Journal o/ Neurochemislry
Raven Press, Ltd.. New York 0 1992 International Society for Neurochemistry
Distinct Mechanisms of Differentiation of SH-SYSY Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors *Ubaldo Leli, *Anne Cataldo, *Thomas B. Shea, *?Ralph A. Nixon, and *?George Hauser *Ralph Lowell Laboratories, McLean Hospital, Belmont, and Department of Psychiatry and TProgrum in Neuroscience, Harvard Medical School, Boston, Massachusetts, U.S.A.
Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SYSY differentiateswhen exposed to 12-tetradecanoyl-13-acetyl-P-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NFH). TPA, 12-deoxy-1 3-tetradecanoyl-P-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy- 13-tetradecanoyl-P-phorbol but not with DAG, staurosporine, or 4-0-methyl-TPA. NFH increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in peri-
karya of TPA- and 12-deoxy-13-tetradecanoyl-P-phorboltreated cells, but much more in the processes than in the perikarya of staurosporinedifferentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SYSY cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation. Key Words: CytoskeletonDifferentiation-Neuroblastoma-Phorbol esters-Protein kinase C-Staurosporine. Leli U. et al. Distinct mechanisms of differentiation of SH-SYSY neuroblastoma cells by protein kinase C activators and inhibitors. J. Neurochem. 58, 11911198 (1992).
Protein kinase C (PKC) was originally described as a Ca2+- and phospholipid-dependent protein kinase (Kikkawaet al., 1982), but is now known to be a family of phospholipid-dependentenzymes that are activated by diacylglycerol (DAG) under physiological conditions (Coussens et al., 1986; Housey et al., 1987; Nishizuka, 1988, 1989;Ono et al., 1988; Osada et al., 1990;Bacher et al., 1991). PKC is involved in a signaling system of general occurrence in animal cells involving generation of the two intracellular second messengers, inositol 1,4,5-trisphosphateand DAG (reviewed in Rana and Hokin, 1990). Phorbol esters are believed to act by substituting for the physiological PKC activator DAG (Nishizuka, 1984). However, many biological effects
of phorbol diesters cannot be duplicated by DAG. For example, in contrast to 12-tetradecanoyl-13-acetyl-Pphorbol (TPA), DAG does not cause morphological differentiation in SH-SYSY cells, but instead elicits only some of the markers that appear during the treatment of the cells with TPA (reviewed in Pihlman et al., 1990). Neuroblastoma cells in culture have been a useful model to study neuronal differentiation (reviewed in Prasad, 1975; Pihlman et al., 1990). SH-SY5Y cells are a purely neuroblastic neuroblastoma adrenergic subclone of human origin, minimally contaminated by epithelioid elements (Biedler et al., 1973), which extend neurites and express neuronal markers under
~~
glycerol; DMSO, dimethyl sulfoxide; DTP, 12-deoxy-13-tetradecanoyl-P-phorbol; 4-0-methyl-TPA, 4-0-methyl- 12-tetradecanoyl-13acetyl-gphorbol; NF-H, high molecular weight subunit of neurofilaments; NSE, neuron-specific enolase; PKC, protein kinase C; TPA, l 2-tetradecanoyl- l 3-acetyl-&phorbol.
Received May 30, 1991; revised manuscript received August 15, 1991; accepted September 3, 1991. Address correspondence and reprint requests to Dr. U. Leli at Ralph Lowell Laboratories, McLean Hospital, 1 15 Mill Street, Belmont, MA 02178, U.S.A. Abbreviations used: ABC, avidin-biotin complex; DAG, diacyl-
