Suppression of Erythroid but Not Megakaryocytic Differentiation of Human K562 Erythroleukemic Cells by Notch-1
2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês
10.1074/jbc.m002866200
ISSN1083-351X
AutoresLloyd T. Lam, Chiara Ronchini, Jason E. Norton, Anthony J. Capobianco, Emery H. Bresnick,
Tópico(s)Developmental Biology and Gene Regulation
ResumoThe Notch signal transduction pathway is a highly conserved regulatory system that controls multiple developmental processes. We have established an erythroleukemia cell model to study how Notch regulates cell fate and erythroleukemic cell differentiation. K562 and HEL cells expressed the Notch-1 receptor and the Notch ligand Jagged-1. The stable expression of the constitutively active intracellular domain of Notch-1 (NIC-1) in K562 cells inhibited erythroid without affecting megakaryocytic maturation. Expression of antisense Notch-1 induced spontaneous erythroid maturation. Suppression of erythroid maturation by NIC-1 did not result from down-regulation of GATA-1 and TAL-1, transcription factors necessary for erythroid differentiation. Microarray gene expression analysis identified genes activated during erythroid maturation, and NIC-1 disrupted the maturation-dependent changes in the expression of these genes. These results show that NIC-1 alters the pattern of gene expression in K562 cells leading to a block in erythroid maturation and therefore suggest that Notch signaling may control the developmental potential of normal and malignant erythroid progenitor cells. The Notch signal transduction pathway is a highly conserved regulatory system that controls multiple developmental processes. We have established an erythroleukemia cell model to study how Notch regulates cell fate and erythroleukemic cell differentiation. K562 and HEL cells expressed the Notch-1 receptor and the Notch ligand Jagged-1. The stable expression of the constitutively active intracellular domain of Notch-1 (NIC-1) in K562 cells inhibited erythroid without affecting megakaryocytic maturation. Expression of antisense Notch-1 induced spontaneous erythroid maturation. Suppression of erythroid maturation by NIC-1 did not result from down-regulation of GATA-1 and TAL-1, transcription factors necessary for erythroid differentiation. Microarray gene expression analysis identified genes activated during erythroid maturation, and NIC-1 disrupted the maturation-dependent changes in the expression of these genes. These results show that NIC-1 alters the pattern of gene expression in K562 cells leading to a block in erythroid maturation and therefore suggest that Notch signaling may control the developmental potential of normal and malignant erythroid progenitor cells. Notch intracellular domain hematopoietic stem cell(s) Iscove's modified Eagle's medium 12-O-tetradecanoylphorbol-13-acetate polymerase chain reaction reverse transcriptase human erythroleukemia interleukin c-Jun NH2-terminal kinase The Notch signal transduction pathway is a highly conserved regulatory system that controls multiple developmental processes (1.Artavanis-Tsakonas S. Matsuno K. Fortini M.E. Science. 1995; 268: 225-232Crossref PubMed Scopus (1417) Google Scholar, 2.Kimble J. Simpson P. Annu. Rev. Cell Dev. Biol. 1997; 13: 333-361Crossref PubMed Scopus (247) Google Scholar). Notch signaling is mediated by a single-pass transmembrane Notch receptor (1.Artavanis-Tsakonas S. Matsuno K. Fortini M.E. Science. 1995; 268: 225-232Crossref PubMed Scopus (1417) Google Scholar). The extracellular domain of Notch contains a ligand-binding site that interacts with transmembrane ligands such as Jagged and Delta (3.Lendahl U. Bioessays. 