Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, including squamous cell carcinoma (SCC), is due to expression and secretion of parathyroid hormone-related protein (PTHrP). Transforming growth factor-beta1 (TGFbeta1), expressed by many tumors, has been demonstrated in vitro to increase the half-life of PTHrP mRNA. In this study, oral squamous carcinoma cells (SCC2/88) had a two-fold increase in PTHrP mRNA stability (from 45 to 90 min) in response to treatment with TGFbeta1. In order to examine the mechanism of TGFbeta1-mediated PTHrP mRNA stability, a cell-free assay of mRNA degradation was utilized in which the degradation of in vitro-transcribed mRNA incubated with cytoplasmic protein extracts from SCC2/88 treated with vehicle or TGFbeta1 was measured. In this assay, full-length PTHrP mRNA was not significantly stabilized in TGFbeta1-treated samples when compared to vehicle treated samples. However, there was a striking (>5-fold) increase in PTHrP mRNA half-life in TGFbeta1-treated samples when PTHrP mRNA lacked the 3'-untranslated region (3'-UTR). In contrast, the degradation of 3'-UTR-truncated PTHrP mRNA using the cell-free assay was not altered in vehicle-treated samples. UV cross-linking of PTHrP mRNA and cytoplasmic proteins from cells treated with either vehicle or TGFbeta1 revealed numerous mRNA-binding proteins. TGFbeta1 treatment resulting in decreased binding of 33, 31, 27, 20 and 18 kDa binding proteins to the terminal coding region. These studies revealed that TGFbeta1-induced PTHrP mRNA stability might be, in part, the result of cis-acting sequences within the coding region of the PTHrP mRNA.