In Vitro Cell. Dev. Biol. 28A:475-478, July-August 1992 © 1992 Tissue Culture Association 0883-8364/92 $01.50+0.00
Letter to the Editor IN VITRO KERATINIZATION
OF NORMAL HUMAN SALIVARY GLAND CELLS
Dear Editor: The histopathological variation of pleomorphic adenoma in the salivary gland has attracted much interest among many pathologist~ Although the tumor is most popular in the salivary gland tumor (4), the mechanism for the morphological variation has remained obscure. And the tumor has been much more popular in the parotid gland than in the submandibular gland from the clinical reports (4). Interestingly the tissue concentration of the salivary hormone (parotin) in the parotid gland of guinea pig has been reported about 7 times as large as that in the submandibutar gland (1). Although no data has been reported concerning the tissue concentration rate other than guinea pig, these reports may suggest some relation between parotin and pleomorphic tumor. On the other hand, the biological effect of over-productive parotin to salivary gland themselves is still unknown. And there was no clinical or pathological report, concerning morphological features of parotin over-producing cells like a sort of 'Parotinoma' and parotin receptors on the normal salivary gland cells, because an appropriate assay system using monoclonal antibody for human parotin or an in vitro dynamic morphogenetic experimental system for human salivary gland has not been available. The hormonal kinetics in the salivary gland is still unknown. But earlier study reported that striated duct cells normally function to reabsorb the salivary hormone specifically excreted by acinar cells (9). And it may suggest parotin receptors on the salivary cells themselves. Thus it is very interesting to evaluate morphologically the in vitro hormonal effect to normal salivary gland cells themselves using long term culture system. For morphogenetic study, three-dimensional culture using collagen gel is very useful for subjecting cellular transforming induction (7). In those points of view, we have tried to subject the in vitro effect morphologically using three-dimensional culture system (Fig. 1) and also to study in vivo effect from the hormonal distribution in the tumor using immunohistochemical technique (Table 1). We have already reported the original re-differentiation in vitro culture model of the normal adult human salivary gland with the irradiated rat tail collagen gel (5,11). And we have also reported in vitro morphological features of the cells in the pleomorphic adenoma and Warthin tumor (11). In the present study, we have succeeded to induce normal salivary cells to keratinized pearl like formation in vitro using bovine parotin with this culture system in the 3 cases. The serum-free medium modified as previously described (11) was supplemented with 100 #g/ml crude parotin purified from bovine parotid gland
according to Aonuma's methods (2) (MP-parotin, purified to 98%: gift from Teikoku Hormone MFG, CO., Ltd., Japan). The primary culture embedded within the gel was performed as described (5,11). Four weeks after three-dimensional culture, squamous like cells (Fig. 2 a, large and small arrowheads) and keratinized pearl like cells (Fig. 2 b arrowheads) were observed within the gels, which were similar to those of pleomorphic tumor cells in collagen gel (Fig. 2 c) as we have already reported (11). On the other hand, ducts formation was observed in the parotin-free medium (Fig. 2 d, large arrowheads) after cellular elimination (Fig. 2 d small arrowheads) as we have previously reported (12). Two immunological and biological active fragments (Fr.AA-1 and Fr.H-1), both of which function to decrease the serum Ca++ level, were isolated from bovine parotin by tryptic digestion (3). And increasing tissue calcium ion level has been reported to have some effect to keratinization using cultured cells (10,13). We have also prepared the polyclonal antibody (pAb) from serum of BALB/C mouse with 8 times of weekly subcutaneous injections of complete adjuvant consist of 1 : 1 (vol) MP-parotin and Freund, and tried to investigate in vivo distribution of human parotid in pleomorphoic tumor using avidin-biotin peroxide method, to support this in vitro cellular transformation. As the result, two different molecular weights (M.W.) of the pAb-positive substances extracted from pleomorphic tumor were detected using immunoblotting analysis. The lower M.W. substance (Fig. 3 a, arrowhead 17K) possibly indicated the human salivary parotin and the higher ones (Fig. 3 a, arrowhead approximately 47K), which is almost same M.W. of bovine parotin subunit (Fig. 3 b, arrowhead 45K), is still unknown. But the antigen (bovine parotin) was purified to 98% and each 2 differing M.W. substances seemed to have common epitopes with bovine parotin. In normal parotid gland, almost the same blotting pattern as in pleomorphic tumor was observed (data not shown). Using this pAb, immunohistochemical study was performed (Table 1). In pleomorphic tumor, keratinized pearl (Fig. 4 a, arrowheads), chondrocyte like cells (Fig. 4 b, arrowheads), oncocyte like cells (Fig. 4 c, arrowheads) were more intensively for the pAb than cells in normal parotid gland (photos not shown). To produce proteoglycan for chondrocytic differentiation in vivo, calcium ion was also reported to be indispensable (6), and recently Ca++-ATPase was suggested to play an important role for pleomorphic differentiation from the ultrastructual localization (8). As for oneocytic cells, mitochondria has relation with calcium ion as in the point of the functions such as calcium ion storing effect and ATP supply for Na+-
475
476
SUZUMURA ET AL.
