The Pro-apoptotic Protein Bim Is a Convergence Point for cAMP/Protein Kinase A- and Glucocorticoid-promoted Apoptosis of Lymphoid Cells

The mechanisms by which cAMP mediates apoptosis are not well understood. In the current studies, we used wild-type (WT) S49 T-lymphoma cells and the kin– variant (which lacks protein kinase A (PKA)) to examine cAMP/PKA-mediated apoptosis. The cAMP analog, 8-CPT-cAMP, increased phosphorylation of the cAMP response element-binding protein (CREB), activated caspase-3, and induced apoptosis in WT but not in kin– S49 cells. Using an array of 96 apoptosis-related genes, we found that treatment of WT cells with 8-CPT-cAMP for 24 h induced expression of mRNA for the pro-apoptotic gene, Bim. Real-time PCR analysis indicated that 8-CPT-cAMP increased Bim RNA in WT cells in <2 h and maintained this increase for >24 h. Bim protein expression increased in WT but not kin– cells treated with 8-CPT-cAMP or with the β-adrenergic receptor agonist isoproterenol. Both apoptosis and Bim expression were reversible with removal of 8-CPT-cAMP after <6 h. The glucocorticoid dexamethasone also promoted apoptosis and Bim expression in S49 cells. In contrast, both UV light and anti-mouse Fas monoclonal antibody promoted apoptosis in S49 cells but did not induce Bim expression. 8-CPT-cAMP also induced Bim expression and enhanced dexamethasone-promoted apoptosis in human T-cell leukemia CEM-C7–14 (glucocorticoid-sensitive) and CEM-C1–15 (glucocorticoid-resistant) cells; increased Bim expression in 8-CPT-cAMP-treated CEM-C1–15 cells correlated with conversion of the cells from resistance to sensitivity to glucocorticoid-promoted apoptosis. Induction of Bim appears to be a key event in cAMP-promoted apoptosis in both murine and human T-cell lymphoma and leukemia cells and thus appears to be a convergence point for the killing of such cells by glucocorticoids and agents that elevate cAMP. The mechanisms by which cAMP mediates apoptosis are not well understood. In the current studies, we used wild-type (WT) S49 T-lymphoma cells and the kin– variant (which lacks protein kinase A (PKA)) to examine cAMP/PKA-mediated apoptosis. The cAMP analog, 8-CPT-cAMP, increased phosphorylation of the cAMP response element-binding protein (CREB), activated caspase-3, and induced apoptosis in WT but not in kin– S49 cells. Using an array of 96 apoptosis-related genes, we found that treatment of WT cells with 8-CPT-cAMP for 24 h induced expression of mRNA for the pro-apoptotic gene, Bim. Real-time PCR analysis indicated that 8-CPT-cAMP increased Bim RNA in WT cells in <2 h and maintained this increase for >24 h. Bim protein expression increased in WT but not kin– cells treated with 8-CPT-cAMP or with the β-adrenergic receptor agonist isoproterenol. Both apoptosis and Bim expression were reversible with removal of 8-CPT-cAMP after <6 h. The glucocorticoid dexamethasone also promoted apoptosis and Bim expression in S49 cells. In contrast, both UV light and anti-mouse Fas monoclonal antibody promoted apoptosis in S49 cells but did not induce Bim expression. 