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Cyclic AMP and growth regulation in rat glioma cells in tissue culture
EXPERIMENTAL
NEUROLOGY
71, 154-160 (1981)
Cyclic AMP and Growth Regulation in Rat Glioma in Tissue Culture1 V. MARES,
V. FLEISCHMANNOVA,
InstitureofPhysiology,
Received
Z. LODIN,
Cz.echoslovakAcademyofSciences, and *K Thomae Werke. BiberachlRiss. February
27, 1980; revision
received
Cells
AND H. UEBERBERG* 14220Prague. F. R. G. June
Czechoslovakia.
13. 1980
C6 glioma cells grown in vitro for 3 to 4 days changed their morphology and showed a decreased rH]thymidine labeling index after adding dibutyryl CAMP, theophylline, isoprenaline, noradrenaline, or adrenaline to the culture medium. Changes in morphology appeared earlier (51 h) than those in the labeling index and/or cell proliferation (520 h). Dibutyryl CAMP and theophylline were found to be more effective in decreasing the rate of cell division than catecholamines. Specific blocking of adrenergic receptors suggests that catecholamines may affect cells via stimulation of both o- and /3-adrenergic receptors.
INTRODUCTION Cyclic nucleotides may take part in the control of cell division of some cell populations under both normal and pathological conditions [for review, see (15)]. A similar effect of adenosine 3’S’-cyclic monophosphate (CAMP) was recently also shown in cells of the central nervous system in tissue culture. Leuschen and Amato (6) found an inverse relationship between the level of CAMP and the proliferation rate of human central nervous system cells transformed by viruses, and Nomura et al. (11) observed a profound decrease in DNA synthesis in a subclone of C6 giioma cells after treatment with dibutyryl CAMP (db-CAMP). Rat C6 glioma cells represent a relatively stable cell line, derived from the chemically induced tumor in vivo (1) and Abbreviations: CAMP, cGMP-adenosine. guanosine 3’,5’-cyclic monophosphate; dbCAMP-dibutyryl CAMP; [3H]TDR-[6-3H]thymidine: L.I.%-percentage labeled cells: PBS-phosphate buffer solution. ’ Preliminary results were presented at the 40th meeting of the Czechoslovak Physiological Society, Prague. February 6-8, 1979 (8). 154 0014-4886/81/010154-07$02.00/O Copyright All rights
Q 1981 by Academic Press. Inc. of reproduction in any form reserved
PROLIFERATION
OF GLIOMA
CELLS
155
exhibiting many properties of normal differentiated glial cells (9). Their sensitivity to db-CAMP (11) suggests that their rate of cell division may be under the control of CAMP, although the effect of db-CAMP was shown to be nonspecific in some cases (18, 19), i.e., due to sodium dibutyrate. The aim of the present experiments, therefore, was to compare the rate of proliferation of C6 glioma cells treated with db-CAMP and some other substances which are known to alter the intracellular level of CAMP via enzymes involved in CAMP metabolism. Preliminary data on the effect of dibutyryl guanosine 3’S’-cyclic monophosphate (db-cGMP) are also reported. MATERIALS
AND METHODS
The C6 rat glioma cells (American Type Culture Collection, Rockville, Maryland) were cultivated in minimal Eagle’s medium supplemented with 10% calf serum and gentamycin (10 pg/ml) on cover glass slips in petri dishes in a humidified air atmosphere with 5% COz. On the third day in vitro, the cultures were treated with one of the substances listed in Table 1, One and 20 h after the administration of a tested substance, living cells were observed and photographed by phase contrast. Then they were exposed 2 h to [6-3H]thymidine ([3H]TdR) (UVVVR, 3 pCi/ml, sp act 19 Ci/mmol), and after being washed in phosphate buffer solution (PBS) they were fixed in Carnoy solution. Slides were covered with Ilford L4 emulsion (l:l, 7 to 9 days of exposure) and stained with 0.05% toluidine blue. The percentage of labeled cells (labeling index, L.I.%) was determined from 500 to 1000 cells in every culture; 5 to 15 cultures were evaluated in every experimental group. Results are given as means + SE. RESULTS Controls. Cultures treated with PBS only, as well as all cultures before treatment with the tested substances, appeared as colonies consisting of a center with a high cell density and a less dense periphery. In the periphery, the cells were usually larger and had longer processes with fine varicosities. Cell nuclei mostly possessed fine dispersed chromatin with one or two distinct nucleoli. After the administration of rH]TdR, the frequency of labeled cells was usually lower in the center of a cell colony than at its periphery. Mitotic figures, regularly present, were mostly unlabeled. Effects of Dibutyryl Cyclic Adenosine Monophosphate, Theophylline, Noradrenaline, or Adrenaline. One hour after the administration of any
