The Inhibition by Colchicine of the Initiation of DNA Synthesis by Hepatocytes in Regenerating Rat Liver and by Cultivated WI-38 and C3H1OTY2 Cells 1 P ROY WALKER, ALTON L BOYNTON AND JAMES F WHITFIELD Animal and Cell Physiology Group, Divlsion of Biolog~calSciences, National Research Council of Canada, Ottawa, Canada K I A OR6
ABSTRACT Colchicine inhibited the initiation of DNA synthesis by hepatocytes in vivo and by WI-38 and C3H10T1hcells in vitro. All three cell types were most sensitive to colchicine when i t was administered a t the time of, or shortly after, proliferative activation (by partial hepatectomy or medium-serum change). WI-38 and C3H10T1h cells became less sensitive to colchicine as the time between proliferative activation and addition of the drug was increased. Hepatocytes, on the other hand, showed a second stage of sensitivity immediately before the onset of DNA synthesis. Ongoing DNA synthesis was not inhibited by colchicine in any of the cell types. The two prereplicative stages of colchicine sensitivity in liver were also sensitive to vincristine but not to lumicolchicine, an analogue of colchicine which does not bind to tubulin. The data obtained with these drugs indicates that microtubules may be involved in the prereplicative stages of cell proliferation, but non-specific binding to other membrane proteins cannot be ruled out. Colchicine (at a dose of 50 pg/lOO g of body weight), which when injected 30 minutes after hepatectomy delayed the initiation of DNA synthesis for at least 14 hours, delayed the induction of ornithine decarboxylase (at the beginning of prereplicative development) by only two hours. Thus, the inhibitory action of colchicine on the initiation of DNA synthesis does not appear to be mediated by inhibition of polyamine synthesis. In 1966, Luyckx and Van Lancker showed t h a t injection of the microtubule disrupter, vinblastine into rats at one hour, or a t one and nine hours, after partial hepatectomy prevented the initiation of DNA synthesis in the remaining liver tissue. Subsequently, Edelman e t al. ('731, Wang e t al. ('75) and Greene e t al. ('761, found that another microtubule disrupter, colchicine, inhibited both the proliferative stimulation of lymphocytes by Concanavalin A and the early prereplicative redistribution of the lectin's receptors on the cell surface. Colchicine completely inhibits the initiation of lymphocyte DNA synthesis when added to the cells at the same time as the mitogen and becomes less effective as the time between addition of mitogen and addition of colchicine is increased indicating that the drug acts at an early prereplicative stage of the cell cycle. (However see Betel and Martijnse '76 for conflicting results). Thus EdelJ. CELL.PHYSIOL., 93: 89-98. NRCC No. 16183
man e t al. ('73) have proposed that microtubules are involved in the redistribution of lectin receptors on the cell membrane which occurs upon activation and that this process is disrupted by colchicine. Further support for an effect of colchicine on the early prereplicative stages of cell proliferation comes from studies which show that colchicine prevents t h e synthesis of ornithine decarboxylase (ODC) in mouse L1210 cells in vitro (Chen et al., '76) and mouse epidermal cells in vivo (O'Brien e t al. '76). The induction of ODC, followed by an accumulation of polyamines, often occurs as an early event following proliferative activation of quiescent cells (see Russell, 1'731 and Tabor "761, for detailed literature). Although the role of polyamines in the metabolism of proliferating cells is not clear, inhibition of polyamine accumulation Received Jan. I , '77. Accepted Mar 30, '11. ' Issued as NRCC No. 16183.
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P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
and/or function by drugs such as methylglyoxal-bislguanylhydrazonel (Holtta e t al., '73; Fillingame and Morris, '73; Boynton e t al., '76b) and diaminopropane (Poso and Janne, '76a,b) prevents, or a t least delays, DNA replication. In the present communication we have examined the effects of colchicine on the early stages of liver regeneration following partial hepatectomy, and on the proliferative activation of human WI-38 and mouse C3HlOT'h cells in culture. The data show that colchicine prevents the initiation of DNA synthesis by hepatocytes in vivo and WI-38 and C3HlOT'h cells in vitro when added a t the time of proliferative activation. The cells in vitro become progressively less sensitive to colchicine when it is added a t later times, whereas the hepatocytes have a second colchicine-sensitive stage shortly before DNA synthesis begins. We will also show that the inhibitory action of colchicine on the initiation of DNA synthesis in hepatocytes is probably not mediated by a n inhibition of ODC activity. MATERIALS AND METHODS
The animals used in this study were males (190-210 g) of a specific-pathogen-free strain of Sprague-Dawley r a t bred in this laboratory. The rats were entrained to a 12-hour 1ight:dark cycle with the dark period being from 7 P.M. to 7 A.M. Food was available ad libitum before the operations, but was withheld afterwards; water was available a t all times. Partial hepatectomy was carried out according to the procedure of Higgins and Anderson ('31) by which 68%of the liver mass was removed. All operations were carried out between 9 A.M. and 11A.M. Colchicine (Sigma Chemical Co., St. Louis, Missouri) and vincristine sulphate (Oncovin, Eli Lilly and Co. [Canada] Ltd., Toronto, Ontario) were dissolved in a solution of 0.9% NaCl or distilled water or diluent, respectively, and injected intraperitoneally. Lumicolchicine, a structural analogue of colchicine which does not bind to tubulin (Wilson e t al., ' 7 4 , was prepared by UV irradiation of colchicine as described by Wilson and Friedkin ('66). DNA synthesis in the liver remnant was determined both autoradiographically and by the incorporation of t3H1-Thymidine into chemically isolated DNA. 13H1-Thymidine(1.0 pcilg body weight; sp. act. 20 ci/mmole; New England Nuclear, Corp., Boston, Massachu-
setts) was injected intraperitoneally one hour prior to sacrifice. Animals were anesthetized with ether and the right anterior lobe removed. Small blocks of tissue were fixed in buffered formalin and processed for autoradiography as described previously (Rixon and Whitfield, '76). A sample (100 mg) of the same liver remnant was homogenized in ice-cold 10% trichloroacetic acid, and DNA was extracted and assayed as described by Hopkins et al. ('73a). A portion of the final extract was added to 10 ml of a scintillation fluid prepared from dioxane (3.8 11, naphthalene (250 g) and omnifluor (30.4 g; New England Nuclear Corp.) and counted in a Beckman LS-255 liquid scintillation system. Samples for the determination of ODC activity were prepared by homogenizing 800 mg of liver in 25 mM Tris-HC1buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM dithiothreitol. The homogenate was centrifuged a t 105,000 g f o r 60 minutes (at 4"C, in a Beckman L2-65B Ultracentrifuge), and 0.4 ml of the supernatant fluid was taken for the assay of enzyme activity a s described by Hopkins e t al. ('73b). The reaction mixture contained 100 pmoles of glycylglycine buffer (pH 7.01, 0.2 pmoles of pyridoxal phosphate, 5.0 Fmoles of dithiothreitol, 2.0 pmoles of Gornithine, 0.4 pci of dLornithine ll-14cl(50 mci/mmole; New England Nuclear COTP.)and liver supernatant fluid in a final volume of 1.0 ml. Incubations were carried out for 45 minutes at 37°C in 10 ml conical flasks containing disposable plastic center-wells (Kontes Glass Co., Vineland, New Jersey). Each center-well contained 0.3 ml of 1 M hyamine hydroxide in methanol (Sigma Chemical Co.). At the end of the incubation period, 1.0 ml of 40% trichloroacetic acid was injected into the flasks, and the incubations continued for a further 60 minutes to ensure complete absorption of 14C02.The center-well and its contents were then transferred to 10 ml of toluene phosphor (15.2 g of Omnifluor in 3.8 litres of toluene) in a scintillation vial and the amount of radioactivity in 14C02measured. Human diploid WI-38 fetal lung cells (passage 20) were obtained from the American Type Culture Collection (Rockville, Maryland). Cultures were maintained in T-75 flasks (Falcon Plastics Co., Oxnard, California) containing 10 ml of a complete, antibiotic-free medium consisting of 90% (vol/vol) Eagle's basal diploid medium (Grand Island Biological Co., Grand Island, New York) and
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COLCHICINE AND CELL PROLIFERATION
7-I B. M-38
o ~ / Ia
Nochanva
..........0........&..-o
0 4 8 P I P o 2 4 WLRS AFTER PARTIAL HEPATECnmY
-
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4 8 P 16 2 0 2 4 H(xNps AFTER AiCDlUAi-SW cIIpM3E
Fig. 1 DNA synthesis in hepatocytes following partial hepatectomy (A), and in WI-38 cells stimulated to proliferate by a medium-serum change (B). Animals were subjected to hepatectomy or laparotomy and injected with PHI-thymidine (1.0 yci/g body weight) one hour prior to sacrifice. A. Samples of liver were fixed, sectioned and prepared for autoradiography as described inMATERIALS AND METHODS. Each point is the mean rt SEM of 8-9 animals. B. Quiescent, confluent monolayers of WI-38 cells (passage 20) maintained in complete medium consisting of BME medium (90%vol/vol) and fetal bovine serum (10%vol/vol) were stimulated to proliferate by the addition of fresh medium consisting of 80%(vol/vol) BME and 20% (vol/vol) fetal bovine serum at 0 hours. The uptake of L3HI-thymidine into DNA was determined at various intervals after the medium-serum change by a one-hour exposure to 5 y d m l PHI-thymidine as described in MATERIALS AND METHODS. Each point is the mean rt SEM of values from four cultures.
