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Inhibition of cell proliferation by glycerol
Life Sciences, Vol. 48, pp. 1511-1517 Printed in the U.S.A.
Pergamon Press
I N H I B I T I O N OF C E L L P R O L I F E R A T I O N BY G L Y C E R O L J.P. Wiebe and C.J. Dinsdale Hormonal Regulatory Mechanisms Laboratory, B&G Building, Department of Zoology, The University of Western Ontario, London, Ontario, Canada N6A 5B7. (Received in final form February Ii, 1991)
Summary
The effect of glycerol on proliferation of BHK, CHO, HBL, MCF-7, and human glioma cells was studied. Cell proliferation was significantly decreased in all the cell lines at glycerol concentrations of 2-4% in the culture medium. The inhibition was dose-dependent, complete suppression of proliferation occurring at a glycerol concentration of 4% for the MCF-7 cell line and 6-8% for the BHK, CHO and human glioma cells. Studies on [3H]thymidine incorporation correlate with the effect on cell proliferation. The viability of the cells was not significantly affected until higher concentrations of glycerol (12% +) were present. Recovery studies with BHK cells indicated that replacement of the glycerol medium with glycerol-free medium resulted in full recovery following exposure to 4% glycerol and only partial recovery (65%) of proliferation rate following exposure to 10-12% glycerol. It is concluded that glycerol, a substance that is normally present in tissues, can serve as a potent inhibitor of cell proliferation. Glycerol (1,2,3-trihydroxypropane) is a low molecular weight, polar molecule that has been used extensively in cryogenics and protein preservation. It has wide-ranging effects at the molecular level including suppression of the induction of heat shock protein synthesis during heat shock conditions (1), inhibiting or increasing relative enzyme activities (2), stimulation of the assembly of microtubules in vitro (3), and induction of differentiation (4). Glycerol is also a free-radical scavenger (5) and prevents DNA damage by genotoxic substances (6) and by harmful electromagnetic radiation (6,7). Studies in our laboratory have shown that an intratesticular injection of glycerol solution suppresses spermatogenesis (meiosis) without any evidence of toxic or endocrine side effects in rats (8), rabbits (9) and monkeys (10). We also observed that the mitotic activity of the spermatogonia had been suppressed. We therefore undertook studies to determine the effects of glycerol on cell proliferation/n vitro. This report describes the results of experiments which show that glycerol effectively suppresses proliferation of Baby Hamster Kidney (BHK), Chinese Hamster Ovary (CHO), Mammary Cancer Fibroblast (MCF-7), Human Breast Line (HBL) and human glioma cells in culture at concentrations which do not significantly reduce viability. The suppression of cell proliferation by a non-toxic substance that is normally present in tissues suggests a new approach to cancer treatment. Materials and Methods
Cell Culture. Serial lines of BHK and CHO cells were grown routinely as monolayer cultures in Medium 199 (Gibco, Grand Island) supplemented with 10% (v/v) fetal bovine serum (FBS) and Correspondence: J.P. Wiebe, Hormonal Regulatory Mechanisms Laboratory, Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
1512
Glycerol Inhibits Cell Proliferation
Vol. 48, No. 16, 1991
5/~g/ml g entamycin sulfate (Sigma, St. Louis) in a humidified atmosphere of 5% CO 2 in air at 37 °C in 25 cm" Corning plastic flasks. The MCF-7 and HBL cell lines were grown in Eagle's Minimum Essential Medium (MEM; Gibco, Grand Island) supplemented with 10% FBS and 5/zg/ml gentamycin sulfate (Sigma) and 20/zg/ml of insulin (Sigma). After allowing the cultures to grow to confluency, with medium changes every 4 days, the cultures were trypsinized and subculture& Human glioma cells were grown in Dulbecco's Minimum Essential Medium (Gibco) supplemented with 15% FBS and 10,000 units of penstrep and streptomycin sulfate. For the experiments, cells were seeded in 35 x 10 mm culture dishes (Falcon, Lincoln Park, NJ) and allowed to attach over a 24-hour period in a glycerol-free medium (1.5 ml). The medium was then replaced with 1.5 ml of the treatment medium. Growth Studies. To test the effect of various concentrations of glycerol on cell proliferation, cells were seeded, allowed to attach (24 hours), and cultured in medium containing 0 (control), 2, 4, 6, 8 or 10% (vN) glycerol (Aldrich Chem., Milwaukee; 4-5 dishes/treatment). At intervals of 24 or 48 h, cells were dispersed by treatment with 0.05% trypsin (Sigma, Type II) and counted using a hemocytometer. Cell viability was measured by staining the cells with 0.25% Trypan Blue. To investigate whether the inhibition of cell proliferation by glycerol is reversible, the cells were subjected to normal growth medium for the first 96 h. The cells were then cultured for 48 h in glycerol-containing medium following which the glycerol-containing medium was removed, the cells were washed with sterile phosphate buffered saline (PBS) and control medium was added. Control (non glycerol-treated) cultures were similarly washed with PBS. Mitotic Index Studies. To visualize the number of cells in mitosis, cultures of BHK cells were grown on sterile coverslips and then fixed with ice-cold absolute methanol for five minutes. The cultures were then stained with Giemsa stain (Sigma), washed, air dried and mounted. The mitotic index was calculated as the number of cells in mitosis per 1000 cells. The time for control cells to pass through mitosis was calculated using the equation (11): tm
loge ( M + I )
T
loge 2
where M is the mitotic index, t is the time spent in mitosis and T is the cell cycle time. (Calculatlons mdmated that BHK cells cycle approxmmately every 27 hours, and mltosm spans almost 35 minutes of the cell cycle). •
•
.