8-CPT-cAMP also induced Bim expression and enhanced dexamethasone-promoted apoptosis in human T-cell leukemia CEM-C7–14 (glucocorticoid-sensitive) and CEM-C1–15 (glucocorticoid-resistant) cells; increased Bim expression in 8-CPT-cAMP-treated CEM-C1–15 cells correlated with conversion of the cells from resistance to sensitivity to glucocorticoid-promoted apoptosis. Induction of Bim appears to be a key event in cAMP-promoted apoptosis in both murine and human T-cell lymphoma and leukemia cells and thus appears to be a convergence point for the killing of such cells by glucocorticoids and agents that elevate cAMP. The second messenger cAMP alters the balance between cell growth and apoptosis in a cell type-dependent manner; cAMP stimulates growth arrest and apoptosis in certain lymphoid cells, especially poorly differentiated lymphoblastic cells, but promotes growth of other cell types (e.g. epithelial cells) and protects other cell types (e.g. neutrophils and eosinophils) from drug- and cytokine-promoted apoptosis (1Roger P.P. Reuse S. Maenhaut C. Dumont J.E. Vitam. Horm. 1995; 51: 59-191Crossref PubMed Scopus (96) Google Scholar, 2Usher L.R. Lawson R.A. Geary I. Taylor C.J. Bingle C.D. Taylor G.W. Whyte M.K. J. Immunol. 2002; 168: 1861-1868Crossref PubMed Scopus (173) Google Scholar, 3Chang H.S. Jeon K.W. Kim Y.H. Chung I.Y. Park C.S. Cell. Immunol. 2000; 203: 29-38Crossref PubMed Scopus (24) Google Scholar). The greater susceptibility of certain tumor cells to cAMP-mediated growth arrest and/or apoptosis has spurred interest in developing cAMP analogs or related agents as anti-cancer drugs (4Lerner A. Kim D.H. Lee R. Leuk. Lymphoma. 2000; 37: 39-51Crossref PubMed Scopus (73) Google Scholar, 5Ogawa R. Streiff M.B. Bugayenko A. Kato G.J. Blood. 2002; 99: 3390-3397Crossref PubMed Scopus (78) Google Scholar, 6Schwede F. Maronde E. Genieser H. Jastorff B. Pharmacol. Ther. 2000; 87: 199-226Crossref PubMed Scopus (211) Google Scholar), including those for glucocorticoid-resistant malignancies (4Lerner A. Kim D.H. Lee R. Leuk. Lymphoma. 2000; 37: 39-51Crossref PubMed Scopus (73) Google Scholar, 5Ogawa R. Streiff M.B. Bugayenko A. Kato G.J. Blood. 2002; 99: 3390-3397Crossref PubMed Scopus (78) Google Scholar, 6Schwede F. Maronde E. Genieser H. Jastorff B. Pharmacol. Ther. 2000; 87: 199-226Crossref PubMed Scopus (211) Google Scholar, 7Krett N.L. Zell J.L. Halgren R.G. Pillay S. Traynor A.E. Rosen S.T. Clin. Cancer Res. 1997; 3: 1781-1787PubMed Google Scholar). The mechanisms of cAMP-mediated apoptosis are poorly understood, although PKA 1The abbreviations used are: PKA, protein kinase A; 8-CPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic monophosphate; Dex, dexamethasone; CREB, cAMP response element-binding protein; CRE, cAMP response element; CBP, CREB-binding protein; 8-Cl-cAMP, 8-chloro-cyclic adenosine monophosphate; Bt2-cAMP, (N6,O2′)-dibutyryl adenosine 3′:5′-cyclic monophosphate; 8-Br-cAMP, 8-bromo-adenosine 3′:5′-cyclic monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; WT, wild type. 1The abbreviations used are: PKA, protein kinase A; 8-CPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic monophosphate; Dex, dexamethasone; CREB, cAMP response element-binding protein; CRE, cAMP response element; CBP, CREB-binding protein; 8-Cl-cAMP, 8-chloro-cyclic adenosine monophosphate; Bt2-cAMP, (N6,O2′)-dibutyryl adenosine 3′:5′-cyclic monophosphate; 8-Br-cAMP, 8-bromo-adenosine 3′:5′-cyclic monophosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; WT, wild type. is known to be involved (5Ogawa R. Streiff M.B. Bugayenko A. Kato G.J. Blood. 2002; 99: 3390-3397Crossref PubMed Scopus (78) Google Scholar, 8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google Scholar, 9Matuszyk J. Cebrat M. Kalas W. Strzadala L. Int. Immunopharmacol. 2002; 2: 435-442Crossref PubMed Scopus (11) Google Scholar). Overexpression of certain anti-apoptotic proteins, such as Bcl-2, can protect lymphoma cells from cAMP-mediated apoptosis; such protection appears to be distinct from the effects of cAMP on cell cycle arrest (10Zhang L. Insel P.A. Am. J. Physiol. 