10% (vol/vol) fetal bovine serum (Colorado Serum Co., Denver, Colorado). Cultures were passaged a t confluency every five to seven days as previously described (Boynton et al., '76a). Murine C3H10T1/zfetal fibroblasts were a gift from Doctor C. H. Heidelberger (McArdie Laboratory for Cancer Research, Madison, Wisconsin). They were maintained in a n antibiotic-free medium consisting of 90%(vol/vol) Eagle's basal diploid medium and 10% (vol/ vol) heat-inactivated fetal bovine serum (Grand Island Biological Co.). Cells were passaged at 10-day intervals as described before (Boynton and Whitfield, '76). Both WI38 and C3HlOT% cells were incubated at 37°C in a humidified atmosphere consisting of 95% air and 5% CO,. The cultures were routinely examined for mycoplasma contamination by exposure to 13H1-thymidine and checking for cytoplasmic labeling by autoradiography. The proportions of DNA-synthesizing cells were determined autoradiographically on confluent cultures grown on plastic coverslips (LUX Scientific Corp., Thousand Oaks, California) contained in 35 mm plastic petri dishes (LUX Scientific Corp.). The cells were exposed to 10pci/ml of I3H1-thymidinefor a 1hour period. The coverslips were then rinsed twice with phosphate-buffered saline (pH 7.21,
fixed in buffered formalin and rinsed twice in a solution of 10 mM non-radioactive thymidine. Autoradiography was performed as described previously (Whitfield et al., '71; Boynton e t al., '76a,b). The amount of I3H1-thymidine incorporated into DNA was determined by exposing monolayers to 5 pci/ml of [3H1-thymidine for one hour, after which time the amount of radioactivity in chemically isolated DNA was estimated as previously described (Boynton e t al., '76b). RESULTS
Partial hepatectomy induces a wave of DNA synthesis in the remaining hepatocytes which commences at 14 to 16 hours and peaks between 20 to 24 hours after the operation (fig. 1A). The administration of a single dose of colchicine, shortly after hepatectomy, inhibited this wave of DNA synthesis. A doseresponse curve for the effect of colchicine, injected at 30 minutes after the operation, on the amount of DNA synthesis observed at 24 hours after hepatectomy, is given in figure 2. A dose of 50 pg/lOO g of body weight was the minimum dose which completely inhibited the initiation of DNA synthesis and this dose was used in all subsequent experiments.
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P. ROY WALKER. ALTON L. BOYNTON AND JAMES F. WHITFIELD
The effect of colchicine injected a t different times after hepatectomy on 24 hour DNA synthesis measured by F3Hl-thymidine incorporation into DNA (fig. 3)and on the percentage of
1
0 5 1 0 20 .m 40 C O L W M &wECTWN€ fNg/rnoV Boor WEIWT~
50
Fig. 2 Dose-response curve for the inhibition of DNA synthesis in hepatocytes by colchicine. Animals were injected with the dose of colchicine indicated 30 minutes after partial hepatectomy. Twentythree hours after the operation the animals were injected with t3H1-thymidine (1.0 FCi/g body weight) and killed one hour later. Tissue samples were prepared for autoradiography or DNA extraction and assay as described in MATERIALS AND METHODS. Each point is the mean -+ SEM of 9-20 determinations.
DNA-synthesizing cells (fig. 4) was studied. Colchicine injected 30 minutes after hepatectomy produced almost complete inhibition of the initiation of DNA synthesis. The animals were less sensitive to colchicine when injected between two and six hours after the operation, but became more sensitive again between 8 and 14 hours. Colchicine injected after the onset of DNA synthesis produced only slight inhibition of synthesis (measured a t 24 hours). Vincristine, which is capable of disrupting microtubule function (Wilson and Bryan, '741,was also found to inhibit the initiation of DNA synthesis when administered either 30 minutes or 10 hours after hepatectomy whereas lumicolchicine has no effect (figs. 3,4). Since the in vivo site of action of colchicine is not confined to liver, and since the drug exerts an effect on the function of most tissues, particularly hormone-secreting cells (Soifer, '75; Borgers and De Brabander, '78, the inhibition of the initiation of DNA synthesis in liver may be produced by an indirect effect of colchicine. To assess this possibility the effect of colchicine on the proliferation of WI-38 and C3H10T1h cells in culture was determined. The proliferatively quiescent cells in confluent monolayers of these strains can be stimulated to proliferate by a complete
Fig. 3 The effect of injection of colchicine, vincristine or lumicolchicineon DNA synthesis in regenerating rat liver. Animals were partially hepatectomized and injected with colchicine 150 pg/lOO g body weight) at the times indicated between 30 minutes and 23 hours after the operation. Some animals were also injected with vincristine sulfate or lumicolchicine 150 Fg/100 g body weight) at either 30 minutes or 10 hours after hepatectomy. Twentythree hours after the operation the animals were injected with PHI-thymidine (1.0 Fci/g body weight) and killed one hour later. Tissue samples were homogenized in cold 10%TCA and the specific radioactivity of the isolated DNA estimated. Each point is the mean rt SEM of eight or nine determinations.