keratinized cells et al
I::!overlay medium [parotin~
(+) ] ::iii::i::~ cells containg layer ~ ~ . / . . ~ , , ~ after 4wks base layer ~ ; . . "..Y"....--" ... .. " primary culture
~ ~:y~,J..;,zl~,:.~.l~~
\
human salivary gland cells after collagenase treatment
over lay medium [parotin~
contracted gel
medium after 4wks primary culture duct re-formation
Fro. 1. The three-dimensional culture system using collagen gel. Parotin was contained in the overlay medium and gel.
Fig. 2. Cells obtained from normal parotid gland showed keratinized like pattern in the gel with 100 #g/ml parotin (a:38y female HEX100; b:25y male HEX400, arrowheads)4 weeks after primary culture, similar to the feature of keratinized cells obtained from pleomorphic tumor in the same gel (c:40y female HEx200 same sample as b, HE )
Letter to the Editor IN VITRO KERATINIZATION
OF NORMAL HUMAN SALIVARY GLAND CELLS
Dear Editor: The histopathological variation of pleomorphic adenoma in the salivary gland has attracted much interest among many pathologist~ Although the tumor is most popular in the salivary gland tumor (4), the mechanism for the morphological variation has remained obscure. And the tumor has been much more popular in the parotid gland than in the submandibular gland from the clinical reports (4). Interestingly the tissue concentration of the salivary hormone (parotin) in the parotid gland of guinea pig has been reported about 7 times as large as that in the submandibutar gland (1). Although no data has been reported concerning the tissue concentration rate other than guinea pig, these reports may suggest some relation between parotin and pleomorphic tumor. On the other hand, the biological effect of over-productive parotin to salivary gland themselves is still unknown. And there was no clinical or pathological report, concerning morphological features of parotin over-producing cells like a sort of 'Parotinoma' and parotin receptors on the normal salivary gland cells, because an appropriate assay system using monoclonal antibody for human parotin or an in vitro dynamic morphogenetic experimental system for human salivary gland has not been available. The hormonal kinetics in the salivary gland is still unknown. But earlier study reported that striated duct cells normally function to reabsorb the salivary hormone specifically excreted by acinar cells (9). And it may suggest parotin receptors on the salivary cells themselves. Thus it is very interesting to evaluate morphologically the in vitro hormonal effect to normal salivary gland cells themselves using long term culture system. For morphogenetic study, three-dimensional culture using collagen gel is very useful for subjecting cellular transforming induction (7). In those points of view, we have tried to subject the in vitro effect morphologically using three-dimensional culture system (Fig. 1) and also to study in vivo effect from the hormonal distribution in the tumor using immunohistochemical technique (Table 1). We have already reported the original re-differentiation in vitro culture model of the normal adult human salivary gland with the irradiated rat tail collagen gel (5,11). And we have also reported in vitro morphological features of the cells in the pleomorphic adenoma and Warthin tumor (11). In the present study, we have succeeded to induce normal salivary cells to keratinized pearl like formation in vitro using bovine parotin with this culture system in the 3 cases. The serum-free medium modified as previously described (11) was supplemented with 100 #g/ml crude parotin purified from bovine parotid gland