one of these substances, many cells changed their morphology. Cell processes became more apparent and more “stringy,” and in some cases the varicosities of the fibers disappeared. In many cells fine dense
156
MARES ET AL. TABLE 1 The List of Substances (and Their Concentrations)
Added to Cultures in Vitro
Treatment
Final cont. (mol/liter)
Time (h)
db-CAMP (Sigma) Theophylline (Sigma) Noradrenaline (Spofa) Isoprenaline (Galena) Adrenaline (Spofa) Theophylline + noradenaline Inderal (Macclesfield) Noradrenaline + Inderal Noradrenaline + Inderal + Regitin Regitin (Ciba-Geigy) Adrenaline + Regitin Phosphate buffer (PBS) db-cGMP (Sigma)
10-3, 2 x 10-S 10-S “10-4, 5 x IO-4 10-4, 5 x 10-S 10-5, 5 x IO-’ 10-3, 5 x IO-’ IO-S, 10-o 5 x 10-4, 10-e 5 x 10-4, IO-G, 3 x IO-5 3 x IO-S, 3 x 10-C 10-s. 3 x IO-5 0.1 ml/ml culture medium 2 x 10-4, 5 x 10-S
1, 20” 1, 20” 29, 205 20” 20” 20” 20” 20” 20” 20” 20” 2.5”, 20° 20”
a Indicates subsequent treatment of cells with [3H]thymidine.
cytoplasmic granules appeared around the nucleus. In some cells nuclear chromatin became more clumped; the latter was especially evident after treatment of cells with db-CAMP and theophylline. After 20 h (the time of t3H]TdR administration), the appearance of many cells reverted either to that of the controls or, especially after theophylline or noradrenaline treatment, to intermediary forms often displaying fine vacuoles in the cytoplasm. The number of labeled cells in both the central and the peripheral parts of cell colonies was significantly lower in all experimental groups after a 20-h treatment with db-CAMP, theophylline, adrenaline, noradrenaline, or isoprenaline (Figs. 1, 3). The highest and most consistent decrease in the L.I.% was induced by db-CAMP or theophylline. Noradrenaline at 1O-4 mol/liter depressed the number of labeled cells only in some cultures and the difference was not significant (P > 0.05). Significant inhibition by noradrenaline was achieved only at 5 x 10e4 mol/liter (P < 0.01). The effect of treatment of cells with noradrenaline and theophylline combined did not significantly differ from the effect of theophylline alone. Isoprenaline, in comparison with noradrenaline, led to a smaller decrease in the L.I.% (Fig. 1). Adrenaline decreased the labeling index to approximately the same degree as noradrenaline (Fig. 3). The labeled cells in the adrenaline- and/or theophylline-treated cultures often possessed a lower number of silver grains per nucleus than in the controls. Mitotic figures were usually unlabeled in all experimental groups. A parallel treatment of cells with noradrenaline and a P-receptor blocking substance, propranolol (Inderal), partly abolished the effect of
PROLIFERATION
OF GLIOMA
CELLS
157
FIG. 1. The labeling index in C6 glioma cells treated 20 h with either dibutyryl CAMP (db-CAMP I, lo+ moPliter; db-CAMP II, 2 x 10m3 moPliter), theophylline (theo;10S3 mol/liter), noradrenaline (nor; 5 X 10m4 mol/liter), isoprenaline (iso; lO-4 mopliter), or dibutyryl cGMP (db-cGMP; 5 x 10e3moPliter). The labeling index values are expressed as a percentage of the controls (indicated by horizontal line = 100%) and are means + SE.
noradrenaline (Fig. 2); propranolol itself exerted a slight inhibitory influence (-7%). A simultaneous treatment of cells with propranolol and an a-receptor blocking substance, phentolamine (Regitin), almost completely prevented the effect of noradrenaline on the labeling index (Fig. 2). Phentolamine also significantly diminished the adrenaline-induced decrease in the L.I.% (Fig. 3). The effect of these substances on cell proliferation at intervals shorter than 20 h has not yet been systematically investigated. In the case of % loo-
i r
p
.
p 50.. z f 5 .. =
o-
b .z -
FIG. 2. The labeling index in C6 glioma cells treated with noradrenaline either for 2.5 h (nor I; 5 x 10m4moPliter) or for 20 h (nor II, lo-“ molkter; nor III, 5 x 10e4 moUliter) or simultaneously with noradrenaline and propranolol (nor + pro; 5 x lo-* and 10e5 moYliter) or noradrenaline and propranolol (Inderal) and phentolamine (Regitin) (nor + reg + pro; 5 X 10-4, 3 x 10m5and lOm6moPliter) for 20 h. Data expressed as in Fig. 1.
