Effects of FR901228 treatment on hematopoietic cells by RT-PCR and Western blot analyses.

(A) RT-PCR analysis of CAR and α_v integrin RNA levels in FR901228-treated cells. K562 cells were incubated without (−) and with the indicated concentrations of FR901228 for 72 hours before the isolation of RNA. Frozen PBMCs and CD34⁺ PBSCs were thawed and expanded in medium for 4 days and then treated without (−) and with the indicated concentrations of FR901228 for 24 hours before isolation of RNA. RNA was isolated, and RT-PCR was performed as previously described25 using β-actin as the loading control. For quantitation, which is shown below the photographs, the density of the bands was determined by densitometry, the brightest was set to a value of 100, and the lighter bands were normalized to it. nd indicates not detectable. (B) Western blot analysis of FR901228-treated cells. K562 cells or CD34⁺ PBSCs were incubated without (−) or with the indicated concentrations of FR901228. K562 cells were treated for 48 hours, and CD34⁺ PBSCs were treated for 24 hours. Total cellular protein was isolated, and Western blot analysis was performed as previously described.25 Histone H3 functions as the loading control. (C) RT-PCR and Western blot analyses of PBMCs from a patient treated with FR901228. RNA and protein were isolated from PBMCs from a patient receiving FR901228 on our phase 1 trial. Time points are before the start of drug infusion (−), at the end of the 4-hour infusion, and after 24 hours. Quantitation, which is shown below the photographs, is as described above. nd indicates not detectable