Survival by Mac-1-mediated Adherence and Anoikis in Phorbol Ester-treated HL-60 Cells
1998; Elsevier BV; Volume: 273; Issue: 25 Linguagem: Inglês
10.1074/jbc.273.25.15345
ISSN1083-351X
AutoresHitoshi Nakamura, Tsukasa Oda, Koichi Hamada, Tetsuo Hirano, Noriaki Shimizu, Hiroyasu Utiyama,
Tópico(s)Phagocytosis and Immune Regulation
ResumoDuring the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence. During the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence. In phorbol ester-induced monocyte/macrophage-like differentiation in human leukemic HL-60 cells (1Collins S.J. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar), the leukocyte integrin receptor Mac-1 is expressed on the cell surface (2Miller L.J. Schwarting R. Springer T.A. J. Immunol. 1986; 137: 2891-2900PubMed Google Scholar), and the cells are growth-arrested at G1 of the cell cycle (3Rovera G. Olashaw N. Meo P. Nature. 1980; 284: 69-70Crossref PubMed Scopus (108) Google Scholar, 4Oda T. Kitajima K. Hirano T. Shimizu N. Utiyama H. Int. J. Hematol. 1993; 58: 125-128PubMed Google Scholar). Mac-1 (CR3, αMβ2, CD11b/CD18) is a member of the β2 integrin subfamily and plays a critical role in numerous physiological functions of monocytes and macrophages that are mediated by cell-cell and cell-substrate interactions (5Anderson D.C. Springer T.A. Annu. Rev. Med. 1987; 38: 175-194Crossref PubMed Scopus (895) Google Scholar, 6Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8968) Google Scholar, 7Arnaout M.A. Blood. 1990; 75: 1037-1050Crossref PubMed Google Scholar, 8Gahmberg C.G. Tolvanen M. Kotovuori P. Eur. J. Biochem. 1997; 245: 215-232Crossref PubMed Scopus (190) Google Scholar, 46Kolanus W. Seed B. Curr. Opin. Cell Biol. 1997; 9: 725-731Crossref PubMed Scopus (128) Google Scholar). Phorbol esters activate Mac-1 receptors on monocyte such that they promote vigorous phagocytosis (9Wright S.D. Silverstein S.C. J. Exp. Med. 1982; 156: 1149-1164Crossref PubMed Scopus (293) Google Scholar, 10Wright S.D. Licht M.R. Craigmyle L.S. Silverstein S.C. J. Cell. Biol. 1984; 99: 336-339Crossref PubMed Scopus (74) Google Scholar). Monocytes die by apoptosis after phagocytosis of bacteria (11Baran J. Guzik K. Hryniewicz W. Ernst M. Flad H.D. Pryjma J. Infect. Immun. 1996; 64: 4242-4248Crossref PubMed Google Scholar). Undifferentiated HL-60 and U937 cells express LFA-1 (αLβ2, CD11a/CD18) (2Miller L.J. Schwarting R. Springer T.A. J. Immunol. 1986; 137: 2891-2900PubMed Google Scholar), another member of the β2 integrin subfamily, although at low levels. CD18 subunit mRNA is thus expressed constitutively, but CD11b subunit mRNA is not expressed at detectable levels (12Rosmarin A.G. Weil S.C. Rosner G.L. Griffin J.D. Arnaout M.A. Tenen D.G. Blood. 1989; 73: 131-136Crossref PubMed Google Scholar). Phorbol ester up-regulates the steady state levels of both CD11b mRNA (13Pahl H.L. Rosmarin A.G. Tenen D.G. Blood. 1992; 79: 865-870Crossref PubMed Google Scholar) and CD18 mRNA (14Rosmarin A.G. Levy R. Tenen D.G. Blood. 1992; 79: 2598-2604Crossref PubMed Google Scholar, 15Bottinger E.P. Shelley C.S. Farokhzad O.C. Arnaout M.A. Mol. Cell. Biol. 1994; 14: 2604-2615Crossref PubMed Google Scholar) by transcriptional activation. Firm adherence of differentiated cells to tissue culture plastic dishes is inhibited by a monoclonal antibody to CD18 (16Skoglund G. Patarroyo M. Forsbeck K. Nilsson K. IngelmanSundberg M Cancer Res. 1988; 48: 3168-3172PubMed Google Scholar) or CD11b (17Hickstein D.D. Smith A. Fisher W. Beatty P.G. Schwartz B.R. Harlan J.M. Root R.K. Locksley R.M. J. Immunol. 1987; 138: 513-519PubMed Google Scholar). Transcription factor NF-κB is constitutively activated by phorbol esters (18Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (438) Google Scholar) and is indispensable for the CD11b gene expression and cell adhesion, because both are suppressed by a dominant negative inhibitor of NF-κB expression (19Eck S.L. Perkins N.D. Carr D.P. