Changes in morphology, cytoskeleton, and substrate dependence of proliferation after transfection of immortalized rat embryonic fibroblasts with E7 gene of type 16 human papilloma virus

Abstract

Transfection of rat embryonic fibroblasts with E7 gene of type 16 human papilloma virus changed the cytoskeleton and cell-cell and cell-matrix interactions in two clones of transformed cells. Cell morphology and substrate-dependent proliferation were also changed. Key Words: human papilloma virus; transformation; actin; fibronectinTransformation is a multistaged genetically determined process of cell change with a number of characteristic features. Phenotypic signs of transformation vary and depend on the inducing agent, cell type, and differentiation stage. The disturbance of substrate dependence of proliferation occurs in cultured transtbrmed cells. Other phenotypic signs of transformation are: 1) decrease in the number of actin microfilament bundles (stress-fibrills) which participate in the formation of focal contacts, i.e., local zones of cell adhesion to the extracellular matrix; 2) tyrosine hyperphosphorilation of proteins involved in focal contacts, which inhibits formation of these contacts and decreases adhesion [5,7,8]; 3) changes in the extracellular matrix structure and in fibronectin (FN) as one of its components [9]. There is structural and functional interaction between the extracellular matrix and the cytoskeleton of an attached cell. Disturbances of this interaction in transformed (malignant) cells potentiate invasion [7,9].Cells transformed by E7 gene of type 16 human papilloma virus (E7 HPV16) are a convenient model The properties of the transformed clones obtained by transfection of rat embryonic fibroblasts (EF) with E7 HPV16 gene have been examined. Cytogenetic analysis of these clones revealed clone expansion of the cells with specific chromosome transformations [11].The aim of the present study was to investigate cell morphology, organization of actin cytoskeleton and FN of extracellular matrix, growth characteristics and substrate dependence of proliferation in immortalized cell clone (IE5) and in trF8 and trB4 clones transformed by E7 HPV16 gene. METHODSParental line of immortalized rat EF was obtained from primary culture transfected with plasmids containing T-antigen polyoma virus gene [3]. IE5 clone was isolated from this line by the final dilution technique. By the start of the present study the virus gene in IE5 clone was lost, as revealed by blot-hybridization technique. IE5 clone was transfected with the E7 HPV16 gene [11]. The transformed clones were grown on selective medium containing geneticin (400 mg/ml, 0007-4888/99/0001-0099 $22.00 9 1999 Kluwer Academic/Plenum Publigher~