Figure 4.

Differential proliferation and differentiation response induced by class I and IV GCSFR. (a) GCSF-induced proliferation of Ba/F3 cells expressing either class I or IV cells was determined at 48 h using MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. Proliferation is quantitated as absorbance at 600 nm. Data are plotted as absorbance vs hGCSF concentration. (b) Comparison of GCSFR isoforms and growth hormone receptor (GHR)/GCSFR chimeric receptors. Three different forms of GCSFR are shown: class I, class IV and the d715 truncation mutant of GCSFR. The three GCSFR receptors differ from each other in their cytoplasmic domain. The full-length receptor has four tyrosine residues, of which the last three are missing in the class IV isoform and d715 mutant. The class IV isoform differs from the d715 mutant in that it has a novel 34 aa C-terminal region containing a single tyrosine. Chimeric receptors were created that have the extracellular domain of the rabbit GHR and intracellular domain of either class I, class IV or d715. The figure shows a schematic representation of the structure of the GHR and GCSFR and the generated chimeric receptors. (c) Proliferation of 32D cells expressing the chimeric receptors GHR/GRI and GHR/GRIV was induced by increasing concentrations of growth hormone (GH). Proliferation was determined using the MTT assay, by quantitating absorbance at 600 nm. Data are plotted as absorbance vs hGH concentration. (d) 32D cells expressing either the chimeric GHR/GRI (class I), GHR/GRIV (class IV) and GHR/d715 (d715, truncation mutant) cells were grown in medium containing 100 ng/ml hGH. Cytospin samples (5 × 10⁴ cells per condition) were stained using Wright-Giemsa staining. After 6 days or later, class I cells differentiated into mature granulocytes with segmented nuclei and secondary granules. Class IV cells showed dysplasia with ring-shaped nuclei and lacked secondary granules. d715 cells did not show dysplasia, but differentiation was inhibited. (e) Total 200 cells were counted under a microscope and categorized into myeloblast/promyelocyte, myelocytes/metamyelocytes and band/neutrophils. The cell types are represented as percent of total cells counted. (f) Immunophenotyping was carried out as a measure of differentiation. Ly6G and 6C (Gr-1) expression on surface were determined using PE-conjugated rat anti-mouse Ly6G and 6C antibody on cells treated with 100 ng/ml hGH for days 0, 3, 6 and 9. Cells were incubated with the antibody for 1 h at 4 °C in dark. Samples were run on a flow cytometer to determine PE-labeled cells. Analysis was carried out using FlowJo software. PE, phycoerythrin; TM, transmembrane.