Biochemical Purification of a CSF-1 like Molecule Released during Malignant Transformation of IL-3 Dependent Hematopoietic Progenitor cell lines Cocultivated with Gamma Irradiated Clonal Marrow Stromal cell lines
Cocultivation of IL-3 dependent hematopoietic progenitor cell line FDC-P1JL26 with 5000 cGy irradiated clonal bone marrow stromal cell line D2XRII has been demonstrated to stimulate selection of factor independent hematopoietic cell lines that produce tumors in vivo (1,2). Hematopoietic stem cell specific and stromal cell specific variables in this experiment have been described (3). The precise molecular mechanism of the malignant transformation of hematopoietic cells and the growth factor or cell membrane contact which is responsible for the transformation have not yet been elucidated. Biochemical purification of several liters of conditioned medium from D2XRII cells revealed a 75,000 molecular weight protein that was neutralized by a polyclonal antiserum to M-CSF. This growth factor stimulated formation of macrophage colonies in fresh mouse bone marrow cells in vitro. A biochemical purification scheme utilizing a Pellicon cassette system concentration, followed by lentil lectin chromatography, ion exchange high pressure liquid chromatography, gel filtration high pressure liquid chromatography, and reverse phase HPLC yield biological activity using tritiated thymidine incorporation into microwell cultures of FDC-P1JL26 cells (4). Active fractions were run out on NaDodSO4/PAGE gel electrophoresis and revealed a band consistent in size with 75,000 molecular weight.
KeywordsHPLC Filtration Electrophoresis Polyacrylamide Thymidine
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