Relative amount of MPO mRNA detected by RT-PCR in primary AML cells of the M3 or M2 subclasses. (A) Autoradiograph shows radiolabeled RT-PCR products from primary AML cells. Lanes 1 to 10, AML-M3 or -M4 cases from Table 1. The top panel shows 550-bp PCR products derived from random primed cDNA using MPO primers. The bottom panel shows products from a separate PCR using actin primers. The reactions in lanes 1 to 4 and 5 to 10 are from different experiments. (B) APL cells from patient no. 803 were cultured for 20 hours in the presence (+RA) or absence (−RA) of RA (10−6 mol/L). RT-PCR was performed with MPO primers. Lane 1 is the same as lane 5 in A. The actin control reactions were repeated to include both cDNAs. (C) Range of MPO transcript levels in the different AML donor cells in multiple experiments is shown with standard deviations indicated. Time in culture ranged from 48 hours to 2 weeks before RNA isolation. Different RT-PCR experiments were normalized to β-actin cDNA levels and also to the MPO signal from a cDNA standard prepared from the NB4 cell line (APL-derived). Relative amount of MPO protein in the different AML cell samples was determined by SDS gel electrophoresis of whole-cell extracts. Proteins were blotted onto nylon membrane, reacted with anti-MPO antibodies, and quantified. Values shown are an average of 3 separate experiments.
