Erythropoietin: new approaches to improved molecular designs and therapeutic alternatives.

Erythropoietin (Epo) is a glycoprotein hormone that is the prime regulator of erythropoiesis. Recombinant Epo is a highly effective pharmaceutical used to correct anemias associated with renal insufficiency, cancer and other diseases. Efforts to increase its efficacy in vivo by manipulating the protein's structure have met with some success, and novel Epo-like agents are in development. Additionally, efforts to create Epo mimetic agents are underway, as is the design of agents to increase endogenous production. Because Epo has tissue protective actions outside of erythropoiesis, other designs have focused on producing erythropoietically inactive molecules that still retain extra-hematopoietic activity. The demonstration that Epo can trigger signaling in some cancer cells with, potentially, adverse effects on patient health has raised warning signs in the medical community and has gained the attention of regulatory authorities.

Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc.

Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

Selenium utilization in humans--a long-term, self-labeling experiment with stable isotopes.

A stable (nonradioactive) isotope of selenium in a chemical form common in foods (selenomethionine) or inorganic selenite was taken orally (200 micrograms/d) for 3 wk to label deep body pools. By deep body pools we mean selenium compartments that are large and/or have a slow turnover (exchange) rate. Blood plasma was removed, stored for 11 mo, and later reinfused as a labeled tracer dose with the selenium label in all of the biologically significant chemical forms. Accessible tissues such as red blood cells were highly labeled (20-25%) in the subjects receiving selenomethionine. Selenium from deep body pools is excreted primarily via the urine (80%). Reexcretion of previously absorbed selenium back into the gastrointestinal tract can be measured, avoiding a major source of error in conventional balance studies used to estimate nutrient absorption.