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• W2032251372 abstract "The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-ΔY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-ΔY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-ΔY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblΔY371 cells arrested in G1 when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G1 fraction, while only 4% of the 32D/Cbl-ΔY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G1 fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-ΔY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-ΔY317 cells. These data suggest that the Cbl-ΔY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-ΔY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions. The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-ΔY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-ΔY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-ΔY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblΔY371 cells arrested in G1 when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G1 fraction, while only 4% of the 32D/Cbl-ΔY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G1 fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-ΔY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-ΔY317 cells. These data suggest that the Cbl-ΔY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-ΔY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions. interleukin-3 B-cell lymphoma 2 extracellular signal-regulated kinase Janus kinase phosphatidylinositol 3-kinase signal transducer and activator of transcription murine IL-3 recombinant murine IL-3 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The c-Cbl proto-oncogene has attracted considerable attention in recent years, since it becomes phosphorylated on tyrosine residues following activation of a wide variety of cell surface receptors. Receptors whose activation induces the phosphorylation of Cbl include the T cell receptor (1Sawasdikosol S. Chang J.-H. 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Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), granulocyte-macrophage colony-stimulating factor (17Odai H. Sasaki K. Iwamatsu A. Hanazono Y. Tanaka T. Mitani K. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 10800-10805Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), thrombopoietin (19Oda A. Ozaki K. Druker B.J. Miyakawa Y. Miyazaki H. Hanada M. Morita H. Ohashi H. Ikeda Y. Blood. 1996; 88: 1330-1338Crossref PubMed Google Scholar), and prolactin (20Hunter S. Koch B.L. Anderson S.M. Mol. Endocrinol. 1997; 11: 1213-1222Crossref PubMed Scopus (41) Google Scholar). Cbl is a 120-kDa protein with 22 tyrosine residues, an amino-terminal region able to bind to phosphotyrosine (21Lupher M.L. Reedquist K. Miyake S. Langdon W.Y. Band H. J. Biol. Chem. 1997; 271: 24063-24068Abstract Full Text Full Text PDF Scopus (166) Google Scholar,22Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Current Biology. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and a proline-rich region with numerous Src homology 3 binding sites. The large size of the protein suggests that it may function as a large adapter or scaffolding molecule that could regulate the activation of other downstream signaling molecules in a manner similar to that of insulin-receptor substrate-1 (23White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). Other studies have suggested that Cbl functions as a negative regulator of signaling, perhaps by stimulating the down-regulation of growth factor receptors (24Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes and Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar, 25Miyake S. Lupher M.L. Druker B. Band H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7927-7932Crossref PubMed Scopus (237) Google Scholar). Several recent studies have suggested that down-regulation of growth factor receptors requires the RING finger motif of Cbl functioning as an E2-dependent ubiquitin-protein ligase inducing the ubiquitination of growth factor receptors (24Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes and Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (720) Google Scholar, 26Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 27Joazeiro C.A.P. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.