The Effect of Potassium on the Cell Membrane Potential and the Passage of Synchronized Cells through the Cell Cycle PETER J. STAMBROOK,] HOWARD G. SACHSZ AND J. D. EBERT Carnegie Institution of Washington, Department of Embryology, I1 5 W. University Parkway, Baltimore, Maryland 21210
ABSTRACT The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the GI phase. A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K+. Cells placed directly into high K+ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K+ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase m isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early GI which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.
Manipulation of the ionic composition of the medium supporting cell growth can result in profound changes in cell proliferation and phenotypic expression. Raising the external potassium ion concentration at the expense of the sodium ion concentration results in a reduction in cell multiplication rate in a CHO (Chinese hamster ovary) cell line (Cone, '71), a BHK (baby hamster kidney) cell line and its polyoma virus transformed variant (Orr et al., '72), and 3T3 and SV40 transformed 3T3 mouse cell lines (Yoshikawa-Fukada and Nojima, '72).Changes in potassium levels have also been shown to reduce puff formation in J. CELL.PHYSIOL., 85: 283-292.
Chironimus salivary glands and to promote chondrogenesis in embryonic cells in vitro (Lash et al., '73). One of the most rapid cellular changes following elevation of the external potassium level is a reduction in the cell membrane potential. Pardee ('71) has proposed that changes at the membrane level can profoundly influence cell division, and others have proposed that the cell membrane Received May 6, '74. Accepted Aug. 29, '74. Present address: Case Western Reserve University, Department of Biology, Cleveland, Ohio 44106. Present address: University of Illinois Medical Center, Department of Anatomy, P.O. Box 6998, Chicago, Illinois 60680.
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PETER J. STAMBROOK, HOWARD G. SACHS AND J. D. EBERT
potential or the transmembrane ionic concentrations which underly the membrane potential may act as a specific mitotic regulator (Cone, '71;Cone and Tongier, '73; McDonald et al., '72). We have recently shown, however, that while the membrane potential of isolated V79 Chinese hamster cells in a synchronized culture varies in a predictable fashion through the cell cycle, the observed periodic increase does not appear to play a causal role in regulating the traverse of cells through the cell cycle (Sachs et al., '73). In the present study we have performed further experiments on V79 cells to confirm our initial observation that the cell membrane potential does not play a causal role in mitotic regulation even though in isolated cells it displays a marked periodicity during the cell cycle. Since isolated cells display an abrupt increase in the cell membrane potential at the start of the S phase, we have examined whether such an increase in membrane potential is essential for the initiation of DNA synthesis and traverse of the cell cycle. Specifically we have asked whether cells with an artificially depressed membrane potential can enter the S phase and carry out DNA synthesis in a normal fashion. In addition, we have examined whether the reduction in growth rate observed in cells cultured in medium containing high levels of potassium is due to a random retardation of traverse through the cell cycle or whether there are specific periods of the cell cycle that are particularly sensitive to the high potassium regimen. The latter possibility was suggested by experiments in which BHK cells were cultured in medium containing high levels of potassium (Om et al., '72). When the high potassium medium was replaced with normal medium, the cells underwent a wave of DNA synthesis and mitosis indicating that a degree of synchrony had been imposed upon the culture. MATERIALS AND METHODS
Cell line and culture conditions The cells used in this study were from a subclone of the Chinese hamster V79 line originated by Ford and Yerganian ('59). Cell culture conditions for cell propagation were identical to those previously described (Stambrook and Sisken, '72). Media containing various potassium concentrations
were prepared by molar replacement of Na+ by K+ ( O n et al., '72). The osmolarity of normal and K+-substituted media was maintained between 315 and 325 milliosmoles/l. Synchronization of cells Populations of synchronized mitotic cells were collected as described by Stambrook and Sisken ('72). Briefly, cells were grown in roller bottles and exposed to colcemid (0.06 pg/ml) for the last 90 minutes. The colcemid-containing medium was poured off and replaced with Gey's balanced salt solution (Gey, '36)which was gently swirled over the cell layer, dislodging mitotic cells. The mitotic cell suspension was centrifuged at 1500 RPM for two minutes and the cell pellet resuspended in the appropriate medium for cell cycle analysis. In each experiment, an aliquot of the mitotic cell preparation was suspended in 1% sodium citrate for microscopic examination. After ten minutes in the sodium citrate solution the cells were centrifuged, resuspended in methano1:acetic acid (3:1), spotted on a microscope slide and flamedried. The slide was stained with toluidine blue and scanned to determine the initial level of synchrony. At the start of an experiment, 95 to 98% of the cells were in metaphase.
Cell cycle analysis Synchronized cells were plated on a series of 5 cm culture plates and their progress through the cell cycle was followed by labeling the cells for ten minutes with 3H thymidine (1 pclml) at one or two hour intervals after synchronization. The cells were washed and fixed in methano1:acetic acid (3:1),coated with photographic emulsion (NTB-2) and kept in the dark at 4 ° C for one week. The plates were developed and the percent of labeled cells and the mitotic index at each time interval were measured. The time elapsed before the appearance of a mitotic peak measures the generation time; the curve describing the percent of labeled cells defines the GI and S periods. Electrophysiology Exponentially growing cells were cultured in 35 mm plastic tissue dishes (Falcon). The appropriate potassium-containing medium was added to the plate, and
P O T A S S I U M A N D T H E CELL CYCLE
the cells were impaled with a microelectrode. The membrane potential was measured as described previously (Sachs and McDonald, '72; Sachs et al., '74). Briefly, impalement of well isolated cells with no visible contacts to other cells was carried out on the stage of a n inverted phasecontrast microscope. Membrane potentials were determined using high resistance micropipettes with tip potentials (measured by breaking the tip) of less than 4 mV measured in the culture medium. Impalements in which the measured potential failed to return to within 2 mV of the baseline before impalement were rejected. RESULTS
285
of a cell in early GI (Sachs et al., '74). The change in membrane potential in response to exposure to 115 mM potassium is rapid, occurring within five minutes, and is maintained for at least 24 hours. Measurements were made on 15-20 separate cells for each time interval and for each potassium concentration ensuring that measurements were made on cells representing all stages of the cell cycle. Since S + Gz occupy almost 70% of the cell cycle, the values obtained with 115 mM potassium must represent a population with a significant proportion of cells in the S or Gf phases. The ratio of potassium permeability to sodium permeability determined by the Goldman equation (Williams, '70) was 0.33 as compared to 0.082 for BHK cells with a resting potential of -55 mV (Sachs and McDonald, '72).
