Effects of GATA-2 on the growth of normal hematopoietic cells.

(A) Normal MNCs and Sca-1⁺Lin⁻ cells were isolated from bone marrow of 5-FU–treated mice and infected with retrovirus containing pMX-GATA-2/ER-neo, pMX-neo, or pMX-GFP. The expression of GFP was examined in pMX-GFP–infected MNCs and Sca-1⁺Lin⁻ cells. Dashed-line histograms indicate mock-infected cells; solid-line histograms, pMX-GFP–infected cells. (B) The cells infected with retrovirus containing GATA-2/ER or an empty vector (Mock) were cultured with G418 for 72 hours and subjected to immunoblot analysis with an anti–GATA-2 Ab. IB indicates immunoblot; Ect, ectopic GATA-2/ER; End, endogenous GATA-2. (C) After 72 hours of culture with G418, retrovirus-transduced cells were also cultured with or without estradiol for 72 hours and subjected to an MTT colorimetric assay. The results are shown as the means ± SD of triplicate cultures. NS indicates statistically not significant. (D) Cell cycle analysis of the cultured cells was performed by PI staining. (E) The cultured cells were subjected to semiquantitative RT-PCR analysis on the expression of c-mycand Cul1 mRNA. Total cellular RNA was isolated at the time indicated and cDNA was synthesized. The amounts of cDNA products were normalized according to the amounts of PCR products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The adjusted amounts of cDNA products from each sample were subjected to PCR for c-myc, Cul1, and GAPDH. After the PCR cycles indicated, the PCR products were size-fractionated on 3.5% polyacrylamide gels, dried, and autoradiographed. (F) The expression levels of p21^WAF1 and p27^Kip1proteins in the cultured cells were examined by Western blot analysis. Results representative of 3 independent experiments are shown.