The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of alpha-amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120-140 population doublings. The serially passaged epithelial cell cultures produced and secreted alpha-amylase, a major component of parotid gland acinar cell secretion. The beta-adrenergic agonist, isoproterenol (ISP), stimulated alpha-amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP-induced stimulation of amylase and cAMP was blocked by the beta-adrenergic antagonist, propranolol. Further, dibutyryl cAMP also enhanced the secretion of amylase. Thus we have established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact beta-adrenergic receptor system.

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Biomedical research into concepts of senescence, apoptosis, and carcinogenesis is almost entirely based on the paradigm that human diploid normal cells are programmed to die after undergoing a certain predetermined number of cell divisions. The paradigm of limited life span is based on observations made almost 30 years ago that normal human cells are difficult to cultivate for extended periods, whereas tumor cells that invariably exhibit numerical and/or structural chromosomal alterations continue to divided in vitro for an infinite period. It should be noted, however, that tumor cells from various human tumors have also been difficult to cultivate for extended periods. Recently, as a result of greater understanding of the nutritional requirements of cultured cells and the identification of growth inhibitory factors, many refinements in tissue culture technology have occurred that have allowed the serial passage of normal cells from a variety of human tissues. Our hypothesis is that, under optimal conditions, diploid normal epithelial cells inherently have an unlimited, not a limited, life span in vitro and that the difficulty of culturing the normal cells for extended periods is due to suboptimum culture conditions. Furthermore, normal cells are not programmed to die after undergoing a predetermined number of divisions, an assumption that was based on the paradigm of limited life span of normal human cells. In support of this hypothesis, for the first time, we have established two normal diploid epithelial cell lines, HPAMl and HPAF1, from the human salivary gland of a male and a female patient, respectively. Both cell lines exhibit diploid karyotypes, normal phenotypes, and are not tumorigenic. Furthermore, the cells produce salivary gland-specific proteins, i.e., a-amylase 1, proline-rich protein (PRP), and cystatin, and express the respective genes.Histologically normal human parotid gland tissues were obtained at surgery from two different patients. The tissues were thoroughly rinsed in cold Leibovitz's L-15 medium and cut into explants (1 X 1 mm) composed primarily of acinar tissue. Primary cultures were prepared by the explant-outgrowth procedure [ 1,2] in serum-free medium comprised of keratinocyte basal medium (KBM) supplemented with insulin (5 pg/ml), hydrocortisone (0.5 kg/ml), EGF (10 ng/m), bovine pituitary extract (25 kg/ ml), penicillin (100 U/ml), and streptomycin (100 pg/ml). The cultures were passaged by using a low concentration of trypsin (0.125%). Serial passage cultures were maintained in collagen-coated dishes at 37°C in an atmosphere of 5% C 0 2 in air. The culture medium was changed three times per week.The epithelial nature and salivary gland origin of the cells was established by ultrastructural, immunocytochemical, and biochemical analyses. For electron microscopy, cultures were fixed in situ with 2% glutaraldehyde and processed as described previously [I]. Immunocytochemical procedures were employed to determine the presence of cytokeratins and salivary-specific proteins in the cells...