External potassium Concentration and the cell membrane potential The cell membrane potential of Chinese hamster V79 cells in exponential growth External potassium concentrations and was measured after varying lengths of excell growth posure to media containing increasing potassium concentrations. As the potassium The growth rate of cells exposed to varconcentration was raised, the membrane ious external concentrations of potassium potential decreased (fig. 1) and at 115 was measured. Between 1 mM and 30 mM mM potassium exhibited a value below that potassium there was little effect on the growth rate (fig. 2a). However, when the -30 potassium concentration was raised to 60 mM or higher, there was a marked decrease in both the growth rate and the density to which the cells would grow (figs. 2a,b). At a concentration of 115 mM potasc sium, the growth rate was very rapidly in> hibited, yet when the potassium was reE -20 moved after a 12-hour exposure, cell growth .-c0 resumed at a rate and to a density similar c Q, to that of control cultures (fig. 2b). The rec versibility of the potassium effect indicates 2 that even though cell growth has stopped, C the cells remain viable. e -10 In an experiment similar to that den scribed in figure Za, cells were collected $ I after 60 hours of exposure to the various exmnal potassium concentrations, fixed, seeded on slides and flame-dried. A count \ \ of mitotic cells revealed an inverse relationship between the growth rate and the 5 10 20 100 200 mitotic index (table 1). suggesting that the Potassium Concentration (mM) progress of cells through mitosis is retarded Fig. 1 Cell membrane potential as a function when incubated with high levels of potasof external potassium concentration. Exponentially sium.
0)
growing cells were transferred to media containing
increasing concentrations of potassium. Five minutes after addition of the potassium media, cells were impaled and their membrane potentials recorded. Bar represents mean S.E.
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Effect of high potassium levels on cell cycle traverse To further examine the effect of high
286
PETER J. STAMBROOK, HOWARD G. SACHS AND J. D. EBERT lo7-
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Fig, 2 Effect of external potassium on cell growth. (a) Inhibition of cell proliferation by exogenous potassium. Cells were seeded into a series of 60 mm culture plates at 105 cells per plate. After 24 hours, the medium was replaced with media containing increasing concentrations of potassium, and at subsequent intervals, the cell number per plate was determined in duplicate. 1 mM K+ (crosses); 10 mM K + (solid triangles); 30 mM K+ (solid circles); 60 mM K+ (open squares); 100 mM K+ (solid squares). (b) Recovery of cell growth. Cells were treated as in figure 2a After 12 hours, half of the cells which had been exposed to 115 mM potassium was transferred to fresh medium containing 10 mM potassium. The number of cells per plate was determined i n duplicate at different times during the culture period. 10 mM K+ (open circles, solid line); 100 mM K+ (solid squares, solid line); 115 mM K + (open triangles, solid line); 115 mM K+ transferred to 10 mM K+ (solid triangles, dashed line).
TABLE 1
The relationship between exogenous potassium concentration and mitotic index Potassium concentration (mM)
1.0
10 30 60 100 115
Mitotic index
1.8 2.2 2.4
3.6 6.2
8.8
Cells seeded at lo5cells per 60 mm culture plate were grown for 24 hours in control medium and transferred to media containing 1.0, 10, 30, 60, 100, or 115 mM K + . After 60 hours of additional culture (24 hours for the 115 mM K + sample) the media were aspirated and the cells washed and fixed in methano1:acetic acid (3:l). One thousand cells were counted from several random fields from each plate and the percentage of mitotjc cells determined.
levels of potassium on the cell cycle, and specifically the ability of cells to enter the S phase, mitotic cells were collected and their passage through the cell cycle was followed in the presence or absence of high potassium medium. A population of mitotic cells was divided into four series of aliquots, each series containing 18 replicate culture plates. The f m t series contained cells seeded directly into medium containing 10 mM potassium which served as the control (fig. 3a). In the second series the cells were placed directly into medium containing 115 mM potassium (fig. 3b). The third group consisted of cells first suspended in 115 mM potassium and after two hours transferred to 10 mM potassium (fig. 3c). And the last group was first placed in 10 mM potassium
28 7
POTASSIUM AND THE CELL CYCLE
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TIME (hrs) Fig. 3 Response of synchronized cells to different exogenous potassium regimens. A synchronized cell population was prepared by selective detachment of mitotic cells and divided into four different aliquots. Each series consisted of 18 replicate60 mm culture plates with equal numbers of cells per plate. (a) Cells incubated in 10 mM K+. (b) Cells incubated in 115 mM K+. (c) Cells incubated for two hours in 115 mM K+ then transferred to 10 mM K+.(d) Cells incubated for two hours in 10 mM K + then transferred to 115 mM K+. In each series, duplicate plates were labeled with 3H thymidine (1 pclml) for ten minutes at successive intervals after synchronization. The plates were aspirated and washed, and the cells fixed with methano1:acetic acid (3:l). The plates were then coated with emulsion for autoradiography. Percentage labeled cells (open circles, solid line); mitotic cells per 1000 cells (crosses, dashed line).
