Parathyroid hormone-related peptide stimulates proliferation of highly tumorigenic human SV40-immortalized breast epithelial cells

Parathyroid hormone-related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy (HHM) and it is produced by many tumors, inclu ding breast cancers, Breast epithelial cells as well as breast cancer tumor s and cell lines have been reported as expressing PTHrP and the PTH/PTHrP r eceptor, suggesting that PTHrP may act as an autocrine factor influencing p roliferation or differentiation of these cell types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mitogenesi s in three immortalized human mammary epithelial cell lines that exhibit di fferential tumorigenicity, The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We used reverse-transcri ption polymerase chain reaction (RT-PCR) and immunoblotting to detect the P TH/PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1-34) [hPTHrP(1-34)] and hPTH(1-34) increased in tracellular cyclic adenosine monophosphate (cAMP) but not free Ca2+ in the nontumorigenic line. These agonists increased both cAMP and free Ca2+ level s in the moderately tumorigenic line, but only increased free Ca2+ in the h ighly tumorigenic line. Application of the PTH/PTHrP receptor antagonist [A sn(10),Leu(11),D Trp(12)]PTHrP(7-34) or PTHrP antibodies reduced [H-3]thymi dine incorporation in a dose-dependent fashion in the highly tumorigenic ce ll line but did not affect the other lines. Thus, treatment with a PTH/PTHr P receptor antagonist reduced cell proliferation, suggesting that PTHrP sig naling mediated by the phospholipase C (PLC) pathway stimulates proliferati on of a highly tumorigenic immortalized breast epithelial cell line.