COLCHICINE AND CELL PROLIFERATION
93
Fig. 4 The effect of time of injection of colchicine, vincristine or lumicolchicine (UV-inactivatedcolchicine) on the percentage of DNA-synthesizing cells in regenerating rat liver. Animals were partially hepatectomized and injected with colchicine (5Opgi100 g body weight) a t the times indicated between 30 minutes and 23 hours after the operation. Some animals were also injected with vincristine sulfate or lumicolchicine (50 pgi100 g body weight) a t either 30 minutes or 10 hours after partial hepatectomy. Twenty-three hours after the operation the animals were injected with PHI-thymidine (1.0 pcilg body weight) and killed one hour later. Tissue samples were fixed in buffered formalin and processed for autoradiography as described in MATERIALS AND METHODS. Each point is the mean 2 SEM of eight or nine determinations.
Fig. 5 Autoradiographic demonstration of the effect of various concentrations of colchicine on DNA synthesis in proliferatively activated cultures of WI-38 (A) and C3HIOT'h iB) cells. Confluent monolayers of both cell types were subjected to a medium-serum change a t time "0".For WI-38 the medium-serum consisted of 80% (vol/vol) BME plus 20% (volivol) fetal bovine serum. For C3HIOT'h cells the medium-serum consisted of 80% (volivol) BME plus 20%(vol/vol) heat-inactivated fetal bovine serum. Various concentrations of colchicine in phosphate-buffered saline (PBS; pH 7.2) were added a t the time of medium-serum change ( 0 1 or 16 hours later (01. Control cultures received either a complete medium-serum change plus PBS, or received no change. The proportion of cells synthesizing DNA a t 20 hours was determined autoradiographically as described in MATERIALS AND METHODS. Each point is the mean -C SEM of four cultures.
medium-serum change. WI-38 cells, for example, commence DNA synthesis approximately ten hours later and show a peak of synthesis a t 20 hours after stimulation (fig. 1B). As
shown in figure 5, colchicine, added a t the same time as the medium-serum change, inhibited the initiation of DNA synthesis in both WI-38 and C3H10T1hcells whereas addi-
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P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
Fig. 6 The effect of time of addition of colchicine on DNA synthesis in proliferatively activated WI-38 cells. Confluent monolayers were activated by the addition of fresh medium consisting of 80Y3 (vol/vol) BME and 20% (volivol) fetal h v i n e serum. Colchicine was added to the cultures at the time of medium-serum change or various times afterwards a t a final concentration of l O + M (in PBS; pH 7 . 2 ) .Twenty hours after the medium-serum change the proportion of DNA-synthesizing cells were determined autoradiographically as described in MATERIALS AND METHODS. Each point is the mean & SEM of four cultures.
tion of colchicine a t 16 hours had no effect. The concentrations of colchicine producing inhibition of the initiation of DNA synthesis are in the range normally associated with the specific disruption of microtubules (Wilson and Bryan, '74; Wilson e t al., '74). The maximum inhibition of the initiation of DNA synthesis by colchicine was observed if the drug was added to the cells during the first two hours after the medium-serum change (fig. 6). However, the drug was less effective when added at later times and showed little or no effect on cells after ten hours when DNA synthesis had already commenced. Thus the inhibitory action of colchicine a t the early stage of proliferation was found to occur in the cell lines in uitro as well as in hepatocytes in liver in vivo. However, the cultured cells were not sensitive immediately before the onset of DNA synthesis. The effect of colchicine (at a dose of 50 p g / 100 g of body weight, injected 30 minutes after hepatectomy) on the induction of ODC
in hepatocytes is illustrated in figure 7. In normal animals, ODC activity is increased 6fold by two hours and 20-fold by four hours after hepatectomy followed by a decrease in activity. This early increase in ODC activity is similar to that observed by others (Russell and Snyder, '68; Janne and Raina, '68; Fausto, '69; Schrock e t al., '70; Barbiroli e t al., '74). The injection of colchicine delays the induction process by approximately two hours, but does not produce any long-term inhibition. Colchicine was without effect on the activity of ODC when added to the in vitro assay (data not shown). The dose of colchicine (on a body weight basis) used in these experiments is much less than t h a t used by O'Brien et al., '76) to produce maximum inhibition. Complete inhibition of the enzyme a t four hours was observed in this study using doses of 100-200 p g l l 0 0 g body weight (data not shown). To assess whether a dose of 50 pg/lOO g body weight only produced a delay in the initiation of DNA synthesis, the rate of synthesis a t 30 hours after hepatectomy was compared with that a t 24 hours (fig. 8). I t is clear that colchicine inhibits the initiation of the synthesis of DNA and that. the onset of synthesis is delayed for at least 14 to 16 hours
z
0 mwIps
A F l I R P-L
4
6
8
HE.0ATECWY
Fig. 7 The effect of colchicine on the induction of ornithine decarboxylase in hepatocytes following partial hepatectomy. Animals were subjected to either partial hepatectomy or laparotomy and sacrificed at 2, 4, 6 or 8 hours after the operation. Some of the animals received a n injection of colchicine (50 pg/lOO g body weight) 30 minutes after the operation. Tissue samples were homogenized in buffer, centrifuged and assayed for ornithine decarboxylase activity as described in MATERIALS AND METHODS. Each point is the mean +- SEM of eight or nine determinations.
COLCHICINE AND CE:LL PROLIFERATION
J-y
LAP HPXt Colchiciine
EHPX
u
\--yi
24 HOURS
30 hwRS
Fig. 8 The effect of colchicine on the synthesis of DNA in hepatocytes following partial hepatectomy. Animals were injected with colchicine 30 minutes after hepatectomy and sacrificed a t either 24 or 30 hours after the operation. One hour prior to sacrifice, the animals were injected with i3H1-thymidine (1.0pc/g body weight). Tissue samples were homogenized in cold 10%TCA, the DNA was isolated and its specific radioactivity estimated. Each point is the mean rt. SEM of nine determinations.