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6530-6536Crossref PubMed Google Scholar) and by antisense oligonucleotides to RelA subunit (20Sokoloski J.A. Sartorelli A.C. Rosen C.A. Narayanan R. Blood. 1993; 82: 625-632Crossref PubMed Google Scholar).Phorbol esters also induce apoptosis of HL-60 and U937 cells (21Gunji H. Hass R. Kufe D. J. Clin. Invest. 1992; 89: 954-960Crossref PubMed Scopus (67) Google Scholar), which is accompanied by a marked decrease in bcl-2 mRNA and protein levels (22Delia D. Aiello A. Soligo D. Fontanella E. Melani C. Pezzella F. Pierotti M.A. Della Porta G. Blood. 1992; 79: 1291-1298Crossref PubMed Google Scholar). Ectopic expression of Bcl-2 protein inhibits apoptosis but has no significant effect on differentiation (23Naumovski L. Cleary M.L. Blood. 1994; 83: 2261-2267Crossref PubMed Google Scholar, 24Terui Y. Furukawa Y. Sakoe K. Ohta M. Saito M. J. Biochem. (Tokyo). 1995; 117: 77-84Crossref PubMed Scopus (31) Google Scholar). Apoptosis of HL-60 cells induced by treatment with phorbol esters has been correlated with an increase in the steady-state level of sphingosine and elevation of the ceramidase activity (25Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar). In fact, exposure of HL-60 cells to sphingosine induced apoptosis (25Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar, 26Jarvis W.D. Fornari F.A. Traylor R.S. Martin H.A. Kramer L.B. Erukulla R.K. Bittman R. Grant S. J. Biol. Chem. 1996; 271: 8275-8284Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Co-exposure of HL-60 cells to phorbol ester and sphingosine blocked phorbol ester-induced differentiation (27Kolesnick R.N. J. Biol. Chem. 1989; 264: 7617-7623Abstract Full Text PDF PubMed Google Scholar, 28Merrill Jr., A.H. Sereni A.M. Stevens V.L. Hannun Y.A. Bell R.M. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 12610-12615Abstract Full Text PDF PubMed Google Scholar). Interestingly, the cell adherence to plastic substrate protected HL-60 cells from apoptosis induced with phorbol esters (29Solary E. Bertrand R. Pommier Y. Leukemia (Baltimore). 1994; 8: 792-797PubMed Google Scholar). Recently, sphingosine-1-phosphate (SPP) 1The abbreviations used are: SPP, sphingosine-1-phosphate; TNF-α, tumor necrosis factor-α; FCS, fetal calf serum; PDB, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate-13-acetate; DMS,N,N-dimethylsphingosine; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole. 1The abbreviations used are: SPP, sphingosine-1-phosphate; TNF-α, tumor necrosis factor-α; FCS, fetal calf serum; PDB, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate-13-acetate; DMS,N,N-dimethylsphingosine; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole. has been shown to prevent apoptosis in HL-60 cells or U937 cells induced by tumor necrosis factor-α (TNF-α) or Fas ligand (30Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). The principal mediator of apoptosis in these cases is ceramide, not sphingosine, but SPP could be equally effective in the protection of adherent HL-60 cells from phorbol ester-induced apoptosis.The several lines of experimental evidence summarized above suggested to us the following possibilities. 1) Apoptosis by sphingosine occurs by default (31Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar) in phorbol ester-treated HL-60 cells; 2) adherence to plastic by Mac-1 elicits an anchorage-dependent survival signal, and adherent cells may undergo anoikis by forced suspension (32Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2750) Google Scholar, 33Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (970) Google Scholar); 3) apoptosis in nonadherent cells and anoikis, as well, may be suppressed by the addition of SPP; and 4) if the commitment signal to apoptosis is lost faster than the survival signal, phorbol ester-differentiated and adherent HL-60 cells may de-differentiate and proliferate again by removing phorbol ester without perturbing the adherent state. Here we demonstrate that our model is substantiated by a line of experimental evidence that was obtained by studying adherent and suspended cell fractions separately after isolation.EXPERIMENTAL PROCEDURESCell Culture and Drug TreatmentThe human acute myeloblastic leukemia cell line HL-60 (34Collins S.J. Gallo R.C. Gallagher R.E. Nature. 