Hiroshi Ideguchi

A genetic defect of erythrocyte band 4.2 protein associated with hereditary spherocytosis

We report two patients with hereditary spherocytosis associated with band 4.2 protein deficiency from a Japanese family. The defect of band 4.2 protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) not only in freshly prepared white ghosts but also in washed whole erythrocytes. The finding was quite reproducible and was also recognized postsplenectomy. The interaction of ankyrin with band 3 in the patients’ghosts was stable both at low ionic strength and at acidic pH. Our results suggested that band 4.2 protein might not be essential for the structural stability of band 3‐ankyrin interaction. On the other hand, membrane protein phosphorylation studies revealed an increased phosphorylation of spectrin/ankyrin, band 3 and band 4.1 in the patients’erythrocytes as compared with normal cells. The finding might be related to a dysregulation of protein phosphorylation which could result in membrane instability in affected cells. Band 4.2 deficiency is an inherited disorder in association with hereditary haemolytic anaemias and seems to be relatively prevalent in the Japanese population.

Enhanced synthesis of heat shock proteins and augmented thermotolerance after induction of differentiation in HL-60 human leukemia cells

The effects of the induction of differentiation were investigated on the expression of heat shock proteins (hsps) and thermotolerance. The synthesis of the major hsps in response to heat stress was markedly enhanced in HL‐60 human leukemia cells after differentiation. An increased amount of mRNA transcripts for hsp70 was also noted. In addition, induction of differentiation resulted in acquisition of greater resistance to heat, which may be advantageous since cells in the peripheral blood must survive many stresses.

Mechanisms involved in the development of adriamycin resistance in human leukemic cells

We have developed three adriamycin (ADR)-resistant K562 sublines with different degrees of resistance. These sublines show a decreased accumulation and an increased efflux of ADR in proportion to the degree of resistance. Two membrane proteins (mol. wt 170,000 and 230,000) reactive with monoclonal antibody against P-glycoprotein were highly expressed in both the K562/ADR200 and the K562/ADR500 subline. Less resistant K562/ADR80 cells contained only small amounts of mol. wt 230,000 protein. Thus, the level of P-glycoprotein expression was not proportionate to the degree of ADR efflux. Verapamil treatment could not completely reverse ADR resistance. No significant change of glutathione-s-transferase activity nor in the level of DNA topoisomerase II was detected in resistant sublines. In our sublines it seems that P-glycoprotein is one of the mechanisms for resistance, but additional mechanisms may be involved.

Absence of correlation between cytotoxicity and drug transport by P‐glycoprotein in clinical leukemic cells

Abstract: Development of resistance to cytotoxic agents is a common problem in the treatment of acute leukemia. In cell lines having multidrug resistance (MDR) phenotype, a decrease in the intracellular accumulation of drugs has been closely related to the overexpression of P‐glycoprotein/mdrl genes. We analyzed the relationship between the cytotoxicity of adriamycin (ADR) in vitro, intracellular accumulation of ADR, and the expression of P‐glycoprotein on fresh leukemic cells from 19 patients at their initial presentation and from 9 relapsed patients. Pretreatment patients showed significantly higher ratio of complete remission than relapsed patients, and mean value of IC50 for adriamycin in initial presentation was higher than at relapse. But we found no significant relationship between in vitro cytotoxicity and drug transport. In addition, only 2 of the 5 relapsed patients examined by monoclonal antibody C219 expressed the P‐glycoprotein. These results suggest that the acquisition of clinical drug resistance may involve various mechanisms other than the reduction of drug accumulation with P‐glycoprotein expression.

Regulation of biosynthesis and phosphorylation of P210bcr/abl protein during differentiation induction of K 562 cells

The changes of P210bcr/abl and other tyrosine phosphorylated proteins in K562 cells during growth and differentiation were studied by metabolic labeling with 32PO4 and immunoprecipitation with anti-phosphotyrosine sera. The anti-phosphotyrosine sera recognized P210bcr/abl and other phosphoproteins with mol wt of 150, 115, 100, 70 and 64 kD. The 2-day incubation of K562 cells with inducers for differentiation, hemin, sodium butyrate and TPA, decreased the level of P210bcr/abl protein and other phosphoproteins. Inhibitors of tyrosine protein kinase, amiloride and genistein, also reduced the phosphorylation of P210bcr/abl protein and other substrates, but did not induce differentiation of the cells. Although most of these additives inhibited the cell growth, cytotoxic agents such as adriamycin, vincristine and Ara-C did not affect the level of P210bcr/abl protein. The experiments using 35S-methionine labeled cells and the immunoprecipitation with anti-abl sera suggested that reduced biosynthesis but not dephosphorylation of P210bcr/abl protein mainly accounted for the reduction of P210bcr/abl protein reacting to anti-phosphotyrosine sera in differentiation-induced cells. These results indicate that the reduction of P210bcr/abl protein synthesis plays some roles in cellular differentiation of K562 cells.

Possible mechanism of ineffective erythropoiesis by an altered transferrin receptor cycle in erythroleukemia

Involvement of the transferrin receptor cycle was noted in erythroblasts from a patient with erythroleukemia (FAB classification M6). The kinetics of transferrin receptor cycle in bone marrow erythroblasts was obtained by pulse‐chase experiments before the initiation of therapy. Internalization of transferrin was impaired and resulted in a delayed peak of internalized transferrin, as compared with the kinetics pattern seen in healthy subjects. The subsequent exocytosis of the internalized ligand was also delayed. Thus, transferrin receptor cycle seems to be influenced all along the transferrin pathway, hence transferrin travels more slowly in erythroblasts in erythroleukemia. The altered transferrin receptor cycle led to a diminished iron uptake per surface transferrin receptor (approximately 30% of that in healthy subjects), and the incorporation of iron into heme was greatly reduced. Our observations suggest a possible role for the altered transferrin receptor cycle in the pathogenesis of defective heme synthesis and ineffective erythropoiesis in erythroleukemia.

Specific phosphorylation of 22-kD:proteins by various inducers for granuloid differentiation in myeloid leukemic cells

We studied the changes of protein phosphorylation in human leukemic cells by granuloid inducers, using two-dimensional electrophoresis. The phosphorylation of 22 kD, pI 6.0 and 5.8 proteins (pp22) in HL-60 cells or myeloid leukemic cells from patients, was enhanced by treatment with granuloid inducers such as retinoic acid, dimethyl sulfoxide or G-CSF, in common with prostaglandin E2 and theophylline, or dibutyryl c-AMP, which increased intracellular c-AMP. In contrast, pp22 phosphorylation was not induced by the monocytes/macrophages inducer in HL-60 cells, or by the granuloid inducers in lymphoid cells. This phosphorylation occurred within 30 min and continued for more than 48 h. These pp22 proteins were present in the cytosol and phosphorylated on the serine residues. We now present a possibility that granuloid differentiation in myeloid cells is closely linked with these pp22 phosphorylation.

Abnormality of platelet membrane glycoprotein GPIIb in a myelodysplastic syndrome with 3q inversion presenting with marked dysmegakaryopoiesis.

Platelet membrane glycoproteins were analyzed in a case of myelodysplastic syndrome with inv(3) (q21q26) presenting with prominent dysmegakaryopoiesis by three different labelling techniques for surface proteins. Markedly decreased level of platelet membrane glycoprotein GPIIb was observed in the patients platelets by terminal sialic acid labelling method, whereas no significant changes in the levels of glycoproteins including GPIIb could be detected either by penultimate galactose labelling or by tyrosine/histidine labelling. These results indicate a decreased sialylation of GPIIb in the patients platelets, implying aberrant process in thrombopoiesis in the disease.