Masato Higuchi

Effect of recombinant human erythropoietin on anticancer drug-induced anaemia.

Summary. Anaemia was induced in rats with fluorouracil (5‐FU) or cisplatin (CDDP) and the mechanisms of anaemia induction were analysed. Furthermore, the therapeutic effects of recombinant human erythropoietin (rHu Epo) on these anticancer drug‐induced anaemias were investigated.

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2.

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.

Quantitative expression of erythropoietin receptor (EPO‐R) on acute leukaemia cells: relationships between the amount of EPO‐R and CD phenotypes, in vitro proliferative response, the amount of other cytokine receptors and clinical prognosis

Expression of erythropoietin (EPO) receptor (EPO‐R) was analysed in leukaemia cells from 150 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). EPO‐R was expressed in 81 (60%) out of 136 AML, and in vitro treatment with EPO led to proliferation of leukaemia cells in 13 (16%) out of 81 AML examined. EPO‐R expression and in vitro response to EPO were observed in all subtypes of AML according to the French–American–British (FAB) classification. All eight patients with FAB‐M6 expressed EPO‐R, and one out of four showed an in vitro response to EPO. Although there was no significant correlation (r = 0.2522) between the amount of EPO‐R and the in vitro response to EPO, all of the AML patients who showed in vitro response expressed EPO‐R. Stem cell factor significantly enhanced both EPO‐R expression and in vitro response to EPO. Interleukin‐3 tended to increase in vitro response to EPO. CD phenotypes, the amount of granulocyte colony‐stimulating factor (G‐CSF) receptors and the amount of TPO receptors had no significant relationship with the amount of EPO‐R. Patients with both EPO‐R expression and in vitro response to EPO had shorter duration of complete remission than those without EPO‐R (P = 0.0053). EPO‐R was expressed in four (29%) out of 14 ALL, and none out of five ALL showed in vitro response to EPO.

Oncostatin M and leukemia inhibitory factor increase hepcidin expression in hepatoma cell lines

Overproduction of hepcidin by interleukin-6 (IL-6) is considered to be the main factor responsible for the development of anemia in inflammatory conditions. Since oncostatin M (OSM), a member of the IL-6 family, plays an important role in immune and inflammatory responses, we assessed the effect of OSM on hepcidin expression, as well as that of leukemia inhibitory factor (LIF), another member of the IL-6 family. We found that hepcidin expression was markedly induced by OSM and LIF in a time- and dose-dependent manner in hepatoma cell lines, and this expression was induced independent of IL-6/IL-6 receptor signaling. Luciferase assay revealed that OSM and LIF stimulated a −1.3-kb hepcidin promoter. This effect was markedly reduced when the signal transducer and activator of transcription (STAT) site of the promoter was mutated, and was almost completely abolished in the presence of AG-490, a Janus kinase (JAK) inhibitor. Hence, the JAK/STAT pathway plays a major role in OSM- and LIF-induced activation of the hepcidin promoter. In conclusion, we demonstrated that OSM and LIF can induce hepcidin expression mainly through the JAK/STAT pathways. Further studies are warranted to evaluate the clinical significance of OSM and LIF in the development of anemia in various inflammatory diseases.

Erythropoietin Receptor in Myelodysplastic Syndrome and Leukemia

Erythropoietin (Epo) is one of the main regulators of growth and differentiation of hematopoietic cells. In normal bone marrow cells, the amount of erythropoietin receptor (EpoR) was highest in the CD34 + CD38 m subset, and decreased on lineage committed progenitor cells expressing CD38 antigens. Among the erythroid cells expressing GpA antigens, CD34-positive fractions expressed more EpoR than CD34-negative fractions. Although the amounts of EpoR of bone marrow cells from patients with refractory anemia (RA) were less than those of normal bone marrow cells in all phenotypes examined, there was no statistical significant difference. EpoR was detected on leukemia cells from 60% of acute myeloblastic leukemia (AML) cases and 29% of acute lymphoblastic leukemia (ALL) cases, and distributed widely among all FAB-subtypes. In spite of the presence of EpoR, in vitro proliferative response to Epo was not observed in a large proportion of AML. And there was no correlation between the amount of EpoR and the in vitro response to EPO. Patients with both EpoR expression and in vitro response to Epo had shorter remission duration than those without EpoR.