-C. Science. 1999; 286: 309-312Crossref PubMed Scopus (916) Google Scholar, 28Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Molecular Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 29Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Src-like kinases (3Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 7Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 18Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Ojaniemi M. Martin S.S. Dolfi F. Olefsky J.M. Vuori K. J. Biol. Chem. 1997; 272: 3780-3787Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 31Hartley D. Corvera S. J. Biol. Chem. 1996; 271: 21939-21943Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 32Tsygankov A.Y. Mahajan S. Fincke J.E. Bolen J.B. J. Biol. Chem. 1996; 271: 27130-27137Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), Syk and/or ZAP-70 (4Fournel M. Davidson D. Weil R. Veillette A. J. Expt. Med. 1997; 183: 301-306Crossref Scopus (123) Google Scholar, 21Lupher M.L. Reedquist K. Miyake S. Langdon W.Y. Band H. J. Biol. Chem. 1997; 271: 24063-24068Abstract Full Text Full Text PDF Scopus (166) Google Scholar, 33Ota Y. Beitz L.O. Scharenberg A.M. Donovan J.A. Kinet J.-P. Samelson L.E. J. Expt. Med. 1996; 184: 1713-1723Crossref PubMed Scopus (86) Google Scholar), phosphatidylinositol 3-kinase (PI 3-kinase) (2Hartley D. Meisner H. Corvera S. J. Biol. Chem. 1995; 270: 18260-18263Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 3Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 7Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 18Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 31Hartley D. Corvera S. J. Biol. Chem. 1996; 271: 21939-21943Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,34Ueno H. Sasaki K. Honda H. Nakamoto T. Yamagata T. Miyagawa K. Mitani K. Yazaki Y. Hirai H. Blood. 1998; 91: 46-53Crossref PubMed Google Scholar, 35Sattler M. Salgia R. Shrikhande G. Verma S. Pisick E. Prasad K.V.S. Griffin J.D. J. Biol. Chem. 1997; 272: 10248-10253Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and several small adapter proteins including Crk, Shc, and Grb2 (1Sawasdikosol S. Chang J.-H. Pratt J.C. Wolf G. Shoelson S.E. Burakoff S.J. J. Immunol. 1996; 157: 110-116PubMed Google Scholar, 3Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 7Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 8Smit L. van Der Horst G. Borst J. Oncogene. 1996; 13: 381-389PubMed Google Scholar, 35Sattler M. Salgia R. Shrikhande G. Verma S. Pisick E. Prasad K.V.S. Griffin J.D. J. Biol. Chem. 1997; 272: 10248-10253Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36Donovan J.A. Ota Y. Langdon W.Y. Samelson L.E. J. Biol. Chem. 1996; 271: 26369-26374Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 37Sattler M. Salgia R. Shrikhande G. Verma S. Uemura N. Law S.F. Golemis E.A. Griffin J.D. J. Biol. Chem. 1997; 272: 14320-14326Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) have been observed to be associated with Cbl in either a constitutive or ligand-induced manner. It has been suggested that either the Syk/ZAP-70 family of tyrosine kinases (38Deckert M. Elly C. Altman A. Liu Y.-C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) or Src-like kinases (38Deckert M. Elly C. Altman A. Liu Y.-C. J. Biol. Chem. 1998; 273: 8867-8874Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 39Feshchenko E.A. Langdon W.Y. Tsygankov A.Y. J. Biol. Chem. 1998; 273: 8323-8331Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 40Dombrosky-Ferlan P.M. Corey S.J. Oncogene. 1997; 14: 2019-2024Crossref PubMed Scopus (40) Google Scholar, 41Hunter S. Burton E.A. Wu S.C. Anderson S.M. J. Biol. Chem. 1999; 274: 2097-2106Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) might be responsible for the phosphorylation of Cbl. We have suggested that the phosphorylation of Cbl could regulate the activation of PI 3-kinases in response to certain stimuli such as cytokines like IL-3 and prolactin (18Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 20Hunter S. Koch B.L. Anderson S.M. Mol. Endocrinol. 