Erythroleukemic Cell Differentiation Factor (Edf): Biochemical, Cloning, Molecular Structure, and Functional Studies

This dissertation, "Erythroleukemic Cell Differentiation Factor (EDF): Biochemical, Cloning, Molecular Structure, and Functional Studies" by 陳思潁, Sze-wing, Scarlet, Chan, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: ABSTRACT OF THESIS ENTITLED "ERYTHROLEUKEMIC CELL DIFFERENTIATION FACTOR (EOF): BIOCHEMICAL, CLONING, MOLECULAR STRUCTURE, AND FUNCTIONAL STUDIES" Submitted by SCARLET SZE WING CHAN For the Degree of Doctor of Philosophy At the University of Hong Kong in December, 2000 A series of methods and technologies, including the "Protein band-fishing by cells," MTT cell proliferation assay, Wright's stain differential cell count, monoclonal antibody, cDNA library screening, gene cloning and sequencing and manipulation, gene expression and protein purification, and animal models, were used in this research in order to detect, identify, and clone putative erythroid differentiation factors existed in erythropoiesis. One of them was named erythroleukemic cell differentiation factor (EDF). hi culturing murine erythroleukemic (MEL) cells with fetal rat liver extract, we have found that the extract inhibited cell proliferation and growth for 5.5-folds as compared to controls without extract as assayed by MTT method. Differential cell count of these cultures revealed that 72% cells cultured with extract as compared to 4.5% in controls without extract were differentiated and denucleated. We have used "Protein band-fishing by cells" method to identify putative EDF(s) and found two protein bands of molecular weight 17kDa and 94kDa, where significant numbers of differentiated and denucleated cells were found. These two protein bands were excited in gel strips and added into MEL cell cultures; about 80-90% of cells were differentiated and denucleated as compared to -5% in control cultures added with empty gel strip. Specific blocking monoclonal antibodies for 17 kDa and 94kDa protein bands were developed to reveal that about 95% MEL cells remained undifferentiated in the culture with the presence of both the extract and the specific blocking MAb. The results suggested that we have obtained specific MAbs capable of blocking the EDF activities.Followed the immuno-screening of EDF clones from the human bone marrow cDNA library, a positive Xphage clone with EDF sequence insert was selected by the blocking monoclonal antibody for 17kDa. DNA sequence analysis of this pure clone revealed a sequence of 288 base pairs, which is identical to the sequence of the exon 8 of platelet endothelial cell adhesion molecule-1. After cloning, gene manipulation, protein expression and affinity protein purification, human EDF protein was obtained. It contains 96 amino acids with a molecular weight of 11.5 kDa. In vitro efficacy tests for this purified human EDF protein showed that it reduced -30% growth of MEL cells in MTT cell proliferation assay after 2 days incubation when compared to controls. Results for differential cell count also showed that over 57% of MEL cells differentiated and denucleated in EDF as compared to 10% in controls. Three animal models for in vivo efficacy tests of EDF were set up. Two anemic mice models were: 1) Severely bled model, and 2) Phenylhydrazine-induced anemic model. The changes of hematocrit level in these models were observed in the presence of EDF, Epo, or saline. 3) The MEL cell-infected nude mice model was used to compare the efficacy of EDF and Epo in extending the life span of these erythroleukemic nude mice. hi both in vitro and in vivo experiments, EDF shows the following characteristics. (1) In vitro, it inhibits cell proliferation and ini