Control of smooth muscle cell (SMC) number is of importance in the development and pathology of the vasculature. Recent evidence suggests this control is exerted both at the level of cell proliferation and cell death. We have described a phenotype of abnormal SMC proliferation in transgenic mice expressing a temperature-sensitive SV4G T-antigen (TAg, isA58) targeted to SMC utilizing the mouse vascular SMC a-actin promoter. Clonal SMC lineages from a variety of tissues with this genotype manifest enhanced growth at the permissive temperature (33°C) in comparison to the restrictive temperature (39.5 °C); while comparison with SMC derived from non-transgemc siblings demonstrated that mis was due not only to a shortened population doubling time (PDT) at 33 °C in the transgenic cells, but also a markedly lengthened PDT seen at 39.5 °C for transgenic cells vs. non-transgenic cells. In order to further understand the basis for this conditional proliferation, we evaluated several transgenic aortic SMC clones with respect to cell cycle (DNA content) distribution, apoptosis, and changes in protein content following temperature shifting. Inununoblot assessment demonstrated TAg expression at 33 °C to vary over a tenfold range among isolated clones; p53 protein expression was correlated tightly with TAg level (R=0,86). Total immunoreactive TAg and p53 disappeared with half-lives of approximately 18 hours following shift to the restrictive temperature. Such temperature shift resulted in a decrease in the fraction of cells in S-phase (39±2 to 28±5%) at confluence in 10% FBS, and in increase in the Gj/M fraction (20±1 to 34±2%); as well as the appearance of cells with apparent DNA contented, suggestive of apoptosis. This was confirmed by intermicleosomal DNA fragmentation observed after temperature shift, with in situ detection demonstrating apoptosis within 8 hours following shift, peak frequencies of 40-50% found at 16 hours, and lower frequencies subsequently. Although differences in apoptosis frequencies among clones accounted in part for variability in clonal sensitivity to temperature, this variability did not correlate simply with Tag or p53 expression. This transgenic model has thus permitted the derivation of several marine aortic SMC clones with a range of conditionally-modulated apoptosis frequencies. These cells may provide a useful reagent for study of proliferation and apoptosis in SMC.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)