2001; 281: C1642-C1647Crossref PubMed Google Scholar). Although over-expression of Bcl-2 can protect cells from cAMP-mediated apoptosis, whether endogenous levels of Bcl-2 or other Bcl-2 family members are involved in cAMP-induced apoptosis is not known. In the current study, we used murine S49 lymphoma cells and human CEM cells as model systems to investigate changes in gene expression that contribute to cAMP-promoted apoptosis. S49 cells, which arose in a Balb/c mouse as a transplantable T-cell tumor, grow in suspension culture with a doubling time of 16–18 h and respond to agonists that elevate cAMP by undergoing G1 arrest and apoptotic cell death (8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google Scholar, 10Zhang L. Insel P.A. Am. J. Physiol. 2001; 281: C1642-C1647Crossref PubMed Google Scholar, 11Coffino P. Bourne H.R. Friedrich U. Hochman J. Insel P.A. Lemaire I. Melmon K.L. Tomkins G.M. Recent Prog. Horm. Res. 1976; 32: 669-684PubMed Google Scholar). kin– S49 cells, isolated as a clonal variant by virtue of resistance to killing by cAMP analogs, lack PKA activity, cAMP-promoted G1 growth arrest, and apoptosis (8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google Scholar, 10Zhang L. Insel P.A. Am. J. Physiol. 2001; 281: C1642-C1647Crossref PubMed Google Scholar, 11Coffino P. Bourne H.R. Friedrich U. Hochman J. Insel P.A. Lemaire I. Melmon K.L. Tomkins G.M. Recent Prog. Horm. Res. 1976; 32: 669-684PubMed Google Scholar, 12Insel P.A. Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 190: 896-898Crossref PubMed Scopus (117) Google Scholar, 13Steinberg R.A. O'Farrell P.H. Friedrich U. Coffino P. Cell. 1977; 10: 381-391Abstract Full Text PDF PubMed Scopus (129) Google Scholar). Thus, WT and kin– S49 cells are a useful system to use in defining PKA-dependent events involved in cell growth and death. CEM cells provide a model of human lymphoid tumor cells with glucocorticoid sensitivity or resistance, one in which cAMP can be used to enhance glucocorticoid sensitivity (14Thompson E.B. Medh R.D. Zhou F. Ayala-Torres S. Ansari N. Zhang W. Johnson B.H. J. Steroid Biochem. Mol. Biol. 1999; 69: 453-461Crossref PubMed Scopus (23) Google Scholar). We show here that the Bcl-2 family member, Bim, may be a key protein in both cAMP/PKA- and glucocorticoid-promoted apoptosis of both murine S49 and human leukemia cells. Materials—Cell culture reagents and a Superscript II reverse transcriptase kit were obtained from Invitrogen, mouse apoptosis GEArray Q series kit (MM-001 N) from SuperArray, annexin V-fluorescein isothiocyanate from BD Pharmingen, Master Mix SYBR green from Applied Biosystems, propidium iodide, 8-CPT-cAMP, (–)-isoproterenol, IBMX, and dexamethasone (Dex) from Sigma, caspase-3 colorimetric assay kit from R&D Systems, biotin-16-dUTP from Roche Applied Science, RNase inhibitor and Maloney murine leukemia virus reverse transcriptase from Promega, Bim primers and GAPDH primers from Integrated DNA Technologies, Inc., subcellular proteome extraction kit from Calbiochem, Restore™ Western blot stripping buffer and BCA Protein Assay kit® from Pierce, and Immobilon-P® polyvinylidine difluoride membrane from Millipore. The following antibodies were used: CREB antibody, phospho-CREB antibody, anti-mouse IgG-horseradish peroxidase, and anti-rabbit IgG-horseradish peroxidase from Cell Signaling Technology; anti-mouse Fas monoclonal antibody, Jo2, and polyclonal rabbit anti-Bim from BD Pharmingen; and actin (mouse monoclonal IgG1) from Santa Cruz Biotechnology. Cell Culture—S49 lymphoma cells (2–3 × 105 cells/ml) were grown