1998; 20: 103-107Crossref PubMed Scopus (35) Google Scholar). Ligand binding induces the site-specific proteolytic cleavage of the intracellular domain of Notch (NIC),1 liberating NIC from the residual membrane-bound polypeptide (4.Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1382) Google Scholar, 5.De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1825) Google Scholar). NIC then enters the nucleus and associates with the DNA-bound transcription factor suppressor of hairless or CBF1. NIC binding converts CBF1 from a transcriptional repressor to an activator and alters target gene expression (6.Hsieh J.J. Hayward S.D. Science. 1995; 268: 560-563Crossref PubMed Scopus (258) Google Scholar, 7.Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israel A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1223) Google Scholar). The presence of multiple Notch homologs (Notch 1–4) (8.Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (5024) Google Scholar), multiple ligands (e.g. Jagged-1 (9.Lindsell C.E. Shawber C.J. Boulter J. Weinmaster G. Cell. 1995; 80: 909-917Abstract Full Text PDF PubMed Scopus (544) Google Scholar), Jagged-2 (10.Shawber C. Boulter J. Lindsell C.E. Weinmaster G. Dev. Biol. 1996; 180: 370-376Crossref PubMed Scopus (195) Google Scholar), and Delta (11.Kopczynski C.C. Alton A.K. Fechtel K. Kooh P.J. Muskavitch M.A. Genes Dev. 1988; 2: 1723-1735Crossref PubMed Scopus (192) Google Scholar)), and additional components that modulate Notch signaling (e.g. deltex (12.Xu T. Artavanis-Tsakonas S. Genetics. 1990; 126: 665-677Crossref PubMed Google Scholar), suppressor of deltex (13.Cornell M. Evans D.A. Mann R. Fostier M. Flasza M. Monthatong M. Artavanis-Tsakonas S. Baron M. Genetics. 1999; 152: 567-576Crossref PubMed Google Scholar), and lunatic fringe (14.Zhang N. Gridley T. Nature. 1998; 394: 374-377Crossref PubMed Scopus (364) Google Scholar)) endow considerable complexity to the Notch signaling system. Moreover, certain biological actions of Notch are CBF1-independent (15.Shawber C. Nofziger D. Hsieh J.J. Lindsell C. Bogler O. Hayward D. Weinmaster G. Development. 1996; 122: 3765-3773Crossref PubMed Google Scholar, 16.Nofziger D. Miyamoto A. Lyons K.M. Weinmaster G. Development. 1999; 126: 1689-1702Crossref PubMed Google Scholar), adding further diversity to Notch signaling. In addition to the diverse biochemical aspects of Notch signaling, Notch serves multiple developmental functions (2.Kimble J. Simpson P. Annu. Rev. Cell Dev. Biol. 1997; 13: 333-361Crossref PubMed Scopus (247) Google Scholar, 8.Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (5024) Google Scholar). Developmental processes regulated by Notch include somitogenesis, myogenesis, neurogenesis, and hematopoiesis. Seydoux and Greenwald (17.Seydoux G. Greenwald I. Cell. 1989; 57: 1237-1245Abstract Full Text PDF PubMed Scopus (179) Google Scholar) have described the lateral inhibition hypothesis of Notch function, in which one cell conveys inhibitory signals to its neighbor through Notch ligand-receptor interactions. These signals suppress differentiation of one lineage and permit differentiation into a distinct lineage. This activity to control cell fate is exemplified by analysis of neurogenesis in Drosophila (18.Artavanis-Tsakonas S. Delidakis C. Fehon R.G. Annu. Rev. Cell Biol. 1991; 7: 427-452Crossref PubMed Scopus (111) Google Scholar). In the ventral blastoderm of Drosophila, precursor cells develop into either neuroblasts or epidermal cells. When Notch signaling is impaired byNotch mutations, the precursor cells develop exclusively into neuroblasts. The Notch ligand Delta expressed in neuroblasts generates signals