Synthesis and Accumulation of (cid:97) B Crystallin in C6 Glioma Cells Is Induced by Agents That Promote the Disassembly of Microtubules*

When C6 cells in culture were exposed at 37 °C to 1 (cid:109) M colchicine or to 1 (cid:109) M colcemid, a tubulin-binding antimitotic alkaloid, levels of (cid:97) B crystallin in cells began to increase after about 10 h, reaching a maximum of more than 1 (cid:109) g/mg protein after 24 h. The level of (cid:97) B crystallin returned to near the control level within two subse- quent days of culture in the normal medium. Northern blot analysis showed that the accumulation of (cid:97) B crystallin was preceded by an increase in the level of the mRNA for (cid:97) B crystallin. Nuclear run-off transcription assays showed that colchicine induced new synthesis of mRNA for (cid:97) B crystallin. Immunofluorescence staining revealed that (cid:97) B crystallin accumulated in the peripheral areas of cells, as did the depolymerized tubulin, after several hours of treatment with colcemid, and then it gradually became more conspicuous in the cytoplasm. Vinblastine and nocodazole, which also promote the disassembly of microtubules by binding to tubulins, also induced the synthesis of (cid:97) B crystallin. Furthermore, induction of (cid:97) B crystallin by these drugs was observed in quiescent cells that had been cultured in serum-free medium. However, taxol, a microtubule-stabilizing antimitotic agent, did not stimulate the synthesis of (cid:97) B crystallin, preincubated in 10% normal goat serum min block nonspecific staining. staining of microtubules or (cid:97) B-crystallin, the incubated with mouse monoclonal antibody against (cid:98) -tubulin or affinity-purified rabbit anti- bodies against carboxyl-terminal peptide of rat (cid:97) B-crystallin 30 min at room temperature. washing with PBS, they were stained with fluorescein isothiocyanate-labeled antibodies against mouse IgG or Texas red-labeled antibodies against rabbit IgG. All specimens were observed under a confocal laser scanning microscope equipped with a krypton-argon laser (MRC1024; Bio-Rad, Watford, UK). Other Methods— Concentrations of soluble protein in extracts were estimated with a protein assay kit (Bio-Rad) with bovine serum albu- min as the standard. Rat HSP27 and (cid:97) B crystallin, which were used as the standards for immunoassays and electrophoresis, were purified from skeletal muscle (20, 25).

␣B crystallin, a major structural protein of vertebrate lenses, is also expressed in various nonlenticular tissues such as central nervous tissues (1)(2)(3)(4)(5)(6), and it is a member of the family of small stress or heat shock proteins. The expression of ␣B crystallin in cells and tissues is stimulated under various stressful conditions, as is that of HSP27 1 and HSP70 (7)(8)(9)(10). However, increases in the level of expression of ␣B crystallin have also been observed in mouse NIH3T3 fibroblasts upon exposure to dexamethasone (11), in dog lens epithelial cells and kidney glomerular endothelial cells in response to hypertonicity (12), and in rat astrocytes exposed to tumor necrosis factor or hypertonic conditions (13). The increased expression of ␣B crystallin has also been observed under certain pathologic conditions (2, 14 -17). In brains of patients with Alexander's disease (2), ␣B crystallin accumulates destructively in astrocytes. However, the cause of the abnormal expression and accumulation of ␣B crystallin in these patients is not known. It has also been shown that the expression of stress proteins is under the control of the cell cycle and that the transcription of genes for HSP70 (18) and HSP90 (19) increases during the synthetic (S) phase of the cell cycle.
We report here that agents that promote the depolymerization of microtubules also induce the synthesis and accumulation of ␣B crystallin, but not of HSP27 and HSP70, in rat C6 glioma cells in culture and that this process is sensitive to staurosporine, an inhibitor of protein kinases.

EXPERIMENTAL PROCEDURES
Reagents-Colchicine, colcemid, vinblastine sulfate, taxol, aphidicolin, hydroxyurea, staurosporine, and phorbol 12-myristate 13-acetate were obtained from Wako Pure Chemicals Co., Osaka, Japan. Nocodazole and forskolin were obtained from Sigma. Affinity-purified Texas red-labeled goat antibodies against rabbit IgG and affinity-purified fluorescein isothiocyanate-labeled goat antibodies against mouse IgG that had been absorbed with rat serum were purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL) and Protos Immunoresearch (San Francisco, CA), respectively. The mouse monoclonal antibody against ␤-tubulin from physarum polycephalum myxamoebae was obtained from Boehringer Mannheim (Tokyo, Japan).