compared with a delay of ODC induction of only two hours. DISCUSSION
The present observations confirm and extend those of Luyckx and Van Lancker ('66) and Wang e t al. ('75) t h a t microtubule disrupters, such a s colchicine, vinblastine and vincristine, block the earliest stages of the prereplicative development of a variety of human and rodent cells, but do not affect ongoing DNA synthesis. However, we have found that the hepatocytes in regenerating liver, unlike the peripheral lymphocytes of Wang e t al. ('75) and the C3HlOT"/zand WI-38 cells of the present study, have varying degrees of sensitivity to colchicine (and vincristine) throughout the prereplicative phase with the most sensitive periods being immediately after partial hepatectomy and immediately before the onset of DNA synthesis. In fact, this seems to be the first demonstration of an effect of colchicine immediately before the onset of DNA synthesis. Work with colchicine is complicated by the fact that the binding and release of the drug to tubulin (and other proteins) is a slow process (Wilson, '70; Lagnado et al., '71). Le
95
Marchand e t al. ('75) have demonstrated that the administration of a single dose of colchicine (50 gI100 g body weight) to mice inhibits the release of triglyceride from the liver with 50%inhibition occurring four hours after injection and almost complete inhibition is observed 11hours after the single injection. Triglyceride release returns to normal by 20 hours. This data illustrates the long-term nature of colchicine-induced changes. Thus it is not possible to determine precisely the prereplicative stages at which colchicine arrests cells, except that there is an early stage in all cells and a second stage before the onset of DNA synthesis in hepatocytes while ongoing DNA synthesis itself is not affected. The low concentrations of colchicine required to inhibit the initiation of DNA synthesis, coupled with the lack of effect of lumicolchicine and an inhibition by vincristine, suggest that microtubules may be involved in the proliferative activation process. The cytoplasmic microtubules are involved in many aspects of cellular function such as phagocytosis, the transport of vesicles such as lysosomes, pigment granules and secretory granules, as well as cell locomotion and the maintenance of cell shape (Soifer, '75; Borgers and De Brabander, '75). Evidence, based on colchicine binding, has been presented which suggests that microtubules are also associated with cellular membrane fractions (Lagnado et al., '76; Stadler and Franke, '72; Oliver e t al., '74). Tubulin has also been shown to be a major component of the nonhistone chromosomal fraction of rat liver nuclei (Le Stourgeon et al., '74; Douvas e t al., '75). However, the binding of colchicine to nuclear and plasma membrane fractions may not be specific for tubulin (Stadler and Franke, '74; Wunderlich e t al., '731, and the drug may interfere with other membrane processes such as the transport of ions and other molecules necessary for proliferation. For example, we have shown that calcium ions regulate the transition of hepatocytes from the GI stage of the cell cycle to S (Rixon and Whitfield, '76) and potassium ions appear to be involved in the proliferative activation of 3T3 cells (Tupper et al., '77). Moreover the inhibitory action of ouabain on proliferation of 3T3 cells is similar to that of colchicine on the cells in this study (Tupper e t al., '77). I t is possible that colchicine may interfere with ion transport either by non-specific binding to the plasma membrane or by interference with
96
P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
microtubules which may be involved in the transport process. Alternatively Niemann and Webb (‘77) have recently shown that colchicine inhibits the release of ribosomal RNA from liver nuclei which may prevent the onset of DNA synthesis. However, in the present study it is not possible to locate the site of colchicine binding which could be at plasma membrane, or cytoplasmic or nuclear binding sites. Finally, we have shown t h a t the dose of colchicine used to inhibit the initiation of DNA synthesis for a t least 14 to 16 hours produces only a 2-hour delay in the synthesis of ODC. Colchicine also inhibits the initiation of DNA synthesis when injected between 8 and 14 hours after hepatectomy, which is after the period of ODC induction and polyamine accumulation (Walker, Sikorska and Whitfield, unpublished observations). Thus the inhibitory action of colchicine on the initiation of hepatocyte DNA synthesis, a t both the early and late periods of sensitivity, does not appear to be due to a n inhibition of polyamine accumulation. ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of J. Bayliss and D. J. Gillan who also prepared the illustrations. LITERATURE CITED Barbiroli, B., M. S. Moruzzi, B. Tadolini and M. G. Monti 1974 Ornithine decarboxylase activity in regenerating liver from rats adapted to controlled feedingschedules. J. Nutr., 105:408-412. Betel, I., and J. Martijnse 1976 Drugs that disrupt microtubuli do not inhibit lymphocyte activation. Nature, 261: 318-319. Borgers, M., and M. De Brabander, eds. 1975 Microtubules and Microtubule Inhibitors. North-Holland Publishing Co. Ltd., Amsterdam. Boynton, A. L., and J. F. Whitfield 1976 Different calcium requirements for proliferation of conditionally and unconditionally tumorigenic mouse cells. Proc. Natl. Acad. Sci. (U.S.A.), 73:1651-1654. Boynton, A. L., J. F. Whitfield and R. J. Isaacs 1976a A possible involvement of polyamines in the initiation of DNA synthesis by human WI-38 and mouse BALBi3TB cells. J. Cell. Physiol., 89:481-488. 1976b Calcium. dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA). J. Cell. Physiol., 87:25-32. Chen, K., J. Heller and E. S. Canellakis 1976 Studies on the regulation of ornithine decarboxylase activity by the microtubules: The effect of colchicine and vinblastine. Biochem. Biophys. Res. Commun., 68:401-408. Douvas, A. S., C. A. Harrington and J. Bonner 1975 Major nonhistone proteins of r a t liver chromatin: Preliminary identification of myosin, actin, tubulin, and tropomyosin. Proc. Natl. Acad. Sci. (USA.), 72:3902-3906.
Edelman, G. M., I. Yahara and J. L. Wang 1973 Receptor mobility and receptor-cytoplasmic interaction in lymphocytes. Proc. Natl. Acad. Sci. (U.S.A.), 70: 1442-1446. Fausto, N. 1969 Studies on ornithine decarboxylase activity in normal and regenerating livers. Biochim. Biophys. Acta, 190: 193-201. Fillingame, R. H., and D. R. Morris 1973 Polyamine accumulation during lymphocyte transformation and its relation to the synthesis, processing, and accumulation of ribonucleic acid. Biochemistry, 12:4479-4487. Greene, W. C., C. M. Parker and C. W. Parker 1976 Colchicine-sensitive structures and lymphocyte activation. J. Immunol., 11 7:1015-1022. Higgins, G. M., and R. M. Anderson 1931 Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch. Path., 12: 186-202. Holtta, E., P. Hannonen, J. Pispa and J. Janne 1973 Effect of methylglyoxal Bis(guanylhydrazone1 on polyamine metabolism in normal and regenerating rat liver and r a t thymus. Biochem. J., 136: 669-676. Hopkins, H. A., R. J. Bonney, P. R. Walker, J. D. Yager, Jr. and V. R. Potter 1973b Food and light as separate entrainment signals for r a t liver enzymes. Adv. Enz. Reg., 11: 169-191. Hopkins, H. A,, H. A. Campbell, B. Barbiroli and V. R. Potter 1973a Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules. Biochem. J., 136: 955966. Janne, J., and A. Raina 1969 On the stimulation of ornithine decarboxylase and RNA polymerase activity in rat liver after treatment with growth hormone. Biochim. Biophys. Acta, 174: 769-772. Lagnado, J. R., C. Lyons and G. Wickremasinghe 1971 The subcellular distribution of colchicine-binding protein (microtubule protein) in rat brain. FEBS lett., 15: 254258. Le Marchand, Y., A. Singh, C. Patzelt, L. Orci and B. Jeanrenaud 1975 In uiuoand in uztroevidences for a role of microtubules in the secretory processes of liver. In: Microtubule and Microtubule Inhibitors. M. Borgers and M. De Brabander, eds., North Holland Publishing Co., Amsterdam, pp. 153-164. LeStourgeon, W. M., R. Totten and A. Forer 1974 The nuclear acidic proteins in cell proliferation and differentiation. In: Acidic Proteins of the Nucleus. I. L. Cameron and J. R. Jeter, Jr., eds. Academic Press, Inc., New York, pp. 159-190. Luyckx, A., and J. L. Van Lancker 1966 Metabolic effects of vinblastine. 11. The effect of vinblastine on deoxyribonucleic acid and ribonucleic acid synthesis of regen. erating liver. Lab. Investig., 15: 1301-1303. Neimann, M., and T. E. Webb 1977 Processing and transport of ribosomal RNA in a cell-free system. Fed. Proc., 36: 730a. OBrien, T. G., R. C. Simsiman and R. K. Boutwell 1976 The effect of colchicine on the induction of ornithine decarboxylase by 12-o-tetradecanoyl-phorbal-13-acetate. Cancer Res., 36: 3766-3770. Oliver, J. M., T. E. Ukena and R. D. Berlin 1974 Effects of phagocytosis and colchicine on the distribution of lectinbinding sites on cell surfaces. Proc. Natl. Acad. Sci. (U.S.A.1, 71:394.398. Poso, H., and J. Janne 1976a Inhibition of ornithine decarboxylase activity and spermidine accumulation in regenerating rat liver. Biochem. Biophys. Res. Commun., 69: 885-892.
