Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stablv transfected with a vector constructed with multiple copies of ibe HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1 alpha (hypoxia-inducible factor-1 alpha) is stabilized, the binding of HIEF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfecled monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1 alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (< 100 mu m) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy
Collini M;Chirico G;
2007
Abstract
Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stablv transfected with a vector constructed with multiple copies of ibe HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1 alpha (hypoxia-inducible factor-1 alpha) is stabilized, the binding of HIEF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfecled monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1 alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (< 100 mu m) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