in suspension in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum, 10 mm HEPES in a humidified atmosphere containing 10% CO2 at 37 °C. The CEM-C7–14 and CEM-C1–15 human acute T-cell leukemia cell lines were provided by Dr. E. Brad Thompson (University of Texas, Galveston) and were cultured in RPMI medium supplemented with 2 mm l-glutamine, 10% fetal bovine serum. Cultures were initiated at a density of 2 × 105 cells/ml and grown in a humidified 5% CO2 incubator at 37 °C. Apoptosis Assay and Analysis of DNA Content and Cell Cycle by Flow Cytometry—Apoptosis was evaluated by assessment of annexin V binding or by propidium iodide analysis of cells for apoptotic sub-G1 levels of DNA (10Zhang L. Insel P.A. Am. J. Physiol. 2001; 281: C1642-C1647Crossref PubMed Google Scholar). Caspase-3 Activity Assay—Cells treated with 8-CPT-cAMP were collected by centrifugation at the appropriate times. Caspase-3 activity was assessed according to the manufacturer's instructions. cDNA Microarray Analysis—Total cellular RNA was isolated by using a Qiagen RNeasy mini-column with extract prepared from cells treated with 8-CPT-cAMP (100 μm) or vehicle for 24 h. The biotin-dUTP-labeled cDNA probe was generated in the presence of a designed set of gene-specific primers using total RNA (5 μg/filter) and 200 units of Maloney murine leukemia virus reverse transcriptase. Hybridization of the array filters, washing conditions, and chemiluminescent detection steps were performed according to the manufacturer's instructions. Chemiluminescent signals were captured with a UVP BioImaging system. WT and kin– cDNA were performed time with filters, and RNA was isolated at times. 8-CPT-cAMP-treated cDNA were were with a GEArray and and to GAPDH mRNA of cDNA and Real-time of total RNA was performed as cDNA was from total RNA with Superscript II reverse transcriptase kit according to the manufacturer's instructions. analysis of cDNA was assessed by of SYBR mouse Bim we used as the and as the reverse mouse GAPDH we used as the and as the reverse PCR containing of cDNA of and reverse and SYBR PCR Master Mix was performed in a total of were as of 10 at by of at at and °C. cDNA were in using the Applied and with Applied were using the The which is to the is the cycle the is or was to and after for GAPDH using and treatment J. I. Biochem. 1999; PubMed Scopus Google Scholar). and used a subcellular proteome extraction kit according to the manufacturer's to extract and of S49 lymphoma cells. were treated with 8-CPT-cAMP or × in of containing of inhibitor and for 10 at with The suspension was at × at for 10 The was used as the the was in of II containing of inhibitor and for at °C. was at × at and the used as the were at the appropriate time after in and × in of buffer mm mm mm and containing inhibitor Bim protein or of buffer 10% mm CREB for on for 10 and (5 at the were used as cell Protein using a BCA protein assay was by the of were treated and on according to the manufacturer's instructions. were with in for 2 h at or at and with anti-Bim antibody or phospho-CREB antibody at °C. in containing were with antibodies at a of and times. Protein was by using a UVP BioImaging system. were in Restore™ and with actin antibody (mouse monoclonal IgG1) or CREB antibody for were performed in or and was at times. are the were by analysis of by between was on a A of was 8-CPT-cAMP PKA to Caspase-3 Activity and Apoptosis of WT S49 S49 cells apoptosis in response to the agents that increase cAMP levels (8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google Scholar). We assessed the pro-apoptotic effects of the cAMP 8-CPT-cAMP (100 μm) on both WT and kin– S49 cells. Recent has indicated that cAMP analogs (e.g. 8-Cl-cAMP, 8-Br-cAMP, and as a on S49 kin– but growth of S49 WT cells R.A. J. Cell. Physiol. 