Culture and Treatment of Cells-C6 cells (obtained from the Japanese Cancer Research Resource Bank, Tokyo) were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., Tokyo), supplemented with 10% fetal calf serum (Life Technologies, Inc., Tokyo) at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The cells were seeded in 35-or 90-mm dishes for large scale culture, and the medium was changed every 2 or 3 days. Cells that had grown to about 80% confluency (1.5-2 ϫ 10 6 cells/35-mm dish) were treated as follows. Quiescent cells were obtained by washing cells with phosphate-buffered saline (PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2 HPO 4 , and 0.2 g of KH 2 PO 4 in 1,000 ml of H 2 O) and by subsequent culture for 24 h in Dulbecco's modified Eagle's medium without serum. Various chemicals, dissolved in dimethyl sulfoxide or PBS, were added to the culture medium of growing or quiescent cells, and incubations were continued at 37°C until cells were harvested. Cells in each dish were rinsed twice with PBS and frozen at Ϫ20°C for a few days prior to analysis. The frozen cells on each dish were collected and suspended in 0.3 ml of PBS, and each suspension was sonicated and centrifuged at 125,000 ϫ g for 20 min at 4°C. The supernatants were used for assays of ␣B crystallin and HSP27.
Immunoassays of ␣B Crystallin and HSP27-Concentrations of ␣B crystallin (6) and HSP27 (20) in extracts of cells were determined by specific immunoassays as described previously.
Electrophoresis and Western Blot Analysis-SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (21) in 12.5% polyacrylamide gels. Western blot analysis was carried out as described * This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
previously (20) using affinity-purified antibodies (0.05 g/ml) raised in rabbits against the carboxyl-terminal decapeptide of ␣B crystallin (6) and peroxidase-labeled antibodies raised in goat against rabbit IgG as second antibodies. Peroxidase activity on nitrocellulose sheets was visualized on x-ray film by use of a Western blot chemiluminescence reagent (Renaissance; DuPont NEN).
Northern Blot Analysis and in Vitro Nuclear Run-off Transcription Assay-Cells cultured in 90-mm dishes were treated in duplicate. Total RNA was isolated from cells with an RNeasy total RNA kit (Qiagen Inc., Hilden, Germany). Twenty g of total RNA were subjected to electrophoresis on a 1.0% agarose, 2.2 M formaldehyde gel and blotted onto a nitrocellulose membrane. For Northern blots, membranes were allowed to hybridize as described by Wahl et al. (22) with cDNA probes that had been labeled with a Multiprime DNA labeling system (Amersham Corp., Buckinghamshire, U.K.). A PstI fragment of cDNA for bovine ␣B crystallin (23) was kindly provided by Dr. H. Bloemendal of the University of Nijmegen. Nuclear run-off analysis was performed by the published procedure of Ausubel et al. (24). Briefly, nuclei from the lysed cells were incubated with 0.1 mCi of [␣-32 P]UTP, and then nuclear RNA was prepared by phenol/chloroform extraction. A linearized cDNA probe for bovine ␣B crystallin was prepared as a set of slot blots, and it was allowed to hybridize with samples of nuclear RNA that contained equal amounts of radioactivity (about 2 ϫ 10 5 cpm).
Immunofluorescence-Cells cultured on coverslips were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min at room temperature. Fixed cells were preincubated in 10% normal goat serum for 30 min to block nonspecific staining. For the staining of microtubules or ␣B-crystallin, the cells were incubated with mouse monoclonal antibody against ␤-tubulin or affinity-purified rabbit antibodies against carboxyl-terminal peptide of rat ␣B-crystallin for 30 min at room temperature. After washing with PBS, they were stained with fluorescein isothiocyanate-labeled antibodies against mouse IgG or Texas red-labeled antibodies against rabbit IgG. All specimens were observed under a confocal laser scanning microscope equipped with a krypton-argon laser (MRC1024; Bio-Rad, Watford, UK).
Other Methods-Concentrations of soluble protein in extracts were estimated with a protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Rat HSP27 and ␣B crystallin, which were used as the standards for immunoassays and electrophoresis, were purified from skeletal muscle (20,25).