1977; 270: 347-349Crossref PubMed Scopus (1707) Google Scholar, 35Gallagher R. Collins S. Trujillo J. McCredie K. Ahearn M. Tsai S. Metzgar R. Aulakh G. Ting R. Ruscetti F. Gallo R. Blood. 1979; 54: 713-733Crossref PubMed Google Scholar, 36Dalton Jr., W.T. Ahearn M.J. McCredie K.B. Freireich E.J. Stass S.A. Trujillo J.M. Blood. 1988; 71: 242-247Crossref PubMed Google Scholar) (CCL240; ATCC, Manassas, VA) was cultured in plastic dishes at 37 °C in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA) and 100 μg/ml kanamycin. The HL-60 cells used in this work were restricted to early passages characterized by a doubling time of 47 h. Cells were passaged before cell density reached 2 × 106 cells/ml and seeded at 3 × 105 cells/ml. The cell viability determined by trypan blue exclusion was scored during the course of culture and found to be higher than 90%. For the differentiation induction we used phorbol 12,13-dibutyrate (PDB; Sigma) rather than phorbol 12-myristate-13-acetate (PMA), because PDB can be easily removed from cells by simple washings (37Cooper R.A. Braunwald A.D. Kuo A.L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2865-2869Crossref PubMed Scopus (83) Google Scholar), provided that FCS or BSA is contained in the washing medium (38Lee L.-S. Weinstein I.B. J. Environ. Pathol. Toxicol. 1978; 1: 327-339PubMed Google Scholar). An ethanol stock solution of PDB (0.2 mm) was diluted before use to 10 μm with the culture medium and added to a cell culture of 5 × 105 cells/ml at a final concentration of 100 nm(4Oda T. Kitajima K. Hirano T. Shimizu N. Utiyama H. Int. J. Hematol. 1993; 58: 125-128PubMed Google Scholar). In using sphingolipids as effectors, cells in logarithmic growth were first precultured for 24 h in a serum-free RPMI 1640 medium supplemented with insulin (5 μg/ml) and transferrin (5 μg/ml) and treated with drugs in the same medium (30Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 39Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell. Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar). An ethanol stock solution (10 mm) ofN,N-dimethylsphingosine (DMS; Biomol) was diluted with the serum-free medium before use. SPP (Biomol) was added as BSA complexes (39Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell. Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar) at a concentration of 125 μm in 4 mg/ml BSA (fatty acid-free). In suppression of anoikis of HL-60 cells with SPP, adherent cells prepared by pretreatment with 100 nmPDB for two days were detached by forced suspension by pipetting three times with the serum-free medium containing 20 μm SPP. PDB was removed from cells by washing three times with RPMI 1640 containing 5% FCS, and FCS was then removed by washing with serum-free medium. The cells were finally suspended in serum-free medium containing 20 μm SPP.Separation of Adherent and Nonadherent Cell PopulationsDuring the PDB treatment of HL-60 cells, suspended cells were separated by gentle pipetting. Culture dishes with the adherent cells were gently washed three times with 10 ml of fresh medium. A small number of cells contained in the wash fractions were discarded. The number of adherent cells were counted without suspending them from dishes by using an inverted microscope fitted with an ocular lens with grid, because the cells formed large aggregates after suspension. A unit area of the