c-Maf Induces Monocytic Differentiation and Apoptosis in Bipotent Myeloid Progenitors by Shrikanth P. Hegde, JingFeng Zhao, Richard A. Ashmun, and Linda H. Shapiro Blood Volume 94(5):1578-1589 September 1, 1999 ©1999 by American Society of Hematology

c-Maf in myeloid cell lines and stable cell clones. c-Maf in myeloid cell lines and stable cell clones. (A) Endogenous expression of c-Maf mRNA expression levels in human myeloid cell lines as determined by Northern blot analysis of polyA+ RNA from the HL-60 promyelocytic or U937 monoblastic cell lines. A single blot was initially probed with the c-Maf specific cDNA probe, stripped, and reprobed with c-Myb, and finally, β-actin as a control for RNA loading and integrity. Exposure times for optimal detection of c-Maf mRNA were routinely significantly longer than for other probes (1 week v 1 day). (B) Conditional expression c-Maf in stably transfected clones. Individual HL-60 or U937 clonal cell lines containing either CB6 vector alone (left 2 lanes) or c-Maf (right lanes) encoding plasmids were cultured for 24 hours with 150 μmol/L ZnSO4. Cell lysates were analyzed beside control lysates from a previously established c-Maf inducible cell line (+).9 The gels were transferred and probed for c-Maf expression with an antiserum that recognized the c-Maf protein. (C) Myb:Maf complexes increase on c-Maf induction. An end-labeled 129-bp promoter fragment probe containing the functionally definedCD13/APN myeloid promoter elements9 was incubated with uninduced (-) or induced (+) whole-cell lysates from control vector-containing (CB6, lanes 2 and 3), c-Maf–containing (c-Maf clone 6, lanes 4 to 8) stable HL-60 clones. Unlabeled competitor oligonucleotides containing either the consensus Myb site from themim-1 promoter (mimAmyb, lane 6) or the consensus Maf binding site (MARE, lane 8) were added to the assays in 100-fold molar excess before addition of probe. Maf binding to its consensus oligonucleotide interferes with its ability to complex with Myb.9Antibodies recognizing the c-Maf protein (lane 7) were added to binding reactions before probe addition. (D) The increase in Myb:Maf complexes is dose-dependent and comparable to induction on monocytic differentiation. The identical promoter fragment probe used in (C) was incubated with uninduced (0) or induced (indicated concentrations) whole-cell lysates from control vector-containing (CB6, lanes 10 and 11), c-Maf–containing (c-Maf, lanes 12 to 16) stable HL-60 clonal lines or from untreated (lane 17), TPA-treated (lane 18) HL-60 parental cells. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

Morphologic and lineage analysis of cloned U937 cell lines conditionally expressing c-Maf. Morphologic and lineage analysis of cloned U937 cell lines conditionally expressing c-Maf. Cytospin preparations of parental U937 cells either untreated (top row) or treated with TPA (second row); or U937 clonal lines containing either vector control (U937 CB6, third row) or c-Maf expression constructs (U937 c-Maf, bottom row) incubated for 48 hours with 150 μmol/L ZnSO4. Left column: Wright-Giemsa stain for morphological examination; right column: -napthyl butyrate esterase stain specific for monocytic lineage. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

c-Maf induces the expression of maturation markers in HL-60 cell lines. c-Maf induces the expression of maturation markers in HL-60 cell lines. (A) Fluorescence-activated cell sorting (FACS) analysis of the expression of the CD14 and CD11b myeloid maturation markers on untreated HL-60 cells, HL-60 cells treated with TPA for 24 hours, and 4 individual c-Maf expressing HL-60 clones treated with zinc for 24 hours. Negative control antibody binding is indicated by dashed lines. (B) Expression of c-Maf mRNA (top panel), CSF-1 receptor (CSF-1R, center panel), or control G3PDH (lower panel), as determined by Northern blot analysis of total RNA from uninduced (−, lane 1) or zinc-induced (+) control vector-containing (CB6, lane 2), c-Maf–containing (lanes 1, 3, and 4) stable HL-60 clones; or TPA-treated HL-60 parental cells (lane 5). A single blot was initially probed with the c-Maf–specific cDNA probe, stripped, and reprobed with CSF-1R and G3PDH; G3PDH served as a control for RNA loading and integrity. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