1997; 11: 1213-1222Crossref PubMed Scopus (41) Google Scholar, 41Hunter S. Burton E.A. Wu S.C. Anderson S.M. J. Biol. Chem. 1999; 274: 2097-2106Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Since phosphatidylinositol 3-kinase is thought to regulate the antiapoptotic protein kinase Akt (42Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 43Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes and Dev. 1997; 11: 701-713Crossref PubMed Scopus (980) Google Scholar, 44Skorski T. Bellacosa A. Nieborowska-Skorska M. Majewski M. Martinez R. Choi J.K. Trotta R. Wlodarski P. Perrotti D. Chan T.O. Wasik M.A. Tsichlis P.N. Calabretta B. EMBO J. 1997; 16: 6151-6161Crossref PubMed Scopus (558) Google Scholar, 45Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 46Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2222) Google Scholar, 47Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (966) Google Scholar, 48Songyang Z. Baltimore D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (323) Google Scholar), Cbl might represent an integration point for proliferative and antiapoptotic signaling pathways. Evidence demonstrating that Cbl plays a critical role in these functions is lacking, particularly since mice bearing homologous loss of both alleles encoding c-Cbl have a relatively mild phenotype (49Murphy M.A. Schnall R.G. Venter D.J. Barnett L. Bertoncello I. Thien C.B. Langdon W.Y. Bowtell D.D. Mol. Cell. Biol. 1998; 18: 4872-4882Crossref PubMed Scopus (332) Google Scholar). Cbl was first discovered as the oncogene present in the Cas NS-1 retrovirus, which has been shown to induce B-cell lymphomas and myeloid leukemia in mice (50Langdon W.Y. Hartley J.W. Klinken S.P. Ruscetti S.K. Morse H.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1168-1172Crossref PubMed Scopus (283) Google Scholar). The v-cbl oncogene is also capable of transforming NIH3T3 cells in vitro, and these transformed cells are tumorigenic when injected into nude mice (51Andoniou C.E. Thein C.B.F. Langdon W.Y. J. Biol. Chem. 1995; 270: 4515-4523Google Scholar). The v-Cbl oncogene consists of the amino-terminal end of the gag gene fused to the amino-terminal end of c-Cbl encompassing the amino-terminal phosphotyrosine binding region but lacking the ring finger motif, the proline-rich region, and the C-terminal end, which contains several major phosphorylation sites (50Langdon W.Y. Hartley J.W. Klinken S.P. Ruscetti S.K. Morse H.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1168-1172Crossref PubMed Scopus (283) Google Scholar, 52Blake T.J. Shapiro M. Morse H.C. Langdon W.Y. Oncogene. 1991; 6: 653-657PubMed Google Scholar). Other oncogenic forms of c-Cbl have been discovered (51Andoniou C.E. Thein C.B.F. Langdon W.Y. J. Biol. Chem. 1995; 270: 4515-4523Google Scholar). The murine pre-B cell line 70Z expresses an oncogenic form of Cbl in which 17 amino acids, from 366 to 382, have been deleted (51Andoniou C.E. Thein C.B.F. Langdon W.Y. J. Biol. Chem. 1995; 270: 4515-4523Google Scholar). Studies by Langdon and colleagues have demonstrated that deletion of either of two single amino acids within this 17-amino acid region results in the oncogenic activation of c-Cbl (51Andoniou C.E. Thein C.B.F. Langdon W.Y. J. Biol. Chem. 1995; 270: 4515-4523Google Scholar). In this study, we have utilized one of these mutants, CblΔY371, in which Tyr371 has been deleted. The expression of oncogenes in hematopoietic cells results in numerous changes, including induction of growth factor-independent proliferation (53Anderson S.M. Carroll P.M. Lee F.D. Oncogene. 1989; 5: 317-325Google Scholar, 54Anderson S.M. Mladenovic J. 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Recent studies have demonstrated that the expression of oncogenic forms of Cbl in fibroblasts results in an increase in the number of tyrosine-phosphorylated proteins, suggesting that one or more tyrosine kinases have been activated by v-Cbl (61Bonita D.P. Miyake S. Lupher M.L. Langdon W.Y. Band H. Mol. Cell. Biol. 1997; 17: 4597-4610Crossref PubMed Google Scholar). We have previously studied the ability of two activated tyrosine kinases, v-Src and BCR-ABL, to induce growth factor-independent proliferation of growth factor-dependent cell lines (53Anderson S.M. Carroll P.M. Lee F.D. Oncogene. 1989; 5: 317-325Google Scholar, 54Anderson S.M. Mladenovic J. Blood. 