grid with an appropriate size was chosen so that a total of 200 or more adherent cells were contained in it. The cell density/unit area was measured at 8–16 different places selected at random in a dish. The total number of adherent cells/dish was estimated by multiplying the average cell density and the total area of a dish.Assay of Cell ProliferationTo a cell culture of 100 μl (3–10 × 105cells/ml), [3H]thymidine (55 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) was added to a final activity of 0.5 μCi/ml. After incubation for 1 h at 37 °C in a CO2 incubator, the cells were washed 3 times and fixed on a slide glass. Autoradiography was done by immersing the slide glass in autoradiographic emulsion (type NR-M2; Konica, Tokyo, Japan) and exposing it for 4–6 days. Developed and fixed slides were counterstained with Giemsa, and more than 400 cells were counted under a light microscope. The labeling index was calculated from the number of cells with silver grains divided by the total cell counts.Assay of ApoptosisNuclear Fragmentation105 cells were fixed with an equal volume of 5% paraformaldehyde, neutralized by one-tenth volume of 1 m Tris-HCl (pH 7.2) and centrifuged onto a glass slide using a cytospin apparatus. The cells were further fixed in cold methanol (−20 °C, 5 min) followed by cold acetone (−20 °C, 5 min), and the plates were allowed to dry. Dried plates were stained with 2 μg/ml 4,6-diamidino-2-phenylindole (DAPI). Alternatively, cytospin preparations were fixed in 90% (v/v) cold methanol (−20 °C, 5 min) and dried. Dried plates were stained with 125 μg/ml acridine orange in phosphate buffer (pH 6.9). Nuclei were assessed in an Olympus OMT2 inverted fluorescence microscope equipped with the appropriate epifluorescence filters at a final magnification of 1500×.DNA Fragmentation (40Sellins K.S. Cohen J.J. J. Immunol. 1987; 139: 3199-3206PubMed Google Scholar)Briefly, 107 cells were lysed in a lysis buffer containing 0.5% Triton X-100, 10 mm EDTA, 10 mm Tris-HCl (pH 7.4) and centrifuged. The supernatant containing the fragmented DNA molecules was recovered and digested with RNase A followed by proteinase K. Nucleic acids were precipitated in 50% isopropanol and 0.5m NaCl. The precipitates were pelleted by repeated high speed centrifugation and used for analyses by electrophoresis on a 1.5% agarose gel and staining with ethidium bromide. The DNA migration was assessed in an image analyzer (FAS-II, Toyobo, Osaka, Japan).Suspension of Adherent CellsForced SuspensionAfter separation of nonadherent cells, adherent cells were washed four times with RPMI 1640 medium containing 5% FCS. The adherent cells were then suspended by the following three different methods. 1) The adherent cells were incubated for 10 min in phosphate buffered saline with no Ca2+ or Mg2+and then suspended by pipetting; 2) the adherent cells were first overlaid with 5 ml of 0.05% trypsin, and 4.5 ml of the supernatant was removed and incubated at 37 °C for 5 min to allow the cells to round up. The cells were suspended by repeating the addition of RPMI 1640 containing 10% FCS and pipetting; 3) addition of RPMI 1640 containing 10% FCS and pipetting were repeated several times until the cells were completely suspended. Culture of the suspended cells was started by seeding at 5 × 105 cells/ml in normal medium of RPMI 1640 containing 10% FCS.Spontaneous SuspensionAfter the treatment of HL-60 cells with 100 nm PDB for 2 days, nonadherent cells were separated by gentle pipetting. PDB was then removed by a procedure that consisted of 1) medium changes for 3 times with fresh medium containing 5% FCS, 2) incubation at 37 °C for 1 h in a CO2incubator, and 3) further medium changes for 3 times. The medium change was made as gently as possible such that the cell adherence was not disturbed. During further incubation in fresh medium, the adherent cells spontaneously