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.

Erythropoietin (Epo)-responsive anemia is a common and debilitating complication ofchronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 ,ug of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 ± 0.4% to levels of 64 ± 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 ,ug of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent 10876The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The carbohydrate composition of human erythropoietin (epo) was determined by micro-GLC. Enzymic removal of most of the sugars results in aggregation of glycosidase-treated epo, loss of biological activity when assayed in mice, and retention of activity when assayed in marrow cell cultures or by RIA. Endoglycosidase F causes the removal of most of the carbohydrates indicating that the oligosaccharides are asparagine linked. The lack of O-linked sugar is confirmed by the absence of N-acetylgalactosamine. These findings indicate that the oligosaccharide portion of epo, although required for action in vivo, is not required for interaction with the target cells of the blood-forming system.

The relationship of serum immunoreactive erythropoietin to haemoglobin concentration was defined for 54 patients with rheumatoid arthritis (RA) and 41 patients with anaemia of varying aetiology (excluding pregnancy and renal insufficiency), not associated with RA. Significant inverse correlations between the logarithm of serum immunoreactive erythropoietin and the haemoglobin concentration were noted for the anaemic patients in both groups. The regression line for the RA patients had a similar slope, but a significantly lower y-intercept as compared to that for the non-RA patients. Erythropoietin levels were also significantly lower for the group of RA patients than for the group of non-RA patients when matched for comparable haemoglobin concentrations. These studies suggest that the erythropoietin response to anaemia in RA is intact but blunted relative to that for anaemia of other aetiologies. Lower levels of serum erythropoietin in anaemic RA patients may contribute to the pathogenesis of their anaemia.

Injection of cobalt into rats resulted in erythropoietin (EPO) mRNA accumulation in the kidney. The same response was obtained upon bleeding. No EPO mRNA was detected in the spleen, salivary gland, or thymus following cobalt injection or bleeding. In some animals, but not in others, EPO mRNA was also expressed in the liver in response to cobalt injection. Time course studies showed that message appearance begins sometime between 3 and 6 h after cobalt injection. This correlated very well with the EPO concentration in the circulation; EPO levels in the circulation were the same as those of controls at 3 h but increased to six-to sevenfold that of controls by 6 h after cobalt injection. The mature EPO mRNA in the rat and mouse comigrated with the 18S rRNA, indicating that it is about 1,850 nucleotides in length.Abundant evidence indicates that, in the adult mammal, the kidney is a major source of erythropoietin (EPO) (2,5,10,11,17,18 These questions can now be addressed more directly by the use of cloned mouse EPO probes (15). We have induced EPO appearance in the circulation by cobalt injection (7) or bleeding and have monitored both EPO levels in the serum and EPO mRNA transcription in various tissues as a function of time. The data indicate that there is accumulation of the EPO mRNA in the kidney after stimulation and that, except for the liver, no other tissue examined shows the presence of the mRNA.MATERIALS AND METHODS Animals and cells. Male Long Evans rats, 2 to 4 months old (Charles Rivers Breeding Laboratories Inc., Wilmington, Mass.), were used in these experiments. In the experiments that used CoCl2, the rats were injected with 1 ml of 75 mM CoCl2 solution in saline subcutaneously in the back and killed by carbon dioxide asphyxiation at specified time intervals. Controls were injected with saline and killed immediately. In the bleeding experiments, 3 ml of blood (1.5 % of body weight) was drawn by cardiac puncture, and the animals were killed 10 h later. The kidneys, livers, thymuses, salivary glands, and spleens were dissected out and frozen in a dry ice-ethanol bath. Once frozen, the organs were stored at -70°C until RNA was extracted from them.The Assay of EPO. Where EPO levels in the serum were measured, blood was obtained by cardiac puncture and allowed to clot for 48 h at 4°C, and serum was collected after centrifugation. EPO was assayed in the serum by radioimmunoassay (19). The assays were done on four different volumes of serum, each one in triplicate.RNA preparation. Frozen tissues were crushed to a powder and homogenized in 4 M guanidine thiocyanate, using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, N.Y.). RNA was extracted by the method of Chirgwin et al. (3), as modified by Feramisco et al. (6). Cell pellets, in contrast to tissues, were simply suspended in 4 M guanidine thiocyanate, and the liberated DNA was sheared by repeatedly taking the lysate up in a syringe and ejecting through an 18-gauge needle before proceeding with the RNA extraction.After precipitation with e...