m
.
.
.
[3H]-Thymidine Incorporation Studies. The BHK cultures were allowed 48 hours to recover from subculturing before [3H]-thymidine incorporation and pool size experiments began. Tritium labelled thymidine ([6- 3 H]-thymidine, No. NET-355; 743.7 GBq/mmol; Dupont Canada) was used at a concentration of 18.5 MBq/ml (0.5/~Ci/ml) of medium. The cultures were exposed to the [3H]thymidine for 45 min. After exposure, the cells were washed with PBS and trypsinized with 0.05% trypsin. Once suspended, the cells were placed in a vial and centrifuged at 100 x g for 5 min and the supernatent was removed. Trichloroacetic acid (1 ml of a 10% solution) was added to the cell pellet and the pellet was sonicated for 15 seconds. The DNA was then spun down at 1500 x g for 15 minutes and the supernatant was discarded. To this solution, 0.5 ml of 1N N a O H was added and the solution placed in a 55 °C water bath for 1 hour. After this time, 0.5 ml of double distilled water was added and the contents were decanted into a scintillation vial. Hydrophilic scintillation fluid (5.0 ml; 25% Triton-X 100 (BDH, Toronto), 0.2 gm/l POPOP (BDH), 3 gm/l PPO (BDH) in xylene) was added to the contents of the vial and radioactivity was measured in a Philips Model PW4700 liquid scintillation counter. The [3H]thymidine pool size was determined by standard procedures (12). Briefly, cells were exposed to [°H]thymidine for 10 minutes (time enough to reach plateau values; data not shown), washed 3x in ice-cold PBS, treated with 1.0 ml/dish of 10% trichloroacetie acid, scraped, sonicated and centrifuged at 1500 x g for 15 min. The supernatant, containing the acid-soluble fraction of the [3H]thymidine, was decanted directly into scintillation vials and radioactivity measured by scintillation spectrometry using the hydrophilic scintillation fluid.
Vol. 48, No. 16, 1991
Glycerol Inhibits Cell Proliferation
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Results
Effect of Glycerol on Cell Growth Kinetics of Established Cell Lines. Untreated BHK cells proliferated rapidly, increasing about 5-fold between days 1-3 and about 3-fold between days 3-5, while the addition of glycerol suppressed cell proliferation in a dose-dependent manner (Figure 1A). At a concentration of 2%, glycerol did not significantly suppress growth of the BHK cell line. A 4% concentration of glycerol in the growth medium resulted in a marked suppression; between 1-3 days post-seeding, the number of cells grown in the 4% glycerol medium had increased by 65.8% while the control cells had increased by 438%. At concentrations of 6-10% glycerol, there was no increase in cell numbers. The viablity of the control and treated cells, judged by trypan blue exclusion, was generally >90%. As viability was measured by examining trypan blue exclusion from the cell cytosol, it also demonstrated that glycerol did not breach the integrity of the cell membrane. Figures 1B and 1C show that in vitro proliferation of CHO and MCF-7 cells is similarly suppressed by exposure to glycerol. The number of CHO cells per culture dish in the 2% glycerol treatment group was significantly less (p<0.05) than the control group by day 7. The 4% glycerol treatment resulted in significant suppression (p<0.001) of cell numbers by day 3 in culture (Fig 1B). At 6% glycerol, CHO cells did not increase in numbers during the 7 days in culture. The number of MCF cells was significantly less (p<0.01-0.001) with the 2%, 4%, and 6% glycerol treatments than with the control medium after 4 days in culture (Figure 1C). Treatment with 4% and 6% glycerol completely suppressed increases in cell numbers during the 4 day culture period, while the controls increased by 148% and the 2% glycerol treated cells increased by 82%. Treatment with glycerol (4%) had essentially the same effect on (HBL) cells (data not shown) as on CHO and MCF cells resulting in a significant suppression of cell proliferation in culture. After 7 days in culture at 4% glycerol, the glycerol-containing dishes had only 41.8% of the numbers of cells in control dishes. The viability of CHO, MCF, and HBL cells was not significantly affected by the glycerol treatments. Effect of Glycerol on Human Glioma Cells. Primary cultures of human glioma