and after two hours transferred to 115 mM potassium, prior to the onset of DNA synthesis. In each series, duplicate plates of cells were labeled with 3H thymidine for ten minutes at varying times after mitosis, and at every time interval the percent of cells which were labeled was determined by autoradiography. The mitotic index was calculated from the same plates. The control series displayed an approximate 10hour generation time with about a %hour GI and a 6-hour S phase (fig. 3a). Cells placed directly into 115 mM potassium me-
dium were retarded in their exit from mitosis and exhibited a greatly impaired DNA synthetic activity (fig. 3b). At no time interval examined were more than 60 percent of the cells labeled and the density of label over nuclei was considerably lower than in the control series. By 14 hours, four hours after the mitotic peak in the control series, no mitotic cells were evident. However, at least a fraction of the population was able to traverse the entire cycle, albeit abnormally, since at 24 hours nearly 20 percent of the cells were in metaphase,
288
PETER J. STAMBROOK, HOWARD G . SACHS AND J. D. EBERT
apparently blocked at that stage. Cells which were first suspended in high potassium medium and two hours later transferred to control medium (fig. 3c), were retarded in their exit from mitosis. Once fresh medium was added, the cells progressed normally through the remainder of the cycle although all marker events were delayed by two hours compared to the control series. The marker events included the times of onset of DNA synthesis, the minimum in the DNA synthetic curve and the mitotic peak. In the last series (fig. 3d), the cells, which were first suspended in control medium and transferred to 115 mM potassium medium at two hours, exited mitosis normally and began DNA synthesis on schedule. The duration of the S phase appeared slightly lengthened and the cells arrived at mitosis with minor delay. A block in mitosis was again evident since cells accumulated in mitosis at 12 and 14 hours, but by 24 hours the mitotic index had dropped below control values. Three conclusions can be drawn from the above series of experiments. (1) V79 cells can initiate and continue DNA synthesis with a membrane potential artificially depressed to a level below that of a normal GI cell (figs. 1, 3d). Synchronized cells incubated with control medium but transferred to 115 mM potassium medium prior to the initiation of DNA synthesis can enter and traverse the S phase and Gf and proceed to the next division with little delay. (2) The high potassium medium impedes the passage of cells through mitosis (table 1, figs. 3b, 3d). (3) There is an event (or events) during GI that is particularly sensitive to the 115 mM potassium medium. Effect of high potassium levels on chromosome structure Since cells incubated in high levels of potassium are impeded in mitosis as well as in GI, mitotic cells were examined for cytological abnormalities. When exponentially growing cells, incubated for 30 hours in high potassium medium, were fixed in methano1:acetic acid (3:1) chromosomes in about 30 percent of the metaphase figures displayed a marked banding pattern after staining with toluidine blue. When chromosome preparations were made with cells from the 24-hour time interval in experiment 3b or subsequent similar experiments,
chromosomes in 92 percent of the metaphase complements were condensed compared to chromosomes in control preparations (fig. 4). In addition to the deeply staining bands apparent in varying degrees
Fig. 4 Effect of prolonged exposure to 115 mM K+ on chromosome morphology. Mitotic cells were collected by mitotic selection and exposed to 115 mM K+ for 24 hours (as in fig. 312). All cells were trypsinized, washed, and suspended in 1 % sodium citrate for 15 minutes. After fixation in methanol: acetic acid (3:1), metaphase chromosomes were spread on slides and stained with toluidine blue. (a) Metaphase chromosomes from a control culture. (b) Metaphase chromosomes from a potassiumtreated culture showing densely staining bands. ( c ) Metaphase chromosomes from a potassiumtreated culture displaying distinct coiling.
POTASSIUM AND THE CELL CYCLE
in most of the condensed chromosomes (fig. 4b), about 15 percent of the mitotic cells contained chromosomes which displayed a marked super-coiled structure (figs. 4c, 5) never evident in control cultures.
289
mosomal puffing patterns in ChiTonomus. Lezzi ('70) subsequently argued that alterations in intracellular Na+/K+ratios act in the same manner, and that a change in the external ratio modifies the internal ratio. The possibility that changes in intracelluDISCUSSION lar calcium ion levels can affect cellular A key question raised by experiments activities should also be considered since which mod* cellular functions by manip- depolarization of the cell membrane alters ulating the cell membrane potential is the membrane permeabilities to a variety of whether alterations in extracellular ionic molecules including calcium ions (e.g., regimens affect cellular activities by act- Baker et al., '71). The stimulation of culing directly on the cell membrane or by pri- tured lymphocytes by phytohemaglutinin, marily altering the intracellular ionic bal- for example, is accompanied by an immediance. McDonald et al. ('72) have shown ate increase in calcium uptake (Whitney that treatment of BHK cells with high lev- and Sutherland, '73) and subsequently an els of potassium (1 14 mM) not only reduces increase in the uptake of potassium (Quasthe membrane potential but also increases tel and Kaplan, '70). Fertilization of sea urthe intracellular potassium content, even chin embryos is also accompanied by memafter a short incubation. That altered Na+/ brane depolarization (Tupper, '73) and by K+ ratios can affect nuclear activity was an increase in calcium uptake (Mazia, demonstrated by Kroeger ('63 and '66) who '37) both of which can be mimicked by found that changes in the extracellular treating eggs with a calcium ion-specific Na+/K+ ratio dramatically altered the chro- ionophore (Steinhardt and Epel, '74). Al-
Fig. 5
Enlargement of figure 4c detailing the coiled morphology of the chromosomes.