Effects of Exposure to Inhibitors of Progression of the Cell
Cycle on the Levels of ␣B Crystallin and HSP27 in C6 Cells-Proliferating C6 rat glioma cells in culture were exposed for 20 h at 37°C to aphidicolin or hydroxyurea, inhibitors that act at the S phase of the cell cycle (26), or to colchicine, colcemid, vinblastine (27), nocodazole (28), or taxol (29), which are inhibitors that act at the mitotic (M) phase of the cell cycle. As shown in Table I, levels of ␣B crystallin in soluble extracts of cells increased markedly in the case of cells that had been treated with colchicine, colcemid, vinblastine, or nocodazole, all of which interrupt the cell cycle at the early M phase by promoting the depolymerization of microtubules. In contrast, the levels of ␣B crystallin did not increase in cells that had been treated with taxol, which also interrupts the cell cycle at the M phase but which stabilizes and prevents the depolymerization of microtubules (29). Aphidicolin and hydroxyurea also failed to increase levels of ␣B crystallin. Western blot analysis clearly revealed the presence of ␣B crystallin in the insoluble fraction of cells treated with colchicine but not in that of cells that had been treated with taxol, aphidicolin, or hydroxyurea (data not shown). Colchicine and other drugs that enhance the disassembly of microtubules did not induce the accumulation of HSP27, another small stress protein (Table I), or of HSP70, which was quantitated by Western blot analysis (data not shown).
The time course of the colcemid-induced accumulation of ␣B crystallin in cells is shown in Fig. 1A. After cells had been cultured for 9 -10 h with 1 M colcemid, the level of ␣B crystallin in the soluble extracts began to increase, reaching a maximal level after 48 -72 h of treatment. A similar time course for the accumulation of ␣B crystallin was observed in the presence of 1 M colchicine. When cells that had been exposed to colcemid for 24 h were washed and then cultured in normal medium, the level of ␣B crystallin returned to close to the control level within 2 days (Fig. 1A). The maximal accumulation of ␣B crystallin in cells that had been exposed to colcemid (Fig. 1B) or colchicine was seen when these drugs were present at 1-3 M.
Northern Blot Analysis and in Vitro Nuclear Run-off Transcription Assays-Levels of mRNA for ␣B crystallin were determined by Northern blot analysis of cells that had been treated with colchicine. The accumulation of ␣B crystallin was preceded by increases in the level of mRNA for ␣B crystallin, and the mRNA was clearly detected after 7 h of exposure to colchicine ( Fig. 2A). By contrast, mRNAs for HSP27 and HSP70 were barely detectable in the same samples (not shown).
In order to determine whether the increase in the level of mRNA for ␣B crystallin was due to increased synthesis de novo or to the accumulation of mRNA by some other mechanism, run-off transcription assays were performed with nuclei from C6 cells. As shown in Fig. 2B, exposure of cells to colchicine as well as to arsenite, which was included as a positive control, stimulated new transcription of the gene for ␣B crystallin. These results indicate that at least part of the increase in the  level of mRNA for ␣B crystallin was due to the production of new mRNA.
Indirect Immunofluorescence Localization of ␣B Crystallin-When C6 glioma cells were treated with 1 M colcemid, the profiles of the cells changed from spindle shaped to polygonal. Depolymerization of microtubules in the colcemid-treated cells was confirmed by indirect immunofluorescence staining of ␤-tubulin. The depolymerized tubulin was concentrated in the peripheral areas of the cytoplasm (Fig. 3B), in contrast to the typical appearance of the microtubules in nontreated cells (Fig.  3A). In nontreated C6 cells, ␣B crystallin was detected only in the nucleoli (Fig. 3C). When the cells were treated with colcemid for 6 h, ␣B crystallin accumulated in the peripheral areas of some but not all cells (Fig. 3D). During incubation for 10 h, the amount of ␣B crystallin increased throughout the cytoplasm of most cells although it was still abundant in the peripheral areas (Fig. 3E). ␣B crystallin in C6 cells that had been treated with colcemid for 24 h was distributed in the cytoplasm of most cells (Fig. 3F).
