459
Biochimca et Biophysica Acta, 539 ( 1 9 7 8 ) 4 5 9 - - 4 6 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28491
INDUCTION OF CYCLIC AMP-BINDING PROTEINS BY DIBUTYRYL CYCLIC AMP IN MOUSE NEUROBLASTOMA CELLS
N A G I N D R A P R A S H A D * a n d R O G E R N. R O S E N B E R G
Department o f Neurology, Southwestern Medical School, University o f Texas Health Science Center at Dallas, Dallas, Texas 75235 (U.S.A.) (Received J u l y 7 t h , 1 9 7 7 )
Summary Mouse neuroblastoma cells grown in the presence of 1 mM N6,O2'-dibutyryl. cyclic AMP showed a 3-fold increase in cyclic AMP-binding proteins. The role of dibutyryl cyclic AMP in the introduction of cyclic AMP-binding proteins in these cells has been studied. Induced cyclic AMP-binding proteins were observed in the cytoplasm 15 h after dibutyryl cyclic AMP treatment. The increase in cyclic AMP-binding proteins required RNA and protein synthesis. It is suggested that the 15-h lag occurs at the post-transcriptional and/or translational level. Cyclic AMP-binding proteins are found in both soluble and particulate cell fractions. Dibutyryl cyclic AMP increased binding prot6ins in both fractions. The control and dibutyryl cyclic AMP-induced binding proteins showed similar affinity for cyclic AMP. The data indicate that dibutyryl cyclic AMP caused the following sequential events: a 12-fold increase in cyclic AMP levels; a 40% increase in phosphodiesterase activity; and a 300% (3-fold) increase in cyclic AMP-binding proteins. It is suggested that the differentiation of mouse neuroblastoma cells involves increased levels of cyclic AMP and cyclic AMP-binding proteins.
Introduction In eukaryotic cells intracellular levels of cyclic AMP are elevated by a variety of chemicals, such as prostaglandin E1 [1], phosphodiesterase inhibitors [2] and N6,O2'-dibutyryl cyclic AMP (dibutyryl cyclic AMP) [3] which also * To whom correspondence and reprint requests should be addressed. Abbreviations: dibutyryl cyclic AMP, N6,O2'-dibutyryl cyclic AMP; cyclic AMP, adenosine 3',5'monophosphate: 2'-AMP, adenosine 2'-monophospnate; 3'-AMP, adenosine 3'-monophosphate~ 5'-AMP, adenosine 5'-monophosphate.
460 increases the activity of phosphodiesterase in several eukaryotic cells [3--5]. In neuroblastoma cells dibutyryl cyclic AMP increases the intracellular cyclic AMP levels and causes differentiation [3]. To understand the role of cyclic AMP in mediating cellular functions in the differentiation of neuroblastoma cells, it is essential to determine the levels of cyclic AMP-binding proteins from the cells grown in the presence of dibutyryl cyclic AMP. The most extensively characterized binding proteins for cyclic AMP in eukaryotic cells are cyclic AMP-dependent protein kinases [6--8] ; and it has been proposed that in higher organisms many, if not all, of the biological effects of cyclic AMP are mediated through activation of this class of enzyme [9]. Cyclic AMP-dependent protein kinases are composed of regulatory and catalytic subunits. Cyclic AMP binds to the regulatory subunit which splits from the kinase holoenzyme, leaving an activated catalytic subunit [10--13]. In bacterial cells cyclic AMP binds to a specific cyclic AMP receptor protein which in turn binds to DNA and facilitates the initiation of transcription of certain catabolite repressible genes [14,15]. The presence of cyclic AMP-binding proteins in neuroblastoma cells has been reported [3]; their level increases in cells when treated with phosphodiesterase inhibitors [16] and prostaglandin E1 [17]. Also, treatment with dibutyryl cyclic AMP causes an 8-fold increase in cyclic AMP-binding proteins in mouse neuroblastoma cells as analyzed by isoelectric focusing, in addition to causing protein modifications [18]. In this study, we investigated the role of dibutyryl cyclic AMP in the induction of cyclic AMP-binding proteins in these cells. Experimental procedures
Cell culture. The cholinergic $20 clone [19] of mouse neuroblastoma cells was grown in a monolayer culture. Confluent cells were trypsinized and 3 . 