{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T08:44:32Z","timestamp":1791276272523,"version":"4.1.0"},"reference-count":45,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5531","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2001,8,3]]},"abstract":"<jats:p>\n                    Many hematopoietic cells undergo apoptosis when deprived of specific cytokines, and this process requires de novo RNA\/protein synthesis. Using DNA microarrays to analyze interleukin-3 (IL-3)\u2013dependent murine FL5.12 pro\u2013B cells, we found that the gene undergoing maximal transcriptional induction after cytokine withdrawal is\n                    <jats:italic>24p3<\/jats:italic>\n                    , which encodes a secreted lipocalin. Conditioned medium from IL-3\u2013deprived FL5.12 cells contained 24p3 and induced apoptosis in na\u0131\u0308ve FL5.12 cells even when IL-3 was present. 24p3 also induced apoptosis in a wide variety of leukocytes but not other cell types. Apoptotic sensitivity correlated with the presence of a putative 24p3 cell surface receptor. We conclude that IL-3 deprivation activates\n                    <jats:italic>24p3<\/jats:italic>\n                    transcription, leading to synthesis and secretion of 24p3, which induces apoptosis through an autocrine pathway.\n                  <\/jats:p>","DOI":"10.1126\/science.1061075","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:39:52Z","timestamp":1027748392000},"page":"829-834","source":"Crossref","is-referenced-by-count":307,"title":["Induction of Apoptosis by a Secreted Lipocalin That is Transcriptionally Regulated by IL-3 Deprivation"],"prefix":"10.1126","volume":"293","author":[{"given":"Laxminarayana R.","family":"Devireddy","sequence":"first","affiliation":[{"name":"Howard Hughes Medical Institute, Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose G.","family":"Teodoro","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabien A.","family":"Richard","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael R.","family":"Green","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/356397a0"},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","unstructured":"S. R. Datta et al. Genes Dev. 13 2905 (1999).","DOI":"10.1101\/gad.13.22.2905"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/S0092-8674(00)80564-4","volume":"96","author":"Vaux D. L.","year":"1999","unstructured":"Vaux D. L., Korsmeyer S. J., Cell 96, 245 (1999).","journal-title":"Cell"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7878464"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1146\/annurev.immunol.17.1.781","volume":"17","author":"Rathmell J. C.","year":"1999","unstructured":"Rathmell J. C., Thompson C. B., Annu. Rev. Immunol. 17, 781 (1999).","journal-title":"Annu. Rev. Immunol."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","unstructured":"K. Polyak et al. Nature 389 300 (1997).","DOI":"10.1038\/38525"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"38","DOI":"10.4049\/jimmunol.132.1.38","volume":"132","author":"Cohen J. J.","year":"1984","unstructured":"Cohen J. J., Duke R. C., J. Immunol. 132, 38 (1984).","journal-title":"J. Immunol."},{"key":"e_1_3_1_9_2","first-page":"1433","volume":"42","author":"Galili U.","year":"1982","unstructured":"Galili U., et al., Cancer Res. 42, 1433 (1982).","journal-title":"Cancer Res."},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.17.1.221"},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","unstructured":"Ishida et al. EMBO J. 11 3887 (1992).","DOI":"10.1002\/j.1460-2075.1992.tb05481.x"},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1016\/0092-8674(93)90508-N","volume":"74","author":"Boise L. H.","year":"1993","unstructured":"Boise L. H., et al., Cell 74, 597 (1993).","journal-title":"Cell"},{"key":"e_1_3_1_13_2","unstructured":"J. McCubrey et al. Oncogene Res. 4 97 (1989)."},{"key":"e_1_3_1_14_2","unstructured":"Supplementary Web material is available on Science Online at www.sciencemag.org\/cgi\/content\/full\/293\/5531\/829\/DC1."},{"key":"e_1_3_1_15_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/bj3180001","volume":"318","author":"Flower D. R.","year":"1996","unstructured":"Flower D. R., Biochem. J. 318, 1 (1996).","journal-title":"Biochem. J."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1074\/jbc.274.1.7","volume":"274","author":"Chen G.","year":"1999","unstructured":"Chen G., et al., J. Biol. Chem. 274, 7 (1999).