Oxygen radicals and the cellular antioxidant enzymes may play a role in cellular senescence. We studied the feasibility of altering oxygen radical metabolism in a normal differentiated cell that undergoes senescence in culture by transfection of an expression vector containing human CuZn-SOD cDNA. Plasmid pRSV-2-cSOD was constructed to contain the cDNA for human CuZn-SOD under the regulation of the Rous sarcoma virus long terminal repeat. Early passage cultures of bovine adrenocortical cells were cotransfected with pRSV2-cSOD and a plasmid (pSV3neo) allowing initial selection and continued growth of transfectants. Three passages after isolation of the polyclonal population, as cells grew to confluence, cultures showed focal cell death that spread outward to affect neighboring cells, so that by 72 h most cells had detached from the culture dish. Long term growth of the polyclonal population of transfectants without extensive cell death was achieved by continuous maintenance of low cell density during growth. Southern blot analysis of DNA from the pooled polyclonal population of transfected cells showed the presence of the expected 625 bp band from human CuZn-SOD. However, the intensity of this band indicated that only a minority of cells in the population had integrated the SOD plasmid, and DNA isolated from cells after 25 passages at low cell density showed plasmid sequences only of an altered form, suggesting that cells containing intact human SOD cDNA had been selectively lost from the population. When early passage low density transfectants were allowed to grow back to high cell density, cell death foci were again observed. Additionally, cell fusion with the formation of giant cells with massive multinucleation was observed by flourescence microscopy after staining cultures with a DNA binding dye. In later stages of this process, the large nuclear mass in such a giant cell became fragmented as the cell detached from the dish and formed the center of a focus of cell death in the surrounding cells. Because cell death prevented the growth of large numbers of transfected cells, it was not possible to demonstrate the involvement of CuZn-SOD in the cytotoxic effect by direct means, but the control plasmid without the CuZn-SOD cDNA insert had no cytotoxic effect. Thus, the introduction of a vector for human CuZn-SOD in a normal differentiated cell caused a cytotoxic effect involving cell death, cell fusion, and nuclear fragmentation.
ASJC Scopus subject areas
- Molecular Biology