1996; 87: 238-244Crossref PubMed Google Scholar). In both cases, we were able to provide evidence that these oncogenes induced the autocrine production of growth factors such as IL-3 or granulocyte-macrophage colony-stimulating factor. These oncogenes are also able to block the ability of granulocyte colony-stimulating factor to induce terminal differentiation of hematopoietic progenitor cell lines such as 32Dcl3 (53Anderson S.M. Carroll P.M. Lee F.D. Oncogene. 1989; 5: 317-325Google Scholar, 57Rovera G. Valtieri M. Mavilio F. Reddy E.P. Oncogene. 1987; 1: 29-35PubMed Google Scholar). Therefore, we hypothesized that oncogenic forms of Cbl would induce growth factor-independent proliferation of factor-dependent cells in a manner like that observed with v-Src. Contrary to our expectation, we observed instead that the CblΔY371 deletion mutant suppressed apoptosis induced by growth factor withdrawal. The mechanism by which apoptosis is suppressed appears to involve an increase in the level of the antiapoptotic protein Bcl-2. The 32Dcl3 cell line was obtained from Dr. Joel Greenberger (University of Pittsburgh), and their cultivation has been described recently (18Anderson S.M. Burton E.A. Koch B.L. J. Biol. Chem. 1997; 272: 739-745Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Fetal calf serum was from Summit Biotechnology (Fort Collins, CO). Recombinant murine IL-3 (mIL-3) was obtained from Becton-Dickinson/Collaborative Biotechnology, Inc. (Franklin Lakes, NJ). All other media components were from Life Technologies, Inc. 32Dcl3 cells expressing the various mutants of c-Cbl were generated by introducing the plasmid DNAs of interest into 32Dcl3 cells with a Cell-Porator (1000 V/cm at 800 microfarads) (Life Technologies). Electroporation chambers used had a 0.4-cm gap between the electrodes, and cells were resuspended in Dulbecco's modified Eagle's medium without any additional supplements at room temperature. The CblΔY371 mutant was expressed in the pZEN-Neo vector (51Andoniou C.E. Thein C.B.F. Langdon W.Y. J. Biol. Chem. 1995; 270: 4515-4523Google Scholar), which contains the neomycin resistance marker as a dominant selectable marker. A Bcl-2 expression vector was constructed in which the coding sequence of Bcl-2 was inserted into the pcDNA3 expression vector (InVitrogen, Carlsbad, CA). Following electroporation, the cells were cultured in the presence of IL-3 for 2–3 days. Transfected cells were then selected by the addition of 1.0 mg/ml G418 (Life Technologies). G418-resistant cells were allowed to expand in the presence of IL-3 for 3 days, after which the drug was removed. Conditions that allow the isolation of growth factor-independent cells have been previously described (53Anderson S.M. Carroll P.M. Lee F.D. Oncogene. 1989; 5: 317-325Google Scholar, 54Anderson S.M. Mladenovic J. Blood. 1996; 87: 238-244Crossref PubMed Google Scholar). Single cell clones were then isolated following growth of the cells at limiting dilution in semisolid media containing 0.6% SeaPlaque agarose (FMC Corp., Freeport, ME). Isolated colonies were picked from soft agar, expanded in liquid culture, and used in the described experiments. Multiple clones of 32D/CblΔY371cells were obtained from two separate electroporations to eliminate the possibility of examining “sister” clones. All clones used in these studies were negative for mycoplasma; this is important, since a recent report indicates that mycoplasma can suppress apoptosis of 32Dcl3 and induce growth factor-independent proliferation (62Feng S.-H. Tsai S. Rodriguez J. Lo S.-C. Mol. Cell. Biol. 1999; 19: 7995-8002Crossref PubMed Scopus (93) Google Scholar). Cells were cultured for 16 h in media supplemented with 7.5% fetal calf serum to reduce the basal level of tyrosine-phosphorylated proteins prior to stimulation with recombinant mIL-3 for the indicated periods of time. In one set of experiments, cells were removed from IL-3 and starved for 0–8 h to determine the kinetics with which phosphorylated proteins disappeared. Cells to be immunoprecipitated were lysed in EB (50 mm NaCl, 10 mm Tris, pH 7.4, 5 mm EDTA, 50 mmNaF, 1% Triton X-100, 1 mm sodium orthovanadate with 100 units/ml Kallikrein inhibitor), and the lysates were clarified by spinning at 13,000 rpm in a Savant RCF13K refrigerated microcentrifuge for 30 min. A 1-μg amount of the indicated antibody was added to a cell lysate made from 2 × 107 32Dcl3 cells in a final volume of 1 ml and placed on a rocking platform for 1 h at 4 °C. The immune complexes