A. B. Kriegler

A growth factor produced by WEHI-3 cells for murine high proliferate potential GM-progenitor colony forming cells

Abstract Media conditioned by a myelomonocytic leukemic cell line (WEHI-3) have been found to produce a haemopoietic growth factor (referred to as “synergistic activity” (SA)), which in combination with pregnant mouse uterus extract (PMUE) acts on a primitive granulocyte — macrophage progenitor cell to promote the growth of large colonies in agar. The highest levels of SA have been obtained by conditioning serum free medium with WEHI-3 cells which had been grown in BALB/c mice. This activity was unaffected by storage at 4 °C and −20 °C for 6 months, and was stable between pH 2.0 and 10.6 and stable to heat up to 50 °C but not above. The SA was however, unstable to trypsin digestion, 8 M urea at pH 8.0 with and without Mercaptoethanol (ME, 4mM), and to ME alone at pH 8.0. The SA has an apparent molecular weight between 9,000 and 25,000, it has an isoelectric point in the range of pH 4.0 to 6.5 and does not bind to Con-A.

A colorimetric liquid culture assay of a growth factor for primitive murine macrophage progenitor cells.

Synergistic factors from media conditioned (CM) by human placentas or the 5637 human bladder carcinoma cell line (SFH-HPCM and SFH-5637 respectively) have the ability to stimulate early progenitor cells in mouse bone marrow to form large colonies in agar cultures after 12-14 days, in the presence of CSF-1. Culture conditions have been examined and a quicker and more convenient liquid culture assay has been developed for this factor, using a tetrazolium salt to quantitate cell proliferation. The use of flat-bottomed vessels, high cell density, supra-optimal doses of CSF-1 or the addition of WEHI-3-CM to these cultures, all resulted in a decrease in the required incubation time. In combination, these modifications reduced the assay time to 4 days.

Detection of synergistic factor and interleukin-3 activity in the serum and ascites fluid of mice bearing the WEHI-3 tumour

Media conditioned (CM) by WEHI-3 cells (a myelomonocytic leukemia cell line) contains a number of haemopoietic growth factors, including synergistic factor (SF) and interleukin-3 (IL3). We have investigated the production of SF and IL3 in vivo in mice bearing the WEHI-3 tumour. SF and IL3 activity were detected in both the sera and ascites fluids of these mice. SF from the ascites fluid was partially purified by a four-step purification schedule consisting of ammonium sulphate fractionation, DEAE-cellulose, hydroxylapatite, and Sephadex G-100 chromatography. This purification sequence resulted in approximately a 250- and 187-fold purification of SF and IL3 respectively on the initial starting material with a yield of 13 and 9.7% respectively of the initial activity. At each stage of purification, the fractions containing SF co-purified with IL3 activity, further supporting our previous report that SF and IL3 are probably identical molecules. The characteristics of the in-vivo derived (sera and ascites fluid) activities were found to be similar to those of the factors produced in vitro in WEHI-3 cell conditioned media. These results support the conclusion that SF and IL3 are produced in vivo in WEHI-3 tumour bearing mice and are not in vitro artifacts.