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.

Agency
National Institute of Health (NIH)
Institute
Division of Cancer Epidemiology And Genetics (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CP005379-02
Application #
4692410
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Cancer Epidemiology and Genetics
Department
Type
DUNS #
City
State
Country
United States
Zip Code