to surrounding cells, suppressing further neurogenesis. Embryos with gain-of-function Notch mutations show increased numbers of epidermal cells and reduced numbers of neuroblasts. Thus, Notch signaling regulates cell fate by controlling asymmetric cell division during stem/progenitor cell differentiation. Because Notch has multiple developmental functions, it is not surprising that components of the Notch pathway are critical for survival. Disruption of murine genes encoding Notch-1 (19.Swiatek P.J. Lindsell C.E. del Amo F.F. Weinmaster G. Gridley T. Genes Dev. 1994; 8: 707-719Crossref PubMed Scopus (616) Google Scholar), the ankyrin repeats of Notch-2 (20.Hamada Y. Kadokawa Y. Okabe M. Ikawa M. Coleman J.R. Tsujimoto Y. Development. 1999; 126: 3415-3424Crossref PubMed Google Scholar), Jagged-1 (21.Xue Y. Gao X. Lindsell C.E. Norton C.R. Chang B. Hicks C. Gendron-Maguire M. Rand E.B. Weinmaster G. Gridley T. Hum. Mol. Genet. 1999; 8: 723-730Crossref PubMed Scopus (646) Google Scholar), or CBF1 (22.Oka C. Nakano T. Wakeham A. de la Pompa J.L. Mori C. Sakai T. Okazaki S. Kawaichi M. Shiota K. Mak T.W. Honjo T. Development. 1995; 121: 3291-3301Crossref PubMed Google Scholar) have embryonic lethal phenotypes. Although components of the Notch pathway are expressed in multiple hematopoietic cell types (7.Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israel A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1223) Google Scholar, 22.Oka C. Nakano T. Wakeham A. de la Pompa J.L. Mori C. Sakai T. Okazaki S. Kawaichi M. Shiota K. Mak T.W. Honjo T. Development. 1995; 121: 3291-3301Crossref PubMed Google Scholar, 23.Lam L.T. Bresnick E.H. J. Biol. Chem. 1998; 273: 24223-24231Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 24.Li L. Milner L.A. Deng Y. Iwata M. Banta A. Graf L. Marcovina S. Friedman C. Trask B.J. Hood L. Torok-Storb B. Immunity. 1998; 8: 43-55Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 25.Milner L.A. Kopan R. Martin D.I. Bernstein I.D. Blood. 1994; 83: 2057-2062Crossref PubMed Google Scholar, 26.Varnum-Finney B. Purton L.E., Yu, M. Brashem-Stein C. Flowers D. Staats S. Moore K.A. Le Roux I. Mann R. Gray G. Artavanis-Tsakonas S. Bernstein I.D. Blood. 1998; 91: 4084-4091Crossref PubMed Google Scholar), the role of Notch in hematopoiesis is ill defined (27.Milner L.A. Bigas A. Blood. 1999; 93: 2431-2448Crossref PubMed Google Scholar). Considering that hematopoietic stem (HSC)/progenitor cells interact functionally with stromal and endothelial cells in the hematopoietic microenvironment (28.Whetton A.D. Spooner E. Curr. Opin. Cell Biol. 1998; 10: 721-726Crossref PubMed Scopus (51) Google Scholar), it seems logical that Notch may be important for this intercellular communication. Several lines of evidence implicate Notch as a regulator of hematopoiesis. First, infection of murine bone marrow with a retrovirus expressing constitutively active Notch-1 induced T-cell leukemia in a bone marrow reconstitution assay (29.Pear W.S. Aster J.C. Scott M.L. Hasserjian R.P. Soffer B. Sklar J. Baltimore D. J. Exp. Med. 1996; 183: 2283-2291Crossref PubMed Scopus (600) Google Scholar). Second, constitutively active Notch-1 and Notch-2 inhibited the myeloid differentiation of the murine 32D cell line (30.Bigas A. Martin D.I. Milner L.A. Mol. Cell. Biol. 1998; 18: 2324-2333Crossref PubMed Scopus (183) Google Scholar). Third, expression of constitutively active Notch in T-cells of transgenic mice induced thymocytes to form CD8+ T-cells rather than CD4+ T-cells (31.Robey E. Chang D. Itano A. Cado D. Alexander H. Lans D. Weinmaster G. Salmon P. Cell. 1996; 87: 483-492Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Lastly, a conditional knock-out of mouse Notch-1 (32.Radtke F. Wilson A. Stark G. Bauer M. van Meerwijk J. MacDonald H.R. Aguet M. Immunity. 