|Budget Amount *help
¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1999 : ¥1,100,000 (Direct Cost : ¥1,100,000)
The Eph-family receptors are the largest known family of receptor protein tyrosine kinases, and are expressed in the embryo and nervous system as well as in human malignancies of various tissues. Receptor protein tyrosine kinases play key roles in cellular proliferation and differentiation in a wide variety of cell types. Although most kinase-defective growth factor receptor proteins are associated with pathogenic conditions, a kinase-defective Eph-family receptor protein, EphB6, is expressed in normal human tissues.
In the present study, we examined the expression of the kinase-defective EphB6 receptor in normal blood cells and human hematopoietic malignancies to clarify whether EphB6 is differentially expressed between normal and transformed hematopoietic cells and to ascertain the specific roles of EphB6 in the hematopoietic system. We generated monoclonal antibodies specific for human EphB6 to characterize its expression on human hematopoietic cells. A very small population of norma
l human peripheral white blood cells (0.57±0.07%) expressed EphB6. The EphB6-positive cells were CD2ィイD1+ィエD1, CD7ィイD1+ィエD1, CD3ィイD1+ィエD1 and CD4ィイD1+ィエD1 or CD8ィイD1+ィエD1 lymphocytes, but they did not express CD19 or CD11b. In human bone marrow, only 1.5±0.19% of lymphocytes expressed EphB6. Compared with the expression in peripheral lymphocytes, prominent expression of EphB6 protein was demonstrated in CD4ィイD1+ィエD1 CD8ィイD1+ィエD1 double-positive mouse thymocytes. The T-cell lineage-specific expression was strictly conserved in human leukemia/lymphoma cells. Among T-cell-derived leukemia cells, the expression level of EphB5 seemed to decrease with maturation of the cells. These results suggest that EphB6 expression is regulated in T-cell development.
We further examined the binding affinities of known ligands (ephrins) for the kinase-defective Eph receptor family member, EphB6. Although only T-cell derived leukemia cell lines expressed EphB6 receptor protein, soluble EphB6/Fc fusion protein bound several human leukemic cell lines derived from all lineages including B-cell and myeloid progenitors. Among the known ligands (ephrins) for Eph receptors, only ephrin-B2 mRNAs, bound recombinant EphB6 expressed on CH0-K1 cells. Both ephrin-B1 and ephrin-B2 mRNAs, but not ephrin-B3 mRNA, were expressed in human leukemia cell lines, only the cells highly expressing ephrin-B2 mRNA bound EphB6/Fc fusion protein. Analysis of ephrin-B2/Fc fusion protein binding to EphB6 transfected CHO-K1 cells revealed a high-affinity saturable binding between EphB6 and EphB6 and ephrin-B2/Fc, but not with other ephrins/Fc. Mice thymocytes lacking the EphB6 gene clearly demonstrated the lack of the binding site of eprin-B2 on wild-type mouse thymocytes. Therefore, ephrin-B2 may be the only physiological ligand for the EphB6 among the known ephrins. Less