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Glycosaminoglycan synthesis by normal human mammary epithelial cells in primary culture

University of British Columbia

The extracellular matrix (ECM) influences cell growth and differentiation. Glycosaminoglycans (GAGs), alone or complexed with protein (proteoglycans), are a major component of the ECM affecting cell behavior. GAG synthesis has been studied extensively in animal models and malignant cells. This research centres on studying GAG production by normal human mammary epithelial cells in culture. Mammary tissue obtained from reduction mammoplasties were dissociated to single cells. The epithelial cell population was seeded onto hydrated collagen gels at 2-2.5x10⁵ cells/cm² in medium containing 5% FCS and 5μg/ml of insulin. Ultrastructural studies confirmed the epithelial nature of the cultures. To measure GAG synthesis, cultures were incubated with ³H-glucosamine for 24 hours at 3 time points; days 3-4, 9-11 and 17-18. The cultures were proliferating at the early time point and had reached a stationary phase at the later time points. Cell, ECM and medium fractions were analyzed for GAGs as identified by enzyme degradation and cellulose acetate electrophoresis. At day 4, when cells were actively growing, the majority of GAGs produced were released into the medium fraction (75-80%). The predominant GAG was the nonsulfated GAG, hyaluronic acid (HA). Of the sulfated GAGs chondroitin sulfate (CS) 4 and 6 comprised only 18% of total GAGs; dermatan sulfate (DS) synthesis was negligible. At the later time periods, when cultures had ceased growing a higher percentage of total GAG was incorporated into an ECM (50-65%). The sulfated GAGs were preferentially incorporated into the ECM, CS 4 and 6 comprising 70% and DS comprising 30%. The marked difference in type and location of GAGs produced was not merely a function of time in culture. Cultures seeded at high densities (5x10⁵ cells/cm²) were not proliferating when terminated at day 4. Their GAG profile was similar to that of lower density cultures at day 10. This data provides a baseline from which we can determine if cell-synthesized GAGs, play a role in maintaining differentiated and malignant phenotypes.

Text

2010-06-20

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http://hdl.handle.net/2429/25903

Anatomy, Cell Biology and Physiology

University of British Columbia

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