1999; 10: 547-558Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar) and disruption of Jagged-2 (33.Jiang R. Lan Y. Chapman H.D. Shawber C. Norton C.R. Serreze D.V. Weinmaster G. Gridley T. Genes Dev. 1998; 12: 1046-1057Crossref PubMed Scopus (351) Google Scholar) caused defective T-cell differentiation. While studying protein components of the β-globin locus control region, we identified CBF1 as a protein in K562 nuclear extracts that binds a conserved, functionally important region of the locus control region (23.Lam L.T. Bresnick E.H. J. Biol. Chem. 1998; 273: 24223-24231Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). As no previous studies had investigated the role of Notch signaling in erythroid cell differentiation and function, we have now asked whether other components of the Notch pathway are expressed in erythroleukemic cells and whether a constitutively active Notch receptor influences erythroleukemia cell maturation. The constructs encoding sense and antisense human Notch-1 were described previously (34.Garces C. Ruiz-Hidalgo M.J. de Mora J.F. Park C. Miele L. Goldstein J. Bonvini E. Porras A. Laborda J. J. Biol. Chem. 1997; 272: 29729-29734Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) (gifts from Jorge Laborda, Food and Drug Administration). The sense and antisense constructs were subcloned into the PvuII site of the plasmid pEBVHisA (Invitrogen), which contains a hygromycin resistance gene. Transcripts encoded by the constructs correspond to amino acids 1176–2232 of human Notch-1. The pBabe-NIC-1 expression vector encoding constitutively active Notch-1 (NIC-1) was described previously (35.Capobianco A.J. Zagouras P. Blaumueller C.M. Artavanis-Tsakonas S. Bishop J.M. Mol. Cell. Biol. 1997; 17: 6265-6273Crossref PubMed Scopus (210) Google Scholar). This vector was derived from the pBabe-puro retroviral vector (36.Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1921) Google Scholar) and includes a cDNA sequence of human Notch-1 encoding amino acids 1758–2556. The expression vector encoding amino acids 1758–2556 of human Notch-1 with a Myc epitope tag fused to its carboxyl terminus was constructed from pcDNA3.1 by standard techniques. The Notch-dependent reporter plasmid containing four CBF1-binding sites and a simian virus 40 promoter fused to luciferase (4×wtCBF1Luc) was described previously (37.Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (398) Google Scholar) (gift of Diane Hayward, Johns Hopkins Medical School). The human erythroleukemia cell lines K562 and HEL were propagated in Iscove's modified Eagle's medium (Biofluids) containing 25 μg/ml gentamycin and 10% fetal calf serum (Life Technologies, Inc.) (complete IMEM). The cell lines were grown in a humidified incubator at 37 °C, in the presence of 5% carbon dioxide. K562 and HEL cells were treated with 40 μm hemin for 48 h or 50 nm12-O-tetradecanoylphorbol-13-acetate (TPA) for 6 days prior to RNA preparation. In certain experiments, TPA treatments were for shorter times as specified in the figure legends. Erythroid differentiation of K562 cells was also induced with sodium butyrate (0.5 mm) by treatment of cells for 3 days. The benzidine stock solution contained 0.2% w/v benzidine hydrochloride in 0.5 m acetic acid. Cells (1 × 105) were washed twice with ice-cold phosphate-buffered saline. The cell pellets were resuspended in ice-cold phosphate-buffered saline (27 μl). The benzidine solution (3 μl) containing hydrogen peroxide (final concentration, 0.0012%) was