Peter P. Dukes

Relationship between erythropoietin effect and reduced DNA synthesis in marrow cell cultures

Abstract 1. Erythropoietin effects were studied in cell cultures with greatly reduced DNA synthesis brought about by the addition of arabinosylcytosine. DNA synthesis, glucosamine incorporation, and heme synthesis were determined at different periods of incubation. 2. DNA synthesis did not appear to be a requirement at the time the intial event of erythropoietin action took place. 3. Erythropoietin stimulated glucosamine incorporation into cells did not seem to require increased DNA synthesis in the cultures. 4. The erythropoietin induced increase in early DNA synthesis bore a different relationship to initial and late stimulated heme synthesis in sensitive cells. Only the full expression of the late heme synthesis required the previous occurrence of stimulated DNA synthesis.

A clinical trial of prostaglandin E2 to increase erythropoiesis in anemia of end stage renal disease. A preliminary report

Prostaglandin E2 is known to stimulate erythropoiesis by different mechanisms. A clinical trial of prostaglandin E2 to stimulate erythropoiesis in four patients with anemia of end stage renal disease resulted in an increment in peripheral blood Burst Forming Units-Erythroid (BFU-E). This increase in erythroid progenitors returned to baseline with cessation of therapy. A significant increase in serum erythropoietin (EPO) activity was demonstrated in one patient and was noticeable in another. Side effects mainly consisted of local pain at the site of the infusion and vomiting.

The relationship between human spleen and blood erythroid burstforming units (BFU-E)

The influence of splenectomy on erythroid burst colony formation by peripheral blood mononuclear cells from 10 patients (four with hereditary spherocytosis, two with β‐thalassaemia major, two with Hodgkins disease and two with idiopathic thrombocytopenic purpura) was studied. In every instance splenectomy was followed by a lowering of blood BFU‐E. The post‐splenectomy levels ranged from 0 to 30% of the preoperative levels. Mononuclear cells from the spleens of eight patients were cultured and found to contain numerous BFU‐E. The total quantity of BFU‐E in the whole blood and in the spleen of the patients was generally of the same order of magnitude. The number of splenic BFU‐E did not correlate with spleen size.

A beneficial effect of the in situ kidney on in vitro marrow erythropoiesis in chronic renal failure.

The effect of the in situ kidney on transfusion requirements and in vitro erythropoiesis was investigated in 20 patients with end stage renal disease undergoing hemodialysis. 6 of the 12 patients with in situ kidneys did not require transfusion, whereas the other 6 had an average monthly transfusion requirement of 277 ml of sedimented RBCs. All 8 anephric patients required transfusions with an average requirement of 352 ml of sedimented RBCs per month. Serum erythropoietin activity was inappropriately low for the degree of anemia in all but 1 patient, and bone marrow was uniformly hypocellular. Marrow cells from patients with in situ kidneys exhibited a greater response to erythropoietin than marrow cells from their anephric counterparts. The response was not improved by hemodialysis.

Erythropoietin action in rat marrow cell cultures in complete absence of DNA synthesis I. Early effect on RNA synthesis

Summary Incubation of rat marrow cells with hexachloroiridate (4.5–5.5 × 10 −4 M) at 36.5° for 45 hours totally abolishes their DNA synthesis. One hour exposure to erythropoietin considerably stimulates the rate of RNA synthesis of such Iridium treated marrow cells. Since hexachloroiridate is reported to be an agent which inhibits mammalian cell division by blocking cells in the G 1 phase, it seems that early erythropoietin action resulting in stimulated RNA synthesis is independent of DNA synthesis and can take place in cells arrested in G 1 .

Incomplete Erythropoietin Activity in Normal Plasma Components

Summary Cohn fractions of normal human plasma were surveyed for erythropoietin activity by an in vivo and two in vitro assay systems. Fractions II + III, II + III W, and especially fraction III, were found to stimulate glucosamine incorporation and heme synthesis of marrow cells in culture. Log dose-log response regression lines of plasma fractions and of an erythropoietin standard were found to be parallel. Only traces of activity could be detected by the exhypoxic polycythemic mouse assay. Fraction III from several different sources and species was found to be active in vitro. A human fraction III was shown to have a different specific activity relative to a common erythropoietin standard in the two in vitro assays. Subfractionation of fraction III by extraction procedures demonstrated low stability for the activity measured by the 59Fe-heme assay, whereas it was possible to obtain without loss a preparation enriched in the activity stimulating glucosamine incorporation.

Erythropoietin action in rat marrow cell cultures in complete absence of DNA synthesis. II. Long term effects on macromolecular synthesis

Abstract Rat marrow cells were preincubated for 45 hours with 5.5 × 10 −4 M sodium hexachloroiridate. This treatment abolished DNA synthesis whilst improving cell survival over that of controls. The synthesis of RNA, protein and glycoprotein continued and could be further increased by the addition of erythropoietin for up to 44 more hours. Heme synthesis also continued in the absence of DNA synthesis but could not be stimulated by erythropoietin.