were collected by adding 30 μl of Pansorbin (Calbiochem) to each immunoprecipitate for 1 h. The bound proteins were washed three times with lysis buffer, and the immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis. The resolved proteins were electrotransferred to Immobilon membranes (Millipore Corp., Bedford, MA). Detection of proteins by immunoblotting was conducted using the enhanced chemiluminescence lighting (ECL) system according to the manufacturer's recommendations (Amersham Pharmacia Biotech). Agarose-conjugated anti-phosphotyrosine monoclonal antibody 4G10, rabbit anti-JAK2, and sheep anti-Akt were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-Akt was obtained from New England Biolabs (Beverly, MA), and anti-phospho-ERK was obtained from Promega Biotechnology (Madison, WI). Polyclonal antibodies directed against Cbl, ERK1/2, Bcl-2, Bax, Bcl-XL, Bak, and A1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibodies to Mcl1 and Bad were obtained from Transduction Laboratories (Lexington, KY). A monoclonal antibody directed against the influenza HA epitope tag (clone 12CA5) was obtained from Roche Molecular Biochemicals. A monoclonal antibody 4G10 directed against phosphotyrosine was kindly provided by Dr. Brian Druker (University of Oregon Health Sciences Center, Portland, OR). A rabbit polyclonal antibody directed against STAT5 was provided by Andrew Larner (Cleveland Clinic, Cleveland, OH). Nonimmune rabbit serum was obtained from our own nonimmunized animals. Cultures were initiated at 2 × 105 cells/ml in medium that either contained or lacked 50 units/ml recombinant murine IL-3 (rmIL-3). The proliferation of 32Dcl3 cells was monitored by direct counting of viable cells, which excluded trypan blue. Results were plotted as the number of viable cells at each time point examined. In one study, 100 nmwortmannin (Calbiochem) was added to each set of cells, and the proliferation of cells was monitored as described. Controls for this study included cultures with the same concentration of ethanol, the solvent in which the wortmannin was dissolved, as that present in the wortmannin-treated cultures. Cells were cultured in the presence or absence of 50 units/ml rmIL-3 for varying periods of time. Samples of cells were withdrawn at varying time points, and the cells were pelleted by centrifugation at 1,500 rpm for 5 min in a refrigerated centrifuge. The supernatant fluid was removed, and the pellet was washed once with phosphate-buffered saline. The supernatant fluid was removed, and the pellet was vortexed briefly. A 0.5-ml volume of saponin/propridium iodide solution was added (PBS containing 0.3% saponin, 25 μg/ml propidium iodide, 10 mm EDTA, 0.2 mg/ml RNase) and incubated for 10 min at room temperature. Stained cells were stored for up to 24 h at 4 °C before analysis. Ten thousand stained cells were analyzed with a Coulter XL flow cytometer (Hialeah, FL). Cell cycle modeling was performed with the ModFit software package (Verity House Software, Topsham, ME). The Caspase-3 Cellular Activity Assay Kit PLUS (Catalogue number AK-703) from BIOMOL (Plymouth Meeting, PA) was used according to the manufacturer's suggestions. At varying time points, a sample of cells used was removed and pelleted by centrifugation. The cells were lysed in cell lysis buffer (50 mm HEPES, pH 7.4, 100 mm NaCl, 0.1% CHAPS, 10 mm dithiothreitol, 1 mm EDTA, 10% glycerol). Following complete lysis of the cells, the lysate was clarified by centrifugation at 10,000 × g for 10 min at 4 °C, and the supernatant was frozen at −70 °C until all the samples were collected. Caspase assays were performed as outlined by the manufacturer, and the cleavage of the substrate,N-acetyl-Asp-Glu-Val-Asp-p-nitroanaline), was monitored at 1-min intervals on a microtiter plate reader at 405 nm. Data were plotted as A405 versus time for each sample. The initial time period over which change in ODversus time was linear was used to" @default.
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• W2032251372 title "Suppression of Apoptosis Induced by Growth Factor Withdrawal by an Oncogenic Form of c-Cbl" @default.
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• W2032251372 doi "https://doi.org/10.1074/jbc.m009386200" @default.
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