Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium

Abstract

A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.

Studies on the in vivo production of a lymphokine activity, interleukin 3 (IL-3) elaborated by lymphocytes and a myeloid leukaemic line in vitro and the fate of IL-3 dependent cell lines

“…wt average for IL-3 has recently been given . In common with previous experience (Bazill et al, 1983) WEHI-3b CM eluted over a broader slightly higher mol. wt range with an average of 32 Kd (mean peak height).…”

“…Thus, the factor (or a group of very similar ones) promotes many cell lineages in vitro (Garland & Dexter, 1982b;Bazill et al, 1983;Garland & Crompton, 1983;Iscove et al, 1982) and "immortalises" cell lines with characteristics of the WEHI-3b leukaemic line (Dexter et al, 1980). "Misplaced" elaboration of IL-3 internally could therefore be a universal route to leukaemia induction within a range of haemopoietic cells.…”

“…Sonicates were prepared by lysing 5 x 106 to I07 washed nucleated cells mlP-on ice using 20 second bursts of ultrasound (MSE ultrasonicator 6-12 p peak height). All sonicates were checked for breakage, the debris spun out at 20,000 rpm (30,000 g) for 1 h and then sterilised by Half-life of IL-3 activity in vivo A preparation of partially-purified factor with very high IL-3 activity was kindly provided by Dr. G. Bazill (Bazill et al, 1983). An amount equivalent to approximately 10ml WEHI-3b CM (active at 10%) was injected i.v.…”

“…Somewhat similar cell lines have also been described by Greenberger (1980) and Hapel et al (1981) in the latter case classified as lymphoid largely on the criteria of Thy antigen and expression of 20a steroid dehydrogenase . The factor promoting these "lymphoid" lines has been termed "Interleukin-3" (IL-3) and appears to be the same as HCGF (reviewed in ; both activities are associated with a 28-3OKd glycoprotein (Bazill et al, 1983; and purified IL-3 optimally promotes FDCP. Additionally, HCGF and IL-3 promote the proliferation of multipotential stem cells, (Bazill et al, 1983;Garland & Crompton, 1983) and basophil/mast cells in vitro (Schrader et al, 1981; J.…”

“…The factor promoting these "lymphoid" lines has been termed "Interleukin-3" (IL-3) and appears to be the same as HCGF (reviewed in ; both activities are associated with a 28-3OKd glycoprotein (Bazill et al, 1983; and purified IL-3 optimally promotes FDCP. Additionally, HCGF and IL-3 promote the proliferation of multipotential stem cells, (Bazill et al, 1983;Garland & Crompton, 1983) and basophil/mast cells in vitro (Schrader et al, 1981; J. Ihle, personal communication).…”