Induction of ␣B Crystallin by Colchicine in Serum-starved or Aphidicolin-treated Cells-In order to clarify whether or not the induction of ␣B crystallin by the tubulin-binding agents was linked to progression of the cell cycle, cells that had been cultured for 24 h in serum-free medium or in normal medium that contained 6 M aphidicolin were exposed to 1 M colchicine in normal or serum-free medium. As shown in Fig. 4, the colchicine-induced synthesis of ␣B crystallin was observed in serum-starved cells and in aphidicolin-treated cells with a similar time course in each case although the final level in the serum-starved culture (Fig. 4A) was significantly lower than that in the culture that included serum (Fig. 4, B and C). Control cultures of synchronized cells that were not exposed to colchicine did not produce ␣B crystallin in normal medium (Fig. 4, B and C). Colcemid, vinblastine and nocodazole also induced the accumulation of ␣B crystallin in cells in serumdepleted quiescent cultures (data not shown).
These results indicate that the expression of ␣B crystallin was not enhanced at a specific stage (G2 or the early stage of the M phase) of the cell cycle and, moreover, that the synthesis of ␣B crystallin induced by colchicine or by drugs that promote the disassembly of microtubules was due to a process that was not directly linked to a specific stage of the cell cycle.
Suppression by Taxol and Staurosporine of the Synthesis of ␣B Crystallin That Was Induced by Tubulin-binding Antimitotic Drugs-When cells were exposed to 1 M colchicine, 1 M colcemid, 1 M nocodazole, or 1 M vinblastine, each one together with 4 M taxol that had been added 3 h before the FIG. 2. Northern blot analysis of mRNA for ␣B crystallin (A) and results of nuclear run-off transcription assays of ␣B crystallin in cells that had been exposed to colchicine or colcemid (B). A, C6 cells cultured in 90-mm dishes were exposed in duplicate to 1 M colchicine for 0 (lanes 1 and 2), 7 (lanes 3 and 4), 12 (lanes 5 and 6), and 24 h (lanes 7 and 8) in the normal medium. Twenty-g aliquots of total RNA isolated from cells in each dish were subjected to electrophoresis, blotted on a nitrocellulose membrane, and allowed to hybridize with a cDNA probe for ␣B crystallin. Bands of 28 S RNA are shown for reference. B, C6 cells cultured in 90-mm dishes were exposed in duplicate to 1 M colchicine (lane 2) for 13 h. As a positive control, cells were exposed for 1 h to 100 M sodium arsenite and then cultured for 12 h in normal medium (lane 3). Control cells were cultured in normal medium (lane 1). Nuclear RNA was labeled with [ 32 P]dCTP. An aliquot (200,000 cpm) of each sample was allowed to hybridize with previously prepared slot blots of cDNA for ␣B crystallin, and the blots were exposed to x-ray film.
FIG. 3. Intracellular distribution of the depolymerized ␤-tubulin and ␣B crystallin in colcemid-treated C6 glioma cells. A, microtubules in nontreated control cells. B, depolymerized tubulin in C6 cells treated with 1 M colcemid for 2 h. Tubulin was concentrated in the peripheral areas of polygonal cells. C, ␣B crystallin in control cells in which only nucleolar staining was detected. D, ␣B crystallin in C6 cells exposed to 1 M colcemid for 6 h. ␣B crystallin was concentrated in the peripheral areas of some but not all cells. E, ␣B crystallin in cells exposed to 1 M colcemid for 10 h. ␣B crystallin accumulated in the cytoplasm of most cells but was still abundant in peripheral areas. F, ␣B crystallin in C6 cells exposed to colcemid for 24 h. ␣B crystallin was distributed in the cytoplasm of most cells.

FIG. 4. Effects of the serum starvation and of the treatment of cells with aphidicolin on the colchicine-induced accumulation
of ␣B crystallin. Cells that had been cultured for 24 h in serum-free medium were incubated in the presence (closed circles) or absence (open circles) of 1 M colchicine in serum-free medium (A) or in medium plus serum (B). C, cells that had been exposed for 24 h to 6 M aphidicolin in normal medium were washed and then cultured in normal medium in the presence (closed circles) or absence (open circles) of 1 M colchicine. Cells were harvested at the indicated times, and concentrations of ␣B crystallin in the soluble extracts were determined, as described in the legend to Fig. 1. addition of tubulin-binding antimitotic drugs, the synthesis of ␣B crystallin was strongly suppressed. In the case of the colcemid-and nocodazole-induced accumulations, suppression was almost complete (Fig. 5A). Exposure of cells to taxol alone barely affected the control level of ␣B crystallin. Since the induction of ␣B crystallin by arsenite (10) was scarcely affected by taxol under the same conditions (Fig. 5, A and B), the suppressive effect of taxol on the colcemid-and nocodazoleinducible syntheses of ␣B crystallin seemed not to be due to a nonspecific effect of taxol on the synthesis of DNA or protein.
