Gloria Juan

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology.

Mitochondrial damage may be a major cause of cellular aging. So far, this hypothesis had only been tested using isolated mitochondria. The aim of this study was to investigate the involvement of mitochondria in aging using whole liver cells and not isolated mitochondria only. Using flow cytometry, we found that age is associated with a decrease in mitochondrial membrane potential (30%), an increase in mitochondrial size, and an increase in mitochondrial peroxide generation (23%). Intracellular peroxide levels were also increased. The number of mitochondria per cell and inner mitochondrial membrane mass did not change. Gluconeogenesis from glycerol or fructose (mitochondrial-independent) did not change with age, whereas it did from lactate (mitochondrial-dependent). The change in the rate of gluconeogenesis was not accompanied by changes in any of the following parameters: phosphoenolpyruvate carboxykinase or pyruvate carboxylase activities or mitochondrial ATP/ADP or cytosolic NADH/NAD+ ratios. This was caused by a decreased rate of malate export (to 20% of the controls) from mitochondria. The impairment of the mitochondrial malate transporter is posttranscriptional because its expression in Xenopus oocytes using polyadenylated RNA from livers of young or old animals did not change. Ketogenesis from oleate also fell in hepatocytes from old rats. Our results show, for the first time in intact cells, a correlation between age-associated impairment of cell metabolism and specific changes in mitochondrial function and morphology, supporting the hypothesis that mitochondrial damage plays a key role in aging.

Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 micrograms/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000-300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dual-staining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed.