Radiation-induced apoptosis in human sarcoma and glioma cell lines.

Six human soft-tissue sarcoma and 14 glioma cell lines, exhibiting considerable differences in radioresponsiveness and histological grade of differentiation of the parental tumour, were examined with respect to apoptosis development after irradiation with 60Co gamma-rays. After test doses of 6 and 25 Gy, significant changes characteristic of apoptosis occurring within 6 to 30 hr were exhibited by only 2 differentiated sarcoma cell lines, EL7 and ESS2. The characteristic internucleosomal fragmentation of DNA was detected as early as 6 hr after exposure of subconfluent monolayer cultures to 6 Gy. It was limited to cells that had detached from the culture plate, whereas adherent cells showed random degradation of DNA, namely after higher doses (25Gy) or longer incubation times (30 hr). As assessed by fluorescence microscopy of unfixed cultures stained with Hoechst 33342 and propidium iodide, the proportion of cells showing apoptotic bodies in non-irradiated controls was < 0.1% and 0.3% for EL7 and ESS2, respectively. The dose-response relationship for apoptosis was determined at 9 hr post-irradiation. After 2 Gy, the percentage of apoptotic cells was elevated to 3.4% in EL7 and 4.5% in ESS2 cultures. Saturation was obtained above 6 Gy, with 8.4% apoptosis in EL7 and 15% in ESS2 after 25 Gy. Taken together, rapid ionizing-radiation-induced apoptosis seems to be limited to a subgroup of sarcomas and is unlikely to occur in gliomas.

Retinoic acid increases CD15 expression in immortalized rat astrocytes.

We have studied the expression of the CD15 (3-fucosyl-N-acetyl-lactosamine) epitope on immortalized astroglial cells derived from embryonic (E 19/20) rat brain. Immortalization was achieved by pulse-treatment of primary culture with 5-azacytidine. Seventy-three permanent cell lines were established by repeated cell cloning. Clones expressing GFAP, A2B5, and vimentin were regarded as immature astrocytes. One of these clones expressing CD15 was selected for manipulation studies. Monoclonal antibody was used for immunocytochemical detection of CD15 epitope and in immunoblot analysis. CD15 expression was visible in about 20% of the cells and was associated with a special morphological appearance. In the presence of retinoic acid the proportion of CD15-positive cells increased in a time-dependent manner, reaching about 90% within four days. Again, this expression was associated with the formation of distinct morphological features, including immunoreactive perinuclear granula, tips of processes and contact sites. After treatment with neuraminidase, all cells showed CD15-positive immunoreaction, revealing the presence of the epitope masked by sialylation. Immunoblot patterns of glycoproteins from trypsinized and mechanically detached cell preparations suggest that proteins, carrying sialylated CD15, might represent intracellular precursors of extracellularly active molecules.

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations.

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D-myo-[2-3H]inositol, addition of alpha-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of alpha-antagonists (phentolamine, prazosin). beta-Agonists and/or beta-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of beta-receptor mediated processes was suggested by the beta-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under alpha-adrenergic control.