Purpose: The transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulatory element of the hypoxic response of cells. High protein levels have been linked to poor prognosis in several tumor types, and HIF-1alpha has been suggested as a potential endogenous marker of tumor hypoxia and associated radioresistance.
Methods and materials: HIF-1alpha expression following in vitro hypoxia was measured in U87 MG glioblastoma cells by Western blot and flow cytometry. Cell suspensions from U87 MG xenograft tumors grown in SCID mice were assayed by flow cytometry for HIF-1alpha and for pimonidazole as a reference hypoxia marker. After 1 h, 6 h, and 18 h of in vitro hypoxia, a constant increase in HIF-1alpha protein levels with decreasing oxygen concentrations between 20% and <0.02% was observed by both Western blot and flow cytometry, correlating with the pattern of pimonidazole labeling after in vitro hypoxia. In U87 MG xenograft tumors, flow-cytometric analysis of HIF-1alpha and pimonidazole showed a significant correlation of the two markers, but distinction of a HIF-1alpha-positive population was affected by a low dynamic range of the signal. As in published studies for HIF-1alpha and the hypoxic marker EF5, the colocalization of HIF-1alpha and pimonidazole in double-staining experiments was low.
Conclusions: While the in vitro data in U87 MG human glioblastoma cells support the use of HIF-1alpha as an endogenous hypoxia marker, comparison with the standard pimonidazole makes its application to clinical material appear questionable.