Detection of Hypoxia-Inducible Gene Manipulation.

Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.