added and incubated for 10 min at room temperature. Benzidine-positive cells were quantitated by light microscopy. At least 100 cells were counted in triplicate for each condition. K562 cells were stably transfected by electroporation with a Bio-Rad Gene pulser electroporator. Cells (5 × 106) were washed, resuspended in 0.5 ml of ice-cold phosphate-buffered saline, mixed with linearized plasmid DNA (5 μg), and subjected to electroporation (960 microfarad; 220 V) in a 0.4-cm-wide electroporation cuvette (BTX). pBabe and pBabe-NIC-1 were linearized with NotI. Cells were then added to 10 ml of complete IMEM, grown for 2 days, and diluted in complete IMEM containing 1.5 μg/ml puromycin. Cells were propagated in complete IMEM containing 1.5 μg/ml puromycin (pools of K562-Babe and K562-NIC-1 cells). Importantly, stably transfected cells were analyzed for erythroid differentiation as soon as the pools were generated (approximately 2–3 weeks) to reduce the probability of phenotypic changes that may result from prolonged growth. For retroviral infection of K562 cells, pBabe-NIC-1 (5 μg) and pMD.G (2 μg) were cotransfected into modified 293 human embryonic kidney cells (2 ml, 105/ml) by the calcium phosphate transfection method as described previously (38.Miyamoto S. Seufzer B.J. Shumway S.D. Mol. Cell. Biol. 1998; 18: 19-29Crossref PubMed Google Scholar). The medium was changed once after 8 h of transfection to remove the calcium phosphate. The pMD.G expression vector encodes the viral envelope protein VSV-G. The modified 293 cells were previously stably transfected withpol and gag genes (gift of Shigeki Miyamoto, University of Wisconsin Medical School). K562 cells (3 ml, 2 × 105/ml) were added with polybrene (4 μg/ml) in complete IMEM and incubated for 48 h. The infected cells were separated from adherent 293 cells and then selected with puromycin (1.5 μg/ml). K562 cells (5 × 105) stably transfected with pBabe-NIC-1 (see Fig.3 C) or control cells (see Fig. 2) were collected by centrifugation at 240 × g for 6 min at 4 °C and resuspended in complete IMEM containing 1.5 μg/ml puromycin. Plasmid DNA (1 μg of 4×wtCBF1Luc or 5xGAL4luc) was suspended in 150 μl of complete IMEM, incubated with 4 μl of Superfect (Qiagen) for 15 min at room temperature and then added to cells. For the experiment of Fig.2, 1 μg of pBabe or pBabe-NIC-1 was cotransfected with the reporter vectors. After incubating for 40 h, cells were harvested and assayed for luciferase activity. The luciferase activity was normalized by the protein content of the lysates, determined by Bradford assay using γ-globulin as a standard.Figure 2CBF1-dependent Notch signaling is functional in K562 cells. K562 cells were transiently transfected with reporter vectors containing four CBF1 (p4xCBF1luc) or five GAL4 (p5xGAL4luc)-binding sites, with or without pBabe or pBabe-NIC-1. A constitutively active β-galactosidase expression vector (pCH110) was included in all conditions to allow for normalization of transfection efficiency. The luciferase activity was also normalized by the protein content of the lysate. Note that strong luciferase activity was only apparent when pBabe-NIC-1 was cotransfected with the CBF1 reporter. The graph depicts averaged data from three independent transient transfection experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Total RNA from K562 or HEL cells was extracted with Triazol (Life Technologies, Inc.). RT-PCR was carried out with a Promega RT-PCR kit. Total RNA (0.2, 2, 20, or 200 ng) was reverse transcribed at 48 °C for 45 min with 0.25 unit of avian myeloblastosis virus reverse transcriptase in a 25-μl mixture containing 0.2 mm nucleotide