1191
U. LELI ET AL.
1192
the effect of co-carcinogenic phorbol esters such as TPA (Pihlman et al., 1981) and PKC inhibitors such as staurosporine (Shea and Beermann, 199 I). Events accompanying the differentiation of SHSYSY cells by TPA have been described in detail (Pihlman et al., 1990). Downregulation of the expression of c-myc and enhancement of the expression of c-fos (Jalava et al., 1988)are very early events, followed by a decrease in affinity and number of muscarink cholinergic receptors, as well as their coupling with intracellular free Ca2+elevation (Heikkila, et al., 1987). These events are followed by appearance of voltagedependent CaZ+channels (Akerman et al., 1984), increases in neuron-specific enolase (NSE) (Pihlman et al., 1981; Akerman et al., 1984), elevation of norepinephrine content (Pihlman et al., 1983), and extension of long neurites (Pihlman et al., 1981). At the same time, the total PKC activity is markedly downregulated (Heikkila et al., 1989). Because both long-term exposure to phorbol diesters and treatment with PKC inhibitors result in SH-SY5Y cell differentiation, downregulation or inhibition of PKC has been proposed to be correlated directly with the appearance of a differentiated phenotype, voltage-dependent Ca2+channels and NSE (Heikkila et al., 1989). Using SH-SYSY cells as a model system, we studied the structure-activity relationship of phorbol diester and DAG molecules, and their capacity to induce differentiation of these cells, as measured by the extension of neuntes, the expression of NSE, and the appearance of the high molecular weight subunit of neurofilaments (NF-H) to pinpoint the importance of specific areas of the phorbol molecule that are not present in DAG. Previous immunocytochemical studies using a monoclonal antibody (SMI-3 1) directed against phosphorylated neurofilament epitopes have revealed that extensively phosphorylated forms of NF-H and the medium molecular weight subunit of neurofilaments are typically segregated within axons (Sternberger and Sternberger, 1983; Carden et al., 1985, 1987; Foster et al., 1987; Lee et al., 1987; Oblinger, 1987) and can therefore be considered a marker of the development of a stabilized axonal cytoskeleton. The appearance of phosphorylated neurofilaments in axons also appears to be a useful marker of differentiation in neuroblastoma cells (Shea et al., 1990). In this article, we report that PKC activators cause differentiation of SH-SY 5Y cells by different mechanisms than PKC inhibitors and we provide evidence suggestingthat certain molecular features of the terpene moiety of TPA that are not present in DAG are important for their action in causing neuroblastoma cells to differentiate.
York, NY, U.S.A.). Polyclonal antibodies against NSE, and a-tubulin were obtained from Dakopatts, Dako (Santa Barbara, CA, U.S.A.) and ICN Biochemicals (Lisle, IL, U.S.A.), respectively. The monoclonal antibody SMI-3 1, which is directed against the phosphorylated epitopes of human NF-H, was purchased from Sternberger-Meyer Immunochemicals, Inc. (Jarrettsville, MD, U.S.A.). Diofein was from Serdary Laboratories (London, Ontario, Canada), TPA from Sigma (St. Louis, MO, U.S.A.). Staurosporine was a generous gift of Dr. Yazuru Matsuda of Kyowa Hakko Kogyo (Tokyo, Japan); 4-O-methyl-TPA, 12-deoxy-l3-tetradecanoyl-/3phorbol (DTP), and mezerein were purchased from LC Services (Woburn, MA, U.S.A.). All other chemicals and reagents were of the purest grade commercially available.
Methods Cells were maintained in RPMI 1640 with L-glutamine, supplemented with 10% fetal bovine serum, 100 IU/ml of penicillin, and 100 pg/ml of streptomycin, in a 95/5% (vol/ vol) atmosphere of air/CO, at 37°C (Heikkila et al., 1989). Drugs were dissolved in dimethyl sulfoxide (DMSO) and added after equilibrating the cells for 24 h in culture medium containing 7% fetal bovine serum. Final concentrations of DMSO never exceeded 0.1%. Untreated controls always contained the same amount of solvent as the experimental samples. Immunocytochemistry was performed with the avidinbiotin complex (ABC) method (Hsu et al., 1981) using Vectastain kits (Vector Labs., Burlingame, CA, U.S.A.) as described (Cataldo et al., 1990). In brief, cell cultures were fixed for 15 min in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde. After fixation cultures were rinsed first with Tris-buffered saline
'OH
MATERIALS AND METHODS
Materials SH-SY5Y neuroblastoma cells were obtained from Dr. June L. Biedler (Memorial Sloan-Kettering Center, New
J. Nrurorhem.. Vol. 58, No. 4, 1992
FIG. 1. Chemical structures of TPA. mezerein, staurosporine, and DAG.
PROTEIN KINASE C IN SH-SYS Y CELL DIFFERENTIATION TABLE 1. Effects of PKC activators or inhibitors on neurite extension Cells with neurites 48 h
1193
indicated. For statistical analysis, six random fields were photographed from each culture. Cells with processes longer than one body length or positively immunostained were scored blindly. Data were analyzed with Student’s t test.
