Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5-50 x 103 cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100-500 μg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 μg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 μg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 103 MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 103 cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma prolifer- ation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10-30 x 103 cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 μg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 μg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin α1 (Tα1), thymosin β4 (Tβ4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.
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