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Enzyme-Linked Immunosorbent Assay for Pharmacological Studies Targeting Hypoxia-Inducible Factor 1

Oncopharmacology Unit of the Centre Antoine Lacassagne,¹ Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543,² Medical School, Nice, France,³ University of Thessaly, Larissa, Greece⁴

Received 2 December 2004/ Returned for modification 3 January 2005/ Accepted 23 February 2005

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of a wide range of genes related to oxygen delivery and metabolic adaptation under hypoxic (low-oxygen) conditions. HIF-1 is, in fact, a heterodimer of two subunits, HIF-1 and HIF-1ß. The only analytical methods available for measuring HIF-1 levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1-expressing cells, but has the intrinsic limitation, as for Western blotting, of being nonquantitative. We developed and validated an enzyme-linked immunosorbent assay (ELISA) approach to measure HIF-1 levels in cultured tumor cell lines in vitro. HIF-1 was expressed in thirteen tumor cell lines grown under hypoxic conditions; however, the levels differed strongly between cell lines. These data point to intrinsic differences between cell lines for the induction of HIF-1 under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1 protein levels and should be useful in preclinical pharmacological studies targeting HIF-1.