Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.
Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.
Wirth
P J
PJ
Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Luo
L D
LD
Fujimoto
Y
Y
Bisgaard
H C
HC
D000199:Actins / Q000032:analysis; D016604:Aflatoxin B1; D000818:Animals; D002461:Cell Line, Transformed; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000187:drug effects; D005353:Fibronectins / Q000032:analysis; D007381:Intermediate Filament Proteins / Q000032:analysis; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D008715:Methionine / Q000378:metabolism; D009857:Oncogenes / Q000235:genetics*; D010455:Peptides / Q000032:analysis*; D010710:Phosphates / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D014335:Tropomyosin / Q000032:analysis
D000199:Actins; D005353:Fibronectins; D007381:Intermediate Filament Proteins; D010455:Peptides; D010710:Phosphates; D014335:Tropomyosin; D016604:Aflatoxin B1; D008715:Methionine