MEK inhibition causes cell cycle arrest and modulates p27^Kip1 and p21^Waf1/CIP1 expression. (a) OCI-AML3 (open circles), HL-60 (filled circles), and freshly isolated leukemic blasts from patient 3 (filled squares) were cultured for 48 hours in the presence of DMSO or PD98059 at the indicated concentrations, and then stained for DNA content. Results are expressed as percentage of S-phase cells in the DMSO-treated group. Cell line results are representative of one of three independent experiments. (b) OCI-AML3, HL-60, and U937 cells were cultured for 24 hours in the presence of DMSO or PD98059 (20 μM), and subjected to Western blot analysis with mAb’s specific for the indicated cell cycle–regulating proteins. Results are representative of one of three independent experiments. (c) OCI-AML3 cells were cultured for the indicated times in the presence of DMSO or PD98059 (20 μM), and subjected to quantitative real-time PCR analysis of p21^Waf1/CIP1 (filled circles) and p27^Kip1 (open circles) RNA transcripts. Results are expressed as percentage of specific RNA transcripts in the DMSO-treated group and are representative of one of two independent experiments. (d) OCI-AML3 cells were cultured for the indicated times in the presence of DMSO or PD98059 (20 μM) and assessed for DNA content. Results are expressed as percentage of S-phase cells in the DMSO-treated group. Two hours after the addition of PD98059, cells were lysed, and the phosphorylation status of p27 ^Kip1 was analyzed by Western blot using T187-phosphospecific Ab’s (p-p27^Kip1).