The kinetics of S phase entry by FMP2.1: effect of IL-3 and GM-CSF receptor expression and ligand affinity.
FMP2.1, a cloned cell line which has morphological characteristics of mast/basophil cells, requires either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for both survival and proliferation. IL-3 and GM-CSF were equally effective as proliferative stimuli. FMP2.1 cells were sensitive to growth factor stimulation in the G1 phase, which has a duration of 9.5 h. G1 cells were selected from FMP2.1 in log phase growth on the basis of Hoechst 33324 staining using a fluorescence activated cell sorter (FACS). It was found that G1 phase cells had to be exposed to either IL-3 or GM-CSF for approximately 1 h for cells to enter S (greater than 20%); without growth factor, FMP2.1 remained in G1 unable to progress into S. Receptor expression was analyzed to further understand this rapid activation of FMP2.1 into cycle. Autoradiography using either 125I-IL-3 or 125I-GM-CSF showed that most cells express both receptor types. In the presence of saturating concentrations of IL-3, FMP2.1 have a relatively high number of IL-3 receptors (42,000/cell) compared to other cell lines (e.g., 32D cl23; 13,000 receptors/cell), and far outnumber GM-CSF receptors on the same cells (600 receptors/cell). Although average IL-3 receptor expression differed for FMP2.1- and IL-3-dependent 32D cl23, the concentration-dependent proliferative response to IL-3 was essentially identical for both cell types. Scatchard plot analysis for 125I-IL-3 and 125I-GM-CSF binding to FMP2.1 cells at 4 degrees C revealed a single type of binding site for both ligands, with dissociation constants (Kd) of approximately 1 nM for GM-CSF and 8 pM for IL-3. The relatively high affinity IL-3 binding to a large number of available IL-3 receptors was associated with a shallow dose response of the FMP2.1 cells to IL-3, compared to the steep GM-CSF dose response which was mediated through fewer receptor sites of relatively low affinity. Mitogenic stimulation of G1 phase cells was observed with either IL-3 or GM-CSF, and appeared to be unaffected by differences in receptor number or binding affinity.
Hasthorpe
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Peter MacCallum Cancer Institute, Cell Biology Group, Melbourne, Australia.
Akinci
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Bartelmez
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D000818:Animals; D001491:Basophils / Q000166:cytology* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000378:metabolism* / Q000494:pharmacology; D007377:Interleukin-3 / Q000032:analysis / Q000494:pharmacology*; D007399:Interphase / Q000187:drug effects; D008407:Mast Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000502:physiology*; D016184:Receptors, Colony-Stimulating Factor
D003115:Colony-Stimulating Factors; D007377:Interleukin-3; D011956:Receptors, Cell Surface; D016184:Receptors, Colony-Stimulating Factor