The primate mammary epithelial cell in vitro, growth properties, antigen expression and cell survival studies

Abstract

Primary tissue cultures of primate mammary epithelial cells (PMEC) in tissue culture were evaluated to serve as a link between rodent models and in vitro human mammary epithelial cell (HMEC) systems used to study mammary carcinogenesis. PMEC growth in vitro, expression of cytokeratin and milk fat globule proteins (MFGP) and the effect of dirnethylbenz(a)anthracene (DMBA) was investigated. Organoids wer~ isolated from primate (Cercopitl1ecus aethfops pygerythrus) and classified according to the d~~greeof lobular development namely less, moderately and well differentiated. Organoid attachment and growth in cell culture reflected mammary gland development. Rapid organoid attachment and growth ln cell culture was associated with organoids derived from less and moderately differentiated tissue. A reduction in in vitro calcium extended the lifespan of PMEC and induced dramatic morphological changes. New cells shed into the medium were passaged. Immunoblotting identified Kat K7 anc' 1<6and/or K11 as the predominant keratins in primate mammary tissue. PMEC in vitro expressed the same keratins as in vivo, the expression of K8 Wc':1S reduced and K7 and 1<6and/or K11 was increased. The milk fat globule protein (MFGP) fraction was isolated from human milk and lactating primate mammary tissue. Immunoblotting revealed that human antiwMFGP detected primate Ml=GP-70 while HMFG-2 did not detect primate sialomucins. Trypstnlsatlon and subsequent thlol reduction resulted in the enrichment of MFGP-70 from human and primate MFGP fractions. MFGP ..70 was not detected in cell lysates and tryptic digests of MCF ..7 and PMEC cells following thiol reduction. A 53 ~~Daprotein was detected in human and prirrate MFGP fractions, tryptic digests of the same fractions and MCF ..7 and PMEC Iysates under nonreducing conditions. The effect of DMBA on the PMEC survival was determined by measuring succinate dehydrogenase inhibition and cell numbers in vitro. The use of Hoechst to determine ii cellular DNA is limited due to nonspecific fl40rescence caused by SDS which was effectively reduced by increasing the counterton concentration and adding chelate, A dose and time related sigmoidal decrease in cell survival was observed. Differences in cell survival and cell numbers in vitro occurs following 3 hours exposure to DMBA, while longer exposure times revealed that cell survival determined by all methods was the same. PMEC in vitro, exhibit groW\r properties that reflect mammary gland development, express the same keratins as in vivo, and antigenic determinants associated with the primate MFGP fraction. PMEC growth in vitro, expression of antigen*, and response to the cytotoxic effects of DMBA is analogous to the HMEC in vitro. This indicates that the PMEC can contnbute significantly to understanding the mechanisms of mammary carcinogenesis,