Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.
OBJECTIVE
The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy.


METHODS
Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting.


RESULTS
Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax.


CONCLUSIONS
Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.
Hyman
T
T
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Rothmann
C
C
Heller
A
A
Malik
Z
Z
Salzberg
S
S
D000081:Acetamides / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000473:pathology*; D004920:Erythropoiesis; D005622:Friend murine leukemia virus; D016193:G1 Phase / Q000187:drug effects; D006454:Hemoglobins / Q000096:biosynthesis; D007091:Image Processing, Computer-Assisted; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D008533:Megakaryocytes / Q000473:pathology*; D051379:Mice; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009363:Neoplasm Proteins / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000473:pathology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology; D051028:bcl-2-Associated X Protein
D000081:Acetamides; C494827:Bax protein, mouse; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D006454:Hemoglobins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; D051028:bcl-2-Associated X Protein; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; C014026:hexamethylene bisacetamide