Characterization of parathyroid hormone/parathyroid hormone-related protein receptor and signaling in hypercalcemic Walker 256 tumor cells.

Parathyroid hormone (PTH)-related protein (PTHrP) is the main factor responsible for humoral hypercalcemia of malignancy. Both PTH and PTHrP bind to the common type I PTH/PTHrP receptor (PTHR), thereby activating phospholipase C and adenylate cyclase through various G proteins, in bone and renal cells. However, various normal and transformed cell types, including hypercalcemic Walker 256 (W256) tumor cells, do not produce cAMP after PTHrP stimulation. We characterized the PTHrP receptor and the signaling mechanism upon its activation in the latter cells. Scatchard analysis of PTHrP-binding data in W256 tumor cells revealed the presence of high affinity binding sites with an apparent K(d) of 17 nM, and a density of 90 000 sites/cell. In addition, W256 tumor cells immunostained with an anti-PTHR antibody, recognizing its extracellular domain. Furthermore, reverse transcription followed by PCR, using primers amplifying two different regions in the PTHR cDNA corresponding to the N- and C-terminal domains, yielded products from W256 tumor cell RNA which were identical to the corresponding products obtained from rat kidney RNA. Consistent with our previous findings on cAMP production, 1 microM PTHrP(1-34), in contrast to 10 microg/ml cholera toxin or 1 microM isoproterenol, failed to affect protein kinase A activity in W256 tumor cells. However, in these cells we found a functional PTHR coupling to G(alpha)(q/11), whose presence was demonstrated in these tumor cell membranes by Western blot analysis. Our findings indicate that W256 tumor cells express the PTHR, which seems to be coupled to G(alpha)(q/11). Taken together with previous data, these results support the hypothesis that a switch from the cAMP pathway to the phospholipase C-intracellular calcium pathway, associated with PTHR activation, occurs in malignant cells.

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

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.

Proliferative effect of parathyroid hormone-related protein on the hypercalcemic Walker 256 carcinoma cell line.

The rat Walker 256 carcinoma is an animal model for humoral hypercalcemia of malignancy. This tumor produces and secretes parathyroid hormone (PTH)-related protein (PTHrP), a likely mediator for this syndrome. In this study, we investigated the effect of PTHrP on Walker 256 tumor cell proliferation. We found that [Tyr36]human (h)PTHrP (1-36)NH2 and hPTHrP (1-86), unlike hPTHrP (38-64)NH2, stimulate DNA synthesis dose-dependently in these cells. A similar mitogenic effect was also observed with bovine (b)PTH (1-34) or (Nle8.18, Tyr34)bPTH (3-34)NH2. Moreover, addition of anti-hPTHrP (1-34) neutralizing antibodies decreased tumor cell growth. Conversely, 10(-4)M dibutyryl cAMP or Sp-cDBIMPS (a cAMP analogue) inhibited DNA synthesis in these cells, being incompetent at lower doses. PTHrP or PTH failed to stimulate cAMP production, but they induced a cytosolic calcium transient increase in these cells. These findings support an autocrine role of PTHrP in the regulation of this tumor growth.