106 cells were plated in 100-mm plates and grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum, 200 pg/ml kanamycin, 125 pg/ml spectinomycin at 37°C with 90% air/10% CO2 at 100% humidity. In the dibutyryl cyclic AMP-treated cells, 1 mM dibutyryl cyclic AMP was included in the medium at the time the cells were plated. Media and dibutyryl cyclic AMP were changed every day. Cell fractionation. Cells grown in a monolayer were rinsed with cold 0.9% NaC1, and I ml of cold homogenization buffer, containing 0.05 M Tris • HC1, pH 7.4, 0.25 M sucrose, 2.0 mM MgCl2 and 4 mM 2-mercaptoethanol, was added to each plate and cells were scraped with a rubber spatula. Cells were homogenized in a glass Dounce homogenizer with a tight fitting " B " pestle (Bellco), Lysis of cells and intact nuclei was monitored under a light microscope. The homogenate was centrifuged in 15-ml corex tubes at 480 × g for 15 min at 5°C in a Sorvall centrifuge. The supernatant was centrifuged at 100 000 :~g in a 50 Ti rotor for 90 min at 5°C. The 100 000 ~g supernatant was designated as the soluble fraction and the pellet as the particulate fraction. Cyclic AMP binding proteins were assayed in both fractions. Protein concentrations were determined by the m e t h o d of Lowry et al. [20], with bovine serum albumin as standard. Assay for cyclic AMP-binding proteins. Cyclic AMP-binding proteins were assayed by determining the a m o u n t of cyclic [3H]AMP bound per mg cellulax
461 protein as described b y Gilman [21] with slight modifications. The standard binding reaction was done in a volume of 400 pl with a final concentration of 50 mM sodium acetate, pH 4.0, 1 mM 2-mercaptoethanol, 37.5 mM papaverine, 333 pM 2'-AMP, 3'-AMP, 5'-AMP and 50--100 pg of protein. Papaverine was included to inhibit phosphodiesterase activity. Unlabelled nucleotides were included in the assays to lower the non-specific binding of cyclic [3H]AMP [22]. The reactions were initiated by the addition of 40 nM cyclic [3H]AMP (37 Ci/mmol). Reaction mixtures were incubated in ice for 1 h, then diluted with 3 ml of 20 mM potassium phosphate buffer, pH 6.0. This diluted mixture was filtered through Miliipore filters (0.45 pm). Tubes were rinsed with 4 ml of buffer and the filters were washed with 6 ml of buffer. The filters were dried in scintillation vials under infrared light, then 2 ml of Filter-Solv (Beckman) was added, and the vials were shaken for 10 min. 10 ml of scintillation liquid containing 5 g PPO and 0.5 g dimethyl POPOP in 1 1 of toluene was added and radioactivity was determined. Cyclic AMP assay. The cyclic AMP content was determined by the method of Gilman [21], using the Amersham/Searle cyclic AMP Assay Kit, T R K 432. Cells in monolayer were rinsed with cold 0.9% NaC1 and scraped in 1 ml of cold 5% trichloroacetic acid and homogenized. The homogenate was centrifuged for 10 min at 3000 rev./min. The trichloroacetic acid supernatant was treated with 0.1 ml of 1 M HC1 and extracted five times with 2 ml of ether. The aqueous phase was lyophilized. The trichloroacetic acid-insoluble pellet was homogenized in 1 ml of 0.4 M NaOH and the protein concentration was determined [20]. The lyophilized aqueous phase was redissolved in 4 mM EDTA plus 0.05 M Tris • HC1, pH 7.5, and cyclic AMP was assayed as described in the Amersham/Searle cyclic AMP Assay Kit, TRK 432. Assay for phosphodiesterase activity. Phosphodiesterase activity was assayed in aliquots of the same supernatant fractions used for cyclic AMP-binding protein determinations. Phosphodiesterase activity was assayed with cyclic [3H]AMP using an anion exchange resin [23]. The reaction volume was 400 pl and contained 40 mM Tris • HC1, pH 8.0, 10 mM MgCI:, 1 mM 2-mercaptoethanol, 5 ~M unlabelled cyclic AMP and 40 nM cyclic [3H]AMP. The reactions were initiated b y the addition of 50 t~g of protein. Materials. Tissue culture media were purchased from GIBCO and fetal calf serum was purchased from Rheis. Cyclic [3H]AMP (37 Ci/mmol) was purchased from New England Nuclear. Dibutyryl cyclic AMP, actinomycin D and cycloheximide were purchased from Sigma. The cyclic AMP Assay Kit was purchased from Amersham/Searle. Anion exchange resin (AGI-X2, 200--400 mesh) was obtained from BioRad. Results
Optimum concentration of cyclic [3H]AMP in measuring cyclic AMP-binding proteins The soluble fractions from untreated control and dibutyryl cyclic AMPtreated mouse neuroblastoma cells were incubated with several concentrations of cyclic [3H]AMP to determine the maximum binding of cyclic [3H]AMP to the binding proteins. In these reactions the unlabelled nucleotides 2'-AMP,
462 3'-AMP and 5'-AMP were included in 500-fold excess to lower the non-specific binding of cyclic [SH]AMP. These nucleotides did not inhibit specific binding of cyclic [3H] AMP to binding proteins. The results presented in Fig. 1 show that the concentrations of cyclic [3H]AMP giving one-half the maximum binding in control and dibutyryl cyclic AMP treated cells is 14 nM and 19 nM, respectively. The control cells show maximum binding at 25 nM cyclic [-~H]AMP, dibutyryl cyclic AMP-treated cells show maximum binding at 50 nM cyclic [SH]AMP. Dibutyryl cyclic AMP-treated cells also show a 3-fold higher level of binding of cyclic AMP to protein than do the control cells.