according to Aonuma's methods (2) (MP-parotin, purified to 98%: gift from Teikoku Hormone MFG, CO., Ltd., Japan). The primary culture embedded within the gel was performed as described (5,11). Four weeks after three-dimensional culture, squamous like cells (Fig. 2 a, large and small arrowheads) and keratinized pearl like cells (Fig. 2 b arrowheads) were observed within the gels, which were similar to those of pleomorphic tumor cells in collagen gel (Fig. 2 c) as we have already reported (11). On the other hand, ducts formation was observed in the parotin-free medium (Fig. 2 d, large arrowheads) after cellular elimination (Fig. 2 d small arrowheads) as we have previously reported (12). Two immunological and biological active fragments (Fr.AA-1 and Fr.H-1), both of which function to decrease the serum Ca++ level, were isolated from bovine parotin by tryptic digestion (3). And increasing tissue calcium ion level has been reported to have some effect to keratinization using cultured cells (10,13). We have also prepared the polyclonal antibody (pAb) from serum of BALB/C mouse with 8 times of weekly subcutaneous injections of complete adjuvant consist of 1 : 1 (vol) MP-parotin and Freund, and tried to investigate in vivo distribution of human parotid in pleomorphoic tumor using avidin-biotin peroxide method, to support this in vitro cellular transformation. As the result, two different molecular weights (M.W.) of the pAb-positive substances extracted from pleomorphic tumor were detected using immunoblotting analysis. The lower M.W. substance (Fig. 3 a, arrowhead 17K) possibly indicated the human salivary parotin and the higher ones (Fig. 3 a, arrowhead approximately 47K), which is almost same M.W. of bovine parotin subunit (Fig. 3 b, arrowhead 45K), is still unknown. But the antigen (bovine parotin) was purified to 98% and each 2 differing M.W. substances seemed to have common epitopes with bovine parotin. In normal parotid gland, almost the same blotting pattern as in pleomorphic tumor was observed (data not shown). Using this pAb, immunohistochemical study was performed (Table 1). In pleomorphic tumor, keratinized pearl (Fig. 4 a, arrowheads), chondrocyte like cells (Fig. 4 b, arrowheads), oncocyte like cells (Fig. 4 c, arrowheads) were more intensively for the pAb than cells in normal parotid gland (photos not shown). To produce proteoglycan for chondrocytic differentiation in vivo, calcium ion was also reported to be indispensable (6), and recently Ca++-ATPase was suggested to play an important role for pleomorphic differentiation from the ultrastructual localization (8). As for oneocytic cells, mitochondria has relation with calcium ion as in the point of the functions such as calcium ion storing effect and ATP supply for Na+-
475
476
SUZUMURA ET AL.
keratinized cells et al
I::!overlay medium [parotin~
(+) ] ::iii::i::~ cells containg layer ~ ~ . / . . ~ , , ~ after 4wks base layer ~ ; . . "..Y"....--" ... .. " primary culture
~ ~:y~,J..;,zl~,:.~.l~~
\
human salivary gland cells after collagenase treatment
over lay medium [parotin~
contracted gel
medium after 4wks primary culture duct re-formation
Fro. 1. The three-dimensional culture system using collagen gel. Parotin was contained in the overlay medium and gel.
Fig. 2. Cells obtained from normal parotid gland showed keratinized like pattern in the gel with 100 #g/ml parotin (a:38y female HEX100; b:25y male HEX400, arrowheads)4 weeks after primary culture, similar to the feature of keratinized cells obtained from pleomorphic tumor in the same gel (c:40y female HEx200 same sample as b, HE )