cells were maintained in medium containing either 0, 1.5%, 3.5%, or 7% glycerol (Figure 1D). In the absence of glycerol the cells increased about 2.24-fold in 24 hours for the period studied (72 hours). At 1.4% .glycerol, cell proliferation was about 1.5 fold during the first 24-hour period and thereafter the increase was parallel to that of the control group. At 3.5% glycerol, cell proliferation was significantly inhibited and cell proliferation was halted at 7% glycerol. The viability of glioma cells at the various treatments ranged from 88% to 95% (data not shown). The Effect of Glycerol on the Mitotic Index of BHK Fibroblasts. To determine if glycerol stops the cell cycle during M phase (mitosis), the mitotic index of cultures both with and without glycerol was determined. The number of mitotic cells in cultures grown in medium containing 4%, 6%, and 10% glycerol was significantly less (p<0.001) than the number in control cultures. Treatment with 4%, 6% and 10% glycerol resulted in reductions in mitotic index values from 14.0 (Controls) to 5.25 (4% glycerol), 3.5 (6% glycerol) and 1.25 (10% glycerol). The Reversibility of Glycerol-Induced Suppression of Proliferation. To determine if the effect of glycerol is reversible following removal of glycerol from the medium, BHK cells were allowed to grow in normal growth medium for 4 days and then were exposed to either 5%, 10%, 12%, or 15% glycerol for 48 hours after which the medium was replaced with normal medium (Figure 2). During the glycerol treatment period, inhibition of cell proliferation occurred; the 5% concentration resulted in partial suppression while the 10% and 12% concentration resulted in complete suppression of cell proliferation. Treatment with 15% glycerol reduced BHK cell viability to 22% but treatment at the lower concentrations did not significantly reduce viability. Replacement with normal medium resulted in the recovery of proliferation by cells treated with 5% glycerol and in a partial recovery of cell proliferation by the cells treated with 10% or 12% glycerol (about a 45% reduction in the doubling time as compared to control cells). Cells treated with 15% glycerol showed a further reduction in viability to 16% during the 'recovery' phase.
1514
Glycerol Inhibits Cell Proliferation
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Dose-dependent inhibitory effect of glycerol on proliferation of (A) BHK, (B) CHO, (C) MCF-7, and (D) primary human glioma cells. Glycerol was added at time 0 and the total number of cells per dish was determined at various times (days) after the start of treatment. Data shown are the mean number of cells (--.S.E.; n = 3-5) from one of two or more replicate experiments with essentially the same results.
Vol. 48, No. 16, 1991
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Figure 2. The reversibility of glycerolinduced suppression of proliferation of BHK cells. BHK cells were grown for 4 days after which the medium was replaced with medium containing glycerol (cross-hatched bar). After 2 days, the medium containing glycerol was removed and replaced with glycerol-free medium. Results are expressed as mean n u m b e r of cells (__S.E.; n = 4) after a specified period of time (days).
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The Effect of Glycerol on [3H]-Thymidine Incorporation into DNA. Incorporation of [3H]thymidine by BI-IK cells was reduced following a 24 h exposure to glycerol and the effect was dosedependent (Figure 3). Thymidine incorporation was reduced by 19% in cells treated with 4% glycerol, 39% (p < 0.05) in cells treated with 6% glycerol, 83% (p < 0.001) in cells treated with 8%
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Figure 3. Dose response of the effect of glycerol on [3H]thymidine incorporation (solid bars; 100% = 100,750 dpm) and pool size (open bars; 100% = 20,400 dpm) in B H K cells. The incorporated activity was determined after a 24 h exposure to a particular concentration of glycerol. [3 I-I]thymidine is presented as the mean ( - S.E.; n = 4) in percent of that in non-treated (control) cells. The incorporation values for 6%, 8%, and 10% glycerol, are significantly lower than controls at p < 0.05, 0.01, and 0.001, respectively.