Taxol also suppressed the colchicine-induced response of ␣B crystallin but to a lesser extent (about 50%) than the effect of taxol on the other drug-induced response. Northern blot analysis indicated that the suppressive effect of taxol was apparent at the level of the mRNA for ␣B crystallin (Fig. 5C). Taxol is a potent stabilizer of microtubules. Thus, these results suggest that the expression of ␣B crystallin might be stimulated under conditions that promote the disassembly of microtubules.
The induction of ␣B crystallin by tubulin-binding antimitotic drugs was very sensitive to staurosporine, an inhibitor of protein kinases (30). The presence of staurosporine inhibited the accumulation of ␣B crystallin in cells exposed to colcemid in a dose-dependent manner (Fig. 6A), and the presence of 10 nM staurosporine completely suppressed induction of the synthesis of ␣B crystallin by colchicine, colcemid, nocodazole, or vinblastine (Fig. 6B). This effect was also detected at the mRNA level (Fig. 6C). DISCUSSION The expression of HSP70 (18) and HSP90 (19) is regulated during the cell cycle, and the levels of mRNAs for HSP70 and HSP90 increase rapidly upon entry of cells into the S phase, declining by the late S and G2 phases. The levels of ␣B crystallin in C6 cells were significantly elevated during the growing phase (50 -100 ng/mg of protein), as compared with those in confluent cultures (Ͻ10 ng/mg of protein). Therefore, the possible regulation of the expression of ␣B crystallin during the cell cycle was examined by the exposure of C6 cells to various inhibitors. Among the inhibitors tested, drugs that promote the disassembly of the microtubule by binding to tubulin and that interrupt the cell cycle at the early M phase induced the accumulation of ␣B crystallin but not that of HSP27 and HSP70.
However, the induction of ␣B crystallin by these drugs was also observed in quiescent cultures of cells without serum and, moreover, the level of ␣B crystallin barely increased in cells during synchronous culture in normal medium. These results suggest that the increased accumulation of ␣B crystallin was not due to the continuous expression at the early M phase but FIG. 5. Suppression by taxol of the synthesis of ␣B crystallin induced by drugs that promote the disassembly of microtubules. A, cells that had been cultured for 3 h in the presence (ϩ) or absence (Ϫ) of 4 M taxol were exposed to 1 M colchicine (Col), 1 M colcemid (Ccd), 1 M nocodazole (Noc), or 1 M vinblastine (Vbt) with or without taxol, and cultures were continued for 20 h. Cells that had been preincubated with or without taxol were also exposed to 100 M sodium arsenite (As) for 1 h and then cultured for 19 h in normal medium in the presence or absence of 4 M taxol. None refers to control cultures without treatment. Concentrations of ␣B crystallin in the soluble extracts of cells were determined as described in the legend to Fig. 1. B, aliquots, containing 20 g of protein, of the same extracts as those that had been analyzed by immunoassay were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis with antibodies against ␣B crystallin (␣B cry). C, total RNA was isolated from control cells (C) and from cells that had been exposed for 13 h to 1 M colcemid (Ccd), nocodazole (Noc), colchicine (Col), or vinblastine (Vbt) in the presence (ϩ) or absence (Ϫ) of taxol, as described above. Northern blot analysis of mRNA was performed as described in the legend to Fig. 2A. Bands of 28 S RNA are shown for reference.
FIG. 6. Suppression by staurosporine of the synthesis of ␣B crystallin induced by drugs that promote the disassembly of microtubules. A, cells were exposed for 20 h to 1 M colcemid together with staurosporine (STP) at the indicated concentrations. B, cells were exposed to 1 M colchicine (Col), colcemid (Ccd), nocodazole (Noc), or vinblastine (Vbt) with (ϩ) or without (Ϫ) 10 nM staurosporine. Concentrations of ␣B crystallin in the soluble extracts of cells were determined as described in the legend to Fig. 1. C, total RNA was isolated from control cells (C) and from cells that had been exposed for 13 h to 1 M colchicine (Col), colcemid (Ccd), nocodazole (Noc), or vinblastine (Vbt) with (ϩ) or without (Ϫ) 10 nM staurosporine. Northern blot analysis of mRNA was performed as described in the legend to Fig. 2A. Bands of 28 S RNA are shown for reference. was due to an unknown mechanism that was not directly related to progression of the cell cycle. Stimulation of the synthesis of ␣B crystallin was also observed in rat BRL-3A and 3Y-1 cells that had been exposed to 1 M colchicine (data not shown).