290
PETER J. STAMBROOK, HOWARD G. SACHS A N D J. D. EBERT
though the present study does not examine whether intracellular ion levels change as cells traverse the cell cycle, the data do argue strongly against a major regulatory role for the cell membrane potential during the cell cycle traverse. In a previous report (Sachs et al., '74) we demonstrated that the cell membrane potential of isolated Chinese hamster cells exhibits cyclic variations during the cell cycle. A somewhat similar study on L cells by Cone ('69) also showed cyclical changes, but in a different direction than we found for V79 cells. At that time we suggested that these potential changes played no regulatory function in mitotic activity. The basis for this argument was that the membrane potential of cells physically apposed in clusters displayed remarkably constant values with very slight variation, even though they were obtained from cells in all stages of the cell cycle. The present study further demonstrates that isolated cells with membrane potentials artificially depressed by high external potassium levels can initiate DNA synthesis and traverse the S phase and G r . Under these experimental conditions the cells traverse late Gz with a membrane potential lower than that of normal isolated GI cells (Sachs et al., '74). There is no increase in membrane potential which normally occurs at the onset of the S phase. The observation that synchronized cells, artificially maintained in a depolarized state, can initiate and carry on DNA synthesis is consistent with the findings of Tupper ('73) who has reported that in early sea urchin development protein synthesis, DNA synthesis and growth are unaffected by maintaining embryos in a depolarized state with high potassium levels. The data indicate that Chinese hamster cells are particularly sensitive to high potassium levels at two unique points of the cell cycle: mitosis and early GI. The two lesions are closely linked in time, but appear to be distinctly separate events. This conclusion derives from the data described in figure 3. Table 1 and figures 3b-d show that cells in mitosis are impeded by a high potassium regimen. Synchronized mitotic cells incubated in the high potassium medium behave abnormally in two ways. In addition to being retarded in their exit from
mitosis, these cells also display severely asynchronous, defective and delayed entry into the S phase (fig. 3b). The observed asynchrony is not due to a loss of synchrony as cells exit from mitosis, since if the high potassium medium is replaced by control medium after two hours, the cells initiate DNA synthesis as synchronously as control populations, but with a two hour delay. Most of the asynchrony must therefore arise during the GI period. If mitotic cells are first cultured for two hours in control medium before being placed into high potassium, they pass through the remainder of G I , initiate DNA synthesis and traverse S and G2 normally. These results suggest that in addition to a block in mitosis there is a second lesion sensitive to the high potassium regimen which occurs in early GI which may impair the start of DNA synthesis, and which is bypassed during the first two hours of culture in control medium. Orr et al. ('72) have previously reported that high external levels of potassium imposed a degree of synchrony on BHK cells. Following release from the 114 mM potassium block, they observed two waves of DNA synthesis and mitosis. They concluded that there were two sensitive periods in the cell cycle: a minor block (15% of the population) either in late GI or early S, and major block in the mid GI period. Cone and Tongier ('71) have previously noted that CHO cells can also be blocked by high potassium medium with the block occurring in the latter half of the GI period. Thus the data obtained with V79 cells are consistent with those obtained with other cells and may indicate a general sensitivity to high extracellular potassium ion concentration during some part of the GI period. The block in mitosis which occurs in the presence of high levels of potassium is likely due to an alteration in chromosome architecture (figs. 4b,c, 5) which in turn may impede chromosome segregation and decondensation. The morphology of these chromosomes resembles the structures described by Ohnuki ('65) who obtained chromosomes with similar spiral architecture by treating colchicine-derived mitotic human peripheral leucocytes with a hypotonic salt solution containing 31 mM potassium. He argued, however, that the coils represented a despiralization of the chromosome
POTASSIUM AND THE CELL CYCLE
rather than a coiling superimposed over the normal structure of the chromosome which results in chromosome shortening. ACKNOWLEDGMENTS
We thank Drs. D. Fambrough and A. Ritchie for their critical reading of the manuscript and Delores Sommerville and Bessie Smith for their excellent technical assistance. LITERATURE CITED Baker, P. F., A. L. Hodgkin and E. B. Ridgeway 1971 Depolarization and calcium entry in squid giant axons. J. Physiol. (London), 218: 709-755. Cone, C . D. 1969 Electroosmotic interactions and accompanying mitosis initiation in sarcoma cells in vitro. Trans. N. Y. Acad. Sci., 31 : 404427. 1971 Unified theory on the basic mechanism of normal mitotic control and oncogenesis. J. Theor. Biol., 30: 151-181. Cone, C. D., and M. Tongier 1971 Control of somatic cell mitosis by simulated changes in the transmembrane potential level. Oncology, 25 : 168-182. 1973 Contact inhibition of division: involvement of the electrical transmembrane potential. J. Cell, Physiol., 82: 373-386. Ford, D. K., and G. Yerganian 1959 Observations on the chromosomes of Chinese hamster cells in tissue culture. J. Nat. Cancer Inst., 21 : 393-475. Gey, G. 0. 1936 Maintenance of human normal cells and tumor in continuous culture. Amer. J. Canc. Inst., 27: 45-76. Kroeger, H. 1963 Chemical nature of the system controlling gene activities in the cell. Nature, 200: 1234-1235. Kroeger, H. 1966 Potential differenz und Puffmuster. Electrophysiologische und cytologische Untersuchungen an den Speicheldruesen von Chironomus thummi. Exp. Cell Res., 41 : 64-80. Lash, J. W., K. Rosene, R. R. Minor J. C. Daniel and R. A. Kosher 1973 Environmental enhancement of in vitro chondrogenesis. 111. The influence of external potassium ions and chondrogenic differentiation. Develop. Biol., 35: 370375. Lezzi, M. 1970 Differential gene activation in iso-
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lated chromosomes. Int. Rev. Cytol., 29: 127168. Mazia, D. 1937 The release of calcium in Arbacia eggs on fertilization. J. Cell. Comp. Physiol., 10:291-304. McDonald, T. F., H. G. Sachs, C. W. M. Orr and and J. D. Ebert 1972 External potassium and baby hamster kidney cells: intracellular ions, ATP, DNA synthesis and membrane potential. Develop. Biol., 28: 290-303. Ohnuki, Y. 1965 Demonstration of the spiral structure of human chromosomes. Nature, 208: 9 16-9 18. Orr, C. W., M. Yoshikawa-Fukada and J. D. Ebert 1972 Potassium: Effect on DNA synthesis and multiplication of baby hamster kidney cells. Roc. Nat. Acad. Sci. (U.S.A.), 69: 243-247. Pardee, A. 1971 The cell surface as a regulator of animal cell division. In Vitro, 7: 95-104. Quastel, M. R., and J. G. Kaplan 1970 Lymphocyte stimulation: the effect of ouabain on nucleic acid and protein synthesis. Exp. Cell Res., 62 : 407420. Sachs, H. G., and T. F. McDonald 1972 Membrane potentials of BHK (Baby Hamster Kidney) cell line: Ionic and metabolic determinants. J. Cell Physiol., 80:347-358. Sachs, H. G., P. J . Stambrook and J. D. Ebert 1974 Changes in membrane potential during the cell cycle. Exp. Cell Res., 83: 362-366. Stambrook, P. J., and J. E. Sisken 1972 Induced changes in the rates of uridine-ZH uptake and incorporation during the GI and S periods of synchronized Chinese hamster cells. J. Cell Biol., 52: 514-525. Steinhardt, R. A,, and D. Epel 1974 Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. (U.S.A.), 71: 1915-1919. Tupper, J. T. 1973 Potassium exchangeability, potassium permeability, and membrane potential: some observations in relation to protein synthesis in the early echinoderm embryo. Develop. Biol., 32:140-154. Whiteney, R. B., and R. M. Sutherland 1974 Kinetics of calcium transport in lymphocytes b e fore and after stimulation by phyto-hemaglutinin. In: Proc. 7th Leukocyte Culture Conference. F. Daguillard, ed. Academic Press, pp. 63-72. Williams, J. A. 1970 Origin of transmembrane potentials in non-excitable cells. J. Theor. Biol., 28 : 287-296. Yoshikawa-Fukada, M., and T. Nojima 1972 Biochemical characteristics of normal and virally transformed mouse cell lines. J. Cell Physiol., 80: 421430.