1516
Glycerol Inhibits Cell Proliferation
Vol. 48, No. 16, 1991
and 91% (p < 0.001) in cells treated with 10% glycerol. The pool size of [3H]thymidine was not significantly reduced by 4% glycerol treatment, but was reduced by 38-55% (p < 0.001) by the 610% glycerol treatments (Figure 3). Discussion
Glycerol in culture medium results in significant suppression of replication of the five cell lines examined in this study: BHK, HBL, CHO, MCF and human glioma ceils. The suppression is dose-dependent between 2% and 10% glycerol; it is first observed at a glycerol concentration of between 2% and 4% and is complete at 8-10%, depending on the cell type. The inhibition is not due to cell death and the viability remains high (85-95%). At these concentrations, glycerol has been shown not to have genotoxic activity (13). The pattern of the dose-dependant inhibition was similiar to that observed with dimethyl sulfoxide treatment (14,15). Glycerol and dimethyl sulfoxide are similar in that they are both low molecular weight, polar molecules and they are both capable of quickly penetrating biological membranes (16,17). Recovery of cell proliferation was complete after removal of medium containing glycerol at a concentration of 5%. The recovery of cell proliferation suggests a reversal of the inhibitory process. Temporary exposure to concentrations of 10% and 12% glycerol results in a permanent effect on cell proliferation after removal of the glycerol; although cell division was reinitiated in these cultures, rates of replication did not reach control levels. This finding suggests that some of the actions of glycerol may not be completely reversible once the glycerol is removed from the medium. These results are similar to those seen in treatments using dimethyl sulfoxide (14). Cell death at 15% glycerol may be due to irreversible disruption of membrane permeability and alteration of intracellular ions (18). The mechanism of inhibition of cellular proliferation by glycerol is not known. Many drugs that suppress cell proliferation attack a specific point in the cell cycle to prevent the continuation of the cycle (19). Glycerol has been well documented in its ability to affect protein function (1,2) and the possible site of action with respect to inhibition of cell proliferation may be cellular proteins involved in the maintenance of the cell cycle. It has been shown that the cytoskeletai states are involved in the regulation of cell growth (20). Microtubule disruption initiates DNA synthesis (21), while microtubule stabilization inhibits initiation of D N A synthesis (15,22) and blocks cell growth (22). Glycerol is known to bind to tubulin molecules and to stabilize cell microtubules (3) by enhancing polymerization and causing microtubule rearrangement (25). In addition, glycerol is known to change membrane permeability, allowing cellular contents to leak, and this in turn is likely to alter cellular pH (24) which is known to cause the aggregation of microtubules (26). It is interesting that a concentration of glycerol of 4% or greater is needed for microtubule polymerization/n vitro (27). The reduction in the mitotic index of BHK cultures by glycerol is proportional to the concentration of glycerol used. This result lends support to the hypothesis that glycerol does not inhibit cell proliferation by blocking the transition of the cell through mitosis and may block the cell cycle somewhere in interphase. Although the mitotic index is relatively lower than expected for control values, this could be due to the high number of multinucleate cells which were classified as interphase ceils in our calculations. The inhibition in D N A synthesis induced by the glycerol-supplemented medium was directly proportional to the concentration of glycerol and parallels the effect on cell replication. Glycerol can also subject cultured cells to osmotic stress (28) but unlike other compounds that cause hypotonicity and stop cell division at the metaphase plate (29), glycerol appears to stop the cell cycle at some point during interphase. Glycerol has a ubiquitous distribution in animal tissues and is involved in cellular structures and lipid and carbohydrate metabolism. It has been used orally and by intravenous injection in humans, without any adverse symptoms (30). Our in vitro results show that glycerol can act as an inhibitor of cell proliferation. In order to be effective, chemotherapeutic agents are normally administered at concentrations that result in adverse side effects. Combining glycerol with lower
Vol. 48, No. 16, 1991
Glycerol Inhibits Cell Proliferation
1517
concentrations of drugs could result in effective antitumor therapy without the undesirable side effects.
Acknowledgements The expenses of this work were defrayed by a grant from Natural Sciences and Engineering Research Council of Canada (No. A6865) to JPW. We would like to acknowledge the assistance of Summer NSERC Scholars, K.W. Glasgow and L.Y. Wong, in preliminary experiments with HBL, CHO and MCF-7 cell lines and of R.F. Del Maestro and E. Stroud, Department of Clinical Neurological Sciences, Victoria Hospital, London, for culturing the human glioma cells.