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/S1074-7613(00)80363-5","volume":"7","author":"Endres R.","year":"1997","unstructured":"Endres R., et al., Immunity 7, 419 (1997).","journal-title":"Immunity"},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/0014-5793(92)80094-W","volume":"299","author":"Mishizawa M.","year":"1992","unstructured":"Mishizawa M., Nagata S., FEBS Lett. 299, 36 (1992).","journal-title":"FEBS Lett."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","unstructured":"J. S. Greenberger et al. Proc. Natl. Acad. Sci. U.S.A. 80 2931 (1983).","DOI":"10.1073\/pnas.80.10.2931"},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","unstructured":"G. Rodriquez-Tarduchy et al. EMBO J. 9 2997 (1990).","DOI":"10.1002\/j.1460-2075.1990.tb07492.x"},{"key":"e_1_3_1_21_2","unstructured":"Conditioned medium (2 ml) from cells grown in the presence or absence of IL-3 was concentrated in Centricon YM-10 filters (Millipore) and the retentates were collected and fractionated on a 12% SDS\u2013polyacrylamide gel electrophoresis (SDS-PAGE) gel. The membrane was incubated with an antibody to 24p3 (43) and developed with an ECL kit from Amersham."},{"key":"e_1_3_1_22_2","unstructured":"IL-3 withdrawal and cell viability determinations were performed as in (11). For cell death assays 2 \u00d7 10 5 hematopoietic cells or 8 \u00d7 10 5 fibroblasts in 60-mm dishes were incubated with conditioned medium from IL-3\u2013deprived FL5.12 cells or COS-7 cells transfected with pcDNA3 or pcDNA3\/24p3 supplemented with IL-3 (3 ng\/ml). For fibroblasts both floating and adherent cells (after trypsinization) were collected at the indicated time points and analyzed by a trypan blue (Sigma) dye exclusion assay. A minimum of 400 cells were counted and all experiments were performed in duplicate."},{"key":"e_1_3_1_23_2","unstructured":"Apoptosis was assessed by staining with annexin V\u2013FITC\/PI (Calbiochem). Stained cells were analyzed in a Beckton-Dickinson flow cytometer. For DNA fragmentation analysis DNA from 2 \u00d7 10 6 cells was isolated by phenol extraction and analyzed on a 1% agarose gel (19)."},{"key":"e_1_3_1_24_2","unstructured":"For construction of a stable cell line expressing 24p3 the 24p3 cDNA was amplified by polymerase chain reaction and cloned into the Eco RI and Bam HI sites of pcDNA3 vector (Invitrogen). COS-7 cells were transfected with pcDNA3\/24p3 and selected with G418 at 600 \u03bcg\/ml. G418-resistant colonies were isolated and screened for 24p3 expression by Northern blotting and immunoblotting."},{"key":"e_1_3_1_25_2","unstructured":"For establishment of a 24p3 ecdysone-inducible cell line the 24p3 cDNA containing a hemagglutinin (HA) tag at the COOH-terminus was cloned into the ecdysone-inducible vector pIND (Invitrogen). FL5.12 cells were transfected with Superfect (Qiagen). FL5.12 cells were first transfected with pVgRXR expressing the subunits of the receptor and selected with Zeocin (600 \u03bcg\/ml) (Invitrogen). The resulting clones were then transfected with pIND\/24p3-HA and selected with G418 (800 \u03bcg\/ml) (Gibco-BRL). 24p3 expression was induced by addition of 10 \u03bcM ponasterone A (Invitrogen)."},{"key":"e_1_3_1_26_2","unstructured":"Comparable expression of a 24p3 derivative lacking the NH2-terminal signal sequence failed to induce cell death (44) suggesting that secretion of 24p3 is required for apoptosis."},{"key":"e_1_3_1_27_2","unstructured":"Recombinant 24p3 was synthesized as a glutathione S -transferase (GST) fusion protein (pGST-2TK-24p3) and isolated to >90% purity. The GST moiety was removed by thrombin digestion. Purified 24p3 was added to cells to a final concentration of 0.8 to 10 \u03bcg\/ml."},{"key":"e_1_3_1_28_2","unstructured":"Phosphorothioate oligonucleotides were purchased from Genosys. Sense and antisense 1 oligonucleotides span \u201312 to +5 and antisense 2 oligonucleotide spans +585 to +593 of 24p3 mRNA (+1 translation start site). FL5.12 cells were transfected with 2 \u03bcM of each oligonucleotide using lipofectamine (Gibco-BRL). After 24 hours cells were washed with RPMI medium plus 10% fetal calf serum (FCS) and again transfected with 2 \u03bcM of oligonucleotide. Transfected cells were deprived of IL-3 24 hours after the second transfection."},{"key":"e_1_3_1_29_2","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1084\/jem.150.6.1510","volume":"150","author":"Watson J.","year":"1980","unstructured":"Watson J., J. Exp. Med. 150, 1510 (1980).","journal-title":"J. Exp. Med."