COLCHICINE AND CELL PROLIFERATION 1976b Inhibition of polyamine accumulation an d deoxyribonucleic acid synthesis in regenerating r a t liver. Biochem. J., 158: 485-488. Rixon, R. H., and J. F. Whitfield 1976 The control of liver regeneration by parathyroid hormone and calcium. J. Cell. Physiol., 87: 147-156. Russell, D. H. 1973 Polyamines in Normal and Neoplastic Growth. Raven Press, New York. Russell, D. H., and S. H. Snyder 1968 Amine synthesis in rapidly growing tissues: Ornithine decarboxylase activity in regenerating r a t liver, chick embryo and various tumors. Proc. Natl. Acad. Sci. (U.S.A.), 60: 1420-1427. Schrock, T. R., N. J. Oakman and N. L. R. Bucher 1970 Ornithine decarboxylase activity in relation to growth of rat liver. Biochim. Biophys. Acta, 204: 564-577. Soifer, D., Editor 1975 The biology of cytoplasmic microtubules. Annals N. Y. Acad. Sc. New York Academy of Sciences, New York, Vol. 253: 691-1009. Stadler, J., and W. W. Franke 1972 Colchicine-binding proteins in chromatin and membranes. Nature New Biol., 237: 237-238. 1974 Characterization of t he colchicine binding of membrane fractions from r a t and mouse liver. J. Cell Biol., 60: 297-303. Tabor, C. W., and H. Tabor 1976 1,4-diaminobutane (putrescine), spermidine and spermine. Ann. Rev. Biochem., 45: 285-306. Tupper, J. T., F. Zorgniotti and B. Mills 1977 Potassium
97
transport and content during G, and S phase following serum stimulation of 3T3 cells. J. Cell. Physiol., 91: 429-440. Wang, J. L., G. R. Guntber and G. M. Edelman 1975 Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to the S phase. J. Cell Biol., 66: 128-144. Whitfield, J. F., J. P. MacManus, T. Youdale and D. J. Franks 1971 The roles of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocyte proliferation. J. Cell. Physiol., 78: 355-368. Wilson, L. 1970 Properties of colchicine binding proteins from chick embryo brain. Interactions with Vinca alkaloids and podophyllotoxin. Biochemistry, 9: 49995007. Wilson, L., and J. Bryan 1974 Biochemical and pharmacological properties of microtubules. Adv. Cell Molec. Biol., Vol. 3. Academic Press, New York, pp. 21-72. Wilson, L., J. R. Bamburg, S. B. Mizel, L. M. Grisham and K. M. Creswell 1974 Interaction of drugs with microtubule proteins. Fed. Proc., 33: 158-166. Wilson, L., and M. Friedkin 1966 The biochemical events of mitosis. I. Synthesis and properties of colchicine labelled with tritium in its acetyl moiety. Biochemistry, 5: 2463-2468. Wunderlich, F., R. Miiller and V. Speth 1973 Direct evi. dence for a colchicine-induced impairment in the mobility of membrane components, 182: 1136-1138.
ABSTRACT Colchicine inhibited the initiation of DNA synthesis by hepatocytes in vivo and by WI-38 and C3H10T1hcells in vitro. All three cell types were most sensitive to colchicine when i t was administered a t the time of, or shortly after, proliferative activation (by partial hepatectomy or medium-serum change). WI-38 and C3H10T1h cells became less sensitive to colchicine as the time between proliferative activation and addition of the drug was increased. Hepatocytes, on the other hand, showed a second stage of sensitivity immediately before the onset of DNA synthesis. Ongoing DNA synthesis was not inhibited by colchicine in any of the cell types. The two prereplicative stages of colchicine sensitivity in liver were also sensitive to vincristine but not to lumicolchicine, an analogue of colchicine which does not bind to tubulin. The data obtained with these drugs indicates that microtubules may be involved in the prereplicative stages of cell proliferation, but non-specific binding to other membrane proteins cannot be ruled out. Colchicine (at a dose of 50 pg/lOO g of body weight), which when injected 30 minutes after hepatectomy delayed the initiation of DNA synthesis for at least 14 hours, delayed the induction of ornithine decarboxylase (at the beginning of prereplicative development) by only two hours. Thus, the inhibitory action of colchicine on the initiation of DNA synthesis does not appear to be mediated by inhibition of polyamine synthesis. In 1966, Luyckx and Van Lancker showed t h a t injection of the microtubule disrupter, vinblastine into rats at one hour, or a t one and nine hours, after partial hepatectomy prevented the initiation of DNA synthesis in the remaining liver tissue. Subsequently, Edelman e t al. ('731, Wang e t al. ('75) and Greene e t al. ('761, found that another microtubule disrupter, colchicine, inhibited both the proliferative stimulation of lymphocytes by Concanavalin A and the early prereplicative redistribution of the lectin's receptors on the cell surface. Colchicine completely inhibits the initiation of lymphocyte DNA synthesis when added to the cells at the same time as the mitogen and becomes less effective as the time between addition of mitogen and addition of colchicine is increased indicating that the drug acts at an early prereplicative stage of the cell cycle. (However see Betel and Martijnse '76 for conflicting results). Thus EdelJ. CELL.PHYSIOL., 93: 89-98. NRCC No. 16183
man e t al. ('73) have proposed that microtubules are involved in the redistribution of lectin receptors on the cell membrane which occurs upon activation and that this process is disrupted by colchicine. Further support for an effect of colchicine on the early prereplicative stages of cell proliferation comes from studies which show that colchicine prevents t h e synthesis of ornithine decarboxylase (ODC) in mouse L1210 cells in vitro (Chen et al., '76) and mouse epidermal cells in vivo (O'Brien e t al. '76). The induction of ODC, followed by an accumulation of polyamines, often occurs as an early event following proliferative activation of quiescent cells (see Russell, 1'731 and Tabor "761, for detailed literature). Although the role of polyamines in the metabolism of proliferating cells is not clear, inhibition of polyamine accumulation Received Jan. I , '77. Accepted Mar 30, '11. ' Issued as NRCC No. 16183.
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P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
and/or function by drugs such as methylglyoxal-bislguanylhydrazonel (Holtta e t al., '73; Fillingame and Morris, '73; Boynton e t al., '76b) and diaminopropane (Poso and Janne, '76a,b) prevents, or a t least delays, DNA replication. In the present communication we have examined the effects of colchicine on the early stages of liver regeneration following partial hepatectomy, and on the proliferative activation of human WI-38 and mouse C3HlOT'h cells in culture. The data show that colchicine prevents the initiation of DNA synthesis by hepatocytes in vivo and WI-38 and C3HlOT'h cells in vitro when added a t the time of proliferative activation. The cells in vitro become progressively less sensitive to colchicine when it is added a t later times, whereas the hepatocytes have a second colchicine-sensitive stage shortly before DNA synthesis begins. We will also show that the inhibitory action of colchicine on the initiation of DNA synthesis in hepatocytes is probably not mediated by a n inhibition of ODC activity. MATERIALS AND METHODS
The animals used in this study were males (190-210 g) of a specific-pathogen-free strain of Sprague-Dawley r a t bred in this laboratory. The rats were entrained to a 12-hour 1ight:dark cycle with the dark period being from 7 P.M. to 7 A.M. Food was available ad libitum before the operations, but was withheld afterwards; water was available a t all times. Partial hepatectomy was carried out according to the procedure of Higgins and Anderson ('31) by which 68%of the liver mass was removed. All operations were carried out between 9 A.M. and 11A.M. Colchicine (Sigma Chemical Co., St. Louis, Missouri) and vincristine sulphate (Oncovin, Eli Lilly and Co. [Canada] Ltd., Toronto, Ontario) were dissolved in a solution of 0.9% NaCl or distilled water or diluent, respectively, and injected intraperitoneally. Lumicolchicine, a structural analogue of colchicine which does not bind to tubulin (Wilson e t al., ' 7 4 , was prepared by UV irradiation of colchicine as described by Wilson and Friedkin ('66). DNA synthesis in the liver remnant was determined both autoradiographically and by the incorporation of t3H1-Thymidine into chemically isolated DNA. 13H1-Thymidine(1.0 pcilg body weight; sp. act. 20 ci/mmole; New England Nuclear, Corp., Boston, Massachu-
setts) was injected intraperitoneally one hour prior to sacrifice. Animals were anesthetized with ether and the right anterior lobe removed. Small blocks of tissue were fixed in buffered formalin and processed for autoradiography as described previously (Rixon and Whitfield, '76). A sample (100 mg) of the same liver remnant was homogenized in ice-cold 10% trichloroacetic acid, and DNA was extracted and assayed as described by Hopkins et al. ('73a). A portion of the final extract was added to 10 ml of a scintillation fluid prepared from dioxane (3.8 11, naphthalene (250 g) and omnifluor (30.4 g; New England Nuclear Corp.) and counted in a Beckman LS-255 liquid scintillation system. Samples for the determination of ODC activity were prepared by homogenizing 800 mg of liver in 25 mM Tris-HC1buffer (pH 7.0) containing 0.1 mM EDTA and 5 mM dithiothreitol. The homogenate was centrifuged a t 105,000 g f o r 60 minutes (at 4"C, in a Beckman L2-65B Ultracentrifuge), and 0.4 ml of the supernatant fluid was taken for the assay of enzyme activity a s described by Hopkins e t al. ('73b). The reaction mixture contained 100 pmoles of glycylglycine buffer (pH 7.01, 0.2 pmoles of pyridoxal phosphate, 5.0 Fmoles of dithiothreitol, 2.0 pmoles of Gornithine, 0.4 pci of dLornithine ll-14cl(50 mci/mmole; New England Nuclear COTP.)and liver supernatant fluid in a final volume of 1.0 ml. Incubations were carried out for 45 minutes at 37°C in 10 ml conical flasks containing disposable plastic center-wells (Kontes Glass Co., Vineland, New Jersey). Each center-well contained 0.3 ml of 1 M hyamine hydroxide in methanol (Sigma Chemical Co.). At the end of the incubation period, 1.0 ml of 40% trichloroacetic acid was injected into the flasks, and the incubations continued for a further 60 minutes to ensure complete absorption of 14C02.The center-well and its contents were then transferred to 10 ml of toluene phosphor (15.2 g of Omnifluor in 3.8 litres of toluene) in a scintillation vial and the amount of radioactivity in 14C02measured. Human diploid WI-38 fetal lung cells (passage 20) were obtained from the American Type Culture Collection (Rockville, Maryland). Cultures were maintained in T-75 flasks (Falcon Plastics Co., Oxnard, California) containing 10 ml of a complete, antibiotic-free medium consisting of 90% (vol/vol) Eagle's basal diploid medium (Grand Island Biological Co., Grand Island, New York) and
91
COLCHICINE AND CELL PROLIFERATION
7-I B. M-38
o ~ / Ia
Nochanva
..........0........&..-o
0 4 8 P I P o 2 4 WLRS AFTER PARTIAL HEPATECnmY
-
0
4 8 P 16 2 0 2 4 H(xNps AFTER AiCDlUAi-SW cIIpM3E
Fig. 1 DNA synthesis in hepatocytes following partial hepatectomy (A), and in WI-38 cells stimulated to proliferate by a medium-serum change (B). Animals were subjected to hepatectomy or laparotomy and injected with PHI-thymidine (1.0 yci/g body weight) one hour prior to sacrifice. A. Samples of liver were fixed, sectioned and prepared for autoradiography as described inMATERIALS AND METHODS. Each point is the mean rt SEM of 8-9 animals. B. Quiescent, confluent monolayers of WI-38 cells (passage 20) maintained in complete medium consisting of BME medium (90%vol/vol) and fetal bovine serum (10%vol/vol) were stimulated to proliferate by the addition of fresh medium consisting of 80%(vol/vol) BME and 20% (vol/vol) fetal bovine serum at 0 hours. The uptake of L3HI-thymidine into DNA was determined at various intervals after the medium-serum change by a one-hour exposure to 5 y d m l PHI-thymidine as described in MATERIALS AND METHODS. Each point is the mean rt SEM of values from four cultures.
10% (vol/vol) fetal bovine serum (Colorado Serum Co., Denver, Colorado). Cultures were passaged a t confluency every five to seven days as previously described (Boynton et al., '76a). Murine C3H10T1/zfetal fibroblasts were a gift from Doctor C. H. Heidelberger (McArdie Laboratory for Cancer Research, Madison, Wisconsin). They were maintained in a n antibiotic-free medium consisting of 90%(vol/vol) Eagle's basal diploid medium and 10% (vol/ vol) heat-inactivated fetal bovine serum (Grand Island Biological Co.). Cells were passaged at 10-day intervals as described before (Boynton and Whitfield, '76). Both WI38 and C3HlOT% cells were incubated at 37°C in a humidified atmosphere consisting of 95% air and 5% CO,. The cultures were routinely examined for mycoplasma contamination by exposure to 13H1-thymidine and checking for cytoplasmic labeling by autoradiography. The proportions of DNA-synthesizing cells were determined autoradiographically on confluent cultures grown on plastic coverslips (LUX Scientific Corp., Thousand Oaks, California) contained in 35 mm plastic petri dishes (LUX Scientific Corp.). The cells were exposed to 10pci/ml of I3H1-thymidinefor a 1hour period. The coverslips were then rinsed twice with phosphate-buffered saline (pH 7.21,
fixed in buffered formalin and rinsed twice in a solution of 10 mM non-radioactive thymidine. Autoradiography was performed as described previously (Whitfield et al., '71; Boynton e t al., '76a,b). The amount of I3H1-thymidine incorporated into DNA was determined by exposing monolayers to 5 pci/ml of [3H1-thymidine for one hour, after which time the amount of radioactivity in chemically isolated DNA was estimated as previously described (Boynton e t al., '76b). RESULTS
Partial hepatectomy induces a wave of DNA synthesis in the remaining hepatocytes which commences at 14 to 16 hours and peaks between 20 to 24 hours after the operation (fig. 1A). The administration of a single dose of colchicine, shortly after hepatectomy, inhibited this wave of DNA synthesis. A doseresponse curve for the effect of colchicine, injected at 30 minutes after the operation, on the amount of DNA synthesis observed at 24 hours after hepatectomy, is given in figure 2. A dose of 50 pg/lOO g of body weight was the minimum dose which completely inhibited the initiation of DNA synthesis and this dose was used in all subsequent experiments.