IL-4 expression is not induced in c-Maf–expressing cell lines. IL-4 expression is not induced in c-Maf–expressing cell lines. (A) IL-4 mRNA is not induced on c-Maf protein expression. RT-PCR analysis of cDNA templates from induced CB6 vector control (lanes 1, 3, 6, and 8) or c-Maf-containing clone 6 (c-Maf, lanes 2, 4, 7, and 9) using primer pairs detecting β-actin control (lanes 1 and 2), IL-4 (lanes 3 to 5), MafG (lanes 6 and 7), or c-Maf (lanes 8 and 9). M, marker lanes; +, IL-4 positive control DNA template. (B) IL-4 neutralizing antibodies do not affect c-Maf–induced monocytic differentiation. Neutralizing antibodies directed against IL-4 or isotype-matched control antibodies were added to cultures of HL-60 c-Maf clone 6 cells at the time of zinc induction. Top row: Wright-Giemsa stain; bottom row: monocyte-specific ANB esterase stain of cytospin preparations. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

Myb-dependent transcription is functionally impaired in clonal lines of HL-60 cells expressing c-Maf. Myb-dependent transcription is functionally impaired in clonal lines of HL-60 cells expressing c-Maf. HL-60 c-Maf clone 6 (▪) or CB6 vector control clone 3 (▨) were incubated at the indicated ZnSO4 concentrations for 12 hours to induce c-Maf protein expression, and then transiently transfected with 5 μg of the −411luc reporter construct containing sequences sufficient for wild-type level, tissue-appropriate expression from theCD13/APN myeloid promoter. Luciferase activities were assayed at 6 hours and normalized for differences in transfection efficiency with SEAP activity produced by the control β-actin-SEAP plasmid. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

MEnT does not induce the expression of monocyte specific markers in HL-60 cell lines. MEnT does not induce the expression of monocyte specific markers in HL-60 cell lines. Flow cytometric analysis of the expression of the CD14 and CD11b maturation markers on untreated, zinc treated (18 hours), or TPA treated (18 hours) HL-60 MEnT-containing cells. Negative control antibody binding is indicated by dashed lines. Data shown are a representative tracing of clonal line, HL-60 MEnT clone 1 (Western blot showing protein expression Fig 9A). Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

c-Maf–expressing cells die by apoptosis. c-Maf–expressing cells die by apoptosis. (A) c-Maf– and MEnT-containing cells show a sub-G1 peak indicative of apoptotic cell death. Positive control MEnT-containing (MEnT, top two panels) or c-Maf–containing (c-Maf, lower panels) that had been untreated or treated with ZnSO4 for 72 hours, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Representative data is presented. (B) c-Maf– and MEnT-expressing cells show increased TUNEL staining. HL-60 clones overexpressing MEnT, CB6 vector, or c-Maf were treated with zinc for 48 hours (c-Maf, CB6) or 24 hours (MEnT), stained, and assessed by flow cytometry. Representative data are presented. (C) c-Maf– and MEnT-containing cells show DNA “laddering” characteristic of apoptosis. DNA was extracted from negative control CB6 (lanes 1 and 2), c-Maf–containing (c-Maf, lanes 3 to 6), or positive control MEnT-containing cells (lanes 7 to 9) that had been treated with ZnSO4 for the indicated time periods and separated by agarose gel electrophoresis. Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology

c-Maf–expressing cells downregulate Bcl-2 protein levels. c-Maf–expressing cells downregulate Bcl-2 protein levels. (A) Cells expressing MEnT have decreased Bcl-2 protein levels. Western blot analysis of MEnT and Bcl-2 protein expression in uninduced or induced HL-60 clones (clones 1 and 2) containing the ZnSO4 inducible MEnT Myb dominant-interfering construct. (B) Bcl-2 protein levels decrease coordinately with c-Maf expression. Western blot analysis of c-Maf, Bcl-2, and actin control protein levels in HL-60 c-Maf–containing clone 6 incubated with ZnSO4 for the indicated time periods. Vector control cells were treated identically and assayed for Bcl-2 and actin protein levels (CB6 control). Shrikanth P. Hegde et al. Blood 1999;94:1578-1589 ©1999 by American Society of Hematology