2002; PubMed Scopus Google Scholar). We found that 8-CPT-cAMP promoted apoptosis in WT but not kin– S49 cells in a to that of cAMP analog, (8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google Scholar), with a increase in apoptosis after 24 h 8-CPT-cAMP also increased caspase-3 activity (which is activated apoptosis J. Cell. 87: Full Text Full Text PDF PubMed Scopus Google of WT but not kin– S49 cells by 24 h of 8-CPT-cAMP treatment a that this of cAMP is PKA, we the a that not PKA but a for the protein A.E. J. M. F. Genieser J.L. J.L. Cell Biol. 2002; PubMed Scopus Google Scholar). to apoptosis not in and in with detection of apoptosis by propidium iodide were by use of an annexin V binding assay not Bim expression in WT but kin– S49 of the time of caspase-3 and apoptosis, we that apoptosis may from changes in gene expression. with this 8-CPT-cAMP increased phosphorylation of the CREB protein in WT but not kin– S49 cells phosphorylation of CREB by be to gene expression in WT but not kin– S49 cells. this we isolated total RNA from WT and kin– cells, treated with vehicle or with 8-CPT-cAMP for 24 and used a GEArray Q series mouse apoptosis cDNA expression array to gene expression. array the expression of 96 murine involved in apoptosis and has to be as by PCR J. H. M. J. 2002; PubMed Scopus Google Scholar). We found that WT and kin– S49 cells show gene expression as assessed by this array not 8-CPT-cAMP treatment of WT but not kin– cells for 24 the expression of Bim of cell increased this was the increase in mRNA expression of pro-apoptotic in the We did not a in Bcl-2 a by PCR analysis not on we that Bim to cAMP/PKA-mediated apoptosis, Bim as a that can members of the Bcl-2 L. A. S. J. PubMed Scopus Google Scholar). this we expression of Bim mRNA by of cDNA for PCR using total RNA from WT and kin– cells treated with Bim was induced as as 2 h following 8-CPT-cAMP treatment and for at h in WT but not kin– S49 cells Bim a to including and in and of which apoptosis L. A. S. J. PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Mol. Immunol. PubMed Scopus Google Scholar, R. C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Using we and protein in S49 membrane but not and found that 8-CPT-cAMP increased expression of both and in WT but not kin– cells and Thus, Bim expression induced by 8-CPT-cAMP in S49 cells on PKA activity, levels of Bim mRNA and protein expression did not between WT and kin– S49 cells not Bim with cAMP and Apoptosis but with UV or whether a receptor that cAMP in S49 cells to apoptosis and Bim expression in a to 8-CPT-cAMP, we used the β-adrenergic agonist in the presence of mm in increased apoptosis and Bim protein expression in WT but not kin– cells a time to that with but also UV and antibody apoptosis of S49 cells in induced expression of and Although UV light and antibody promoted apoptosis and did 8-CPT-cAMP did not induce Bim protein expression of Dex, 8-CPT-cAMP, UV and anti-mouse Fas on apoptosis and Bim expression in WT S49 cells. WT S49 cells were treated with 8-CPT-cAMP (100 8-CPT-cAMP (100 μm) UV light and anti-mouse Fas for the indicated time are as apoptosis The are from and 8-CPT-cAMP and UV light treatment and treatment Bim protein expression in WT S49 cells treated with 8-CPT-cAMP (100 or 8-CPT-cAMP for 24 h and UV light and for h was by of cell analysis of to actin from is the and with 