92
P. ROY WALKER. ALTON L. BOYNTON AND JAMES F. WHITFIELD
The effect of colchicine injected a t different times after hepatectomy on 24 hour DNA synthesis measured by F3Hl-thymidine incorporation into DNA (fig. 3)and on the percentage of
1
0 5 1 0 20 .m 40 C O L W M &wECTWN€ fNg/rnoV Boor WEIWT~
50
Fig. 2 Dose-response curve for the inhibition of DNA synthesis in hepatocytes by colchicine. Animals were injected with the dose of colchicine indicated 30 minutes after partial hepatectomy. Twentythree hours after the operation the animals were injected with t3H1-thymidine (1.0 FCi/g body weight) and killed one hour later. Tissue samples were prepared for autoradiography or DNA extraction and assay as described in MATERIALS AND METHODS. Each point is the mean -+ SEM of 9-20 determinations.
DNA-synthesizing cells (fig. 4) was studied. Colchicine injected 30 minutes after hepatectomy produced almost complete inhibition of the initiation of DNA synthesis. The animals were less sensitive to colchicine when injected between two and six hours after the operation, but became more sensitive again between 8 and 14 hours. Colchicine injected after the onset of DNA synthesis produced only slight inhibition of synthesis (measured a t 24 hours). Vincristine, which is capable of disrupting microtubule function (Wilson and Bryan, '741,was also found to inhibit the initiation of DNA synthesis when administered either 30 minutes or 10 hours after hepatectomy whereas lumicolchicine has no effect (figs. 3,4). Since the in vivo site of action of colchicine is not confined to liver, and since the drug exerts an effect on the function of most tissues, particularly hormone-secreting cells (Soifer, '75; Borgers and De Brabander, '78, the inhibition of the initiation of DNA synthesis in liver may be produced by an indirect effect of colchicine. To assess this possibility the effect of colchicine on the proliferation of WI-38 and C3H10T1h cells in culture was determined. The proliferatively quiescent cells in confluent monolayers of these strains can be stimulated to proliferate by a complete
Fig. 3 The effect of injection of colchicine, vincristine or lumicolchicineon DNA synthesis in regenerating rat liver. Animals were partially hepatectomized and injected with colchicine 150 pg/lOO g body weight) at the times indicated between 30 minutes and 23 hours after the operation. Some animals were also injected with vincristine sulfate or lumicolchicine 150 Fg/100 g body weight) at either 30 minutes or 10 hours after hepatectomy. Twentythree hours after the operation the animals were injected with PHI-thymidine (1.0 Fci/g body weight) and killed one hour later. Tissue samples were homogenized in cold 10%TCA and the specific radioactivity of the isolated DNA estimated. Each point is the mean rt SEM of eight or nine determinations.
COLCHICINE AND CELL PROLIFERATION
93
Fig. 4 The effect of time of injection of colchicine, vincristine or lumicolchicine (UV-inactivatedcolchicine) on the percentage of DNA-synthesizing cells in regenerating rat liver. Animals were partially hepatectomized and injected with colchicine (5Opgi100 g body weight) a t the times indicated between 30 minutes and 23 hours after the operation. Some animals were also injected with vincristine sulfate or lumicolchicine (50 pgi100 g body weight) a t either 30 minutes or 10 hours after partial hepatectomy. Twenty-three hours after the operation the animals were injected with PHI-thymidine (1.0 pcilg body weight) and killed one hour later. Tissue samples were fixed in buffered formalin and processed for autoradiography as described in MATERIALS AND METHODS. Each point is the mean 2 SEM of eight or nine determinations.
Fig. 5 Autoradiographic demonstration of the effect of various concentrations of colchicine on DNA synthesis in proliferatively activated cultures of WI-38 (A) and C3HIOT'h iB) cells. Confluent monolayers of both cell types were subjected to a medium-serum change a t time "0".For WI-38 the medium-serum consisted of 80% (vol/vol) BME plus 20% (volivol) fetal bovine serum. For C3HIOT'h cells the medium-serum consisted of 80% (volivol) BME plus 20%(vol/vol) heat-inactivated fetal bovine serum. Various concentrations of colchicine in phosphate-buffered saline (PBS; pH 7.2) were added a t the time of medium-serum change ( 0 1 or 16 hours later (01. Control cultures received either a complete medium-serum change plus PBS, or received no change. The proportion of cells synthesizing DNA a t 20 hours was determined autoradiographically as described in MATERIALS AND METHODS. Each point is the mean -C SEM of four cultures.
medium-serum change. WI-38 cells, for example, commence DNA synthesis approximately ten hours later and show a peak of synthesis a t 20 hours after stimulation (fig. 1B). As
shown in figure 5, colchicine, added a t the same time as the medium-serum change, inhibited the initiation of DNA synthesis in both WI-38 and C3H10T1hcells whereas addi-
94
P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
Fig. 6 The effect of time of addition of colchicine on DNA synthesis in proliferatively activated WI-38 cells. Confluent monolayers were activated by the addition of fresh medium consisting of 80Y3 (vol/vol) BME and 20% (volivol) fetal h v i n e serum. Colchicine was added to the cultures at the time of medium-serum change or various times afterwards a t a final concentration of l O + M (in PBS; pH 7 . 2 ) .Twenty hours after the medium-serum change the proportion of DNA-synthesizing cells were determined autoradiographically as described in MATERIALS AND METHODS. Each point is the mean & SEM of four cultures.
tion of colchicine a t 16 hours had no effect. The concentrations of colchicine producing inhibition of the initiation of DNA synthesis are in the range normally associated with the specific disruption of microtubules (Wilson and Bryan, '74; Wilson e t al., '74). The maximum inhibition of the initiation of DNA synthesis by colchicine was observed if the drug was added to the cells during the first two hours after the medium-serum change (fig. 6). However, the drug was less effective when added at later times and showed little or no effect on cells after ten hours when DNA synthesis had already commenced. Thus the inhibitory action of colchicine a t the early stage of proliferation was found to occur in the cell lines in uitro as well as in hepatocytes in liver in vivo. However, the cultured cells were not sensitive immediately before the onset of DNA synthesis. The effect of colchicine (at a dose of 50 p g / 100 g of body weight, injected 30 minutes after hepatectomy) on the induction of ODC
in hepatocytes is illustrated in figure 7. In normal animals, ODC activity is increased 6fold by two hours and 20-fold by four hours after hepatectomy followed by a decrease in activity. This early increase in ODC activity is similar to that observed by others (Russell and Snyder, '68; Janne and Raina, '68; Fausto, '69; Schrock e t al., '70; Barbiroli e t al., '74). The injection of colchicine delays the induction process by approximately two hours, but does not produce any long-term inhibition. Colchicine was without effect on the activity of ODC when added to the in vitro assay (data not shown). The dose of colchicine (on a body weight basis) used in these experiments is much less than t h a t used by O'Brien et al., '76) to produce maximum inhibition. Complete inhibition of the enzyme a t four hours was observed in this study using doses of 100-200 p g l l 0 0 g body weight (data not shown). To assess whether a dose of 50 pg/lOO g body weight only produced a delay in the initiation of DNA synthesis, the rate of synthesis a t 30 hours after hepatectomy was compared with that a t 24 hours (fig. 8). I t is clear that colchicine inhibits the initiation of the synthesis of DNA and that. the onset of synthesis is delayed for at least 14 to 16 hours
z
0 mwIps
A F l I R P-L
4
6
8
HE.0ATECWY
Fig. 7 The effect of colchicine on the induction of ornithine decarboxylase in hepatocytes following partial hepatectomy. Animals were subjected to either partial hepatectomy or laparotomy and sacrificed at 2, 4, 6 or 8 hours after the operation. Some of the animals received a n injection of colchicine (50 pg/lOO g body weight) 30 minutes after the operation. Tissue samples were homogenized in buffer, centrifuged and assayed for ornithine decarboxylase activity as described in MATERIALS AND METHODS. Each point is the mean +- SEM of eight or nine determinations.
