Molecular mechanisms associated with the regulation of apoptosis by the two alternatively spliced products of c-Myb

Abstract

The c-myb proto-oncogene encodes two alternatively spliced mRNAs, which in turn code for proteins of 75 kDa and 89 kDa. It is at present unclear whether the two isoforms of c-Myb perform identical functions or whether they mediate different biological effects. To assess their role in apoptotic death of hematopoietic cells, we expressed the two isoforms of c-Myb in the murine myeloid cell lines 32Dcl3 and FDCP1. Our results show that while ectopic overexpression of p75 c-Myb results in the acceleration of cell death, similar overexpression of p89 c-Myb results in the protection of cells from apoptotic death. An analysis of gene expression changes with mouse cDNA expression arrays revealed that while p75 c-Myb blocked the expression of glutathione S-transferase micro mRNA, p89 c-Myb greatly enhanced the expression of this gene. These results were further confirmed by Northern blot analysis. Ectopic overexpression of the glutathione S-transferase micro gene in 32Dcl3 cells resulted in protection of cells from interleukin-3 withdrawal-induced cell death similar to that seen with the ectopic overexpression of p89 c-Myb. These results suggest that the two isoforms of c-Myb differentially regulate apoptotic death of myeloid cells through differential regulation of glutathione S-transferase micro gene expression.

FIG. 1.

Transcriptional transactivation by p75 c-Myb, p75Δlz c-Myb, p89 c-Myb, and t-Myb. (A) Schematic representations of wild-type p75 c-Myb and p89 c-Myb and mutants p75Δlz c-Myb and t-Myb are depicted. The numbers above each diagram are positions of amino acid residues in each corresponding region. Horizontal arrows represent the three 51- to 52-amino-acid repeats that constitute the DNA-binding domain. The stretch of amino acids below represents the putative leucine zipper motif present in wild-type p75 c-Myb. The downward arrows indicate the two leucine to alanine mutations made in order to construct p75Δlz c-Myb. DBD, DNA binding domain; TA, transactivation domain; NRD, negative regulatory domain; LZ, leucine zipper; E9A, exon 9A. (B) Schematics of the reporter and effector plasmids used in transient transactivation assays. The dotted box represents the promoters, and the arrows in the promoters represent the starting sites of transcription. The arrows in Myb represent the three 51- to 52-amino-acid repeats of the DNA binding domain, and the black box represents the transactivation domain. The boxes labeled A in pTA3-luc represent the three Myb-binding sites. TK, herpes simplex virus thymidine kinase promoter; CMV, immediate-early promoter for cytomegalovirus. (C) Transient expression of effector molecules. Cell lysates from QT6 cells transfected with different myb expression plasmids and expressing equal amounts of firefly luciferase activity were subjected to Western blot analysis and probed with an antibody raised against c-Myb. (D) Transcriptional activation by Myb proteins. Each myb expression plasmid was transfected into QT6 cells with reporter plasmid pTA3-luc or pT81-luc and the pRL-CH110 control plasmid as described in the text. After 2 days, the cells were harvested and assayed for firefly luciferase activity with the dual luciferase system (Promega). The luciferase activities were normalized to Renilla luciferase activity, and the activation was obtained by setting the value of empty vector at 1.0. Shown are the means of activation transcriptional potential from three independent experiments.

FIG. 2.

Constitutive expression of p75 c-Myb, p89 c-Myb, and p75Δlz c-Myb in 32Dcl3 cells. p75^c-myb, p89^c-myb, and p75Δlz-c-myb cDNAs in the pMT-neo vector were transfected into 32Dcl3 cells. and mass cultures as well as single cell clones were established. (A and D) Northern blot analysis of total RNA extracted from different cell lines with a full-length c-myb cDNA probe. Endogenous c-myb transcript (upper band) and exogenous p75^c-myb transcript (lower band in panel A), p89^c-myb transcript (middle band in panel A), and p75Δlz-c-myb transcript (lower band in panel D) are shown. As an internal control for RNA loading, blots were stripped and reprobed with full-length GAPDH cDNA (shown below). (B and E) Expression of c-Myb protein in the presence of IL-3 in empty-vector-, p75^c-myb-, and p89^c-myb-transfected cells (B) and p75Δlz-c-myb-transfected cells (E). (C and F) Expression of c-Myb protein in the presence of G-CSF for 10 days in empty-vector-, p75^c-myb- and p89^c-myb-transfected cells (C) and p75Δlz-c-myb-transfected cells (F). −Zn and +Zn indicate the absence and presence, respectively, of 100 μM ZnCl₂ used to induce the expression of different transgenes in the metallothionein promoter-based constructs.

