Morphologic and temporal analysis of vascular smooth muscle cell apoptosisinduced by c-myc and E1A

Apoptosis is a physiologic form of cell death present in many disease condi tions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in sever al diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. S ince c-myc is upregulated in the pathogenesis of these diseases, we chose t o study the sequential morphologic features of programmed cell death in vas cular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time -lapse videomicroscopy. We observed that both c-myc and E1A-induced apoptos is (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Tran smission electron microscopy analysis revealed the larger blebs contained m ostly nuclear chromatin, whereas the many smaller fragments often had littl e or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably tr ansfected with E1A. The early changes of apoptosis, including cell shrinkag e and intense blebbing, occurred in under 5 min in both cells. Slight alter ations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from mov ement of the culture flask. These studies provide a framework of timed morp hologic events for future mechanistic investigation into the key aspects of myc- and E1A-induced apoptosis in vascular smooth muscle.