158
MARES ET AL.
50-l o-
5 b q
= ;1P &
FIG. 3. The labeling index in C6 glioma cells treated with adrenaline (adr I, lo-” mol/liter; adr II, 5 x lo-’ mol/liter) or Regitin (reg; 3 x 10m5 mol/liter) or simultaneously with adrenaline and Regitin (adr + reg; 10e5 and 3 x 10m5mol/liter) for 20 h. Data expressed as in Fig. 1.
noradrenaline, no significant change in the L.I.% was observed 2.5 h after its administration (Fig. 2). No significant change in the L.I.% could be found after treatment of cells with db-cGMP for 20 h (Fig. 1). DISCUSSION Increased concentrations of CAMP often, but not always, inhibit cell division [for review, see (IS)]. The changes in the [3H]thymidine labeling index observed in the present study show that C6 glioma cells slow the rate of cell division when the concentration of CAMP is increased. In terms of cell kinetics, the decrease in the L.I.% is due to prolongation of the cell cycle, especially the G, and G, phases, as shown recently in glioma cells treated with db-CAMP by Nomura et al. (11). In our experiments this effect appeared both after addition of db-CAMP to the culture medium and after treatment of cells with substances which are known to alter the activity of enzymes involved in the control of CAMP concentrations, e.g., the phosphodiesterase inhibitor, theophylline, or catecholamines, which stimulate adenilate cyclase. The latter result argues against the possibility of a nonspecific action.of db-CAMP (18, 19). The present data thus provide indirect evidence of CAMP involvement in the regulation of cell proliferation in C6 glioma cells, although the actual concentrations of CAMP were not measured in our experiments. The catecholaminedependent increase in CAMP concentration was, however, shown earlier in a subclone of C6 glioma cells ( 12). The latter was found to be caused by the stimulation of P-adrenergic receptors coupled to adenylate cyclase (13). In agreement with this, the noradrenaline-induced inhibition of cell division
PROLIFERATION
OF GLIOMA
CELLS
159
was diminished significantly in our experiments when cells were treated at the same time with propranolol. The complete inhibition of the noradrenaline effect by propranolol and phentolamine, as well as the partial blockade of the adrenaline effect by phentolamine, however, suggest that not only /3- but also a-adrenergic receptors may be involved in the control of proliferation of C6 glioma cells. The presence of cu-adrenergic receptors in C6 glioma cells was recently also suggested byBottenstein and de Vellis (2). Those authors observed, however, an a-adrenergic inhibition of adenylate cyclase. It should therefore be admitted either that the receptor equipment of cells studied under different laboratory conditions varies or that the apparent a-receptor-mediated inhibition of cell division in our experiments is not related exclusively to a primary change in CAMP concentrations. A long-term secondary increase in adenylate cyclase activity, following the initial inhibition of the enzyme induced by stimulation of a-adrenergic receptors, was, for instance, recently observed in neuroblastoma x glioma cell hybrids (16), and such a possibility might explain the changes observed in our preparations. The noradrenaline-induced increase in CAMP takes place within a few minutes and decreases to the basal value within 4 to 5 h (13). The decrease in the labeling index in our experiments is evident only within the time interval >2.5 h and 520 h (Fig. 2). The decrease in the rate of cell division is, however, preceded by changes in the morphology of cells, especially of their processes, as shown earlier (12), as well as by phase-contrast microscopy in our experiments at the l-h interval. According to earlier findings in glioma cells (12), as well as in some immature normal glial cells (4, 7, lo), changes in cell morphology are accompanied by formation of microfilaments and microtubules. The microtubular-microfilamentous structures are presumed to convey the membrane-dependent information to the nucleus (14, 17); at the end of this chain, a change in DNA synthesis and cell division (as indicated by the L.I.%) occurs. Molecular mechanisms involved in the transmission of information from the cell membrane to the cell nucleus are poorly understood. As suggested by Lazo et al. (5), a final signal for the inhibition of DNA synthesis might be the CAMP-dependent decrease in the synthesis of histones and especially of the phosphorylation of their H, fraction. There are, however, many other possible intermediate steps and/or mechanisms which may take part in the transfer of “membrane impression” (3) to the nucleus and lead finally to a change in cell proliferation and/or differentiation. db-cGMP did not influence the labeling index of cells in our experiments. Similarly, Leuschen and Amato (6) could not demonstrate a positive effect of db-cGMP on proliferation of virus-transformed human central nervous system cells. The data on db-cGMP are, however, not yet conclusive; study of the effects of treatment of cultures at earlier or later stages, i.e.,
160
MARES
before or after the growth optimum cGMP, is desirable.