References 1. B.V. EDINGTON, S.A. WHELAN and L.E. HIGHTOWER, J. Cell. Physiol. 13__9.9219-228 (1989). 2. S. YAMAMOTO and K.B. STOREY, Int. J. Biochem. 20 1267-1271 (1988). 3. H.W. DETRICH, S.A. BERKOWlTZ, H. KIM and R.C. WILLIAMS, Biochem. Biophys Res. Comm. 68 961-968 (1976). 4. H.D. PREISLER and G. LYMAN, CellDifferentiation 4 179-185 (1975). 5. M.S. SASAKI and S. MATSUBARA, Int. J. Radial Biol. 32 439-445 (1977). 6. M.O. BRADLEY and L.C. ERICKSON, Biochim. biophys.Acta 654 135-141 (1981). 7. J.G. PEAK and M.J. PEAK, Radial Res. 97 570-575 (1984). 8. J.P. WIEBE and K.J. BARR, Life Sci. 34 1747-1754 (1984). 9. J.P. WIEBE, K.J. BARR, K.D. BUCKINGHAM, P.D. GEDDES and P.A. KUDO, Male Contraception: Advances and Future Prospects, G.I. Zatuchni, A. Goldsmith, J.M. Spieler and J.J. Sciarra (eds), 252-270, Harper and Row, Philadelphia (1986). 10. J.P. WIEBE, K.J. BARR and ICD. BUCKINGHAM, Contraception 39 447-457 (1989). 1I. D. LLOYD, R.IC POOLE and S.W. EDWARDS, The Cell Division Cycle. Temporal Organization and Control of Cellular Growth and Reproduction. Academic Press. New York, N.Y. pp. 8586 (1982). 12. K. NAITO, S. SKOG, B. TRIBUKAIT, L. ANDERSON and H. HISAZUMI, Cell Tissue Kinet. 20 447-457 (1987). 13. D.J. DOOLI'ITLE, D.A. LEE and C.K. LEE, Fd~ Chem. Toxic. 26 631-635 (1988). 14. D.L. BERLINER, and A.G. RUHMANN, Proc. NaL Acad. Sc~ ~ 159-167 (1967). 15. S. KATSUDA, Y. OKADA, I. NAKANISHI and J. TANAKA, Exp. Mol. Pathol. 48 48-58 (1988). 16. R. COLLANDER, Trans. Faraday Soc. 33 985-990 (1937). 17. D.H. RAMMLER and A. Zaffaroni, Ann. N. Y. Acad. Sci. 14___!13-23 (1967). 18. F.D. WESTFALL and B. HOWARTH, Poultry Sc~ 56 1454-1456 (1977). 19. A. BASERGA, The Biology of Cell Reproduction. Harvard University Press. Cambridge, Mass. pp. 91-10 (1985). 20. D.A. McCLAIN and G.M. EDELMAN, Proc. Nat. Acad. ScL 77 2748-2752 (1980). 21. K.L. CROSSIN and D.H. CARNEY, Cell 23 61-71 (1981). 22. K.L. CROSSIN and D.H. CARNEY, Cell 27 341-350 (1981). 23. Y. SHINOHARA, E. NISHIFA and H. SAKAI, Febs LetL 23___6619-22 (1988). 24. S. INDI, G.WAKLEY and H. STEBBINGS, Tissue and Cell. 18 331-339 (1986). 25. F. FRANKS, J. Microscopy, 11! 3-16 (1977). 26. H. STEBBINGS and C. HUNT, Cell Tiss. Res. 227 609-617 (1982). 27. J.C. LEE and S.N. TIMASHEFF, Biochemstry 16 1754-1761 (1977). 28. W.J. ARM1TAGE, Cryobiology 23 116-125 (1986). 29. D.N. WHEATLEY, Exp. Cell Res. 87 219-232 (1974). 30. E.C.C. LIN, Ann. Rev. Biochem. 46 765-795 (1977).
Pergamon Press
I N H I B I T I O N OF C E L L P R O L I F E R A T I O N BY G L Y C E R O L J.P. Wiebe and C.J. Dinsdale Hormonal Regulatory Mechanisms Laboratory, B&G Building, Department of Zoology, The University of Western Ontario, London, Ontario, Canada N6A 5B7. (Received in final form February Ii, 1991)
Summary
The effect of glycerol on proliferation of BHK, CHO, HBL, MCF-7, and human glioma cells was studied. Cell proliferation was significantly decreased in all the cell lines at glycerol concentrations of 2-4% in the culture medium. The inhibition was dose-dependent, complete suppression of proliferation occurring at a glycerol concentration of 4% for the MCF-7 cell line and 6-8% for the BHK, CHO and human glioma cells. Studies on [3H]thymidine incorporation correlate with the effect on cell proliferation. The viability of the cells was not significantly affected until higher concentrations of glycerol (12% +) were present. Recovery studies with BHK cells indicated that replacement of the glycerol medium with glycerol-free medium resulted in full recovery following exposure to 4% glycerol and only partial recovery (65%) of proliferation rate following exposure to 10-12% glycerol. It is concluded that glycerol, a substance that is normally present in tissues, can serve as a potent inhibitor of cell proliferation. Glycerol (1,2,3-trihydroxypropane) is a low molecular weight, polar molecule that has been used extensively in cryogenics and protein preservation. It has wide-ranging effects at the molecular level including suppression of the induction of heat shock protein synthesis during heat shock conditions (1), inhibiting or increasing relative enzyme activities (2), stimulation of the assembly of microtubules in vitro (3), and induction of differentiation (4). Glycerol is also a free-radical scavenger (5) and prevents DNA damage by genotoxic substances (6) and by harmful electromagnetic radiation (6,7). Studies in our laboratory have shown that an intratesticular injection of glycerol solution suppresses spermatogenesis (meiosis) without any evidence of toxic or endocrine side effects in rats (8), rabbits (9) and monkeys (10). We also observed that the mitotic activity of the spermatogonia had been suppressed. We therefore undertook studies to determine the effects of glycerol on cell proliferation/n vitro. This report describes the results of experiments which show that glycerol effectively suppresses proliferation of Baby Hamster Kidney (BHK), Chinese Hamster Ovary (CHO), Mammary Cancer Fibroblast (MCF-7), Human Breast Line (HBL) and human glioma cells in culture at concentrations which do not significantly reduce viability. The suppression of cell proliferation by a non-toxic substance that is normally present in tissues suggests a new approach to cancer treatment. Materials and Methods
