cell death induced by physical agents: morphological features

The aim of this work is to present and discuss in vitro cell death appearing after exposure to physical conditions such as UVB radiation, static magnetic fields, hyperthermia and hypothermia. UVB radiation induces oxidative stress, leading, in most experimental models, to apoptotic death. Generally death occurs through the intrinsic apoptotic pathway, even if the extrinsic one cannot be excluded. UVB radiation also appears effective on cell systems which are normally apoptosis-resistant, such as muscle cells. Static magnetic fields mostly induce plasma membrane and microvilli alterations; occasionally apoptotic cell death appears. Hyperthermic conditions applied were mild, i.e. variable exposures to 43\ub0C, as well as hypothermia, consisting of variable exposures to 0-6\ub0C. Both treatments were followed by incubation at physiological conditions. Heat exposure is a powerful apoptotic inducer in a variety of cells, where it induces classical apoptotic changes and the well known biochemical pathways. The effect of hyperthermia has been described in adherent human tumor cells, which undergo cell rounding and progressively detach from the substrate, in close correlation with the down-regulation of adhesion molecules. Hypothermia, only occasionally triggers apoptosis, more frequently inducing cell necrosis. Therefore, cell death can be induced by physical agents dependently on the treatment and cell model. In particular, UVB and hyperthermia can be considered reliable and reproducible apoptotic triggers

An ultrastructural approach to the study of apoptotic double strand DNA cleavage

Apoptotic DNA cleavage initially produces large fragments (50 kbp), followed by the formation of nucleosomic/ oligonucleosomic ones. On the other hand, apoptosis without DNA fragmentation, at least the nucleosomic one, has been described. To study the correlation between the DNA cleavage and the well known chromatin behavior, we applied TUNEL to electron microscopy, by using a gold-conjugated antidigoxigenin antibody, on U937 and Molt-4 cells, both exposed to UVB or staurosporine. Gold particle density in the different domains of apoptotic nuclei was statistically evaluated. Gold labelling was more intense in dense apoptotic chromatin than in the diffuse one. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not, even if showing larger DNA fragments in vitro. DNA fragment sizes, characterized by gel electrophoresis and by FIGE, appeared closely correlated to gold particle density on apoptotic chromatin domains. TUNEL applied to electron microscopy is an useful tool to highlight mechanisms underlying apoptotic chromatin condensation and DNA cleavage pattern