72 h
RESULTS % oftotal
Control TPA Diolein Staurosporine 4-0-methyl-TPA DTP Mezerein
17.6 t 4.6 25.7 f 3.8
-
24.0 f 6.9 17.0 f 9.1 32.8 f 10.8 23.0 f 10.3
p
Raven Press, Ltd.. New York 0 1992 International Society for Neurochemistry
Distinct Mechanisms of Differentiation of SH-SYSY Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors *Ubaldo Leli, *Anne Cataldo, *Thomas B. Shea, *?Ralph A. Nixon, and *?George Hauser *Ralph Lowell Laboratories, McLean Hospital, Belmont, and Department of Psychiatry and TProgrum in Neuroscience, Harvard Medical School, Boston, Massachusetts, U.S.A.
Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SYSY differentiateswhen exposed to 12-tetradecanoyl-13-acetyl-P-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NFH). TPA, 12-deoxy-1 3-tetradecanoyl-P-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy- 13-tetradecanoyl-P-phorbol but not with DAG, staurosporine, or 4-0-methyl-TPA. NFH increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in peri-
karya of TPA- and 12-deoxy-13-tetradecanoyl-P-phorboltreated cells, but much more in the processes than in the perikarya of staurosporinedifferentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SYSY cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation. Key Words: CytoskeletonDifferentiation-Neuroblastoma-Phorbol esters-Protein kinase C-Staurosporine. Leli U. et al. Distinct mechanisms of differentiation of SH-SYSY neuroblastoma cells by protein kinase C activators and inhibitors. J. Neurochem. 58, 11911198 (1992).
Protein kinase C (PKC) was originally described as a Ca2+- and phospholipid-dependent protein kinase (Kikkawaet al., 1982), but is now known to be a family of phospholipid-dependentenzymes that are activated by diacylglycerol (DAG) under physiological conditions (Coussens et al., 1986; Housey et al., 1987; Nishizuka, 1988, 1989;Ono et al., 1988; Osada et al., 1990;Bacher et al., 1991). PKC is involved in a signaling system of general occurrence in animal cells involving generation of the two intracellular second messengers, inositol 1,4,5-trisphosphateand DAG (reviewed in Rana and Hokin, 1990). Phorbol esters are believed to act by substituting for the physiological PKC activator DAG (Nishizuka, 1984). However, many biological effects
of phorbol diesters cannot be duplicated by DAG. For example, in contrast to 12-tetradecanoyl-13-acetyl-Pphorbol (TPA), DAG does not cause morphological differentiation in SH-SYSY cells, but instead elicits only some of the markers that appear during the treatment of the cells with TPA (reviewed in Pihlman et al., 1990). Neuroblastoma cells in culture have been a useful model to study neuronal differentiation (reviewed in Prasad, 1975; Pihlman et al., 1990). SH-SY5Y cells are a purely neuroblastic neuroblastoma adrenergic subclone of human origin, minimally contaminated by epithelioid elements (Biedler et al., 1973), which extend neurites and express neuronal markers under
~~
glycerol; DMSO, dimethyl sulfoxide; DTP, 12-deoxy-13-tetradecanoyl-P-phorbol; 4-0-methyl-TPA, 4-0-methyl- 12-tetradecanoyl-13acetyl-gphorbol; NF-H, high molecular weight subunit of neurofilaments; NSE, neuron-specific enolase; PKC, protein kinase C; TPA, l 2-tetradecanoyl- l 3-acetyl-&phorbol.
Received May 30, 1991; revised manuscript received August 15, 1991; accepted September 3, 1991. Address correspondence and reprint requests to Dr. U. Leli at Ralph Lowell Laboratories, McLean Hospital, 1 15 Mill Street, Belmont, MA 02178, U.S.A. Abbreviations used: ABC, avidin-biotin complex; DAG, diacyl-