},{"key":"e_1_3_1_30_2","doi-asserted-by":"crossref","unstructured":"A. R. Khaled et al. Proc. Natl. Acad. Sci. U.S.A. 96 14476 (1999).","DOI":"10.1073\/pnas.96.25.14476"},{"key":"e_1_3_1_31_2","unstructured":"The protein kinase C inhibitor staurosporine induced apoptosis in FL5.12 cells but did not transcriptionally activate 24p3 (44)."},{"key":"e_1_3_1_32_2","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1038\/284555a0","volume":"284","author":"Wyllie A. H.","year":"1980","unstructured":"Wyllie A. H., Nature 284, 555 (1980).","journal-title":"Nature"},{"key":"e_1_3_1_33_2","doi-asserted-by":"crossref","unstructured":"E. Garay-Rojas et al. Gene 170 173 (1996).","DOI":"10.1016\/0378-1119(95)00896-9"},{"key":"e_1_3_1_34_2","doi-asserted-by":"crossref","unstructured":"G. Rodriguez-Tarduchy et al. J. Immunol. 149 535 (1992).","DOI":"10.4049\/jimmunol.149.2.535"},{"key":"e_1_3_1_35_2","doi-asserted-by":"crossref","unstructured":"J. Zha et al. Cell 87 619 (1996).","DOI":"10.1016\/S0092-8674(00)81382-3"},{"key":"e_1_3_1_36_2","unstructured":"For analysis of Bad phosphorylation FL5.12 cells were treated with conditioned medium from COS-7 cells transfected with pcDNA3 or the 24p3 expression vector. Cells were lysed in 1% NP-40 lysis buffer containing 20 mM tris-HCl (pH 7.4) 150 mM NaCl 1.5 mM MgCl2 1 mM EDTA 10 mM NaF 0.2 mM Na 3 VO 4 1 mM Na 3 MoO 4 and protease inhibitor tablets from Boehringer-Mannheim. Cell lysates were incubated with 2 \u03bcg of Bad antibody (Transduction Labs Lexington KY). Immune complexes were resolved by 12% SDS-PAGE and transferred onto a polyvinylidene diflouride membrane (Millipore). Blots were incubated with either a phospho-specific antibody to Bad (New England Biolabs) or an antibody to Bad and were developed with an ECL kit (Amersham)."},{"key":"e_1_3_1_37_2","unstructured":"M. G. Vanderheiden et al. Mol. Cell 3 159 (1999)."},{"key":"e_1_3_1_38_2","unstructured":"FL5.12\/Bcl-X L cells synthesize and secrete 24p3 after IL-3 deprivation (44)."},{"key":"e_1_3_1_39_2","doi-asserted-by":"crossref","first-page":"22565","DOI":"10.1074\/jbc.270.38.22565","volume":"270","author":"Liu A.","year":"1995","unstructured":"Liu A., Nilsen-Hamilton M., J. Biol. Chem. 270, 22565 (1995).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_40_2","doi-asserted-by":"crossref","unstructured":"B. S. Nielsen et al. Gut 38 41 (1996).","DOI":"10.1136\/gut.38.Suppl_1.A41"},{"key":"e_1_3_1_41_2","doi-asserted-by":"crossref","unstructured":"Q. Liu et al. Mol. Reprod. Dev. 46 507 (1997).","DOI":"10.1002\/(SICI)1098-2795(199704)46:4<507::AID-MRD9>3.0.CO;2-S"},{"key":"e_1_3_1_42_2","doi-asserted-by":"crossref","first-page":"313","DOI":"10.2741\/A276","volume":"3","author":"Johnson D. E.","year":"1998","unstructured":"Johnson D. E., Front. Biosci. 3, 313 (1998).","journal-title":"Front. Biosci."},{"key":"e_1_3_1_43_2","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1038\/sj.leu.2401493","volume":"13","author":"Blalock W. L.","year":"1999","unstructured":"Blalock W. L., et al., Leukemia 13, 1109 (1999).","journal-title":"Leukemia"},{"key":"e_1_3_1_44_2","doi-asserted-by":"crossref","unstructured":"S.-T. Chu et al. Biochem. J. 316 545 (1996).","DOI":"10.1042\/bj3160545"},{"key":"e_1_3_1_45_2","unstructured":"L. R. Devireddy et al. data not shown."},{"key":"e_1_3_1_46_2","unstructured":"We thank C. Thompson and S. Durum for cell lines; M. Stevenson and P. Newburger for primary cells; M. Kress for the 24p3 cDNA; Y.-H. Chen for 24p3 protein and antibody; K. Rock M. Czech S. Corvera M. Kelliher M. Zapp and members of our laboratory for helpful discussions and comments on this manuscript; and S. Persengiev for advice on Scatchard plot analysis. Supported in part by grants from NIH and Cap Cure (M.R.G.) and by the Leukemia and Lymphoma Society (L.R.D.)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.1061075","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,9]],"date-time":"2024-01-09T17:10:43Z","timestamp":1704820243000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.1061075"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,8,3]]},"references-count":45,"journal-issue":{"issue":"5531","published-print":{"date-parts":[[2001,8,3]]}},"alternative-id":["10.1126\/science.1061075"],"URL":"https:\/\/doi.org\/10.1126\/science.1061075","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2001,8,3]]}}}