triphosphates, 0.25 unit of Tfl DNA polymerase, 1 mm MgSO4, and 25 pmol of sense-antisense primers specific for the PCR products. The resulting cDNA pool was amplified by 35 cycles of PCR. The PCR products were resolved on 1.8% agarose gels and visualized by ethidium bromide staining. The RT-PCR primers used in this study were humanNotch-1 (5′ sense, GCGCAGCGACAAGGTGTTGACGTT; 3′ antisense, CAACGGTAGAAGGGGCTCTCGGAT); human Jagged-1 (5′ sense, ATACTTCAAAGTGTGCCTCAAG; 3′ antisense, TTCCCGTGAGGACCACAGACGTT); humanIntegrin αIIb (5′ sense, AGCTACTGGTGCAAGCTTCAC; 3′ antisense, GCGCCCCGGGGCAGGTGCACG; human Integrin β 3 (5′ sense, GCCTCTGGGCTCACCTCGCTG; 3′ antisense, CTGGGATAGCTTCTCAGTCATCAGCCC); and human HPRT (5′ sense, CAGACTGAAGAGCTATTGTAATG; 3′ antisense, CTTAGATGCTGTCTTTGATGTG). Nuclear extracts were prepared from K562 cells (2.5 × 105) as described previously (39.Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Proteins (15 μg) were resolved by SDS-polyacrylamide gel electrophoresis on a 9% acrylamide gel. The proteins were transferred to an Immobilon P membrane (Millipore), and TAL1 was detected by Western blotting with an anti-TAL1 polyclonal antibody (40.Gould K.A. Bresnick E.H. Gene Exp. 1998; 7: 87-101PubMed Google Scholar). The TAL1 antibody was incubated with the membrane for 12 h at 4 °C, and immunoreactive proteins were visualized by incubation with a mouse anti-rabbit immunoglobulin conjugated to horseradish peroxidase, followed by chemoluminescence detection. To detect stably expressed Myc-tagged NIC-1 (NIC-1-myc), whole cell lysates were prepared in Nonidet P-40 lysis buffer (50 mmHepes, pH 7.4, 1 mm EDTA, 150 mm NaCl, 10% glycerol, 1% Nonidet P-40). Lysates were cleared by centrifugation at 13,000 × g for 20 min at 4 °C. Supernatants were split into two aliquots and immunoprecipitated with either preimmune serum or anti-NIC 925 polyclonal antibody. Anti-Nic 925 is a rabbit polyclonal antiserum directed against amino acids 1759–2095 of human Notch-1. Immune complexes were collected by adsorption to protein A-Sepharose. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, and NIC-1-myc was detected by immunoblotting with the anti-Myc tag monoclonal antibody 9E10. The preparation of K562 cell nuclear extracts and DNA binding analysis were performed as described previously (39.Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). GATA-1 DNA binding activity was measured by electrophoretic mobility shift assay with a double-stranded end-labeled oligonucleotide containing a high affinity GATA-1-binding site (TTCGGTTGCAGATAAACATTGAAT). The specificity of DNA binding was assessed by competition with a 200-fold excess of homologous or unrelated oligonucleotide (EboxGTWT) (39.Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) containing a repetitive GT sequence and a high affinity E-box (GCTTAGGGTGTGTGCCCAGATGTTCTCAGC). DNA binding activity was quantitated by PhosphorImager analysis with ImageQuant software (Molecular Dynamics). Polyadenylated RNA was isolated from untreated and hemin-treated (20 μm, 48 h) K562 cells with the OligoTex (Qiagen) RNA kit. Microarray analysis was done by Incyte Pharmaceuticals Inc. (Palo Alto, CA). Briefly, polyadenylated RNA from untreated and hemin-treated K562 cells was reverse transcribed to generate Cy3- and Cy5-labeled cDNA probes, respectively. cDNA probes were competitively hybridized to a UniGEM1 cDNA microarray (Incyte Pharmaceuticals Inc.) containing 9844 immobilized cDNA fragments (average cDNA length, 500–5000 base pairs). Cy3 and Cy5 fluorescence were imaged individually, and the normalized ratios of Cy3/Cy5 fluorescence at a given spot on the microarray