In Vitro Cell.Dev. Biol.--Animal 31:767-772, November 1995 © 1995 Society for In Vitro Biology 1071-2690/95 $05.00+0.00 CHARACTERIZATION OF HUMAN AND RAT IMMORTALIZED CLONES OF PAROTID ACINAR CELLS WITH RESPECT TO SPECIFIC PROTEINS AND THEIR mRNAs, AND RECEPTOR-LINKED ADENYLATE CYCLASE KEDAR N. PRASAD, ~SANJAYKUMAR, ERIKACARVALHO, JUDITH EDWARDS-PRASAD, R1TAKUMAR, FRANCISCO G. La ROSA, BIRTEB. LARSEN,AND DAVIDANN Centerfor Vitamins and Cancer Research (K. N. P., S. K., E. C., J. E.-P., R K.), Departmentof Radiology (K. IV. P., S. If., E. C., J. E.-P., R. K.) and Pathology (F. G. L), School of Medieine, University of Colorado Health Sciences Center, Denver, Colorado 80262-0278; Department of Molecular Biology, University of Aarhus, Aarhus, Denmark (B. B. L.); Department of Pharmacology and Institute of Human Genetics, Universityof Minnesota, Minnesota 55455 (D. a.) (Received 5 August 1994; accepted 25 April 1995) SUMMARY This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP~G). The levels of a-amylase mRNAs detected when using a-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP~G cells. In contrast to a- amylase mRNAs levels, the u-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HPIG acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the same a-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG ceils; the reverse was observed in 2HPC8 ceils and human parotid glands. The level of PSP in 2HP1G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HPtG clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Pros- taglandin E~ (PGE~) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP~G ceils, suggesting that the PGE~ receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation. Key words: parotid acinar cells; a-amylase; proline-rich protein; parotid secretory protein; tumorigenicity; cAMP. INTRODUCTION Studies on regulation of growth, differentiation, and transformation of parotid acinar cells are difficult to investigate until the clonal lines of such cells become available. Such clones may also be very im- portant in the study of modulation of salivary gland gene expression. Recently, we have established immortalized and nontumorigenic clones of rat parotid acinar cells by insertion of the T-antigen gene into acinar cell genome using the plasmid vector pSVz "~° (16). We have also established immortalized and nontumorigenic clones of acinar cells from a human pleomorphic parotid adenoma (15). The nontumorigenic (2HP) cells spontaneously became tumorigenic (2HP 0 after 30-35 passages. These acinar cells produce ct-amylase mRNAs and a-amylase (15,16). Others have reported the establish- 1 To whomcorrespondenceshouldbe addressed at Centerfor Vitamins and Cancer Research, Departmentof Radiology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue,Denver, Colorado80262-0278. merit of long-term cultures of normal human parotid acinar cells by manipulating the growth medium (6,19). The establishment of long- term primary cultures of salivary acinar cells have been reported by others (13,21). This study reports the isolation and characterization of a rat nontumorigenic acinar clone (2RSG), a human nontumori- genic acinar clone (2HPC8), and a human tumorigenic acinar clone (2HP1G)- These acinar cells have varying levels of a-amylase mRNA, a-amylase, proline-rich protein (PRP) mRNA, parotid secretory pro- tein (PSP) mRNA, PSP, and I~-adrenoceptor- and prostaglandin El- receptor-linked adenylate cyclase. MATERIALS AND METHODS Cell cuhure. A human nontumorigenic acinar clone (2HPCS), a human tumorigenic acinar clone (2HP1G), and a rat nontumorigenic acinar clone (2RSG), which have been developedin our laboratory(15,16), were used in this study. Human parotid acinar cells were grownin F12 medium containing 10% agammaglobulin newborncalf serum, and penicillin (100 unit]ml), and 767