Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells

Objective. The aim of this study was to examine the structural characteriza tion of erythroid and megakaryocytic cell differentiation in Friend erythro leukemic cells using spectral imaging and electron microscopy. Materials and Methods. Two variants of Friend erythroleukemia cells were tr eated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiat ion; and 2) the resistant R1 cells. The cells were analyzed by spectral ima ging along with transmission and scanning electron microscopy, The expressi on of cell cycle regulatory proteins was analyzed by Western blotting. Results. Spectral imaging of HMBA-treated MEL and R1 tells stained by May-G runwald-Giemsa and subjected to spectral similarity mapping revealed five m orphologic cell types: proerythroblast-like cells, normoblast-like cells, r eticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and eryt hroid differentiation characterized by accumulation of hemoglobin were dete cted; Ri cells were not committed to terminal differentiation, HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins i n a similar manner in both types of cells. Expression of cyclin-dependent k inase 4 decreased and expression of p21(WAF1) increased. The level of the u nderphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc, In addition, we detected a decr ease in the expression of the anti-apoptotic regulator, Bcl-2, and an incre ased expression of the pro-apoptotic regulator, Pax, Conclusions, Spectral imaging provides new insight for the morphologic char acterization of erythroid and megakaryocytic cell differentiation as well a s apoptosis, Image analysis was well correlated to cell cycle arrest and th e expression of regulatory proteins. (C) 2001 International Society for Exp erimental Hematology. Published by Elsevier Science Inc.