Altered parathyroid hormone-related protein secretion and mRNA expression in squamous carcinoma cells in vitro

Parathyroid hormone-related protein (PTHrP), a major factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many squamous carcinoma cells (SCCs). Two SCC lines were grown in multilayered culture systems and compared to cells grown as monolayers to evaluate the effects of cell proliferation, confluence and differentiation on PTHrP secretion and mRNA expression. Well-differentiated (SCC 2/88) and poorly differentiated (SCC-A253) SCCs were grown as monolayer and three-dimensional cultures on collagen-coated membranes to compare the regulation of PTHrP expression and secretion by the cell lines in vitro. Parathyroid hormone-related protein secretion was evaluated by radioimmunoassay and immunohistochemistry. Messenger RNA expression was analyzed by RNase protection assay and in situ hybridization. Secretion of PTHrP was greatest in preconfluent SCC 2/88 cells in monolayer culture and decreased after confluence, which was the result of decreased PTHrP mRNA expression. In contrast, PTHrP secretion in cultures of SCC-A253 cells reached maximal levels after confluence. In multilayered cultures, total PTHrP secretion and mRNA expression remained high in both SCC 2/88 and SCC-A253 cells, and secretion by the multi-layered cultures was principally in the basal direction. Parathyroid hormone-related protein was present in all cell layers in three-dimensional cultures as determined by immunohistochemistry. These results indicated that multilayered cultures of SCCs produced PTHrP continuously, whereas decreased proliferation in monolayer cultures was associated with decreased PTHrP production. Multilayered cultures represent a better system to investigate PTHrP secretion and mRNA expression in vitro and PTHrP secretion by SCCs in vivo may be greatest by proliferating cells.