Cyclic AMP-binding proteins from control and dibutyryl cyclic AMP-treated cells Dibutyryl cyclic AMP-treated cells show a 3-fold increase in cyclic AMP binding in both the soluble and particulate fractions (Table I). In control cells the binding of cyclic [3H]AMP to the proteins from the soluble fraction was 4.62 pmol per mg protein and 2.8 pmol per mg protein for the particulate fraction, whereas the binding of cyclic [ 3H] AMP to the proteins of the soluble fraction of dibutyryl cyclic AMP treated is 15.34 pmol per mg protein and 8.7 pmol per mg protein for the particulate fraction (Table I). When examining cyclic AMP binding in crude extract, the presence of intracellular cyclic AMP must be considered. The intracellular cyclic AMP would dilute the cyclic [3H]AMP used to detect binding and might also mask some binding sites. The effect of dialysis on cyclic AMP binding was therefore examined. Extensive dialysis of samples from control and dibutyryl cyclic AMP treated cells had no effect on the cyclic [3H]AMP binding (data not included). Thus, intracellular cyclic AMP levels did not affect the cyclic [3H] AMP binding results as presented in Table I. These results agree with results reported previously [24]. 16 .E
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Biochimca et Biophysica Acta, 539 ( 1 9 7 8 ) 4 5 9 - - 4 6 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28491
INDUCTION OF CYCLIC AMP-BINDING PROTEINS BY DIBUTYRYL CYCLIC AMP IN MOUSE NEUROBLASTOMA CELLS
N A G I N D R A P R A S H A D * a n d R O G E R N. R O S E N B E R G
Department o f Neurology, Southwestern Medical School, University o f Texas Health Science Center at Dallas, Dallas, Texas 75235 (U.S.A.) (Received J u l y 7 t h , 1 9 7 7 )
Summary Mouse neuroblastoma cells grown in the presence of 1 mM N6,O2'-dibutyryl. cyclic AMP showed a 3-fold increase in cyclic AMP-binding proteins. The role of dibutyryl cyclic AMP in the introduction of cyclic AMP-binding proteins in these cells has been studied. Induced cyclic AMP-binding proteins were observed in the cytoplasm 15 h after dibutyryl cyclic AMP treatment. The increase in cyclic AMP-binding proteins required RNA and protein synthesis. It is suggested that the 15-h lag occurs at the post-transcriptional and/or translational level. Cyclic AMP-binding proteins are found in both soluble and particulate cell fractions. Dibutyryl cyclic AMP increased binding prot6ins in both fractions. The control and dibutyryl cyclic AMP-induced binding proteins showed similar affinity for cyclic AMP. The data indicate that dibutyryl cyclic AMP caused the following sequential events: a 12-fold increase in cyclic AMP levels; a 40% increase in phosphodiesterase activity; and a 300% (3-fold) increase in cyclic AMP-binding proteins. It is suggested that the differentiation of mouse neuroblastoma cells involves increased levels of cyclic AMP and cyclic AMP-binding proteins.
Introduction In eukaryotic cells intracellular levels of cyclic AMP are elevated by a variety of chemicals, such as prostaglandin E1 [1], phosphodiesterase inhibitors [2] and N6,O2'-dibutyryl cyclic AMP (dibutyryl cyclic AMP) [3] which also * To whom correspondence and reprint requests should be addressed. Abbreviations: dibutyryl cyclic AMP, N6,O2'-dibutyryl cyclic AMP; cyclic AMP, adenosine 3',5'monophosphate: 2'-AMP, adenosine 2'-monophospnate; 3'-AMP, adenosine 3'-monophosphate~ 5'-AMP, adenosine 5'-monophosphate.
