Role of p15 and TGF-β1 in myelodysplastic megakaryocytic differentiation.

(A) On day 7 the cell output in the MDS cultures was comparable to the cell output in the normal CD34⁺ cultures. In contrast, the proportion of CD41-expressing cells was significantly lower. These findings were not modified by AZA or by anti–TGF-β1 antibody. (B) p15 Amplification bands were detectable in the presence of unmethylated and methylated sequence-specific primer pairs in CD34⁺cells (lanes 1 and 2) and in day 7 CD41⁺ cells (lanes 3 and 4). On the contrary, in CD41⁺ cells cultured in the presence of AZA, p15 amplification bands were detectable only in the presence of unmethylated sequence-specific primer pairs (lanes 5 and 6). Lanes 7 and 8 show negative controls. U indicates unmethylated; M, methylated. (C) Semiquantitative RT-PCR analysis of p15 mRNA expression showed that p15 was not expressed in CD34⁺ cells (lane 1), was expressed after 7 day culture in CD41⁺ cells (lane 2, p15/β-actin ratio 0.4), and was strongly up-regulated in CD41⁺ cells treated with AZA (lane 3, p15/β-actin ratio 1). Neutralizing anti–TGF-β1 antibody abrogated p15 expression both in control (lane 4) and in demethylated (lane 5) CD41⁺cells. Negative and positive controls are shown in lanes 6 and 7, respectively. (D) Semiquantitative RT-PCR analysis of TGF-β1 mRNA expression demonstrated the increase of TGF-β1 during megakaryocytic differentiation. TGF-β1/β-actin ratios are 0.4 in CD34⁺cells (lane 1) and 0.95 in CD41⁺ cells on day 7 (lane 2). Negative and positive controls are shown in lanes 3 and 4, respectively.