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Transformation-sensitive isoactin in passaged chick embryo fibroblasts transformed by Rous sarcoma virus
Abstract:
Transformation by Rous sarcoma virus (RSV) has been reported to block the expression of differentiated cell products in chicken cells. The expression of these proteins may or may not be suppressed when temperature-sensitive mutants are shifted from the nonpermissive to the permissive temperature. A general characteristic of cellular transformation is the disruption of the microfilament system. In passaged chick embryo fibroblast cultures (CEF), this system is principally composed of isomeric forms of actin designated alpha, beta, and gamma by their isoelectric focusing and when subjected to SDS-PAGE behavior. We present evidence that an alpha-actin in CEF cultures, identified by its electrofocusing behavior, retention in the cytoskeleton, and DNase 1 binding properties, is selectively and dramatically reduced in amount upon transformation by RSV. Little or no reduction is observed in the beta- and gamma-isoactins. The reduction of alpha-actin is shown to be reversible and transformation related by use of a temperature-sensitive mutant, tsNY68. The decrease in this transformation-sensitive isoactin is apparently due to a decrease in synthesis, though other possibilities are discussed. A specific decrease in a particular isoactin after transformation may give insight into the mechanism by which the microfilaments are normally maintained.
Insights
Rous sarcoma virus (RSV) transformation selectively reduces alpha-actin in chicken cells, disrupting microfilaments. This specific actin reduction is reversible, offering insights into microfilament maintenance mechanisms.
Area of Science:
- Cell Biology
- Virology
- Biochemistry
Background:
- Cellular transformation by Rous sarcoma virus (RSV) is known to suppress differentiated cell product expression in chicken cells.
- Microfilament system disruption is a hallmark of cellular transformation.
- Chick embryo fibroblast (CEF) microfilaments primarily consist of alpha, beta, and gamma actin isoforms.
Purpose of the Study:
- To investigate the effect of RSV transformation on specific actin isoforms in CEF cultures.
- To determine if the observed changes in actin expression are transformation-dependent and reversible.
Main Methods:
- Isoelectric focusing and SDS-PAGE to analyze actin isoforms.
- Assessment of actin retention in the cytoskeleton.
- DNase 1 binding assays to identify alpha-actin.
- Use of a temperature-sensitive RSV mutant (tsNY68) to study reversibility.
Main Results:
- RSV transformation selectively and significantly reduces the amount of alpha-actin in CEF cultures.
- Beta- and gamma-actin isoforms show little to no reduction upon transformation.
- The decrease in alpha-actin is reversible when using the temperature-sensitive mutant tsNY68, indicating a transformation-related mechanism.
- Evidence suggests the reduction in alpha-actin is primarily due to decreased synthesis.
Conclusions:
- RSV transformation specifically targets and reduces alpha-actin expression in chicken embryo fibroblasts.
- The selective decrease in alpha-actin is reversible and linked to the transformation process.
- Understanding the regulation of alpha-actin during transformation may elucidate mechanisms of microfilament maintenance.
