Browsing by Author "Machulik, A."

The objective of this study was to examine the mode of cell death and the hypoxia inducible factor-1 (HIF-1) expression of human head and neck squamous cell carcinoma (HNSCC) exposed to hypoxia in vitro. Apoptosis and necrosis rates were examined using flow cytometry. The findings suggest that HNSCC cells show a considerable heterogeneity in cell size and in response to hypoxia. A small-cell population showed a high spontaneous apoptosis and necrosis rate which was in-sensitive to hypoxia. A large-cell population responded to hypoxia by increase of apoptosis rate in parallel to recruitment of HIF-1. Hypoxia led to increased HIF-1 alpha protein levels in nuclear extract using ELISA-binding activity. In all cells, accumulation of HIF-1 in the nuclei during hypoxia and a rapid degradation of HIF-1 in the post-hypoxic period were observed immunocytochemically. The HIF-1 alpha mRNA level showed an expression of 10-40 pg/mu g total RNA and remained unchanged in one cell line, while slightly decreasing in the other. Remarkably, no increased luciferase activity response was found on the reporter gene level using pGL3 reporter gene with three erythropoietin hypoxia responsive elements, either by hypoxia or by application of lactacystin, desferrioxamine or CoCl2. These findings suggest that, in HNSCC cells, hypoxia induces HIF-1 alpha to stabilize and accumulate in the cell nuclei but have a cell-specific transcriptional complex.

Hypoxia/ischemia is a major pathogenetic factor in the development of hearing loss. An important transcription factor involved in the signaling and adaptation to hypoxia/ischemia is the hypoxia-inducible factor-1 (HIF-1). To study HIF-1 expression we used an in vitro hypoxia model of explant and dissociated cultures of the stria vascularis, the organ of Corti with limbus and the modiolus from the cochlea of 3-5-day-old Wistar rats. Hypoxia differentially increased HIF-1 activity as measured by a reporter gene. Twenty-four hour hypoxia increased HIF-1 activity 14.1 +/- 3.5-fold in the modiolus, 9.4 +/- 3.0-fold in the organ of Corti with limbus, and 6.4 +/- 1.5-fold in the stria vascularis. The HIF-1alpha mRNA level was measured by quantitative reverse transcription polymerase chain reaction and showed a lower expression in the modiolus (1.3 +/- 0.2 pg/mug RNA) than in both the organ of Corti with limbus and the stria vascularis (2.7-3.2 +/- 1.3, P < 0.01). Hypoxia had no effect on the HIF-1 alpha mRNA levels. The region-specific regulation of HIF-1 expression on the transcriptional and posttranslational levels may expand the possibilities for adaptation of the cochlea to hypoxia. (C) 2003 Elsevier B.V. All rights reserved.