detached from plastic surfaces.Sphingosine Kinase AssaySphingosine kinase activity was measured essentially as described previously (41Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar). Briefly, 107 cells were lysed by freeze-thawing in 0.2 ml of 0.1 m phosphate buffer (pH 7.2) containing 10 mm MgCl2, 20% glycerol, 1 mm mercaptoethanol, 1 mm EDTA, 20 μm ZnCl2, 1 mmNa3VO4, 15 mm NaF, 10 μg/ml leupeptin and aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mm 4-deoxypyrodoxine. Cytosolic fractions were prepared by ultracentrifugation at 105,000 ×g for 90 min. Sphingosine kinase activity in supernatants (50 μl) was measured by incubating with 5 μm sphingosine-BSA complex (42Zhang H. Buckley N.E. Gibson K. Spiegel S. J. Biol. Chem. 1990; 265: 76-81Abstract Full Text PDF PubMed Google Scholar) and [γ-32P]ATP (1 mm, 0.2 Ci/mmol) for 30 min at 37 °C. Labeled lipids were extracted with a mixture of chloroform/methanol/concentrated HCl (100:200:1, by volume), and the extract was partitioned into two phases by adding 2 mKCl/methanol (1:1, v/v) (43Hasegawa-Sakai H. Biochem. J. 1985; 232: 99-109Crossref PubMed Scopus (66) Google Scholar). The lower phase was further washed twice with the same mixture and dried in a vacuum centrifuge at room temperature. The dried lipid was completely dissolved in 100 μl of chloroform/methanol (1:1, v/v) with occasional stirring over 30 min. Lipids were resolved on silica gel plates (Silica Gel 60; Merck) impregnated with 1% potassium oxalate/2 mm EDTA, using the solvent system of butanol/water/acetic acid (3:1:1) (39Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell. Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar). The phospholipid standards were visualized with molybdenum blue spray (Sigma), and the radioactivity was measured by autoradiography in a Bioimaging analyzer (BAS2000, Fuji Film, Tokyo, Japan). In phorbol ester-induced monocyte/macrophage-like differentiation in human leukemic HL-60 cells (1Collins S.J. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar), the leukocyte integrin receptor Mac-1 is expressed on the cell surface (2Miller L.J. Schwarting R. Springer T.A. J. Immunol. 1986; 137: 2891-2900PubMed Google Scholar), and the cells are growth-arrested at G1 of the cell cycle (3Rovera G. Olashaw N. Meo P. Nature. 1980; 284: 69-70Crossref PubMed Scopus (108) Google Scholar, 4Oda T. Kitajima K. Hirano T. Shimizu N. Utiyama H. Int. J. Hematol. 1993; 58: 125-128PubMed Google Scholar). Mac-1 (CR3, αMβ2, CD11b/CD18) is a member of the β2 integrin subfamily and plays a critical role in numerous physiological functions of monocytes and macrophages that are mediated by cell-cell and cell-substrate interactions (5Anderson D.C. Springer T.A. Annu. Rev. Med. 1987; 38: 175-194Crossref PubMed Scopus (895) Google Scholar, 6Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8968) Google Scholar, 7Arnaout M.A. Blood. 1990; 75: 1037-1050Crossref PubMed Google Scholar, 8Gahmberg C.G. Tolvanen M. Kotovuori P. Eur. J. Biochem. 1997; 245: 215-232Crossref PubMed Scopus (190) Google Scholar, 46Kolanus W. Seed B. Curr. Opin. Cell Biol. 1997; 9: 725-731Crossref PubMed Scopus (128) Google Scholar). Phorbol esters activate Mac-1 receptors on monocyte such that they promote vigorous phagocytosis (9Wright S.D. Silverstein S.C. J. Exp. Med. 1982; 156: 1149-1164Crossref PubMed Scopus (293) Google Scholar, 10Wright S.D. Licht M.R. Craigmyle L.S. Silverstein S.C. J. Cell. Biol. 1984; 99: 336-339Crossref PubMed Scopus (74) Google Scholar). Monocytes die by apoptosis after phagocytosis of bacteria (11Baran J. Guzik K. Hryniewicz W. Ernst M. Flad H.D. Pryjma J. Infect. Immun. 1996; 64: 4242-4248Crossref PubMed Google Scholar). Undifferentiated HL-60 and U937 cells express LFA-1 (αLβ2, CD11a/CD18) (2Miller L.J. Schwarting R. Springer T.A. J. Immunol. 