Temperature-Dependent Formation of Dimers and Oligomers of Mitochondrial DNA in Cells Transformed by a Thermosensitive Mutant of Rous Sarcoma Virus

Abstract

In chick-embryo fibroblasts infected by T5, a temperature-sensitive mutant of Schmidt-Ruppin Rous sarcoma virus, the level of catenated dimeric and oligomeric mitochondrial DNA is temperature-dependent and correlates with the phenotypic manifestation of transformation. At the permissive temperature (360), where transformation is expressed, the 2-to 3-fold elevated level characteristic of cells transformed by the wild-type Rous sarcoma virus was observed, but at the nonpermissive temperature (410), at which cells appear normal and behave normally, the oligomer level was characteristic of uninfected cells. Temperature shifts from 41 to 360 and vice versa resulted in phenotypic reversion and reversals of the respective levels of multiple-length DNA. Cellular growth rates were not altered. Treatment of control cells with cycloheximide resulted in a 5-fold increase of oligomeric DNA, whereas exposure to 9-j8-D-arabinofuranosyladenine had little effect. With both inhibitors, nuclear but not mitochondrial DNA synthesis was inhibited. The possible relation of formation of oligomeric mitochondrial DNA to alterations in the mitochondrial membranes of transformed cells and to inhibition of protein synthesis in the cytoplasm is discussed.

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

“…3). Also, the fraction of cells synthesizing nuclear DNA as determined by autoradiography was found to be about the same in all cell types at both temperatures (13,19). This suggests that the quantitative differences observed in the synthesis of mtDNA were not merely a consequence of differences in growth rates between the transformed and nontransformed ceils.…”

“…The cells were harvested by trypsinization and the mitochondria were isolated in the same manner as for the in vivo experiments (19) with one exception: homogenization of the cells was done in a solution of 0.7% bovine serum albumin, 0.025 M Tris-HCI, pH 7.4 (4~ 0.002 M EDTA, and 0.03 M nicotinamide. Centrifugation was carried out in this solution containing 0.3 M sucrose.…”

“…Radioisotopic labeling in vivo was performed by the addition of 5 /~Ci/ml of [methyl-ZH]thymidine (New England Nuclear; sp act 43.1 Ci/mmol) to cells growing in monolayer culture in the logarithmic phase of growth. After 3 h of incubation in the presence of this isotope, the cells were harvested by trypsinization and the mitochondria were isolated according to procedures previously described from this laboratory (19). mtDNA was prepared as described (19) and centrifuged to equilibrium in cesium chloride-ethidium bromide-buoyant density gradients (17,19,20).…”

“…From this laboratory it has recently been demonstrated (19) that there is a two-to threefold increase in the frequency of multiple-length mtDNA molecules in CEF transformed by oncogenic viruses as compared to nontransformed CEF. In view of this evidence, we have initiated a detailed study of the synthesis of mtDNA to determine whether and how this synthesis differs in CEF cells that express normal and in cells that express malignant phenotypes.…”

Genetics of RNA Tumor Viruses

“…A transformation-related change which at present cannot be linked directly to altered growth or surface properties of avian sarcoma cells is the increase in dimeric and oligomeric mitochondrial DNA (Nass, 1973). This change is also temperature dependent in chick embryo fibroblast cultures infected with class T Is mutants of avian sarcoma viruses.…”