8-CPT-cAMP for cAMP-promoted Apoptosis and of Bim to the of the response to 8-CPT-cAMP as well as the time to cells to apoptosis. were with 8-CPT-cAMP for to 8-CPT-cAMP, and to culture for to h. The show that a or with 8-CPT-cAMP was a to apoptosis; cells to h treatment with 8-CPT-cAMP to to apoptosis. in in with the of apoptosis, of 8-CPT-cAMP Bim mRNA expression to between and on Apoptosis and Bim in CEM whether of Bim by cAMP in other lymphoid cells and in other we assessed apoptosis and Bim expression in response to 8-CPT-cAMP, Dex, and the of 8-CPT-cAMP and in the human acute leukemia CEM cell in CEM-C7–14 (glucocorticoid-sensitive) and CEM-C1–15 (glucocorticoid-resistant) cells R.D. Johnson B.H. Thompson E.B. Cancer Res. Google Scholar). that treatment with cAMP analogs glucocorticoid sensitivity in CEM-C1–15 cells R.D. Johnson B.H. Thompson E.B. Cancer Res. Google Scholar). We found that induced apoptosis in CEM-C7–14 but not in CEM-C1–15 cells 8-CPT-cAMP (100 or with Dex, in the cell lines and 8-CPT-cAMP with to apoptosis, in the cell Western that of Bim were induced by 8-CPT-cAMP in CEM-C7–14 and CEM-C1–15 cells but that induced Bim in CEM-C7–14 cells. The of cAMP and also induced Bim expression Thus, the in human leukemia CEM cells show a of cAMP to apoptosis and increase Bim expression as in murine lymphoma S49 cells. Although the second messenger cAMP was to cell growth and cell death (e.g. 11Coffino P. Bourne H.R. Friedrich U. Hochman J. Insel P.A. Lemaire I. Melmon K.L. Tomkins G.M. Recent Prog. Horm. Res. 1976; 32: 669-684PubMed Google and Johnson Biochem. 1975; PubMed Scopus Google Scholar), the of cAMP-promoted cell death are poorly understood. in the S49 cell system that cell death promoted by cAMP analogs apoptosis (8Yan L. Herrmann V. Hofer J.K. Insel P.A. Am. J. Physiol. 2000; 279: C1665-C1674Crossref PubMed Google and that apoptosis and G1 growth arrest are events that in WT but not kin– cells (10Zhang L. Insel P.A. Am. J. Physiol. 2001; 281: C1642-C1647Crossref PubMed Google Scholar, 11Coffino P. Bourne H.R. Friedrich U. Hochman J. Insel P.A. Lemaire I. Melmon K.L. Tomkins G.M. Recent Prog. Horm. Res. 1976; 32: 669-684PubMed Google Scholar), thus of PKA in the apoptotic In the current we show that the cAMP 8-CPT-cAMP phosphorylation of CREB and caspase-3, as well as apoptosis in WT but not kin– S49 cells Using a cDNA to cAMP/PKA-mediated changes in gene we found that mRNA expression for Bim the pro-apoptotic in WT S49 cells in response to Real-time PCR and the of Bim mRNA and protein by 8-CPT-cAMP in WT S49 cells and in human CEM leukemia cells. In we found that the β-adrenergic agonist promoted apoptosis and enhanced expression of Bim protein a time to that of 8-CPT-cAMP, that that increase cAMP levels an to the cAMP the show a between Bim expression and apoptosis in both murine and human lymphoid cells as be are with the that Bim mediates apoptosis. Bim is a of the Bcl-2 which apoptosis. family is by at one of Bcl-2 in Bcl-2 A. L. 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Cancer Res. 1997; 3: 1781-1787PubMed Google Scholar). show that 8-CPT-cAMP and are in apoptosis in both and glucocorticoid-resistant CEM cells and that effects with of Bim. are with the that Bim expression is a convergence point for killing of lymphoid cells by glucocorticoids and agents that elevate cAMP. expression of Bim by 8-CPT-cAMP appears to be a key in both murine and human lymphoid cells. Bim expression thus appears to be a by the and glucocorticoid receptor in apoptosis. We Dr. E. Brad Thompson for CEM cells, for and for with