Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl 2 ) in Hep3B cells. However, neither the mechanism of cellular O 2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O 2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O 2 ) or CoCl 2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA ( 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) 0 cells increase ROS generation in response to CoCl 2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl 2 in wild-type cells, and abolish the response to CoCl 2 in °cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl 2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

The use of replication-defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene-encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses direct against foreign transgene-encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long-term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials.

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Previous studies of the erythropoietic response to hypoxia in high-altitude natives suggest that the hematocrit and hemoglobin values in Himalayan natives (Sherpas) are lower than expected for the altitude, perhaps because of a genetic adaptation. However, differences in sampling techniques and experimental methods make comparisons difficult. Our studies were carried out to compare the erythropoietic response with the same altitude in age-matched natives of the Himalayas and Andes by the same experimental techniques. Healthy male subjects were selected in Ollagüe, Chile (n = 29, 27.3 +/- 5.9 yr) and in Khunde, Nepal (n = 30, 24.7 +/- 3.8 yr). Both of these villages are located at 3,700 m above sea level. Hematologic measurements confirmed lower hematocrit values in Nepal (48.4 +/- 4.5%) than in Chile (52.2 +/- 4.6%) (P less than 0.003). When subjects were matched for hematocrit, erythropoietin concentrations in Chile were higher than in Nepal (P less than 0.01). Detailed measurements of blood O2 affinity in Nepal showed no differences in shape or position of the O2 equilibrium curve between Sherpas and Western sojourners. Our results indicate that although Quechua Indians have higher hematocrits than Sherpas living at the same altitude, nevertheless they may be functionally anemic.