COLCHICINE AND CE:LL PROLIFERATION
J-y
LAP HPXt Colchiciine
EHPX
u
\--yi
24 HOURS
30 hwRS
Fig. 8 The effect of colchicine on the synthesis of DNA in hepatocytes following partial hepatectomy. Animals were injected with colchicine 30 minutes after hepatectomy and sacrificed a t either 24 or 30 hours after the operation. One hour prior to sacrifice, the animals were injected with i3H1-thymidine (1.0pc/g body weight). Tissue samples were homogenized in cold 10%TCA, the DNA was isolated and its specific radioactivity estimated. Each point is the mean rt. SEM of nine determinations.
compared with a delay of ODC induction of only two hours. DISCUSSION
The present observations confirm and extend those of Luyckx and Van Lancker ('66) and Wang e t al. ('75) t h a t microtubule disrupters, such a s colchicine, vinblastine and vincristine, block the earliest stages of the prereplicative development of a variety of human and rodent cells, but do not affect ongoing DNA synthesis. However, we have found that the hepatocytes in regenerating liver, unlike the peripheral lymphocytes of Wang e t al. ('75) and the C3HlOT"/zand WI-38 cells of the present study, have varying degrees of sensitivity to colchicine (and vincristine) throughout the prereplicative phase with the most sensitive periods being immediately after partial hepatectomy and immediately before the onset of DNA synthesis. In fact, this seems to be the first demonstration of an effect of colchicine immediately before the onset of DNA synthesis. Work with colchicine is complicated by the fact that the binding and release of the drug to tubulin (and other proteins) is a slow process (Wilson, '70; Lagnado et al., '71). Le
95
Marchand e t al. ('75) have demonstrated that the administration of a single dose of colchicine (50 gI100 g body weight) to mice inhibits the release of triglyceride from the liver with 50%inhibition occurring four hours after injection and almost complete inhibition is observed 11hours after the single injection. Triglyceride release returns to normal by 20 hours. This data illustrates the long-term nature of colchicine-induced changes. Thus it is not possible to determine precisely the prereplicative stages at which colchicine arrests cells, except that there is an early stage in all cells and a second stage before the onset of DNA synthesis in hepatocytes while ongoing DNA synthesis itself is not affected. The low concentrations of colchicine required to inhibit the initiation of DNA synthesis, coupled with the lack of effect of lumicolchicine and an inhibition by vincristine, suggest that microtubules may be involved in the proliferative activation process. The cytoplasmic microtubules are involved in many aspects of cellular function such as phagocytosis, the transport of vesicles such as lysosomes, pigment granules and secretory granules, as well as cell locomotion and the maintenance of cell shape (Soifer, '75; Borgers and De Brabander, '75). Evidence, based on colchicine binding, has been presented which suggests that microtubules are also associated with cellular membrane fractions (Lagnado et al., '76; Stadler and Franke, '72; Oliver e t al., '74). Tubulin has also been shown to be a major component of the nonhistone chromosomal fraction of rat liver nuclei (Le Stourgeon et al., '74; Douvas e t al., '75). However, the binding of colchicine to nuclear and plasma membrane fractions may not be specific for tubulin (Stadler and Franke, '74; Wunderlich e t al., '731, and the drug may interfere with other membrane processes such as the transport of ions and other molecules necessary for proliferation. For example, we have shown that calcium ions regulate the transition of hepatocytes from the GI stage of the cell cycle to S (Rixon and Whitfield, '76) and potassium ions appear to be involved in the proliferative activation of 3T3 cells (Tupper et al., '77). Moreover the inhibitory action of ouabain on proliferation of 3T3 cells is similar to that of colchicine on the cells in this study (Tupper e t al., '77). I t is possible that colchicine may interfere with ion transport either by non-specific binding to the plasma membrane or by interference with
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P. ROY WALKER, ALTON L. BOYNTON AND JAMES F. WHITFIELD
microtubules which may be involved in the transport process. Alternatively Niemann and Webb (‘77) have recently shown that colchicine inhibits the release of ribosomal RNA from liver nuclei which may prevent the onset of DNA synthesis. However, in the present study it is not possible to locate the site of colchicine binding which could be at plasma membrane, or cytoplasmic or nuclear binding sites. Finally, we have shown t h a t the dose of colchicine used to inhibit the initiation of DNA synthesis for a t least 14 to 16 hours produces only a 2-hour delay in the synthesis of ODC. Colchicine also inhibits the initiation of DNA synthesis when injected between 8 and 14 hours after hepatectomy, which is after the period of ODC induction and polyamine accumulation (Walker, Sikorska and Whitfield, unpublished observations). Thus the inhibitory action of colchicine on the initiation of hepatocyte DNA synthesis, a t both the early and late periods of sensitivity, does not appear to be due to a n inhibition of polyamine accumulation. ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of J. Bayliss and D. J. Gillan who also prepared the illustrations. LITERATURE CITED Barbiroli, B., M. S. Moruzzi, B. Tadolini and M. G. Monti 1974 Ornithine decarboxylase activity in regenerating liver from rats adapted to controlled feedingschedules. J. Nutr., 105:408-412. Betel, I., and J. Martijnse 1976 Drugs that disrupt microtubuli do not inhibit lymphocyte activation. Nature, 261: 318-319. Borgers, M., and M. De Brabander, eds. 1975 Microtubules and Microtubule Inhibitors. North-Holland Publishing Co. Ltd., Amsterdam. Boynton, A. L., and J. F. Whitfield 1976 Different calcium requirements for proliferation of conditionally and unconditionally tumorigenic mouse cells. Proc. Natl. Acad. Sci. (U.S.A.), 73:1651-1654. Boynton, A. L., J. F. Whitfield and R. J. Isaacs 1976a A possible involvement of polyamines in the initiation of DNA synthesis by human WI-38 and mouse BALBi3TB cells. J. Cell. Physiol., 89:481-488. 1976b Calcium. dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA). J. Cell. Physiol., 87:25-32. Chen, K., J. Heller and E. S. Canellakis 1976 Studies on the regulation of ornithine decarboxylase activity by the microtubules: The effect of colchicine and vinblastine. Biochem. Biophys. Res. Commun., 68:401-408. Douvas, A. S., C. A. Harrington and J. Bonner 1975 Major nonhistone proteins of r a t liver chromatin: Preliminary identification of myosin, actin, tubulin, and tropomyosin. Proc. Natl. Acad. Sci. (USA.), 72:3902-3906.
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