were used to calculate differential gene expression. Nothern blotting was used to assess the influence of NIC-1 on gene expression. Total RNA (10 μg) was resolved on 1% agarose/formaldehyde gels and analyzed by standard procedures. Blots were hybridized under stringent conditions with random-primed probes, exposed to a PhosphorImager overnight, and quantitated with ImageQuant software. TheHSP70 and IL-8 cDNA probes were gifts from Rick Morimoto (Northwestern University), and David Denhardt (Rutgers University), respectively. The DD andα-globin cDNA probes were obtained from Genome Systems (IMAGE numbers 298560 and 74275, respectively). Treatment of K562 cells with hemin induces erythroid maturation (41.Dean A. Ley T.J. Humphries R.K. Fordis M. Schechter A.N. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5515-5519Crossref PubMed Scopus (88) Google Scholar), whereas treatment with the phorbol ester TPA induces megakaryocytic differentiation (42.Alitalo R. Leukocyte Res. 1990; 14: 501-514Crossref PubMed Scopus (134) Google Scholar). This system has been used to define factors that regulate leukemic cell differentiation, which may also control normal hematopoiesis (43.Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (206) Google Scholar, 44.Athanasiou M. Clausen P.A. Mavrothalassitis G.J. Zhang X.K. Watson D.K. Blair D.G. Cell Growth Diff. 1996; 7: 1525-1534PubMed Google Scholar, 45.Rosson D. O'Brian T.G. Mol. Cell. Biol. 1995; 15: 772-779Crossref PubMed Scopus (36) Google Scholar, 46.Lebrun J.J. Vale W.W. Mol. Cell. Biol. 1997; 17: 1682-1691Crossref PubMed Scopus (147) Google Scholar, 47.Yamamoto H. Tsukahara K. Kanaoka Y. Jinno S. Okayama H. Mol. Cell. Biol. 1999; 19: 3829-3841Crossref PubMed Scopus (93) Google Scholar, 48.Sella O. Gerlitz G. Le S.-Y. Elroy-Stein O. Mol. Cell. Biol. 1999; 19: 5429-5440Crossref PubMed Google Scholar). To begin to assess whether Notch signaling regulates erythroid or megakaryocytic differentiation of K562 cells, we asked whether components of the Notch pathway are expressed in these cells. We previously detected CBF1 in K562 and mouse erythroleukemia cells (23.Lam L.T. Bresnick E.H. J. Biol. Chem. 1998; 273: 24223-24231Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). RNA isolated from untreated, hemin-treated, or TPA-treated K562 cells and another human erythroleukemia (HEL) cell line was analyzed by RT-PCR with Notch-1, Jagged-1, and HPRT primers. Notch-1 transcripts were detected at similar levels in all conditions (Fig. 1, A and B). Jagged-1 transcripts were detected in undifferentiated K562 and HEL cells, and hemin treatment did not influence Jagged-1 expression. In contrast, treatment of K562 cells with TPA for 6 days enhanced Jagged-1 expression (2.6 ± 0.1 fold; mean ± S.E., n= 3) (Fig. 1 A). We compared the time course for TPA induction of Jagged-1 with the induction of Integrin β3, a megakaryocyte-specific cell surface marker (Fig. 1 C) (49.Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). The induction of Integrin β3 was apparent after 1 day of TPA treatment and was maximal after 3 days (Fig. 1 C). In contrast to K562 cells, TPA treatment of HEL cells did not increase Jagged-1 expression (Fig. 1 B). However, as HEL cells constitutively express Integrin β3 (data not shown), the lack of Jagged-1 induction in this system may be related to the expression of megakaryocytic markers in the uninduced state (50.Tani T. Ylanne J. Virtanen I. Exp. Hematol. 