ALBERT

Description: PML-RARa is very toxic to early myeloid cell lines that express neutrophil elastase. As a consequence, myeloid cell lines that stably express PML-RARa must undergo significant adaptations to avoid toxicity (eg. loss of expression of neutrophil elastase (Lane and Ley, MCB, 2005). To identify the direct target genes of PML-RARa, we decided to bypass the issue of adaptation by profiling naive U937 cells that transiently expressed PML-RARa. To define “immediate-early” expression changes caused by PML-RARa, we harvested U937 cells electroporated with expression vectors containing eGFP-PML-RARa (known to possess all the measurable functional properties of PML-RARa) vs. eGFP alone, at the earliest time point when adequate numbers of eGFP positive cells could be collected via high speed cell sorting (6 hours). RNA obtained from these cells was subjected to linear amplification and hybridized with Affymetrix U133 Plus 2.0 arrays. Probesets that demonstrated the largest differences in expression between cells transfected with eGFP vs. eGFP-PML-RARa were PML and RARa themselves (15-fold increase for PML, 9-fold for RARa), providing an important validation for the experimental system. Differentially expressed genes were selected by three filters for further study: (i) ANOVA statistic (P 〈 0.05); (ii) “present” call and 〉150 average expression units in at least one sample; and (iii) Reproducible (n=2) 〉2-fold differences between eGFP-PML-RARa vs. eGFP vector transfected cells. Only 280 probesets (out of 54,613 total) met all of these criteria. Among them, 37 genes were involved in signal transduction, 15 in cell proliferation and differentiation, 7 in apoptosis, 14 in cell cycle, 15 in transport, 27 in protein metabolism and modification, 13 in oncogenesis, and 13 in immunity and defense (Panther classification system). 15/15 genes selected from a variety of categories were further validated with Q-PCR in duplicate using replicate samples, and all differences were statistically significant (p