Effects of recombinant human erythropoietin on anaemic W/Wv and Sl/Sld mice.

Summary. The effects of recombinant human erythropoietin (rHuEPO) on anaemic W/Wv and SI/SId mice were investigated. rHuEPO was injected every day for a week in doses up to 86000 iu/kg. Wv/+ and SId+ mice, which have genetically a weak anaemia, received 17 or 86 iu/kg of rHuEPO and showed dose‐dependent increases in haemoglobin, PCV, RBC and reticulocytes to the same extent as that in normal mice. W/Wv mice also showed increases in the haematological parameters in response to 8600 iu/kg of rHuEPO but the dose was much higher than that for normal mice. A reticulocyte increase in W/Wv mice appeared later than in normal mice and was not sustained for 2 weeks even though the rHuEPO treatment was continued. SI/SId mice, however, did not show any significant haematological effect from doses up to 86 000 iu/kg. In both W/Wv and SI/SId mice receiving 8600 and 86000 iu/kg of rHuEPO, respectively, an increase in splenic or bone marrow CFU‐E was observed regardless of the defect in their haemopoietic systems. The plasma erythropoietin (EPO) level in W/Wv and SI/SId mice was inversely correlated with the haemoglobin, indicating that EPO production was not influenced by the haemopoietic defect and was regulated by the hypoxic properties of the anaemia. These results indicate that a large dose of exogenous rHuEPO is effective for the anaemia in W/Wv mice caused by a stem cell defect but not for the anaemia in SI/SId mice caused by a defective microenvironment.

Effect of purified recombinant human erythropoietin on anemia in rats with experimental renal failure induced by five-sixth nephrectomy

Recombinant human erythropoietin (rHuEPO) was purified from the conditioned media of Chinese hamster ovary cells with a transfected human erythropoietin gene. We investigated the effects of the rHuEPO in rats with renal anemia induced by partial nephrectomy. Five-sixth nephrectomy resulted in renal failure with anemia. Twenty-five days after the operation plasma urea nitrogen was increased about 2.5 times, and the red blood cell count, hematocrit, and hemoglobin concentration fell to 85% of normal. The reticulocyte count and plasma erythropoietin level did not change such as they do in patients with anemia due to chronic renal failure. Both total red blood cell volume and the plasma iron turnover rate were depressed in five-sixth nephrectomized rats compared with normal rats.The five-sixth nephrectomized rats were injected with rHuEPO (60 IU/kg) intravenously every second day for a total of six injections. After three injections of rHuEPO, circulation volume of total red blood cells was increased from 9.9 ml to 14.6 ml, and the plasma iron turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day, and the reticulocyte count was also increased. After six injections, a marked increase of the red blood cell count, hematocrit, and hemoglobin concentration were observed. Plasma urea nitrogen and the creatinine levels as indications for renal function did not change after rHuEPO administration in both normal and five-sixth nephrectomized rats.In conclusion rHuEPO has a potent erythropoietic action and it is possible to cure the anemia caused by renal failure.