ABSTRACT The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the GI phase. A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K+. Cells placed directly into high K+ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K+ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase m isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early GI which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.
Manipulation of the ionic composition of the medium supporting cell growth can result in profound changes in cell proliferation and phenotypic expression. Raising the external potassium ion concentration at the expense of the sodium ion concentration results in a reduction in cell multiplication rate in a CHO (Chinese hamster ovary) cell line (Cone, '71), a BHK (baby hamster kidney) cell line and its polyoma virus transformed variant (Orr et al., '72), and 3T3 and SV40 transformed 3T3 mouse cell lines (Yoshikawa-Fukada and Nojima, '72).Changes in potassium levels have also been shown to reduce puff formation in J. CELL.PHYSIOL., 85: 283-292.
Chironimus salivary glands and to promote chondrogenesis in embryonic cells in vitro (Lash et al., '73). One of the most rapid cellular changes following elevation of the external potassium level is a reduction in the cell membrane potential. Pardee ('71) has proposed that changes at the membrane level can profoundly influence cell division, and others have proposed that the cell membrane Received May 6, '74. Accepted Aug. 29, '74. Present address: Case Western Reserve University, Department of Biology, Cleveland, Ohio 44106. Present address: University of Illinois Medical Center, Department of Anatomy, P.O. Box 6998, Chicago, Illinois 60680.
283
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PETER J. STAMBROOK, HOWARD G. SACHS AND J. D. EBERT
potential or the transmembrane ionic concentrations which underly the membrane potential may act as a specific mitotic regulator (Cone, '71;Cone and Tongier, '73; McDonald et al., '72). We have recently shown, however, that while the membrane potential of isolated V79 Chinese hamster cells in a synchronized culture varies in a predictable fashion through the cell cycle, the observed periodic increase does not appear to play a causal role in regulating the traverse of cells through the cell cycle (Sachs et al., '73). In the present study we have performed further experiments on V79 cells to confirm our initial observation that the cell membrane potential does not play a causal role in mitotic regulation even though in isolated cells it displays a marked periodicity during the cell cycle. Since isolated cells display an abrupt increase in the cell membrane potential at the start of the S phase, we have examined whether such an increase in membrane potential is essential for the initiation of DNA synthesis and traverse of the cell cycle. Specifically we have asked whether cells with an artificially depressed membrane potential can enter the S phase and carry out DNA synthesis in a normal fashion. In addition, we have examined whether the reduction in growth rate observed in cells cultured in medium containing high levels of potassium is due to a random retardation of traverse through the cell cycle or whether there are specific periods of the cell cycle that are particularly sensitive to the high potassium regimen. The latter possibility was suggested by experiments in which BHK cells were cultured in medium containing high levels of potassium (Om et al., '72). When the high potassium medium was replaced with normal medium, the cells underwent a wave of DNA synthesis and mitosis indicating that a degree of synchrony had been imposed upon the culture. MATERIALS AND METHODS
Cell line and culture conditions The cells used in this study were from a subclone of the Chinese hamster V79 line originated by Ford and Yerganian ('59). Cell culture conditions for cell propagation were identical to those previously described (Stambrook and Sisken, '72). Media containing various potassium concentrations
were prepared by molar replacement of Na+ by K+ ( O n et al., '72). The osmolarity of normal and K+-substituted media was maintained between 315 and 325 milliosmoles/l. Synchronization of cells Populations of synchronized mitotic cells were collected as described by Stambrook and Sisken ('72). Briefly, cells were grown in roller bottles and exposed to colcemid (0.06 pg/ml) for the last 90 minutes. The colcemid-containing medium was poured off and replaced with Gey's balanced salt solution (Gey, '36)which was gently swirled over the cell layer, dislodging mitotic cells. The mitotic cell suspension was centrifuged at 1500 RPM for two minutes and the cell pellet resuspended in the appropriate medium for cell cycle analysis. In each experiment, an aliquot of the mitotic cell preparation was suspended in 1% sodium citrate for microscopic examination. After ten minutes in the sodium citrate solution the cells were centrifuged, resuspended in methano1:acetic acid (3:1), spotted on a microscope slide and flamedried. The slide was stained with toluidine blue and scanned to determine the initial level of synchrony. At the start of an experiment, 95 to 98% of the cells were in metaphase.
Cell cycle analysis Synchronized cells were plated on a series of 5 cm culture plates and their progress through the cell cycle was followed by labeling the cells for ten minutes with 3H thymidine (1 pclml) at one or two hour intervals after synchronization. The cells were washed and fixed in methano1:acetic acid (3:1),coated with photographic emulsion (NTB-2) and kept in the dark at 4 ° C for one week. The plates were developed and the percent of labeled cells and the mitotic index at each time interval were measured. The time elapsed before the appearance of a mitotic peak measures the generation time; the curve describing the percent of labeled cells defines the GI and S periods. Electrophysiology Exponentially growing cells were cultured in 35 mm plastic tissue dishes (Falcon). The appropriate potassium-containing medium was added to the plate, and
P O T A S S I U M A N D T H E CELL CYCLE
the cells were impaled with a microelectrode. The membrane potential was measured as described previously (Sachs and McDonald, '72; Sachs et al., '74). Briefly, impalement of well isolated cells with no visible contacts to other cells was carried out on the stage of a n inverted phasecontrast microscope. Membrane potentials were determined using high resistance micropipettes with tip potentials (measured by breaking the tip) of less than 4 mV measured in the culture medium. Impalements in which the measured potential failed to return to within 2 mV of the baseline before impalement were rejected. RESULTS
285
of a cell in early GI (Sachs et al., '74). The change in membrane potential in response to exposure to 115 mM potassium is rapid, occurring within five minutes, and is maintained for at least 24 hours. Measurements were made on 15-20 separate cells for each time interval and for each potassium concentration ensuring that measurements were made on cells representing all stages of the cell cycle. Since S + Gz occupy almost 70% of the cell cycle, the values obtained with 115 mM potassium must represent a population with a significant proportion of cells in the S or Gf phases. The ratio of potassium permeability to sodium permeability determined by the Goldman equation (Williams, '70) was 0.33 as compared to 0.082 for BHK cells with a resting potential of -55 mV (Sachs and McDonald, '72).
