Two-dimensional polyacrylamide gel electrophoresis in experimental hepatocarcinogenesis studies.
High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in combination with computer-assisted densitometry was used to analyze sequential changes in polypeptide expression during chemically (aflatoxin Bl; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-raflv-myc)-induced experimental rat hepatocarcinogenesis. Two-dimensional mapping of [35S]methionine and [32P]orthophosphate-labeled whole cell lysate and nuclear polypeptides revealed subsets of polypeptides specific for each transformation modality in the in vitro rat liver epithelial (RLE) transformation model. Many of the observed changes in whole cell lysate preparations were localized to specific subcellular organelles. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, cytokeratins 8, 14, and 18, and actin) were observed among the various transformant cell lines. Whereas alterations in the tropomyosin isoforms appeared to be transformation specific, concomitant modulation of intermediate filament expression was related more to the differentiation state of the individual cell lines than to the transformed phenotype. To integrate protein and DNA information of polypeptides believed to be critically involved during cellular transformation, N-terminal amino acid microsequencing of selected nuclear polypeptides was performed. Preliminary results suggest that N-terminal blockage of rat liver epithelial nuclear proteins to be minor (approximately 20%) with sequencing sensitivity of one pmol. These studies extend our on-going efforts toward the establishment of computerized database of rat liver epithelial cellular proteins (Wirth et al., Electrophoresis, 1991, 12, 931-954) to aid in the delineation of polypeptides critically involved in cellular growth and differentiation as well as transformation.
Wirth
P J
PJ
Biopolymer Chemistry Section, National Cancer Institute, Bethesda, MD 20892.
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography / Q000379:methods; D002460:Cell Line; D002461:Cell Line, Transformed; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004848:Epithelium; D005260:Female; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D009857:Oncogenes; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D011916:Rats, Inbred F344; D013462:Sulfur Radioisotopes
D007381:Intermediate Filament Proteins; D009363:Neoplasm Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011506:Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine