Actin mutations in a human fibroblast model for carcinogenesis.
We assessed the modulation of gene expression accompanying neoplastic transformation by computerized microdensitometry of autoradiographic patterns of [35S]-methionine-labeled polypeptides separated by two-dimensional polyacrylamide gel electrophoresis. Nearly 1000 polypeptide species of parent diploid human fibroblasts (KD strain) and clonally-derived malignant fibroblasts (HUT-14 strain) were compared. We found that the neoplastic HUT-14 fibroblasts express a mutation in one of the two functional beta-actin genes. In addition, of the 700 more-abundant polypeptides measured, 13 were lost and 14 new ones gained after this neoplastic transformation. We estimate that although 2% of fewer of the genes expressing abundant polypeptides were either activated or shut off, at least 32% were modulated quantitatively. A substrain of HUT-14--HUT-14T--shows increased tumorigenicity, producing larger, faster-growing fibrosarcomas in the nude mouse than does the present parent HUT-14 strain, and with fewer inoculated cells. This increase in tumorigenicity is accompanied by three subsequent changes in the mutant beta-actin polypeptide expression. A more variant mutant actin species is synthesized in HUT-14T, which differs from the original mutant polypeptide by (i) one additional negative net charge, (ii) a short half-life in the cell, (iii) a greatly diminished ability to incorporate into the detergent-resistant cytoskeleton, (iv) a decrease in affinity for deoxyribonuclease I (EC 3.1.21.1), and (v) a faster rate of synthesis. Our results suggest that a second-site mutation in the mutant beta-actin of HUT-14 was selected for during a subcloning step in the presence of 6-thioguanine before derivation of the HUT-14T substrain. This apparent mutation and two subsequent defective beta-actin expressions are accompanied by incremental increases of malignant potential.
D000199:Actins / Q000032:analysis / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005354:Fibrosarcoma / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007525:Isoelectric Focusing; D051379:Mice; D008819:Mice, Nude; D008957:Models, Genetic; D009154:Mutation; D012513:Sarcoma, Experimental / Q000378:metabolism
D000199:Actins