ET AL.
and in different
concentrations
of
REFERENCES L. LEVINE, AND W. SWEET. 1968. Differentiated rat Science 161: 370-371. BOTTENSTEIN, J. E., AND J. DE VELLIS. 1978. Regulation ofcyclic GMP, cyclic AMPand lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line. Life Sci. 23: 821-834. BRUNNER. G. 1977. Membrane impression and gene expression. Towards a theory of cytodifferentiation. Differentiation 8: 123- 132. KOR~NKOVA, P., AND Z. LODIN. 1977. A transitional differentiation of glial cells of cultured corpus callosum caused by dibutyryl cyclic adenosine monophosphate. Neurosciences 2: 1103- 1114. LAZO, J. S., K. N. PRASAD, AND R. W. RUDDON. 1976. Synthesis and phosphorylation of chromatin-associated proteins in CAMP-induced “differentiated” neuroblastoma cells in culture. Exp. Cell Res. 100: 41-46. LEUSCHEN, M. P., AND R. S. AMATO. 1978. The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue. Exp. Cell Res. 114: 79-84. LODIN, Z., J. FALTIN, AND P. KOR~NKOVA. 1979. The effect of dibutyryl cyclic AMP on cultivated glial cells from corpus callosum of 30-day-old rats. Physiol. BohemosloLf. 28: 105-111. MARES, V., V. FLEISCHMANNOVA. AND Z. LODIN. 1979. Cyclic AMP and cell division in rat ghoma cells in tissue cultures. Physiol. Bohemoslott. 28: 454. MCMORRIS. F. A. 1977. Norepinephrine induces ghal-specific enzyme activity in cultured glioma cells. Proc. N&l. Acad. Sci. U.S.A. 74: 4501-4504. MOONEN, G., Y. CAM, M. SENSENBRENNER, AND P. MANDEL. 1975. Variability of the effects of serum-free medium, db-CAMP or theophyline on the morphology of cultured newborn rat astroblasts. Cell Tissue Res. 163: 365-372. NOMURA, K., T. HASHINO, K. KNEBEL, AND M. BARKER. 1978. Effect of dibutyryl CAMP on cell cycle progression of rat brain tumor cells in vitro. In Vitro 14: 174- 179. OEY, J. 1975. Noradrenaline induces morphological alterations in nucleated and enucleated rat C6 ghoma cells. Nature (London) 257: 3 17-3 19. OEY, J. 1976. Noradrenaline-refractoriness in cultured glioma cells. Exp. Brain Res. 25: 359-368. PUCK, T. T. 1977. Cyclic AMP, the microtubule-microfilament system and cancer. Proc. Natl. Acad. Sci. U.S.A. 74: 4491-4495. REBHUN, L. 1. 1977. Cyclic nucleotides, calcium and cell division. Znt. Rev. Cytol. 49: l-54. SABOL, S. L., AND M. NIRENBERG. 1979. Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. II. Long lived increase of adenylate cyclase activity mediated by alpha receptors. .I. Biol. Chem. 254: 1921- 1926. SEGAL, J. 1979. Biophysikalische Aspekre der elementaren ZeQfunktionen. VEB Georg Thieme, Leipzig. Pp. I-219. WAYMIRE, J. C., K. GILMER-WAYMIRE, AND J. W. HAYCOCK. 1978. Cyclic AMP-induced differentiation in neuroblastoma is independent of cell division rate. Nature (London) 276: 194- 195. WRIGHT, J. A. 1973. Morphology and growth rate changes in Chinese hamster cells cultured in presence of sodium butyrate. Exp. Cell. Res. 78: 456-460.
1. BENDA,
P., J. LIGHTBODY,
glial cell strain
2.
3. 4.
5.
6.
7.
8. 9. 10.
11. 12. 13. 14. 15. 16.