460 increases the activity of phosphodiesterase in several eukaryotic cells [3--5]. In neuroblastoma cells dibutyryl cyclic AMP increases the intracellular cyclic AMP levels and causes differentiation [3]. To understand the role of cyclic AMP in mediating cellular functions in the differentiation of neuroblastoma cells, it is essential to determine the levels of cyclic AMP-binding proteins from the cells grown in the presence of dibutyryl cyclic AMP. The most extensively characterized binding proteins for cyclic AMP in eukaryotic cells are cyclic AMP-dependent protein kinases [6--8] ; and it has been proposed that in higher organisms many, if not all, of the biological effects of cyclic AMP are mediated through activation of this class of enzyme [9]. Cyclic AMP-dependent protein kinases are composed of regulatory and catalytic subunits. Cyclic AMP binds to the regulatory subunit which splits from the kinase holoenzyme, leaving an activated catalytic subunit [10--13]. In bacterial cells cyclic AMP binds to a specific cyclic AMP receptor protein which in turn binds to DNA and facilitates the initiation of transcription of certain catabolite repressible genes [14,15]. The presence of cyclic AMP-binding proteins in neuroblastoma cells has been reported [3]; their level increases in cells when treated with phosphodiesterase inhibitors [16] and prostaglandin E1 [17]. Also, treatment with dibutyryl cyclic AMP causes an 8-fold increase in cyclic AMP-binding proteins in mouse neuroblastoma cells as analyzed by isoelectric focusing, in addition to causing protein modifications [18]. In this study, we investigated the role of dibutyryl cyclic AMP in the induction of cyclic AMP-binding proteins in these cells. Experimental procedures
Cell culture. The cholinergic $20 clone [19] of mouse neuroblastoma cells was grown in a monolayer culture. Confluent cells were trypsinized and 3 . 106 cells were plated in 100-mm plates and grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum, 200 pg/ml kanamycin, 125 pg/ml spectinomycin at 37°C with 90% air/10% CO2 at 100% humidity. In the dibutyryl cyclic AMP-treated cells, 1 mM dibutyryl cyclic AMP was included in the medium at the time the cells were plated. Media and dibutyryl cyclic AMP were changed every day. Cell fractionation. Cells grown in a monolayer were rinsed with cold 0.9% NaC1, and I ml of cold homogenization buffer, containing 0.05 M Tris • HC1, pH 7.4, 0.25 M sucrose, 2.0 mM MgCl2 and 4 mM 2-mercaptoethanol, was added to each plate and cells were scraped with a rubber spatula. Cells were homogenized in a glass Dounce homogenizer with a tight fitting " B " pestle (Bellco), Lysis of cells and intact nuclei was monitored under a light microscope. The homogenate was centrifuged in 15-ml corex tubes at 480 × g for 15 min at 5°C in a Sorvall centrifuge. The supernatant was centrifuged at 100 000 :~g in a 50 Ti rotor for 90 min at 5°C. The 100 000 ~g supernatant was designated as the soluble fraction and the pellet as the particulate fraction. Cyclic AMP binding proteins were assayed in both fractions. Protein concentrations were determined by the m e t h o d of Lowry et al. [20], with bovine serum albumin as standard. Assay for cyclic AMP-binding proteins. Cyclic AMP-binding proteins were assayed by determining the a m o u n t of cyclic [3H]AMP bound per mg cellulax
461 protein as described b y Gilman [21] with slight modifications. The standard binding reaction was done in a volume of 400 pl with a final concentration of 50 mM sodium acetate, pH 4.0, 1 mM 2-mercaptoethanol, 37.5 mM papaverine, 333 pM 2'-AMP, 3'-AMP, 5'-AMP and 50--100 pg of protein. Papaverine was included to inhibit phosphodiesterase activity. Unlabelled nucleotides were included in the assays to lower the non-specific binding of cyclic [3H]AMP [22]. The reactions were initiated by the addition of 40 nM cyclic [3H]AMP (37 Ci/mmol). Reaction mixtures were incubated in ice for 1 h, then diluted with 3 ml of 20 mM potassium phosphate buffer, pH 6.0. This diluted mixture was filtered through Miliipore filters (0.45 pm). Tubes were rinsed with 4 ml of buffer and the filters were washed with 6 ml of buffer. The filters were dried in scintillation vials under