1986; 137: 2891-2900PubMed Google Scholar), another member of the β2 integrin subfamily, although at low levels. CD18 subunit mRNA is thus expressed constitutively, but CD11b subunit mRNA is not expressed at detectable levels (12Rosmarin A.G. Weil S.C. Rosner G.L. Griffin J.D. Arnaout M.A. Tenen D.G. Blood. 1989; 73: 131-136Crossref PubMed Google Scholar). Phorbol ester up-regulates the steady state levels of both CD11b mRNA (13Pahl H.L. Rosmarin A.G. Tenen D.G. Blood. 1992; 79: 865-870Crossref PubMed Google Scholar) and CD18 mRNA (14Rosmarin A.G. Levy R. Tenen D.G. Blood. 1992; 79: 2598-2604Crossref PubMed Google Scholar, 15Bottinger E.P. Shelley C.S. Farokhzad O.C. Arnaout M.A. Mol. Cell. Biol. 1994; 14: 2604-2615Crossref PubMed Google Scholar) by transcriptional activation. Firm adherence of differentiated cells to tissue culture plastic dishes is inhibited by a monoclonal antibody to CD18 (16Skoglund G. Patarroyo M. Forsbeck K. Nilsson K. IngelmanSundberg M Cancer Res. 1988; 48: 3168-3172PubMed Google Scholar) or CD11b (17Hickstein D.D. Smith A. Fisher W. Beatty P.G. Schwartz B.R. Harlan J.M. Root R.K. Locksley R.M. J. Immunol. 1987; 138: 513-519PubMed Google Scholar). Transcription factor NF-κB is constitutively activated by phorbol esters (18Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (438) Google Scholar) and is indispensable for the CD11b gene expression and cell adhesion, because both are suppressed by a dominant negative inhibitor of NF-κB expression (19Eck S.L. Perkins N.D. Carr D.P. Nabel G.J. Mol. Cell. Biol. 1993; 13: 6530-6536Crossref PubMed Google Scholar) and by antisense oligonucleotides to RelA subunit (20Sokoloski J.A. Sartorelli A.C. Rosen C.A. Narayanan R. Blood. 1993; 82: 625-632Crossref PubMed Google Scholar). Phorbol esters also induce apoptosis of HL-60 and U937 cells (21Gunji H. Hass R. Kufe D. J. Clin. Invest. 1992; 89: 954-960Crossref PubMed Scopus (67) Google Scholar), which is accompanied by a marked decrease in bcl-2 mRNA and protein levels (22Delia D. Aiello A. Soligo D. Fontanella E. Melani C. Pezzella F. Pierotti M.A. Della Porta G. Blood. 1992; 79: 1291-1298Crossref PubMed Google Scholar). Ectopic expression of Bcl-2 protein inhibits apoptosis but has no significant effect on differentiation (23Naumovski L. Cleary M.L. Blood. 1994; 83: 2261-2267Crossref PubMed Google Scholar, 24Terui Y. Furukawa Y. Sakoe K. Ohta M. Saito M. J. Biochem. (Tokyo). 1995; 117: 77-84Crossref PubMed Scopus (31) Google Scholar). Apoptosis of HL-60 cells induced by treatment with phorbol esters has been correlated with an increase in the steady-state level of sphingosine and elevation of the ceramidase activity (25Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar). In fact, exposure of HL-60 cells to sphingosine induced apoptosis (25Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar, 26Jarvis W.D. Fornari F.A. Traylor R.S. Martin H.A. Kramer L.B. Erukulla R.K. Bittman R. Grant S. J. Biol. Chem. 1996; 271: 8275-8284Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Co-exposure of HL-60 cells to phorbol ester and sphingosine blocked phorbol ester-induced differentiation (27Kolesnick R.N. J. Biol. Chem. 1989; 264: 7617-7623Abstract Full Text PDF PubMed Google Scholar, 28Merrill Jr., A.H. Sereni A.M. Stevens V.L. Hannun Y.A. Bell R.M. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 12610-12615Abstract Full Text PDF PubMed Google Scholar). Interestingly, the cell adherence to plastic substrate protected HL-60 cells from apoptosis induced with phorbol esters (29Solary E. Bertrand R. Pommier Y. Leukemia (Baltimore). 