1191
U. LELI ET AL.
1192
the effect of co-carcinogenic phorbol esters such as TPA (Pihlman et al., 1981) and PKC inhibitors such as staurosporine (Shea and Beermann, 199 I). Events accompanying the differentiation of SHSYSY cells by TPA have been described in detail (Pihlman et al., 1990). Downregulation of the expression of c-myc and enhancement of the expression of c-fos (Jalava et al., 1988)are very early events, followed by a decrease in affinity and number of muscarink cholinergic receptors, as well as their coupling with intracellular free Ca2+elevation (Heikkila, et al., 1987). These events are followed by appearance of voltagedependent CaZ+channels (Akerman et al., 1984), increases in neuron-specific enolase (NSE) (Pihlman et al., 1981; Akerman et al., 1984), elevation of norepinephrine content (Pihlman et al., 1983), and extension of long neurites (Pihlman et al., 1981). At the same time, the total PKC activity is markedly downregulated (Heikkila et al., 1989). Because both long-term exposure to phorbol diesters and treatment with PKC inhibitors result in SH-SY5Y cell differentiation, downregulation or inhibition of PKC has been proposed to be correlated directly with the appearance of a differentiated phenotype, voltage-dependent Ca2+channels and NSE (Heikkila et al., 1989). Using SH-SYSY cells as a model system, we studied the structure-activity relationship of phorbol diester and DAG molecules, and their capacity to induce differentiation of these cells, as measured by the extension of neuntes, the expression of NSE, and the appearance of the high molecular weight subunit of neurofilaments (NF-H) to pinpoint the importance of specific areas of the phorbol molecule that are not present in DAG. Previous immunocytochemical studies using a monoclonal antibody (SMI-3 1) directed against phosphorylated neurofilament epitopes have revealed that extensively phosphorylated forms of NF-H and the medium molecular weight subunit of neurofilaments are typically segregated within axons (Sternberger and Sternberger, 1983; Carden et al., 1985, 1987; Foster et al., 1987; Lee et al., 1987; Oblinger, 1987) and can therefore be considered a marker of the development of a stabilized axonal cytoskeleton. The appearance of phosphorylated neurofilaments in axons also appears to be a useful marker of differentiation in neuroblastoma cells (Shea et al., 1990). In this article, we report that PKC activators cause differentiation of SH-SY 5Y cells by different mechanisms than PKC inhibitors and we provide evidence suggestingthat certain molecular features of the terpene moiety of TPA that are not present in DAG are important for their action in causing neuroblastoma cells to differentiate.
York, NY, U.S.A.). Polyclonal antibodies against NSE, and a-tubulin were obtained from Dakopatts, Dako (Santa Barbara, CA, U.S.A.) and ICN Biochemicals (Lisle, IL, U.S.A.), respectively. The monoclonal antibody SMI-3 1, which is directed against the phosphorylated epitopes of human NF-H, was purchased from Sternberger-Meyer Immunochemicals, Inc. (Jarrettsville, MD, U.S.A.). Diofein was from Serdary Laboratories (London, Ontario, Canada), TPA from Sigma (St. Louis, MO, U.S.A.). Staurosporine was a generous gift of Dr. Yazuru Matsuda of Kyowa Hakko Kogyo (Tokyo, Japan); 4-O-methyl-TPA, 12-deoxy-l3-tetradecanoyl-/3phorbol (DTP), and mezerein were purchased from LC Services (Woburn, MA, U.S.A.). All other chemicals and reagents were of the purest grade commercially available.
Methods Cells were maintained in RPMI 1640 with L-glutamine, supplemented with 10% fetal bovine serum, 100 IU/ml of penicillin, and 100 pg/ml of streptomycin, in a 95/5% (vol/ vol) atmosphere of air/CO, at 37°C (Heikkila et al., 1989). Drugs were dissolved in dimethyl sulfoxide (DMSO) and added after equilibrating the cells for 24 h in culture medium containing 7% fetal bovine serum. Final concentrations of DMSO never exceeded 0.1%. Untreated controls always contained the same amount of solvent as the experimental samples. Immunocytochemistry was performed with the avidinbiotin complex (ABC) method (Hsu et al., 1981) using Vectastain kits (Vector Labs., Burlingame, CA, U.S.A.) as described (Cataldo et al., 1990). In brief, cell cultures were fixed for 15 min in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde. After fixation cultures were rinsed first with Tris-buffered saline
'OH
MATERIALS AND METHODS
Materials SH-SY5Y neuroblastoma cells were obtained from Dr. June L. Biedler (Memorial Sloan-Kettering Center, New
J. Nrurorhem.. Vol. 58, No. 4, 1992
FIG. 1. Chemical structures of TPA. mezerein, staurosporine, and DAG.
PROTEIN KINASE C IN SH-SYS Y CELL DIFFERENTIATION TABLE 1. Effects of PKC activators or inhibitors on neurite extension Cells with neurites 48 h
1193
indicated. For statistical analysis, six random fields were photographed from each culture. Cells with processes longer than one body length or positively immunostained were scored blindly. Data were analyzed with Student’s t test.
72 h
RESULTS % oftotal
Control TPA Diolein Staurosporine 4-0-methyl-TPA DTP Mezerein
17.6 t 4.6 25.7 f 3.8
-
24.0 f 6.9 17.0 f 9.1 32.8 f 10.8 23.0 f 10.3
p
