ABSTRACT
When cells are treated with cytotoxic agents, they enter apoptosis asynchronously to yield cells at various stages of cellular deterioration. This mixture makes it difficult to study the biochemical pathways leading to cell death. We have fractionated apoptotic mammalian cells in a simple discontinuous bovine serum albumin (BSA) density gradient centrifugation into five layers, each containing cells at different stages of apoptosis, (1) nonapoptotic, (2) undergoing apoptosis, and (3) mature apoptotic cells, as judged by light and electron microscopy of chromatin condensation and by the extent of DNA fragmentation. Modifications of apoptosis markers including c-Jun N-terminal kinase/stress-activated protein kinase and procaspase 3 cleavage were apparent in those cells that are undergoing apoptosis. Apoptosis-specific histone H2B phosphorylation was highly elevated and DNA fragmentation activity in the cytoplasm was observed in those cells that are undergoing apoptosis, but not much was observed in the cells of other fractions. Results show that apoptotic cells can be fractionated easily by the BSA gradient method, and this method will be invaluable for studying the biochemical processes that drive apoptosis.
Subject(s)
Apoptosis , Cell Separation/methods , Centrifugation, Density Gradient/methods , Serum Albumin, Bovine/chemistry , Animals , Cattle , Cell Line, Tumor , Cell Separation/instrumentation , Cell Survival , Cells, Cultured , Centrifugation, Density Gradient/instrumentation , DNA/isolation & purification , DNA/metabolism , DNA Fragmentation , HL-60 Cells , HeLa Cells , Humans , Mice , Rats , Rats, Sprague-Dawley , Thymus Gland/cytologyABSTRACT
Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo, without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis. 3 H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.