Cell Culture. Serial lines of BHK and CHO cells were grown routinely as monolayer cultures in Medium 199 (Gibco, Grand Island) supplemented with 10% (v/v) fetal bovine serum (FBS) and Correspondence: J.P. Wiebe, Hormonal Regulatory Mechanisms Laboratory, Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
1512
Glycerol Inhibits Cell Proliferation
Vol. 48, No. 16, 1991
5/~g/ml g entamycin sulfate (Sigma, St. Louis) in a humidified atmosphere of 5% CO 2 in air at 37 °C in 25 cm" Corning plastic flasks. The MCF-7 and HBL cell lines were grown in Eagle's Minimum Essential Medium (MEM; Gibco, Grand Island) supplemented with 10% FBS and 5/zg/ml gentamycin sulfate (Sigma) and 20/zg/ml of insulin (Sigma). After allowing the cultures to grow to confluency, with medium changes every 4 days, the cultures were trypsinized and subculture& Human glioma cells were grown in Dulbecco's Minimum Essential Medium (Gibco) supplemented with 15% FBS and 10,000 units of penstrep and streptomycin sulfate. For the experiments, cells were seeded in 35 x 10 mm culture dishes (Falcon, Lincoln Park, NJ) and allowed to attach over a 24-hour period in a glycerol-free medium (1.5 ml). The medium was then replaced with 1.5 ml of the treatment medium. Growth Studies. To test the effect of various concentrations of glycerol on cell proliferation, cells were seeded, allowed to attach (24 hours), and cultured in medium containing 0 (control), 2, 4, 6, 8 or 10% (vN) glycerol (Aldrich Chem., Milwaukee; 4-5 dishes/treatment). At intervals of 24 or 48 h, cells were dispersed by treatment with 0.05% trypsin (Sigma, Type II) and counted using a hemocytometer. Cell viability was measured by staining the cells with 0.25% Trypan Blue. To investigate whether the inhibition of cell proliferation by glycerol is reversible, the cells were subjected to normal growth medium for the first 96 h. The cells were then cultured for 48 h in glycerol-containing medium following which the glycerol-containing medium was removed, the cells were washed with sterile phosphate buffered saline (PBS) and control medium was added. Control (non glycerol-treated) cultures were similarly washed with PBS. Mitotic Index Studies. To visualize the number of cells in mitosis, cultures of BHK cells were grown on sterile coverslips and then fixed with ice-cold absolute methanol for five minutes. The cultures were then stained with Giemsa stain (Sigma), washed, air dried and mounted. The mitotic index was calculated as the number of cells in mitosis per 1000 cells. The time for control cells to pass through mitosis was calculated using the equation (11): tm
loge ( M + I )
T
loge 2
where M is the mitotic index, t is the time spent in mitosis and T is the cell cycle time. (Calculatlons mdmated that BHK cells cycle approxmmately every 27 hours, and mltosm spans almost 35 minutes of the cell cycle). •
•
.
m
.
.
.
[3H]-Thymidine Incorporation Studies. The BHK cultures were allowed 48 hours to recover from subculturing before [3H]-thymidine incorporation and pool size experiments began. Tritium labelled thymidine ([6- 3 H]-thymidine, No. NET-355; 743.7 GBq/mmol; Dupont Canada) was used at a concentration of 18.5 MBq/ml (0.5/~Ci/ml) of medium. The cultures were exposed to the [3H]thymidine for 45 min. After exposure, the cells were washed with PBS and trypsinized with 0.05% trypsin. Once suspended, the cells were placed in a vial and centrifuged at 100 x g for 5 min and the supernatent was removed. Trichloroacetic acid (1 ml of a 10% solution) was added to the cell pellet and the pellet was sonicated for 15 seconds. The DNA was then spun down at 1500 x g for 15 minutes and the supernatant was discarded. To this solution, 0.5 ml of 1N N a O H was added and the solution placed in a 55 °C water bath for 1 hour. After this time, 0.5 ml of double distilled water was added and the contents were decanted into a scintillation vial. Hydrophilic scintillation fluid (5.0 ml; 25% Triton-X 100 (BDH, Toronto), 0.2 gm/l POPOP (BDH), 3 gm/l PPO (BDH) in xylene) was added to the contents of the vial and radioactivity was measured in a Philips Model PW4700 liquid scintillation counter. The [3H]thymidine pool size was determined by standard procedures (12). Briefly, cells were exposed to [°H]thymidine for 10 minutes (time enough to reach plateau values; data not shown), washed 3x in ice-cold PBS, treated with 1.0 ml/dish of 10% trichloroacetie acid, scraped, sonicated and centrifuged at 1500 x g for 15 min. The supernatant, containing the acid-soluble fraction of the [3H]thymidine, was decanted directly into scintillation vials and radioactivity measured by scintillation spectrometry using the hydrophilic scintillation fluid.
Vol. 48, No. 16, 1991
Glycerol Inhibits Cell Proliferation
1513
Results
Effect of Glycerol on Cell Growth Kinetics of Established Cell Lines. Untreated BHK cells proliferated rapidly, increasing about 5-fold between days 1-3 and about 3-fold between days 3-5, while the addition of glycerol suppressed cell proliferation in a dose-dependent manner (Figure 1A). At a concentration of 2%, glycerol did not significantly suppress growth of the BHK cell line. A 4% concentration of glycerol in the growth medium resulted in a marked suppression; between 1-3 days post-seeding, the number of cells grown in the 4% glycerol medium had increased by 65.8% while the control cells had increased by 438%. At concentrations of 6-10% glycerol, there was no increase in cell numbers. The viablity of the control and treated cells, judged by trypan blue exclusion, was generally >90%. As viability was measured by examining trypan blue exclusion from the cell cytosol, it also demonstrated that glycerol did not breach the integrity of the cell membrane. Figures 1B and 1C show that in vitro proliferation of CHO and MCF-7 cells is similarly suppressed by exposure to glycerol. The number of CHO cells per culture dish in the 2% glycerol treatment group was significantly less (p<0.05) than the control group by day 7. The 4% glycerol treatment resulted in significant suppression (p<0.001) of cell numbers by day 3 in culture (Fig 1B). At 6% glycerol, CHO cells did not increase in numbers during the 7 days in culture. The number of MCF cells was significantly less (p<0.01-0.001) with the 2%, 4%, and 6% glycerol treatments than with the control medium after 4 days in culture (Figure 1C). Treatment with 4% and 6% glycerol completely suppressed increases in cell numbers during the 4 day culture period, while the controls increased by 148% and the 2% glycerol treated cells increased by 82%. Treatment with glycerol (4%) had essentially the same effect on (HBL) cells (data not shown) as on CHO and MCF cells resulting in a significant suppression of cell proliferation in culture. After 7 days in culture at 4% glycerol, the glycerol-containing dishes had only 41.8% of the numbers of cells in control dishes. The viability of CHO, MCF, and HBL cells was not significantly affected by the glycerol treatments. Effect of Glycerol on Human Glioma Cells. Primary cultures of human glioma cells were maintained in medium containing either 0, 1.5%, 3.5%, or 7% glycerol (Figure 1D). In the absence of glycerol the cells increased about 2.24-fold in 24 hours for the period studied (72 hours). At 1.4% .glycerol, cell proliferation was about 1.5 fold during the first 24-hour period and thereafter the increase was parallel to that of the control group. At 3.5% glycerol, cell proliferation was significantly inhibited and cell proliferation was halted at 7% glycerol. The viability of glioma cells at the various treatments ranged from 88% to 95% (data not shown). The Effect of Glycerol on the Mitotic Index of BHK Fibroblasts. To determine if glycerol stops the cell cycle during M phase (mitosis), the mitotic index of cultures both with and without glycerol was determined. The number of mitotic cells in cultures grown in medium containing 4%, 6%, and 10% glycerol was significantly less (p<0.001) than the number in control cultures. Treatment with 4%, 6% and 10% glycerol resulted in reductions in mitotic index values from 14.0 (Controls) to 5.25 (4% glycerol), 3.5 (6% glycerol) and 1.25 (10% glycerol). The Reversibility of Glycerol-Induced Suppression of Proliferation. To determine if the effect of glycerol is reversible following removal of glycerol from the medium, BHK cells were allowed to grow in normal growth medium for 4 days and then were exposed to either 5%, 10%, 12%, or 15% glycerol for 48 hours after which the medium was replaced with normal medium (Figure 2). During the glycerol treatment period, inhibition of cell proliferation occurred; the 5% concentration resulted in partial suppression while the 10% and 12% concentration resulted in complete suppression of cell proliferation. Treatment with 15% glycerol reduced BHK cell viability to 22% but treatment at the lower concentrations did not significantly reduce viability. Replacement with normal medium resulted in the recovery of proliferation by cells treated with 5% glycerol and in a partial recovery of cell proliferation by the cells treated with 10% or 12% glycerol (about a 45% reduction in the doubling time as compared to control cells). Cells treated with 15% glycerol showed a further reduction in viability to 16% during the 'recovery' phase.
1514
Glycerol Inhibits Cell Proliferation
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Dose-dependent inhibitory effect of glycerol on proliferation of (A) BHK, (B) CHO, (C) MCF-7, and (D) primary human glioma cells. Glycerol was added at time 0 and the total number of cells per dish was determined at various times (days) after the start of treatment. Data shown are the mean number of cells (--.S.E.; n = 3-5) from one of two or more replicate experiments with essentially the same results.