Effects of recombinant human erythropoietin on haemolytic anaemia in mice

The effects of repeated administration of recombinant human erythropoietin (rHuEPO) were investigated in mice with haemolytic anaemia. Mice with haemolytic anaemia induced by phenylhydrazine (PHZ mice) were examined as an acute model and New Zealand black mice (NZB mice) at 13 months of age were examined as a chronic model. The plasma erythropoietin (EPO) level in PHZ mice was high and showed a strong inverse correlation with the Hb in the anaemia development period. However, it was relatively low in the recovery period from anaemia. On the other hand, the plasma EPO level in NZB mice showed a simple inverse correlation with the Hb. The rHuEPO was injected every day for a week into these mice. While a high plasma EPO level was maintained in PHZ mice, no significant effect was observed by injection with rHuEPO at dose of 600 IU/kg. However, in the recovery period from anaemia, RBC and haemoglobin in PHZ mice were increased by the rHuEPO treatment and recovered more quickly to their normal levels. In NZB mice, RBC and haemoglobin were also increased by treatment with rHuEPO at dose of 600 IU/kg. Anti‐RBC autoantibodies and anti‐EPO antibodies did not increase, while RBC and plasma EPO levels were increased by the rHuEPO treatment. These results suggest that some types of haemolytic anaemia are not always combined with high endogenous EPO levels and that exogenous rHuEPO may be effective for use in the treatment of haemolytic anaemia.

Alteration of mRNA expression of molecules related to iron metabolism in adenine-induced renal failure rats: a possible mechanism of iron deficiency in chronic kidney disease patients on treatment

BACKGROUND Recombinant human erythropoietin (rHuEpo) is a definitive treatment for anaemia in chronic kidney disease (CKD). During long-term rHuEpo treatment most patients develop and show persistent iron deficiency in spite of oral iron supplementation. Abnormalities of iron absorption and transport in the duodenum may contribute to this deficiency. METHODS To investigate changes in iron absorption and transport in CKD and iron deficiency against the background of rHuEpo treatment, we used severely anaemic rats with adenine-induced renal failure (adenine rats) and sham-treated control rats given only the vehicle. After 4 weeks on adenine or the vehicle, the rats were divided into four groups according to whether or not they received rHuEpo for the next 4 weeks: rHuEpo(-)-adenine, rHuEpo(-)-control, rHuEpo(+)-adenine and rHuEpo(+)-control. We evaluated the effects of rHuEpo treatment on iron balance, duodenal mRNA expression of molecules related to iron absorption and transport and hepatic mRNA expression of hepcidin. RESULTS Treatment with rHuEpo improved anaemia and induced iron deficiency only in the adenine rats, in whom the expression of mRNAs for ferroportin 1 and hephaestin 1 increased and for divalent metal transporter 1 (DMT1) was unchanged. In contrast, control rats treated with rHuEpo showed no changes. Hepcidin mRNA expression was greater in adenine rats than in control rats. CONCLUSIONS In the adenine rats, rHuEpo treatment improved renal anaemia and induced persistent iron deficiency. An alteration of mRNA expression of molecules related to iron metabolism in renal insufficiency may be one of the reasons for this iron deficiency.

Increased mobilization of c-kit+ Sca-1+ Lin− (KSL) cells and colony-forming units in spleen (CFU-S) following de novo formation of a stem cell niche depends on dynamic, but not stable, membranous ossification

Stem cells are thought to inhabit in a unique microenvironment, known as “niche,” in which they undergo asymmetric cell divisions that results in reproducing both stem cells and progenies to maintain various tissues throughout life. The cells of osteoblastic lineage have been identified as a key participant in regulating the number of hematopoietic stem cells (HSCs). HSCs receive their regulatory messages from the microenvironment in the bone marrow. This would account for a reason why the localization of hematopoiesis is usually restricted in the bone marrow. To clarify the above possibility we employed a cell implantation‐based strategy with a unique osteoblast cell line (KUSA‐A1) derived from a C3H/He mouse. The implantation of KUSA‐A 1 cells resulted in the generation of ectopic bones in the subcutaneous tissues of the athymic BALB/c nu/nu mice. Subsequently the mice obtained a greater amount of the bone marrow than normal mice, and they showed an increased number of HSCs. These results indicate that the newly generated osteoblasts‐derived ectopic bones are responsible for the increase in the number of the HSC population. Furthermore, the increased number of HSCs directly correlates with both the magnitude of dynamic osteogenic process and the size of the newly generated bone or “niche.” J. Cell. Physiol.