External potassium Concentration and the cell membrane potential The cell membrane potential of Chinese hamster V79 cells in exponential growth External potassium concentrations and was measured after varying lengths of excell growth posure to media containing increasing potassium concentrations. As the potassium The growth rate of cells exposed to varconcentration was raised, the membrane ious external concentrations of potassium potential decreased (fig. 1) and at 115 was measured. Between 1 mM and 30 mM mM potassium exhibited a value below that potassium there was little effect on the growth rate (fig. 2a). However, when the -30 potassium concentration was raised to 60 mM or higher, there was a marked decrease in both the growth rate and the density to which the cells would grow (figs. 2a,b). At a concentration of 115 mM potasc sium, the growth rate was very rapidly in> hibited, yet when the potassium was reE -20 moved after a 12-hour exposure, cell growth .-c0 resumed at a rate and to a density similar c Q, to that of control cultures (fig. 2b). The rec versibility of the potassium effect indicates 2 that even though cell growth has stopped, C the cells remain viable. e -10 In an experiment similar to that den scribed in figure Za, cells were collected $ I after 60 hours of exposure to the various exmnal potassium concentrations, fixed, seeded on slides and flame-dried. A count \ \ of mitotic cells revealed an inverse relationship between the growth rate and the 5 10 20 100 200 mitotic index (table 1). suggesting that the Potassium Concentration (mM) progress of cells through mitosis is retarded Fig. 1 Cell membrane potential as a function when incubated with high levels of potasof external potassium concentration. Exponentially sium.
0)
growing cells were transferred to media containing
increasing concentrations of potassium. Five minutes after addition of the potassium media, cells were impaled and their membrane potentials recorded. Bar represents mean S.E.
*
Effect of high potassium levels on cell cycle traverse To further examine the effect of high
286
PETER J. STAMBROOK, HOWARD G. SACHS AND J. D. EBERT lo7-
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Fig, 2 Effect of external potassium on cell growth. (a) Inhibition of cell proliferation by exogenous potassium. Cells were seeded into a series of 60 mm culture plates at 105 cells per plate. After 24 hours, the medium was replaced with media containing increasing concentrations of potassium, and at subsequent intervals, the cell number per plate was determined in duplicate. 1 mM K+ (crosses); 10 mM K + (solid triangles); 30 mM K+ (solid circles); 60 mM K+ (open squares); 100 mM K+ (solid squares). (b) Recovery of cell growth. Cells were treated as in figure 2a After 12 hours, half of the cells which had been exposed to 115 mM potassium was transferred to fresh medium containing 10 mM potassium. The number of cells per plate was determined i n duplicate at different times during the culture period. 10 mM K+ (open circles, solid line); 100 mM K+ (solid squares, solid line); 115 mM K + (open triangles, solid line); 115 mM K+ transferred to 10 mM K+ (solid triangles, dashed line).
TABLE 1
The relationship between exogenous potassium concentration and mitotic index Potassium concentration (mM)
1.0
10 30 60 100 115
Mitotic index
1.8 2.2 2.4
3.6 6.2
8.8
Cells seeded at lo5cells per 60 mm culture plate were grown for 24 hours in control medium and transferred to media containing 1.0, 10, 30, 60, 100, or 115 mM K + . After 60 hours of additional culture (24 hours for the 115 mM K + sample) the media were aspirated and the cells washed and fixed in methano1:acetic acid (3:l). One thousand cells were counted from several random fields from each plate and the percentage of mitotjc cells determined.
levels of potassium on the cell cycle, and specifically the ability of cells to enter the S phase, mitotic cells were collected and their passage through the cell cycle was followed in the presence or absence of high potassium medium. A population of mitotic cells was divided into four series of aliquots, each series containing 18 replicate culture plates. The f m t series contained cells seeded directly into medium containing 10 mM potassium which served as the control (fig. 3a). In the second series the cells were placed directly into medium containing 115 mM potassium (fig. 3b). The third group consisted of cells first suspended in 115 mM potassium and after two hours transferred to 10 mM potassium (fig. 3c). And the last group was first placed in 10 mM potassium
28 7
POTASSIUM AND THE CELL CYCLE
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TIME (hrs) Fig. 3 Response of synchronized cells to different exogenous potassium regimens. A synchronized cell population was prepared by selective detachment of mitotic cells and divided into four different aliquots. Each series consisted of 18 replicate60 mm culture plates with equal numbers of cells per plate. (a) Cells incubated in 10 mM K+. (b) Cells incubated in 115 mM K+. (c) Cells incubated for two hours in 115 mM K+ then transferred to 10 mM K+.(d) Cells incubated for two hours in 10 mM K + then transferred to 115 mM K+. In each series, duplicate plates were labeled with 3H thymidine (1 pclml) for ten minutes at successive intervals after synchronization. The plates were aspirated and washed, and the cells fixed with methano1:acetic acid (3:l). The plates were then coated with emulsion for autoradiography. Percentage labeled cells (open circles, solid line); mitotic cells per 1000 cells (crosses, dashed line).
