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

Abstract

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.

Inherited Defects of Sodium-dependent Glutamate Transport Mediated by Glutamate/Aspartate Transporter in Canine Red Cells Due to a Decreased Level of Transporter Protein Expression

“…These sequences specific to canine glutamate transporter subtypes were derived from those of cDNA fragments amplified by PCR from canine brain cDNAs using degenerate primers designed according to the sequences of rat GLAST (5Ј-ACC AC(C/T) ATC ATT GCT GTG GTG-3Ј and 5Ј-GC(A/G) GTC CCA TCC ATG TTA ATG-3Ј, corresponding to nt 388 -408 and nt 1,208 -1,188 of rat GLAST (1)), rat GLT-1 (5Ј-CTG GAT GCT AAG GCT AGT GGC CGC-3Ј and 5Ј-GC(A/G) GTC CCA TCC ATG TTA ATG-3Ј, corresponding to nt 322-345 and nt 1,202-1,182, respectively), rabbit EAAC1 (5Ј-GG(A/C/G/T) GAA ATC CTG ATG AGG ATG CTG-3Ј and 5Ј-GC(A/G) GTC CCA TCC ATG TTA ATG-3Ј, corresponding to nt 166 -189 and nt 1,112-1,092, respectively) and human EAAT4 (5Ј-GG(A/C/G/T) GAA ATC CTG ATG AGG ATG CTG-3Ј and 5Ј-GGG GAA GGG GTT CCG GTG AGT GAC-3Ј, corresponding to nt 277-300 and nt 1,116 -1,093, respectively). Canine glyceraldehyde 3-phosphate dehydrogenase gene transcripts were also amplified as an internal control using primers (5Ј-TGC TCC TTC TGC TGA TGC CCC CAT-3Ј and 5Ј-TCT GGG TGG CAG TGA TGG CAT GGA-3Ј) prepared according to the sequence reported by Grone et al (24). PCR products were analyzed on 2% agarose gel (see Fig.…”

A New Strategy for Modulating Chemotherapy-Induced Alopecia, Using PTH/PTHrP Receptor Agonist and Antagonist

“…In human and rat skin, PTHrP is produced by keratinocytes (Danks et al, 1989;Lee et al, 1995;Shin et al, 1997), whereas the PTH/PTHrP receptor (PTH/PTHrP-R) is expressed by ®broblasts (Hana®n et al, 1995;Lee et al, 1995). PTHrP synthesis and release from keratinocytes are enhanced under conditions in which keratinocyte differentiation is induced, e.g., by ®broblast-derived growth factors (Kremer et al, 1991;Grone et al, 1996). PTH/PTHrP-R agonists stimulate insulin-like growth factor I production by ®broblasts (Wu et al, 1987;Shin et al, 1997), which acts as a cell cycle progression factor in keratinocytes (Tsuboi et al, 1992;Ristow, 1996;Vogt et al, 1997).…”

Overview of Structural and Functional Lesions in Endocrine Organs of Animals

“…In some forms of HHM, there are increased serum 1,25-dihydroxyvitam in D levels that may increase calcium absorption from the intestine (100). Although excessive secretion of biologically active PTH -rP plays a central role in the pathogenesis of hypercalcemia in most forms of HHM cytokines such as interleukin-1, tumor necrosis factor-alpha, transforming growth factors, and 1,25-dihydroxyvitam in D m ay have synergistic or cooperative actions with PTH-rP (45,65,66).…”