17. 18.
19.
in tissue
G. SATO,
culture.
NEUROLOGY
71, 154-160 (1981)
Cyclic AMP and Growth Regulation in Rat Glioma in Tissue Culture1 V. MARES,
V. FLEISCHMANNOVA,
InstitureofPhysiology,
Received
Z. LODIN,
Cz.echoslovakAcademyofSciences, and *K Thomae Werke. BiberachlRiss. February
27, 1980; revision
received
Cells
AND H. UEBERBERG* 14220Prague. F. R. G. June
Czechoslovakia.
13. 1980
C6 glioma cells grown in vitro for 3 to 4 days changed their morphology and showed a decreased rH]thymidine labeling index after adding dibutyryl CAMP, theophylline, isoprenaline, noradrenaline, or adrenaline to the culture medium. Changes in morphology appeared earlier (51 h) than those in the labeling index and/or cell proliferation (520 h). Dibutyryl CAMP and theophylline were found to be more effective in decreasing the rate of cell division than catecholamines. Specific blocking of adrenergic receptors suggests that catecholamines may affect cells via stimulation of both o- and /3-adrenergic receptors.
INTRODUCTION Cyclic nucleotides may take part in the control of cell division of some cell populations under both normal and pathological conditions [for review, see (15)]. A similar effect of adenosine 3’S’-cyclic monophosphate (CAMP) was recently also shown in cells of the central nervous system in tissue culture. Leuschen and Amato (6) found an inverse relationship between the level of CAMP and the proliferation rate of human central nervous system cells transformed by viruses, and Nomura et al. (11) observed a profound decrease in DNA synthesis in a subclone of C6 giioma cells after treatment with dibutyryl CAMP (db-CAMP). Rat C6 glioma cells represent a relatively stable cell line, derived from the chemically induced tumor in vivo (1) and Abbreviations: CAMP, cGMP-adenosine. guanosine 3’,5’-cyclic monophosphate; dbCAMP-dibutyryl CAMP; [3H]TDR-[6-3H]thymidine: L.I.%-percentage labeled cells: PBS-phosphate buffer solution. ’ Preliminary results were presented at the 40th meeting of the Czechoslovak Physiological Society, Prague. February 6-8, 1979 (8). 154 0014-4886/81/010154-07$02.00/O Copyright All rights
Q 1981 by Academic Press. Inc. of reproduction in any form reserved
PROLIFERATION
OF GLIOMA
CELLS
155
exhibiting many properties of normal differentiated glial cells (9). Their sensitivity to db-CAMP (11) suggests that their rate of cell division may be under the control of CAMP, although the effect of db-CAMP was shown to be nonspecific in some cases (18, 19), i.e., due to sodium dibutyrate. The aim of the present experiments, therefore, was to compare the rate of proliferation of C6 glioma cells treated with db-CAMP and some other substances which are known to alter the intracellular level of CAMP via enzymes involved in CAMP metabolism. Preliminary data on the effect of dibutyryl guanosine 3’S’-cyclic monophosphate (db-cGMP) are also reported. MATERIALS
AND METHODS
The C6 rat glioma cells (American Type Culture Collection, Rockville, Maryland) were cultivated in minimal Eagle’s medium supplemented with 10% calf serum and gentamycin (10 pg/ml) on cover glass slips in petri dishes in a humidified air atmosphere with 5% COz. On the third day in vitro, the cultures were treated with one of the substances listed in Table 1, One and 20 h after the administration of a tested substance, living cells were observed and photographed by phase contrast. Then they were exposed 2 h to [6-3H]thymidine ([3H]TdR) (UVVVR, 3 pCi/ml, sp act 19 Ci/mmol), and after being washed in phosphate buffer solution (PBS) they were fixed in Carnoy solution. Slides were covered with Ilford L4 emulsion (l:l, 7 to 9 days of exposure) and stained with 0.05% toluidine blue. The percentage of labeled cells (labeling index, L.I.%) was determined from 500 to 1000 cells in every culture; 5 to 15 cultures were evaluated in every experimental group. Results are given as means + SE. RESULTS Controls. Cultures treated with PBS only, as well as all cultures before treatment with the tested substances, appeared as colonies consisting of a center with a high cell density and a less dense periphery. In the periphery, the cells were usually larger and had longer processes with fine varicosities. Cell nuclei mostly possessed fine dispersed chromatin with one or two distinct nucleoli. After the administration of rH]TdR, the frequency of labeled cells was usually lower in the center of a cell colony than at its periphery. Mitotic figures, regularly present, were mostly unlabeled. Effects of Dibutyryl Cyclic Adenosine Monophosphate, Theophylline, Noradrenaline, or Adrenaline. One hour after the administration of any
