Transcriptional repression of c-Myb and GATA-2 is involved in the biologic effects of C/EBPalpha in p210BCR/ABL-expressing cells

Abstract

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.

Figure 1

c-Myb and GATA-2 expression in 4-HT–treated 32D-BCR/ABL-C/EBPα-ER cells. Western blots (representative of 3 experiments) show c-Myb (A) and GATA-2 (B) expression in 4-HT–treated 32D-BCR/ABL cells expressing wild-type or mutant C/EBPα-ER. (C) RT-PCR amplification of c-Myb or GATA-2 pre-mRNA in 4-HT–treated 32D-BCR/ABL-C/EBPα-ER cells using pairs of intron 1 c-Myb– or GATA-2–specific primers. RT-PCR amplification of pre-GAPDH RNA was used as control. Representative of 4 different experiments.

Figure 2

c-Myb and GATA-2 expression in 4-HT/siRNA-treated parental or C/EBPα-ER–expressing K562 cells. (A) Western blots show c-Myb and GATA-2 expression in 4-HT–treated wild-type C/EBPα-ER–expressing K562 cells. (B) Western blots show c-Myb or GATA-2 levels in K562 cells transfected with c-Myb or GATA-2 siRNAs. Data are representative of 3 experiments.

Figure 3

c-Myb and GATA-2 expression in 4-HT–treated C/EBPα-ER–transduced normal and CML-BC CD34⁺ cells. Western blots show c-Myb and GATA-2 levels and in normal CD34⁺ cells (A) and in CML-BC CD34-enriched (80%) cells (B). Expression of C/EBPα-ER and GRB2 was also assessed as control. Data are representative of 2 experiments. CD34⁺ normal cells were from 2 donors.

Figure 4

Effect of ectopic c-Myb expression on C/EBPα-induced differentiation of K562 cells. CD11b and CD15 levels (A); G-CSFR mRNA levels (B); morphology (C); and protein levels (D) in 4-HT–treated K562 cells. Data are representative of 3 experiments.

Figure 5

Effect of ectopic GATA-2 expression on C/EBPα-induced differentiation of K562 cells. CD11b and CD15 levels (mean plus SD); G-CSFR mRNA levels (B); morphology (C); and protein levels (D) of 4-HT–treated K562-C/EBPα-ER cells; representative of 3 different experiments.

Figure 6

Effect of ectopic c-Myb or GATA-2 expression on C/EBPα-dependent proliferation inhibition of K562 cells. (A) Cell counts of untreated and 4-HT–treated K562-C/EBPα-ER and Δ(358-452) c-Myb/K562-C/EBPα-ER cells. (B) Cell counts of untreated and 4-HT–treated C/EBPα-ER and GATA-2/K562-C/EBPα-ER cells. Mean plus SD (3 different experiments performed in triplicate).

Figure 7

Cell-cycle distribution of K562-C/EBPα-ER, K562-C/EBPα-ER-Myb, and GATA-2/K562-C/EBPα-ER cells. Histogram shows cell-cycle distribution (DNA content of propidium iodide–stained nuclei) of untreated and 4-HT–treated cells. Representative of 2 experiments.

Figure 8

Effect of c-Myb silencing on C/EBPα-induced differentiation and proliferation inhibition in K562 cells. c-Myb (A) and CD11b and CD15 (B) levels in K562-C/EBPα-ER cells treated with 4-HT and a control or c-Myb siRNAs. (C) Cell counts of siRNA-, 4-HT–, or siRNA and 4-HT–treated K562-C/EBPα-ER cells. Mean plus SD (3 different experiments, performed in triplicate).