and after two hours transferred to 115 mM potassium, prior to the onset of DNA synthesis. In each series, duplicate plates of cells were labeled with 3H thymidine for ten minutes at varying times after mitosis, and at every time interval the percent of cells which were labeled was determined by autoradiography. The mitotic index was calculated from the same plates. The control series displayed an approximate 10hour generation time with about a %hour GI and a 6-hour S phase (fig. 3a). Cells placed directly into 115 mM potassium me-
dium were retarded in their exit from mitosis and exhibited a greatly impaired DNA synthetic activity (fig. 3b). At no time interval examined were more than 60 percent of the cells labeled and the density of label over nuclei was considerably lower than in the control series. By 14 hours, four hours after the mitotic peak in the control series, no mitotic cells were evident. However, at least a fraction of the population was able to traverse the entire cycle, albeit abnormally, since at 24 hours nearly 20 percent of the cells were in metaphase,
288
PETER J. STAMBROOK, HOWARD G . SACHS AND J. D. EBERT
apparently blocked at that stage. Cells which were first suspended in high potassium medium and two hours later transferred to control medium (fig. 3c), were retarded in their exit from mitosis. Once fresh medium was added, the cells progressed normally through the remainder of the cycle although all marker events were delayed by two hours compared to the control series. The marker events included the times of onset of DNA synthesis, the minimum in the DNA synthetic curve and the mitotic peak. In the last series (fig. 3d), the cells, which were first suspended in control medium and transferred to 115 mM potassium medium at two hours, exited mitosis normally and began DNA synthesis on schedule. The duration of the S phase appeared slightly lengthened and the cells arrived at mitosis with minor delay. A block in mitosis was again evident since cells accumulated in mitosis at 12 and 14 hours, but by 24 hours the mitotic index had dropped below control values. Three conclusions can be drawn from the above series of experiments. (1) V79 cells can initiate and continue DNA synthesis with a membrane potential artificially depressed to a level below that of a normal GI cell (figs. 1, 3d). Synchronized cells incubated with control medium but transferred to 115 mM potassium medium prior to the initiation of DNA synthesis can enter and traverse the S phase and Gf and proceed to the next division with little delay. (2) The high potassium medium impedes the passage of cells through mitosis (table 1, figs. 3b, 3d). (3) There is an event (or events) during GI that is particularly sensitive to the 115 mM potassium medium. Effect of high potassium levels on chromosome structure Since cells incubated in high levels of potassium are impeded in mitosis as well as in GI, mitotic cells were examined for cytological abnormalities. When exponentially growing cells, incubated for 30 hours in high potassium medium, were fixed in methano1:acetic acid (3:1) chromosomes in about 30 percent of the metaphase figures displayed a marked banding pattern after staining with toluidine blue. When chromosome preparations were made with cells from the 24-hour time interval in experiment 3b or subsequent similar experiments,
chromosomes in 92 percent of the metaphase complements were condensed compared to chromosomes in control preparations (fig. 4). In addition to the deeply staining bands apparent in varying degrees
Fig. 4 Effect of prolonged exposure to 115 mM K+ on chromosome morphology. Mitotic cells were collected by mitotic selection and exposed to 115 mM K+ for 24 hours (as in fig. 312). All cells were trypsinized, washed, and suspended in 1 % sodium citrate for 15 minutes. After fixation in methanol: acetic acid (3:1), metaphase chromosomes were spread on slides and stained with toluidine blue. (a) Metaphase chromosomes from a control culture. (b) Metaphase chromosomes from a potassiumtreated culture showing densely staining bands. ( c ) Metaphase chromosomes from a potassiumtreated culture displaying distinct coiling.
POTASSIUM AND THE CELL CYCLE
in most of the condensed chromosomes (fig. 4b), about 15 percent of the mitotic cells contained chromosomes which displayed a marked super-coiled structure (figs. 4c, 5) never evident in control cultures.
289
mosomal puffing patterns in ChiTonomus. Lezzi ('70) subsequently argued that alterations in intracellular Na+/K+ratios act in the same manner, and that a change in the external ratio modifies the internal ratio. The possibility that changes in intracelluDISCUSSION lar calcium ion levels can affect cellular A key question raised by experiments activities should also be considered since which mod* cellular functions by manip- depolarization of the cell membrane alters ulating the cell membrane potential is the membrane permeabilities to a variety of whether alterations in extracellular ionic molecules including calcium ions (e.g., regimens affect cellular activities by act- Baker et al., '71). The stimulation of culing directly on the cell membrane or by pri- tured lymphocytes by phytohemaglutinin, marily altering the intracellular ionic bal- for example, is accompanied by an immediance. McDonald et al. ('72) have shown ate increase in calcium uptake (Whitney that treatment of BHK cells with high lev- and Sutherland, '73) and subsequently an els of potassium (1 14 mM) not only reduces increase in the uptake of potassium (Quasthe membrane potential but also increases tel and Kaplan, '70). Fertilization of sea urthe intracellular potassium content, even chin embryos is also accompanied by memafter a short incubation. That altered Na+/ brane depolarization (Tupper, '73) and by K+ ratios can affect nuclear activity was an increase in calcium uptake (Mazia, demonstrated by Kroeger ('63 and '66) who '37) both of which can be mimicked by found that changes in the extracellular treating eggs with a calcium ion-specific Na+/K+ ratio dramatically altered the chro- ionophore (Steinhardt and Epel, '74). Al-
Fig. 5
Enlargement of figure 4c detailing the coiled morphology of the chromosomes.
290
PETER J. STAMBROOK, HOWARD G. SACHS A N D J. D. EBERT
though the present study does not examine whether intracellular ion levels change as cells traverse the cell cycle, the data do argue strongly against a major regulatory role for the cell membrane potential during the cell cycle traverse. In a previous report (Sachs et al., '74) we demonstrated that the cell membrane potential of isolated Chinese hamster cells exhibits cyclic variations during the cell cycle. A somewhat similar study on L cells by Cone ('69) also showed cyclical changes, but in a different direction than we found for V79 cells. At that time we suggested that these potential changes played no regulatory function in mitotic activity. The basis for this argument was that the membrane potential of cells physically apposed in clusters displayed remarkably constant values with very slight variation, even though they were obtained from cells in all stages of the cell cycle. The present study further demonstrates that isolated cells with membrane potentials artificially depressed by high external potassium levels can initiate DNA synthesis and traverse the S phase and G r . Under these experimental conditions the cells traverse late Gz with a membrane potential lower than that of normal isolated GI cells (Sachs et al., '74). There is no increase in membrane potential which normally occurs at the onset of the S phase. The observation that synchronized cells, artificially maintained in a depolarized state, can initiate and carry on DNA synthesis is consistent with the findings of Tupper ('73) who has reported that in early sea urchin development protein synthesis, DNA synthesis and growth are unaffected by maintaining embryos in a depolarized state with high potassium levels. The data indicate that Chinese hamster cells are particularly sensitive to high potassium levels at two unique points of the cell cycle: mitosis and early GI. The two lesions are closely linked in time, but appear to be distinctly separate events. This conclusion derives from the data described in figure 3. Table 1 and figures 3b-d show that cells in mitosis are impeded by a high potassium regimen. Synchronized mitotic cells incubated in the high potassium medium behave abnormally in two ways. In addition to being retarded in their exit from