1996; 24: 158-168PubMed Google Scholar). To assess whether the Notch signaling pathway is functional in K562 cells, we used a reporter gene assay that measures CBF1-dependent Notch signaling. We tested whether a previously described vector encoding the constitutively active cytosolic domain of Notch-1 (NIC-1) could activate a Notch-responsive reporter gene in transient transfection assays in K562 cells. The Notch-responsive reporter contained four binding sites for the Notch-regulated transcription factor CBF1 upstream of the simian virus 40 promoter fused to luciferase (p4×CBF1lluc). Cells were also transfected with a control reporter containing five GAL4-binding sites and lacking CBF1-binding sites (p5×GAL4luc). Strong luciferase activity was apparent when pBabe-NIC-1 was cotransfected with p4xCBF1luc but not with p5×GAL4luc (Fig.2). In contrast, very low luciferase activity was measured with either of the reporters with or without the empty expression vector pBabe. Thus, the activation of p4xCBF1luc by transiently transfected pBabe-NIC-1 confirms that pBabe-NIC-1 encodes functional NIC-1 in K562 cells and that the CBF1-dependent Notch pathway is functional in K562 cells. To determine whether Notch signaling influences erythroid maturation of K562 cells, we generated stably transfected and retrovirally infected pools of K562 cells containing pBabe-NIC-1 or the control vector pBabe. The cells were treated with hemin to determine whether NIC-1 altered their responsiveness to hemin-induced erythroid maturation. After treatment of cells for 48 h with hemin, cells were assayed for hemoglobin accumulation, a marker for erythroid differentiation, by benzidine staining. A representative staining pattern of cells stably transfected with the pBabe vector (K562-Babe) or pBabe-NIC-1 (K562-NIC-1) is shown in Fig.3 A. The percentage of benzidine positive cells was considerably lower for all 11 pools of K562-NIC-1 cells versus K562-Babe cells (Fig. 3 B) (p < 0.001). Similar results were seen with six pools of K562 cells in which pBabe and pBabe-NIC-1 were stably introduced by retroviral infection (Fig. 3 B). Erythroid maturation of K562 cells can be induced by certain chemicals other than hemin, including butyrate, cytosine arabinofuranoside, and hydroxyurea (42.Alitalo R. Leukocyte Res. 1990; 14: 501-514Crossref PubMed Scopus (134) Google Scholar). To determine whether NIC-1 inhibits erythroid differentiation by multiple inducers, we tested whether butyrate-induced differentiation was sensitive to NIC-1. The percentage of benzidine positive K562-Babe cells was 1.9 ± 0.9% (n = 6) and 10.6 ± 1.8% (n = 12) for control and butyrate treated, respectively. Thus, butyrate was less efficient in inducing erythroid maturation than hemin. Similar to the results with hemin, expression of NIC-1 strongly inhibited butyrate-induced erythroid maturation control and butyrate-treated, −1.5 ± 0.8% (n = 6) and 3.5 ± 0.8% (n = 12) benzidine-positive cells, respectively). Even though butyrate is a weaker inducer than hemin, the inhibition was highly significant (p < 0.001). The pBabe-NIC-1 expression vector used in Figs. 2 and 3 was shown previously to overexpress NIC-1 protein after stable transfection into baby rat kidney cells (35.Capobianco A.J. Zagouras P. Blaumueller C.M. Artavanis-Tsakonas S. Bishop J.M. Mol. Cell. Biol. 1997; 17: 6265-6273Crossref PubMed Scopus (210) Google Scholar). However, we have been unable to detect NIC-1 protein expression in K562-NIC-1 cells by Western blot analysis of whole cell extracts using the same antibody used in the previous study, an anti-human Notch-1 rat monoclonal antibody that recognizes the cytosolic domain of Notch-1 (35.Capobianco A.J. Zagouras P. Blaumueller C.M. Artavanis-Tsakonas S. Bishop J.M. Mol. Cell. Biol. 1997; 17: 6265-6273Crossref PubMed
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