FIG.3.

Effect of p75, p75Δlz, and p89 c-Myb proteins on IL-3 withdrawal-induced apoptosis of 32Dcl3 cells. (A) Wild-type, mock-transfected (empty-vector pMTneo) and 32Dcl3 cells expressing exogenous p75, p75Δlz, and p89 c-Myb proteins were washed in IL-3-free medium and incubated up to 8 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent a mean of three experiments. (B and C) Wild-type 32D cells were subjected to IL-3 withdrawal, and the quantity of c-myb mRNA and protein was measured at the indicated time points by Northern blot and Western blot analyses, respectively. As a control for RNA loading, filters were stained with ethidium bromide to compare the levels of 28S and 18S RNAs. (D) Analysis of DNA fragmentation. At the indicated times following IL-3 withdrawal, DNA fragments released from 10 × 10⁶ cells from different 32D cell lines were extracted, separated by electrophoresis, and stained with ethidium bromide. (E) Cell cycle analysis of empty-vector, p75^c-myb and p89^c-myb cells at day 0, 8 h, and day 2 following IL-3 withdrawal. The cells were fixed and stained with propidium iodide, and DNA content was measured with a flow cytometer. The horizontal lines designated B, C, and D in the graphs represent the amount of DNA in the cells (1N, intermediate, and 2N, respectively) and therefore correspond to cells in G₁, S, and G₂ phases, respectively.

FIG. 4.

Analysis of caspase activity in p75-, p75Δlz-, and p89^c-myb-transfected cells. (A) Cleavage of nuclear lamin B in wild-type, p75Δlz-c-myb, p75^c-myb and p89^c-myb cells subsequent to withdrawal of IL-3 from the medium. Cell lysates from different cell lines at the indicated time points were subjected to Western blotting with anti-lamin B antibody. The uncleaved lamin B is represented as a 69-kDa protein. A cleaved product of 32 kDa is detected secondary to lamin B breakdown by caspases. (B) Cytochrome c release during IL-3 withdrawal. Immunoblot analysis of cytosolic and organelle-bound fractions (as described in the text) after subjecting the various 32D cell lines to 0, 12, and 24 h of IL-3 withdrawal. The protein samples were analyzed by Western blotting with anti-cytochrome c monoclonal antibody. wt, wild-type; ev, empty vector. (C) Activation of DEVDase (caspase 3) and LEHDase (caspase 9) by IL-3 withdrawal. Empty-vector (pMTneo), p75^c-myb and p89^c-myb transfected cells were harvested at 0, 6, 12, 24, and 48 h following IL-3 withdrawal. After lysing the cells in Triton X-100-containing buffer as described in the text, the lysates were clarified by centrifugation, and the supernatants (50 μg of protein) were incubated with 50 μM substrate, including DEVD-AFC and LEHD-AFC, at 37°C for 1 h. Levels of released AFC were measured with a fluorescence microplate reader. Data are means of three experiments.

FIG. 5.

Effect of p75- and p89 c-Myb proteins on IL-3 withdrawal-induced apoptosis of FDCP1 cells. Wild-type, mock-transfected (empty-vector neo), and 32Dcl3 cells expressing exogenous p75 and p89 c-Myb proteins were washed in IL-3-free medium and incubated up to 4 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent means of three experiments.

FIG. 6.

