Effects of transforming growth factor beta1 on cell growth and parathyroid hormone-related protein in Walker 256...

Hypercalcemic strains of the rat Walker 256 (W256) tumor synthesize parathyroid hormone-related protein (PTHrP) and at least one of them produces an ill-defined transforming growth factor activity. We tested the production of transforming growth factor (TGF) beta by a hypercalcemic W256 tumor strain, and assessed its effects on tumor cell growth and PTHrP expression. We found that addition of TGF beta1 for 7 days inhibited cell growth ([3H]thymidine incorporation and cell number) dose dependently, between 0.04-20 pM. The antiproliferative effect of TGF beta1 on W256 tumor cell growth was likely mediated by binding to high affinity receptors (Kd = 14 pM) in these cells. At different tumor cell growth stages, acidified cell-conditioned medium contained immunoreactive TGF beta1. However, the nonacidified tumor cell-conditioned medium was found to contain neither immunoreactive nor bioactive TGF beta. Moreover, exposure of W256 tumor cells to a neutralizing anti-TGF beta1 antibody failed to affect tumor cell proliferation. Thus, W256 tumor cells appear to secrete TGF beta in an inactive form. Using reverse transcription followed by PCR, we found that addition of 20 pM TGF beta1 increased its own mRNA expression, apparently by stimulating gene transcription, within 6-12 h in W256 tumor cells. In addition, 20 pM TGF beta1 stimulated PTHrP mRNA in these cells at 24 h; an effect which was mediated, at least in part, by increasing PTHrP mRNA stability. Immunoreactive PTHrP decreased in the W256 tumor cell-conditioned medium after treatment with 20 pM TGF beta1 for 24-48 h. These results support the validity of this W256 tumor strain for in vivo studies to clarify the relative role of TGF beta and PTHrP in the pathogenesis of humoral hypercalcemia of malignancy.