The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems*

Abstract

Cell death in gliomas may occur either by apoptosis, or, in the case of high grade tumours, by necrosis, but questions remain as to the pathogenesis and relationship between these processes. The development of cell death was investigated in multicellular glioma spheroid cultures. Spheroids model the development of cell death due to diffusion gradients in a three-dimensional system without confounding influences of immune response, pressure gradients, etc. Spheroid cultures were established from four malignant glioma cell lines: U87, U373, MOG-G-CCM and A172; harvested from culture at weekly intervals and stained with Haematoxylin and Eosin (H&E), TdT-mediated dUTP-X nick end labelling (TUNEL) and by immunohistochemistry for vimentin, Glial Fibrillary Acidic Protein (GFAP) and Ki67. Annexin V flow cytometry and counts of apoptotic cells on H & E stained sections were performed to assess levels of apoptosis. Modes of cell death were also characterized by electron microscopy. Spatially separate zones of proliferation, differentiation and central cell death developed with increasing spheroid diameter. Central cell death developed at a predictable radius (300-400 microm) for each cell line. Ultrastructural examination showed this to be necrotic in type. Apoptosis was most reliably assayed by morphological counts using H & E. Basal levels of apoptosis were low (< 0.5%), but increased with increasing spheroid diameter (> 2% in U87). In particular, levels of apoptosis rose following development of central necrosis and apoptoses were most abundant in the peri-necrotic zone. There were quantitative differences in the levels of apoptosis and necrosis between glioma cell lines. The predictable onset of necrosis in the spheroids will allow us to investigate the pathogenesis of necrosis and events in prenecrotic cells. There is a relationship between the development of necrosis and apoptosis in this model and these processes can be separately assayed. Further in vitro and genetic studies will enable us to study these events and interactions in greater detail than is possible using other cell culture and in vivo systems.

Cell death in honeybee (Apis mellifera) larvae treated with oxalic or formic acid

“…ISCDDK was found to demonstrate DNA-fragmentation after both apoptotic and necrotic cell death (Matylevitch et al, 1998;Orita et al, 2000), while the Promega apoptosis kit demonstrated apoptosis only (Sperandio et al, 2000). It was also found that apoptosis and necrosis coexist and that an apoptotic process might become necrotic (Bell et al, 2001). …”

A coherent modeling procedure to describe cell activation in biological systems

“…In other words, we are assuming that chemical-induced phenotypic differentiations preserve cell physical dimensions and uniquely determine variations in the intercellular interaction force components (as well as in the sensitivity to chemotactic cues). In particular, according to experimental measurements on tumors (refer to [20] for gliomas and to [21] for ovarian cancer spheroids), we set d r = 30 µm and d a = 60 µm. The specific values of the intercellular adhesive strengths have been instead empirically estimated, after a number of trial simulations and in accordance with the above-explained considerations, equal to Finally, we assume that cell activation is locally induced by a sufficiently high concentration of chemical growth factors (which, as seen, also behave as chemotactic cues for the already metastatic individuals).…”

Glioma Cell Death: Cell–Cell Interactions and Signalling Networks

“…Signalling Networks and Stem Cell Analysis Networks of interacting cells, including vascular endothelial and microglial signalling, provide survival and death signals affecting glioma fate [9,36,63]. The role of endothelial cell signalling in glioma pathogenesis is an important area of glioma research ( Table 2, question 4).…”