Abstract #4283: Erythropoetin receptor (EpoR) was expressed at low to undetectable levels in tumor cell lines: Expression was not regulated by hypoxia and no epo-induced downstream signaling was detectable

EpoR mRNA and protein expression were surveyed in 67 tumor cell lines from ovary, breast, head and neck, lung, brain, prostate, cervix, liver, colon and blood. A sensitive and specific anti-EpoR monoclonal antibody (mAb), A82, was recently discovered allowing analysis of EpoR levels in human cells by immunoblotting. Total EpoR protein levels were estimated by comparing the amount of full length (59 kDa) EpoR protein in total cell lysates to a known amount of purified EpoR protein. The functional form of EpoR is a dimer, so total EpoR protein levels are reported as dimers/cell. The cell lines, UT-7/Epo and OCIM-1 cells were used as positive controls and in repeat experiments the total amount of EpoR varied 2-3-fold (30,000 -100,000 and 8,000 -16,000 EpoR/cell respectively). Primary human erythroid cells (generated from purified CD34 + cells after 4 days in-vitro culture) had 32,000-64,000 EpoR/cell. Scatchard analysis of UT-7/Epo and OCIM-1 cells demonstrated 10-20% of the total EpoR protein was detected on the cell surface, while COLO677 and 769-P cells showed no binding (and also had no detectable EpoR protein). In a survey of 61 solid tumor cell lines, 26 had no detectable EpoR protein ( 125 I-rHuEpo) were performed and specific surface binding was detected in one line (NCI-H661 cells) where the cpms bound were ~6% that of UT-7/Epo cells. Second, Epo/EpoR-mediated signaling was assessed. No stimulation of phosphorylation of STAT5, AKT2, Erk1/2, or p70S6RP was seen in any of the 6 lines when treated with rHuEpo. Stimulation of phosphorylation was observed in these cells using a cocktail of growth factors, and in UT-7/Epo cells treated with rHuEpo. The effect of hypoxia (1% O 2 ) on expression of EpoR mRNA and protein was also investigated in all of the tumor lines. VEGF and BNIP3 mRNA showed an average 4- and 5-fold increase respectively with hypoxia. In contrast the fold change in level of EpoR mRNA clustered around 1.0 as did the fold change for the housekeeping genes cyclophilin B and s-actin. Likewise, EpoR protein was not induced by hypoxia; 26 of the lines had undetectable EpoR protein in both hypoxic and ambient growth conditions. For those where a change could be assessed, the fold change also clustered around 1.0 with a maximum of 2 to 3 - fold. Taken together these results show that, in this panel of cell lines, EpoR is expressed at low to undetectable levels even after hypoxic growth conditions and there was no evidence of intracellular signaling upon rHuEpo addition. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4283.