovery of cells from the mitotic lag (fig. 7). The cells to which CaCl, or putrescine was added following a pulse treatment with estradiol suffered a smaller delay and their rate of mitotic accumulation reached the level of the untreated control faster than those cells that were not rescued (fig. 7). DISCUSSION The data presented in this paper indicate that estradiol induces a reversible mitotic delay in HeLa cells. Simultaneous addition of estradiol and colcemide to a suspension culture of HeLa cells caused an immediate lag in the mitotic accumulation function (figs 1, 3). The duration of the lag period was directly proportional to the concentration of estradiol. The lag was shorter in cultures synchronized by excess thymidine method than in random cultures. The unbalanced growth that is associated with the inhibition of DNA synthesis while the synthesis of RNA and protein was still in progress, seem to reduce the impact of estradiol as manifested by the reduction in the lag period by about 50 % of that of the exponential culture. The effect of estradiol in inducing the mitotic delay was enhanced by the presence of colcemide in the medium. Estradiol (2 X 1O-5M) plus colcemide (6.8 x lo-’ M) induced a lag of 0.5 h in a synchronized culture while estradiol alone did not cause any significant delay (compare curves Q and Q’ with P of fig. 2A, B). Similarly, estradiol at a final dose of 8 x 1O-5M induced a 2 h lag in the presence of colcemide and only of 1 h lag in its absence (fig. 2; curves S and S’). In a previous study it was shown that some of the effects of estradiol on HeLa cells were similar to those of colcemide [12]. As a mitotic inhibitor Exptl Cell Res 57
234 P. N. Rao estradiol was less specific and far less effective than colcemide. Mitotic delay was induced by e&radio1 and not by colcemide even at higher concentrations [13]. Becauseof the fact that both the drugs are mitotic inhibitors they could have some common sites of binding within the cell, such as micro-tubular proteins. Colcemide is highly specific in binding to micro-tubular protein [3] whereas estradiol is not. It is possible that the saturation of these common binding sites by colcemide would relieve more estradiol molecules and thus enhance the effect of e&radio1 in causing mitotic delay. The mitotic lag was seen in all the cells if they were exposed to estradiol continuously. A brief exposure of a random population of HeLa cells to estradiol for 15 min induced a lag period that extended over 10.5 h, after which the curve for mitotic index rose sharply and joined the curve for the control (fig. 4). For this cell line the duration of G2 plus S is equal to 10.5 to 11.0 h. The steep rise of the mitotic index curve to the level of the control was due to the arrival of Gl cells which did not suffer the mitotic delay. This fact was confirmed by the data in fig. 6 which indicate that only those cells that have completed DNA synthesis (in G2) or those that have commenced to do so (in S period) at the time of the estradiol treatment were delayed at the beginning of mitosis. One of the events that distinguishes the interphase nuclei from the prophase nuclei is the condensation of chromatin. The fluctuations in the ionic concentrations in the cells seem to have a great influence on this event [17]. Ionizing radiations are equally effective in inducing mitotic delay [4, 81.The mitotic delay induced by estradiol in HeLa cells is similar to the division delay reported in HeLa S, and hamster cells following X-irradiation [5, 9, 141and in both the cases it is reversed by positively charged compounds like polyamines or CaCl, [15-171. Anderson [l, 21 proposed that the condensation of chromatin which marks the initiation of mitosis is the result of an interaction between positively charged substances like polyamines and the negatively charged DNA strands of the Exptl Cell Res 57
chromosomes, According to this hypothesis one would expect the cationic compounds to enhance the entry of interphase (G2) cells into prophase while anionic substances delay this event. This has been confirmed earlier by the studies on the reversal of radiation-induced mitotic delay in mouse cells and rat thymocytes by calcium salts and agmatine [15-171. Likewise, in the present study CaCl, and putrescine successfully reversed the mitotic delay in HeLa cells induced by estradiol-17p (fig. 7). This fact lends support to Anderson’s hypothesis and would suggest that the ionic equilibrium within the cell plays an important role in the initiation of mitosis. A major portion of this work was done while 1 was with the Department of Physiology and Biophysics, University of Kentucky, Lexington, Ky. I thank Dr James Flesher for performing a pilot experiment to study the extent of reversibility of tritium labeled estradiol binding in HeLa cells. My thanks are also due to Mrs Nora Mitchell and Mrs Mattie Mitchell for their technical assistance. This investigation was aided by PHS grant NIH-CA06835 from the National Cancer Institute. Contribution no. 321, Department of Biophysics, University of Colorado Medical Center, Denver, Colo. 80220. USA.
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Received February 18, 1969 Revised version received April 14, 1969