mitosis, these cells also display severely asynchronous, defective and delayed entry into the S phase (fig. 3b). The observed asynchrony is not due to a loss of synchrony as cells exit from mitosis, since if the high potassium medium is replaced by control medium after two hours, the cells initiate DNA synthesis as synchronously as control populations, but with a two hour delay. Most of the asynchrony must therefore arise during the GI period. If mitotic cells are first cultured for two hours in control medium before being placed into high potassium, they pass through the remainder of G I , initiate DNA synthesis and traverse S and G2 normally. These results suggest that in addition to a block in mitosis there is a second lesion sensitive to the high potassium regimen which occurs in early GI which may impair the start of DNA synthesis, and which is bypassed during the first two hours of culture in control medium. Orr et al. ('72) have previously reported that high external levels of potassium imposed a degree of synchrony on BHK cells. Following release from the 114 mM potassium block, they observed two waves of DNA synthesis and mitosis. They concluded that there were two sensitive periods in the cell cycle: a minor block (15% of the population) either in late GI or early S, and major block in the mid GI period. Cone and Tongier ('71) have previously noted that CHO cells can also be blocked by high potassium medium with the block occurring in the latter half of the GI period. Thus the data obtained with V79 cells are consistent with those obtained with other cells and may indicate a general sensitivity to high extracellular potassium ion concentration during some part of the GI period. The block in mitosis which occurs in the presence of high levels of potassium is likely due to an alteration in chromosome architecture (figs. 4b,c, 5) which in turn may impede chromosome segregation and decondensation. The morphology of these chromosomes resembles the structures described by Ohnuki ('65) who obtained chromosomes with similar spiral architecture by treating colchicine-derived mitotic human peripheral leucocytes with a hypotonic salt solution containing 31 mM potassium. He argued, however, that the coils represented a despiralization of the chromosome
POTASSIUM AND THE CELL CYCLE
rather than a coiling superimposed over the normal structure of the chromosome which results in chromosome shortening. ACKNOWLEDGMENTS
We thank Drs. D. Fambrough and A. Ritchie for their critical reading of the manuscript and Delores Sommerville and Bessie Smith for their excellent technical assistance. LITERATURE CITED Baker, P. F., A. L. Hodgkin and E. B. Ridgeway 1971 Depolarization and calcium entry in squid giant axons. J. Physiol. (London), 218: 709-755. Cone, C . D. 1969 Electroosmotic interactions and accompanying mitosis initiation in sarcoma cells in vitro. Trans. N. Y. Acad. Sci., 31 : 404427. 1971 Unified theory on the basic mechanism of normal mitotic control and oncogenesis. J. Theor. Biol., 30: 151-181. Cone, C. D., and M. Tongier 1971 Control of somatic cell mitosis by simulated changes in the transmembrane potential level. Oncology, 25 : 168-182. 1973 Contact inhibition of division: involvement of the electrical transmembrane potential. J. Cell, Physiol., 82: 373-386. Ford, D. K., and G. Yerganian 1959 Observations on the chromosomes of Chinese hamster cells in tissue culture. J. Nat. Cancer Inst., 21 : 393-475. Gey, G. 0. 1936 Maintenance of human normal cells and tumor in continuous culture. Amer. J. Canc. Inst., 27: 45-76. Kroeger, H. 1963 Chemical nature of the system controlling gene activities in the cell. Nature, 200: 1234-1235. Kroeger, H. 1966 Potential differenz und Puffmuster. Electrophysiologische und cytologische Untersuchungen an den Speicheldruesen von Chironomus thummi. Exp. Cell Res., 41 : 64-80. Lash, J. W., K. Rosene, R. R. Minor J. C. Daniel and R. A. Kosher 1973 Environmental enhancement of in vitro chondrogenesis. 111. The influence of external potassium ions and chondrogenic differentiation. Develop. Biol., 35: 370375. Lezzi, M. 1970 Differential gene activation in iso-
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lated chromosomes. Int. Rev. Cytol., 29: 127168. Mazia, D. 1937 The release of calcium in Arbacia eggs on fertilization. J. Cell. Comp. Physiol., 10:291-304. McDonald, T. F., H. G. Sachs, C. W. M. Orr and and J. D. Ebert 1972 External potassium and baby hamster kidney cells: intracellular ions, ATP, DNA synthesis and membrane potential. Develop. Biol., 28: 290-303. Ohnuki, Y. 1965 Demonstration of the spiral structure of human chromosomes. Nature, 208: 9 16-9 18. Orr, C. W., M. Yoshikawa-Fukada and J. D. Ebert 1972 Potassium: Effect on DNA synthesis and multiplication of baby hamster kidney cells. Roc. Nat. Acad. Sci. (U.S.A.), 69: 243-247. Pardee, A. 1971 The cell surface as a regulator of animal cell division. In Vitro, 7: 95-104. Quastel, M. R., and J. G. Kaplan 1970 Lymphocyte stimulation: the effect of ouabain on nucleic acid and protein synthesis. Exp. Cell Res., 62 : 407420. Sachs, H. G., and T. F. McDonald 1972 Membrane potentials of BHK (Baby Hamster Kidney) cell line: Ionic and metabolic determinants. J. Cell Physiol., 80:347-358. Sachs, H. G., P. J . Stambrook and J. D. Ebert 1974 Changes in membrane potential during the cell cycle. Exp. Cell Res., 83: 362-366. Stambrook, P. J., and J. E. Sisken 1972 Induced changes in the rates of uridine-ZH uptake and incorporation during the GI and S periods of synchronized Chinese hamster cells. J. Cell Biol., 52: 514-525. Steinhardt, R. A,, and D. Epel 1974 Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. (U.S.A.), 71: 1915-1919. Tupper, J. T. 1973 Potassium exchangeability, potassium permeability, and membrane potential: some observations in relation to protein synthesis in the early echinoderm embryo. Develop. Biol., 32:140-154. Whiteney, R. B., and R. M. Sutherland 1974 Kinetics of calcium transport in lymphocytes b e fore and after stimulation by phyto-hemaglutinin. In: Proc. 7th Leukocyte Culture Conference. F. Daguillard, ed. Academic Press, pp. 63-72. Williams, J. A. 1970 Origin of transmembrane potentials in non-excitable cells. J. Theor. Biol., 28 : 287-296. Yoshikawa-Fukada, M., and T. Nojima 1972 Biochemical characteristics of normal and virally transformed mouse cell lines. J. Cell Physiol., 80: 421430.