infrared light, then 2 ml of Filter-Solv (Beckman) was added, and the vials were shaken for 10 min. 10 ml of scintillation liquid containing 5 g PPO and 0.5 g dimethyl POPOP in 1 1 of toluene was added and radioactivity was determined. Cyclic AMP assay. The cyclic AMP content was determined by the method of Gilman [21], using the Amersham/Searle cyclic AMP Assay Kit, T R K 432. Cells in monolayer were rinsed with cold 0.9% NaC1 and scraped in 1 ml of cold 5% trichloroacetic acid and homogenized. The homogenate was centrifuged for 10 min at 3000 rev./min. The trichloroacetic acid supernatant was treated with 0.1 ml of 1 M HC1 and extracted five times with 2 ml of ether. The aqueous phase was lyophilized. The trichloroacetic acid-insoluble pellet was homogenized in 1 ml of 0.4 M NaOH and the protein concentration was determined [20]. The lyophilized aqueous phase was redissolved in 4 mM EDTA plus 0.05 M Tris • HC1, pH 7.5, and cyclic AMP was assayed as described in the Amersham/Searle cyclic AMP Assay Kit, TRK 432. Assay for phosphodiesterase activity. Phosphodiesterase activity was assayed in aliquots of the same supernatant fractions used for cyclic AMP-binding protein determinations. Phosphodiesterase activity was assayed with cyclic [3H]AMP using an anion exchange resin [23]. The reaction volume was 400 pl and contained 40 mM Tris • HC1, pH 8.0, 10 mM MgCI:, 1 mM 2-mercaptoethanol, 5 ~M unlabelled cyclic AMP and 40 nM cyclic [3H]AMP. The reactions were initiated b y the addition of 50 t~g of protein. Materials. Tissue culture media were purchased from GIBCO and fetal calf serum was purchased from Rheis. Cyclic [3H]AMP (37 Ci/mmol) was purchased from New England Nuclear. Dibutyryl cyclic AMP, actinomycin D and cycloheximide were purchased from Sigma. The cyclic AMP Assay Kit was purchased from Amersham/Searle. Anion exchange resin (AGI-X2, 200--400 mesh) was obtained from BioRad. Results
Optimum concentration of cyclic [3H]AMP in measuring cyclic AMP-binding proteins The soluble fractions from untreated control and dibutyryl cyclic AMPtreated mouse neuroblastoma cells were incubated with several concentrations of cyclic [3H]AMP to determine the maximum binding of cyclic [3H]AMP to the binding proteins. In these reactions the unlabelled nucleotides 2'-AMP,
462 3'-AMP and 5'-AMP were included in 500-fold excess to lower the non-specific binding of cyclic [SH]AMP. These nucleotides did not inhibit specific binding of cyclic [3H] AMP to binding proteins. The results presented in Fig. 1 show that the concentrations of cyclic [3H]AMP giving one-half the maximum binding in control and dibutyryl cyclic AMP treated cells is 14 nM and 19 nM, respectively. The control cells show maximum binding at 25 nM cyclic [-~H]AMP, dibutyryl cyclic AMP-treated cells show maximum binding at 50 nM cyclic [SH]AMP. Dibutyryl cyclic AMP-treated cells also show a 3-fold higher level of binding of cyclic AMP to protein than do the control cells.
Cyclic AMP-binding proteins from control and dibutyryl cyclic AMP-treated cells Dibutyryl cyclic AMP-treated cells show a 3-fold increase in cyclic AMP binding in both the soluble and particulate fractions (Table I). In control cells the binding of cyclic [3H]AMP to the proteins from the soluble fraction was 4.62 pmol per mg protein and 2.8 pmol per mg protein for the particulate fraction, whereas the binding of cyclic [ 3H] AMP to the proteins of the soluble fraction of dibutyryl cyclic AMP treated is 15.34 pmol per mg protein and 8.7 pmol per mg protein for the particulate fraction (Table I). When examining cyclic AMP binding in crude extract, the presence of intracellular cyclic AMP must be considered. The intracellular cyclic AMP would dilute the cyclic [3H]AMP used to detect binding and might also mask some binding sites. The effect of dialysis on cyclic AMP binding was therefore examined. Extensive dialysis of samples from control and dibutyryl cyclic AMP treated cells had no effect on the cyclic [3H]AMP binding (data not included). Thus, intracellular cyclic AMP levels did not affect the cyclic [3H] AMP binding results as presented in Table I. These results agree with results reported previously [24]. 16 .E
e J
14
~
°
/
c~ 12 ,~ ~08
e
8 6
