ANALYSIS OF POLYPEPTIDE CHANGES DURING CELLULAR DIFFERENTIATION

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This project was initiated to analyze, both qualitatively and quantitatively, changes in total cellular protein patterns during cellular differentiation and transformation using the technique of quantitative two-dimensional electrophoresis. Results obtained to date include: 1) Mouse epidermal cells were cultured in the presence of either low calcium (0.02-0.1 mM) (mainly basal cells) or high calcium (1.2 mM) (mainly mature keratinocytes) and then treated with 12-0-tetradecanoyl-13-acetate (TPA) for 1, 4 or 24 hours. Differentiation of epidermal basal cells were induced by increasing Ca+2 from 0.05 to 1.2 mM in the medium or by treatment with TPA. Analysis of epidermal polypeptides by 2-D electrophoresis indicated that the synthesis of eight polypeptides were increased and six decreased, and the phosphorylation of at least one polypeptide is altered similarly by Ca+2 and TPA. These common polypeptide changes, both associated with differentiation induced by Ca+2 and TPA, may involve the phorbol ester receptor, protein kinase C. 2) Analysis of polypeptide changes from normal bronchial epithelial cells (NHBE) and the Harvey v-ras transfected lines, TBE-1, TBE-1SA, and TBE-1SAT revealed the progressive appearance of four new polypeptides (pI 5.5-5.75/56-55 kDa; 4.9/55 kDa; 6.4/66 kDa; and 6.7/39 kDa) from NHBE to the most malignant TBE-1SAT line. Numerous quantitative changes were also observed, most notably, the progressive loss of expression of two polypeptides (5.6/40 kDa and 5.7/40 kDa), which were almost completely absent in TBE-1SAT cells. 3) Sodium bisulfite transformed hamster fetal cells were characterized by the shift of two polypeptides 1 and 2 (pI 5.1/52 kDa and 5.1/28 kDa), the expression of two new polypeptides 3 and 4 (6.8/44 kDa and 5.5/46 kDa) and the loss of one polypeptide 5 9(6.0/55 kDa). Early polypeptide changes were analyzed in a series of 20 bisulfite transformed lines. Polypeptide changes in 1, 2, and 5 were associated with early steps in the transformation process and related to morphologic changes, whereas polypeptides 3 and 4 appear to occur later and are more closely associated with the acquisition of tumorigenicity.