1994; 8: 792-797PubMed Google Scholar). Recently, sphingosine-1-phosphate (SPP) 1The abbreviations used are: SPP, sphingosine-1-phosphate; TNF-α, tumor necrosis factor-α; FCS, fetal calf serum; PDB, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate-13-acetate; DMS,N,N-dimethylsphingosine; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole. 1The abbreviations used are: SPP, sphingosine-1-phosphate; TNF-α, tumor necrosis factor-α; FCS, fetal calf serum; PDB, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate-13-acetate; DMS,N,N-dimethylsphingosine; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole. has been shown to prevent apoptosis in HL-60 cells or U937 cells induced by tumor necrosis factor-α (TNF-α) or Fas ligand (30Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). The principal mediator of apoptosis in these cases is ceramide, not sphingosine, but SPP could be equally effective in the protection of adherent HL-60 cells from phorbol ester-induced apoptosis. The several lines of experimental evidence summarized above suggested to us the following possibilities. 1) Apoptosis by sphingosine occurs by default (31Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar) in phorbol ester-treated HL-60 cells; 2) adherence to plastic by Mac-1 elicits an anchorage-dependent survival signal, and adherent cells may undergo anoikis by forced suspension (32Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2750) Google Scholar, 33Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (970) Google Scholar); 3) apoptosis in nonadherent cells and anoikis, as well, may be suppressed by the addition of SPP; and 4) if the commitment signal to apoptosis is lost faster than the survival signal, phorbol ester-differentiated and adherent HL-60 cells may de-differentiate and proliferate again by removing phorbol ester without perturbing the adherent state. Here we demonstrate that our model is substantiated by a line of experimental evidence that was obtained by studying adherent and suspended cell fractions separately after isolation. EXPERIMENTAL PROCEDURESCell Culture and Drug TreatmentThe human acute myeloblastic leukemia cell line HL-60 (34Collins S.J. Gallo R.C. Gallagher R.E. Nature. 1977; 270: 347-349Crossref PubMed Scopus (1707) Google Scholar, 35Gallagher R. Collins S. Trujillo J. McCredie K. Ahearn M. Tsai S. Metzgar R. Aulakh G. Ting R. Ruscetti F. Gallo R. Blood. 1979; 54: 713-733Crossref PubMed Google Scholar, 36Dalton Jr., W.T. Ahearn M.J. McCredie K.B. Freireich E.J. Stass S.A. Trujillo J.M. Blood. 1988; 71: 242-247Crossref PubMed Google Scholar) (CCL240; ATCC, Manassas, VA) was cultured in plastic dishes at 37 °C in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA) and 100 μg/ml kanamycin. The HL-60 cells used in this work were restricted to early passages characterized by a doubling time of 47 h. Cells were passaged before cell density reached 2 × 106 cells/ml and seeded at 3 × 105 cells/ml. The cell viability determined by trypan blue exclusion was scored during the course of culture and found to be higher than 90%. For the differentiation induction we used phorbol 12,13-dibutyrate (PDB; Sigma) rather than phorbol 12-myristate-13-acetate (PMA), because PDB can be easily removed from cells by simple washings (37Cooper R.A. Braunwald A.D. Kuo A.L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2865-2869Crossref PubMed Scopus (83) Google Scholar), provided that FCS or BSA is contained in the washing medium (38Lee L.-S. Weinstein I.B. J. Environ. Pathol. Toxicol. 1978; 1: 327-339PubMed Google Scholar). An ethanol stock solution of PDB (0.2 mm) was diluted before use to 10 μm with the culture medium and added to a cell culture of 5 × 105 cells/ml at a final concentration of 100 nm(4Oda T. Kitajima K. Hirano T. Shimizu N. Utiyama H. Int. J. Hematol. 1993; 58: 125-128PubMed Google Scholar). In using sphingolipids as effectors, cells in logarithmic growth were first precultured for 24 h in a serum-free RPMI 1640 medium supplemented with insulin (5 μg/ml) and transferrin (5 μg/ml) and treated with drugs in the same medium (30Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S.
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