THYMOSIN FRACTION-5 INHIBITS THE PROLIFERATION OF THE RAT NEUROENDOCRINE MMQ PITUITARY-ADENOMA AND C6 GLIOMA CELL-LINES IN-VITRO

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothal amic-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 immu ne system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor ce ll proliferation. Cells of the PRL-secreting rat anterior pituitary ad enoma, MMQ (5-50 x 10(3) cells/well), were exposed to vehicle (RPMI-16 40 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100 -500 mu g/ml) for up to 96 h and the proliferation of MMQ cells monito red using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl te trazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. M inimal reductions in optical densities resulted from exposure to 100 m u g/ml TF5, whereas the highest concentration of this preparation (i.e . 500 mu g/ml) completely blocked MMQ cell division. The concentration -dependent effects of TF5 were particularly striking at initial platin g densities of 25 and 50 x 10(3) MMQ cells/well; in contrast, all conc entrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 1 0(3) cells/well. The antiproliferative actions of TF5 on MMQ cells wer e demonstrable within 24 h and remained for up to 96 h as determined b y the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effect s of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell cond itioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. T he TF5 concentration-dependent inhibition of MMQ cell growth was large ly reversed by the 50% conditioned medium, whereas PRL slightly potent iated the antiproliferative actions of TF5. The proliferation of the r at C6 glioma cell line (10-30 x 10(3) cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 mu g/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was not ed at 200 mu g/ml TF5. Significant reductions in MMQ and C6 cell viabi lities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin alpha(1) (T alpha(1)), thymosin beta(4) (T beta(4)), MB35, and MB40 ha d no effect on either MMQ or C6 cell proliferation, indicating that th ese TF5 components are not the principle active peptides. Therefore, T F5 was further separated into 60 fractions by preparative reverse phas e HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC f ractions had no effect. These data demonstrate a new biological proper ty of TF5: the inhibition of cell proliferation and the induction of a poptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by a n activity present in the MMQ cell conditioned medium. Thus, TF5 and c ytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH(3) cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor format ion, an action opposed by autocrine growth factors secreted by these t umors.