one of these substances, many cells changed their morphology. Cell processes became more apparent and more “stringy,” and in some cases the varicosities of the fibers disappeared. In many cells fine dense
156
MARES ET AL. TABLE 1 The List of Substances (and Their Concentrations)
Added to Cultures in Vitro
Treatment
Final cont. (mol/liter)
Time (h)
db-CAMP (Sigma) Theophylline (Sigma) Noradrenaline (Spofa) Isoprenaline (Galena) Adrenaline (Spofa) Theophylline + noradenaline Inderal (Macclesfield) Noradrenaline + Inderal Noradrenaline + Inderal + Regitin Regitin (Ciba-Geigy) Adrenaline + Regitin Phosphate buffer (PBS) db-cGMP (Sigma)
10-3, 2 x 10-S 10-S “10-4, 5 x IO-4 10-4, 5 x 10-S 10-5, 5 x IO-’ 10-3, 5 x IO-’ IO-S, 10-o 5 x 10-4, 10-e 5 x 10-4, IO-G, 3 x IO-5 3 x IO-S, 3 x 10-C 10-s. 3 x IO-5 0.1 ml/ml culture medium 2 x 10-4, 5 x 10-S
1, 20” 1, 20” 29, 205 20” 20” 20” 20” 20” 20” 20” 20” 2.5”, 20° 20”
a Indicates subsequent treatment of cells with [3H]thymidine.
cytoplasmic granules appeared around the nucleus. In some cells nuclear chromatin became more clumped; the latter was especially evident after treatment of cells with db-CAMP and theophylline. After 20 h (the time of t3H]TdR administration), the appearance of many cells reverted either to that of the controls or, especially after theophylline or noradrenaline treatment, to intermediary forms often displaying fine vacuoles in the cytoplasm. The number of labeled cells in both the central and the peripheral parts of cell colonies was significantly lower in all experimental groups after a 20-h treatment with db-CAMP, theophylline, adrenaline, noradrenaline, or isoprenaline (Figs. 1, 3). The highest and most consistent decrease in the L.I.% was induced by db-CAMP or theophylline. Noradrenaline at 1O-4 mol/liter depressed the number of labeled cells only in some cultures and the difference was not significant (P > 0.05). Significant inhibition by noradrenaline was achieved only at 5 x 10e4 mol/liter (P < 0.01). The effect of treatment of cells with noradrenaline and theophylline combined did not significantly differ from the effect of theophylline alone. Isoprenaline, in comparison with noradrenaline, led to a smaller decrease in the L.I.% (Fig. 1). Adrenaline decreased the labeling index to approximately the same degree as noradrenaline (Fig. 3). The labeled cells in the adrenaline- and/or theophylline-treated cultures often possessed a lower number of silver grains per nucleus than in the controls. Mitotic figures were usually unlabeled in all experimental groups. A parallel treatment of cells with noradrenaline and a P-receptor blocking substance, propranolol (Inderal), partly abolished the effect of
PROLIFERATION
OF GLIOMA
CELLS
157
FIG. 1. The labeling index in C6 glioma cells treated 20 h with either dibutyryl CAMP (db-CAMP I, lo+ moPliter; db-CAMP II, 2 x 10m3 moPliter), theophylline (theo;10S3 mol/liter), noradrenaline (nor; 5 X 10m4 mol/liter), isoprenaline (iso; lO-4 mopliter), or dibutyryl cGMP (db-cGMP; 5 x 10e3moPliter). The labeling index values are expressed as a percentage of the controls (indicated by horizontal line = 100%) and are means + SE.
noradrenaline (Fig. 2); propranolol itself exerted a slight inhibitory influence (-7%). A simultaneous treatment of cells with propranolol and an a-receptor blocking substance, phentolamine (Regitin), almost completely prevented the effect of noradrenaline on the labeling index (Fig. 2). Phentolamine also significantly diminished the adrenaline-induced decrease in the L.I.% (Fig. 3). The effect of these substances on cell proliferation at intervals shorter than 20 h has not yet been systematically investigated. In the case of % loo-
i r
p
.
p 50.. z f 5 .. =
o-
b .z -
FIG. 2. The labeling index in C6 glioma cells treated with noradrenaline either for 2.5 h (nor I; 5 x 10m4moPliter) or for 20 h (nor II, lo-“ molkter; nor III, 5 x 10e4 moUliter) or simultaneously with noradrenaline and propranolol (nor + pro; 5 x lo-* and 10e5 moYliter) or noradrenaline and propranolol (Inderal) and phentolamine (Regitin) (nor + reg + pro; 5 X 10-4, 3 x 10m5and lOm6moPliter) for 20 h. Data expressed as in Fig. 1.