It has been reported (31) that the depolymerization of microtubules early in the cell cycle is sufficient to initiate DNA synthesis and that colchicine and other drugs that promote the disassembly of microtubules enhance the synthesis of DNA, as does thrombin, a growth factor. However, human thrombin (2 g/ml), when added to the culture medium of C6 cells with or without serum, did not induce the synthesis of ␣B crystallin nor did it stimulate the induction by colchicine (data not shown). The induced synthesis of ␣B crystallin by colcemid or nocodazole was strongly suppressed by taxol, a drug that stabilizes cytoplasmic microtubules and prevents their depolymerization by tubulin-binding antimitotic drugs (32). The suppressive effect of taxol on the colchicine-inducible response was not as great as its effect on the colcemid-or nocodazole-induced response. This difference was probably due to the low reversibility of the binding of colchicine to tubulin, as compared with that of colcemid (33) or nocodazole. It is likely that exposure to colcemid or nocodazole increases the pool of free tubulin in cells, whereas taxol reduces the size of this pool by preventing depolymerization. Therefore, it is suggested that the increased depolymerization of microtubules might be a trigger for the increased synthesis of ␣B crystallin.
The results of nuclear run-off assays indicated that the increased synthesis of ␣B crystallin was a result of the increased rate of transcription of the gene for ␣B crystallin. The elevated level of ␣B crystallin protein in cells that had been exposed to colcemid for 24 h decreased rapidly from 24 to 48 h after the addition of cycloheximide or staurosporine (Fig. 7A) and in cultures in the normal medium (Fig. 1A). However, high levels of ␣B crystallin protein (Fig. 7A) and mRNA (Fig. 7B) were maintained for 24 h even after the addition of 1 g/ml actinomycin D, which completely blocked the induction of ␣B crystallin when added simultaneously with colcemid. These results suggest that the microtubule-disrupting drugs enhance not only the transcription of the gene for ␣B crystallin but also the stabilization of the mRNA for ␣B crystallin in cells.
During several hours of exposure of cells to colcemid, the ␣B crystallin in some cells was colocalized with the depolymerized tubulin in the peripheral areas of cells. However, it remains to be determined whether or not the two proteins are associated under these conditions.
The induction of the synthesis of ␣B crystallin by colchicine and by other microtubule-disrupting drugs was completely suppressed in the presence of 3-10 nM staurosporine. It has been reported (34) that the heat-induced synthesis of HSP70 and HSP27 is suppressed in the presence of staurosporine at 2-10 M, concentrations about 1,000-fold higher than the present effective concentrations. Staurosporine is a potent inhibitor of protein kinases, with IC 50 values of 2.7 nM for protein kinase C, 8.2 nM for protein kinase A, and 6.4 nM for the tyrosine protein kinase p60 v-srk (30). The IC 50 value for the colchicine-induced response of ␣B crystallin was about the same as that for protein kinase C (Fig. 4). However, exposure of cells for 20 h to 0.1 M phorbol 12-myristate 13-acetate (an activator of protein kinase C), 20 M forskolin (an activator of protein kinase A), or 30 M sodium orthovanadate (an inhibitor of tyrosine phosphoprotein phosphatase) did not induce the accumulation of ␣B crystallin nor did these treatments stimulate the colchicine-induced accumulation of ␣B crystallin (data not shown).
It is not known which protein kinases are involved in the signal transduction cascade of the colchicine-induced response. However, the present results suggest that the synthesis and accumulation of ␣B crystallin are stimulated, via phosphorylation reactions that are sensitive to staurosporine, when the depolymerization of intracellular microtubules is enhanced. FIG. 7. The messenger RNA for ␣B crystallin in C6 cells is more stable during exposure to colcemid. A, cells were exposed continuously to 1 M colcemid (closed circles), and after 24 h, as indicated by an arrow, 1 g/ml actinomycin D (ϩAcD), 8 g/ml cycloheximide (ϩCHX), or 10 nM staurosporine (ϩSTP) was added to the culture medium. Cells were also exposed to 1 M colcemid together with 1 g/ml actinomycin D (closed squares) or 8 g/ml cycloheximide (closed triangles). After incubation for the indicated time in the CO 2 incubator, cells were harvested for quantitation of ␣B crystallin as described in the legend to