Gene expression profile of 32Dcl3 cells with constitutive expression of p75 c-Myb and p89 c-Myb in the absence of IL-3. Atlas mouse cDNA expression arrays (Clontech) were used to analyze the differential gene expression of 32Dcl3 cells transfected with alternatively spliced c-myb gene products 9 h following IL-3 withdrawal. The arrays include 588 mouse cDNAs, 9 housekeeping control cDNAs, and negative controls immobilized in duplicate on a nylon membrane. Each array is divided in six functional subclasses (A to F) of 98 genes each. The genes on each functional subclass are laid on a 7 by 14 grid (1 to 7 horizontally and a to n vertically). Following hybridization and washing, the array filters were scanned with a Fuji PhosphorImager. (A) Gene expression of empty-vector-transfected 32Dcl3 cells at 9 h afrter IL-3. (B) Gene expression of p75^c-myb-transfected 32Dcl3 cells at 9 h after IL-3. (C) Gene expression of p89^c-myb-transfected 32Dcl3 cells at 9 h after IL-3. The boxes in panels A, B, and C highlight the position and expression of GSTμ in the different cell lines.

FIG. 7.

Activation of GSTμ in 32Dcl3 cells following IL-3 withdrawal. (A) Northern blot analysis of total RNA extracted at the indicated times following IL-3 withdrawal from wild-type 32Dcl3 cells with a full-length GSTμ cDNA probe. As an internal control for RNA loading, blots were stained with ethidium bromide after completion of RNA transfer onto the nitrocellulose filter to compare levels of 18S and 28S rRNAs across lanes (shown below). (B) Comparison of GSTμ expression by Northern blotting in wild-type-, empty-vector-, p75^c-myb-, and p89^c-myb-transfected cells in the presence of IL-3 and at 9 h following IL-3 withdrawal. (C) Expression pattern of GSTμ in empty-vector-, p75^c-myb-, p89^c-myb-, and p75Δlz-c-myb-transfected cells. Total RNA was isolated from each cell line at the indicated time points following IL-3 withdrawal and subjected to Northern blotting.

FIG. 8.

Effect of GSTμ on IL-3 withdrawal mediated apoptosis of 32Dcl3 cells. (A) Constitutive expression of GSTμ cDNA in 32Dcl3 cells with the IPTG-inducible pOPRSVI/MCS vector. Total RNA was extracted from mock-transfected and GSTμ-transfected cells and probed with full-length GSTμ cDNA in the absence or presence of IPTG. As a control for RNA loading, filters were stained with ethidium bromide to compare the levels of 28S and 18S RNAs (shown below). (B) Analysis of the viability of GSTμ-transfected cells in the absence of IL-3. Mock-transfected (32D/E) and GSTμ-transfected (32D/GST) 32Dcl3 cells were washed in IL-3-free medium and incubated up to 8 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent means of three experiments. (C) Analysis of DNA fragmentation. At the indicated times following IL-3 withdrawal, DNA fragments released from 10⁷ cells from the two 32D cell lines were extracted and separated by electrophoresis and stained with ethidium bromide. (D) Analysis of caspase activity by determining the breakdown of nuclear lamin B. Mock-transfected and GSTμ-transfected cells were incubated in IL-3-free medium, and the cell lysates from the indicated time points were subjected to Western blotting with anti-lamin B antibody. The full-length lamin B is detected as a 69-kDa protein. The caspase activity is marked by lamin B cleavage, detected as a 32-kDa product.

FIG. 9.

Effect of GSTμ on 32D/c-myb p75 and p89 cells. (A) 32D/c-myb p89 cells were transfected with GSTμ antisense oligodeoxynucleotides (ODN1 and ODN2) and a nonspecific oligonucleotide (cntrl). At 3 h following transfection, the cells were washed in IL-3-free medium and incubated for 3 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent means of three experiments. (B) 32D/c-myb p75 cells were transfected either with an empty vector or with the GSTμ expression vector, and stable transfectants were selected (GSTμ/1 and GSTμ/2). Cells were washed in IL-3-free medium and incubated for 3 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent means of three experiments.