158
MARES ET AL.
50-l o-
5 b q
= ;1P &
FIG. 3. The labeling index in C6 glioma cells treated with adrenaline (adr I, lo-” mol/liter; adr II, 5 x lo-’ mol/liter) or Regitin (reg; 3 x 10m5 mol/liter) or simultaneously with adrenaline and Regitin (adr + reg; 10e5 and 3 x 10m5mol/liter) for 20 h. Data expressed as in Fig. 1.
noradrenaline, no significant change in the L.I.% was observed 2.5 h after its administration (Fig. 2). No significant change in the L.I.% could be found after treatment of cells with db-cGMP for 20 h (Fig. 1). DISCUSSION Increased concentrations of CAMP often, but not always, inhibit cell division [for review, see (IS)]. The changes in the [3H]thymidine labeling index observed in the present study show that C6 glioma cells slow the rate of cell division when the concentration of CAMP is increased. In terms of cell kinetics, the decrease in the L.I.% is due to prolongation of the cell cycle, especially the G, and G, phases, as shown recently in glioma cells treated with db-CAMP by Nomura et al. (11). In our experiments this effect appeared both after addition of db-CAMP to the culture medium and after treatment of cells with substances which are known to alter the activity of enzymes involved in the control of CAMP concentrations, e.g., the phosphodiesterase inhibitor, theophylline, or catecholamines, which stimulate adenilate cyclase. The latter result argues against the possibility of a nonspecific action.of db-CAMP (18, 19). The present data thus provide indirect evidence of CAMP involvement in the regulation of cell proliferation in C6 glioma cells, although the actual concentrations of CAMP were not measured in our experiments. The catecholaminedependent increase in CAMP concentration was, however, shown earlier in a subclone of C6 glioma cells ( 12). The latter was found to be caused by the stimulation of P-adrenergic receptors coupled to adenylate cyclase (13). In agreement with this, the noradrenaline-induced inhibition of cell division
PROLIFERATION
OF GLIOMA
CELLS
159
was diminished significantly in our experiments when cells were treated at the same time with propranolol. The complete inhibition of the noradrenaline effect by propranolol and phentolamine, as well as the partial blockade of the adrenaline effect by phentolamine, however, suggest that not only /3- but also a-adrenergic receptors may be involved in the control of proliferation of C6 glioma cells. The presence of cu-adrenergic receptors in C6 glioma cells was recently also suggested byBottenstein and de Vellis (2). Those authors observed, however, an a-adrenergic inhibition of adenylate cyclase. It should therefore be admitted either that the receptor equipment of cells studied under different laboratory conditions varies or that the apparent a-receptor-mediated inhibition of cell division in our experiments is not related exclusively to a primary change in CAMP concentrations. A long-term secondary increase in adenylate cyclase activity, following the initial inhibition of the enzyme induced by stimulation of a-adrenergic receptors, was, for instance, recently observed in neuroblastoma x glioma cell hybrids (16), and such a possibility might explain the changes observed in our preparations. The noradrenaline-induced increase in CAMP takes place within a few minutes and decreases to the basal value within 4 to 5 h (13). The decrease in the labeling index in our experiments is evident only within the time interval >2.5 h and 520 h (Fig. 2). The decrease in the rate of cell division is, however, preceded by changes in the morphology of cells, especially of their processes, as shown earlier (12), as well as by phase-contrast microscopy in our experiments at the l-h interval. According to earlier findings in glioma cells (12), as well as in some immature normal glial cells (4, 7, lo), changes in cell morphology are accompanied by formation of microfilaments and microtubules. The microtubular-microfilamentous structures are presumed to convey the membrane-dependent information to the nucleus (14, 17); at the end of this chain, a change in DNA synthesis and cell division (as indicated by the L.I.%) occurs. Molecular mechanisms involved in the transmission of information from the cell membrane to the cell nucleus are poorly understood. As suggested by Lazo et al. (5), a final signal for the inhibition of DNA synthesis might be the CAMP-dependent decrease in the synthesis of histones and especially of the phosphorylation of their H, fraction. There are, however, many other possible intermediate steps and/or mechanisms which may take part in the transfer of “membrane impression” (3) to the nucleus and lead finally to a change in cell proliferation and/or differentiation. db-cGMP did not influence the labeling index of cells in our experiments. Similarly, Leuschen and Amato (6) could not demonstrate a positive effect of db-cGMP on proliferation of virus-transformed human central nervous system cells. The data on db-cGMP are, however, not yet conclusive; study of the effects of treatment of cultures at earlier or later stages, i.e.,
160
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before or after the growth optimum cGMP, is desirable.