Vol. 48, No. 16, 1991
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The Effect of Glycerol on [3H]-Thymidine Incorporation into DNA. Incorporation of [3H]thymidine by BI-IK cells was reduced following a 24 h exposure to glycerol and the effect was dosedependent (Figure 3). Thymidine incorporation was reduced by 19% in cells treated with 4% glycerol, 39% (p < 0.05) in cells treated with 6% glycerol, 83% (p < 0.001) in cells treated with 8%
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1516
Glycerol Inhibits Cell Proliferation
Vol. 48, No. 16, 1991
and 91% (p < 0.001) in cells treated with 10% glycerol. The pool size of [3H]thymidine was not significantly reduced by 4% glycerol treatment, but was reduced by 38-55% (p < 0.001) by the 610% glycerol treatments (Figure 3). Discussion
Glycerol in culture medium results in significant suppression of replication of the five cell lines examined in this study: BHK, HBL, CHO, MCF and human glioma ceils. The suppression is dose-dependent between 2% and 10% glycerol; it is first observed at a glycerol concentration of between 2% and 4% and is complete at 8-10%, depending on the cell type. The inhibition is not due to cell death and the viability remains high (85-95%). At these concentrations, glycerol has been shown not to have genotoxic activity (13). The pattern of the dose-dependant inhibition was similiar to that observed with dimethyl sulfoxide treatment (14,15). Glycerol and dimethyl sulfoxide are similar in that they are both low molecular weight, polar molecules and they are both capable of quickly penetrating biological membranes (16,17). Recovery of cell proliferation was complete after removal of medium containing glycerol at a concentration of 5%. The recovery of cell proliferation suggests a reversal of the inhibitory process. Temporary exposure to concentrations of 10% and 12% glycerol results in a permanent effect on cell proliferation after removal of the glycerol; although cell division was reinitiated in these cultures, rates of replication did not reach control levels. This finding suggests that some of the actions of glycerol may not be completely reversible once the glycerol is removed from the medium. These results are similar to those seen in treatments using dimethyl sulfoxide (14). Cell death at 15% glycerol may be due to irreversible disruption of membrane permeability and alteration of intracellular ions (18). The mechanism of inhibition of cellular proliferation by glycerol is not known. Many drugs that suppress cell proliferation attack a specific point in the cell cycle to prevent the continuation of the cycle (19). Glycerol has been well documented in its ability to affect protein function (1,2) and the possible site of action with respect to inhibition of cell proliferation may be cellular proteins involved in the maintenance of the cell cycle. It has been shown that the cytoskeletai states are involved in the regulation of cell growth (20). Microtubule disruption initiates DNA synthesis (21), while microtubule stabilization inhibits initiation of D N A synthesis (15,22) and blocks cell growth (22). Glycerol is known to bind to tubulin molecules and to stabilize cell microtubules (3) by enhancing polymerization and causing microtubule rearrangement (25). In addition, glycerol is known to change membrane permeability, allowing cellular contents to leak, and this in turn is likely to alter cellular pH (24) which is known to cause the aggregation of microtubules (26). It is interesting that a concentration of glycerol of 4% or greater is needed for microtubule polymerization/n vitro (27). The reduction in the mitotic index of BHK cultures by glycerol is proportional to the concentration of glycerol used. This result lends support to the hypothesis that glycerol does not inhibit cell proliferation by blocking the transition of the cell through mitosis and may block the cell cycle somewhere in interphase. Although the mitotic index is relatively lower than expected for control values, this could be due to the high number of multinucleate cells which were classified as interphase ceils in our calculations. The inhibition in D N A synthesis induced by the glycerol-supplemented medium was directly proportional to the concentration of glycerol and parallels the effect on cell replication. Glycerol can also subject cultured cells to osmotic stress (28) but unlike other compounds that cause hypotonicity and stop cell division at the metaphase plate (29), glycerol appears to stop the cell cycle at some point during interphase. Glycerol has a ubiquitous distribution in animal tissues and is involved in cellular structures and lipid and carbohydrate metabolism. It has been used orally and by intravenous injection in humans, without any adverse symptoms (30). Our in vitro results show that glycerol can act as an inhibitor of cell proliferation. In order to be effective, chemotherapeutic agents are normally administered at concentrations that result in adverse side effects. Combining glycerol with lower
Vol. 48, No. 16, 1991
Glycerol Inhibits Cell Proliferation
1517
concentrations of drugs could result in effective antitumor therapy without the undesirable side effects.
Acknowledgements The expenses of this work were defrayed by a grant from Natural Sciences and Engineering Research Council of Canada (No. A6865) to JPW. We would like to acknowledge the assistance of Summer NSERC Scholars, K.W. Glasgow and L.Y. Wong, in preliminary experiments with HBL, CHO and MCF-7 cell lines and of R.F. Del Maestro and E. Stroud, Department of Clinical Neurological Sciences, Victoria Hospital, London, for culturing the human glioma cells.
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