ET AL.
and in different
concentrations
of
REFERENCES L. LEVINE, AND W. SWEET. 1968. Differentiated rat Science 161: 370-371. BOTTENSTEIN, J. E., AND J. DE VELLIS. 1978. Regulation ofcyclic GMP, cyclic AMPand lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line. Life Sci. 23: 821-834. BRUNNER. G. 1977. Membrane impression and gene expression. Towards a theory of cytodifferentiation. Differentiation 8: 123- 132. KOR~NKOVA, P., AND Z. LODIN. 1977. A transitional differentiation of glial cells of cultured corpus callosum caused by dibutyryl cyclic adenosine monophosphate. Neurosciences 2: 1103- 1114. LAZO, J. S., K. N. PRASAD, AND R. W. RUDDON. 1976. Synthesis and phosphorylation of chromatin-associated proteins in CAMP-induced “differentiated” neuroblastoma cells in culture. Exp. Cell Res. 100: 41-46. LEUSCHEN, M. P., AND R. S. AMATO. 1978. The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue. Exp. Cell Res. 114: 79-84. LODIN, Z., J. FALTIN, AND P. KOR~NKOVA. 1979. The effect of dibutyryl cyclic AMP on cultivated glial cells from corpus callosum of 30-day-old rats. Physiol. BohemosloLf. 28: 105-111. MARES, V., V. FLEISCHMANNOVA. AND Z. LODIN. 1979. Cyclic AMP and cell division in rat ghoma cells in tissue cultures. Physiol. Bohemoslott. 28: 454. MCMORRIS. F. A. 1977. Norepinephrine induces ghal-specific enzyme activity in cultured glioma cells. Proc. N&l. Acad. Sci. U.S.A. 74: 4501-4504. MOONEN, G., Y. CAM, M. SENSENBRENNER, AND P. MANDEL. 1975. Variability of the effects of serum-free medium, db-CAMP or theophyline on the morphology of cultured newborn rat astroblasts. Cell Tissue Res. 163: 365-372. NOMURA, K., T. HASHINO, K. KNEBEL, AND M. BARKER. 1978. Effect of dibutyryl CAMP on cell cycle progression of rat brain tumor cells in vitro. In Vitro 14: 174- 179. OEY, J. 1975. Noradrenaline induces morphological alterations in nucleated and enucleated rat C6 ghoma cells. Nature (London) 257: 3 17-3 19. OEY, J. 1976. Noradrenaline-refractoriness in cultured glioma cells. Exp. Brain Res. 25: 359-368. PUCK, T. T. 1977. Cyclic AMP, the microtubule-microfilament system and cancer. Proc. Natl. Acad. Sci. U.S.A. 74: 4491-4495. REBHUN, L. 1. 1977. Cyclic nucleotides, calcium and cell division. Znt. Rev. Cytol. 49: l-54. SABOL, S. L., AND M. NIRENBERG. 1979. Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. II. Long lived increase of adenylate cyclase activity mediated by alpha receptors. .I. Biol. Chem. 254: 1921- 1926. SEGAL, J. 1979. Biophysikalische Aspekre der elementaren ZeQfunktionen. VEB Georg Thieme, Leipzig. Pp. I-219. WAYMIRE, J. C., K. GILMER-WAYMIRE, AND J. W. HAYCOCK. 1978. Cyclic AMP-induced differentiation in neuroblastoma is independent of cell division rate. Nature (London) 276: 194- 195. WRIGHT, J. A. 1973. Morphology and growth rate changes in Chinese hamster cells cultured in presence of sodium butyrate. Exp. Cell. Res. 78: 456-460.
1. BENDA,
P., J. LIGHTBODY,
glial cell strain
2.
3. 4.
5.
6.
7.
8. 9. 10.
11. 12. 13. 14. 15. 16.
17. 18.
19.
in tissue
G. SATO,
culture.