Validated analysis of HIF-1 alpha expression in cancer cells using a controlled and comparative immunoassay

Abstract

The immunoreactivity of hypoxia inducible factor 1 alpha (HIF-1 alpha) has been considered a reliable indicator of the HIF-1 pathway activation in tissue hypoxia. However, HIF-1 alpha immunoreactivity has been evaluated with different antibodies and heterogeneous protocols. The need to interpret contradictory findings requires, among other things, a comparison of the antibodies. This could be accomplished by using identical, well characterized antigenic targets and by decreasing the influence of other variables. We applied most of the commercially available antibodies, and an antibody developed in our laboratories, to the human cervical cancer HeLa cell line and tissue sections from a renal cell carcinoma systematically, and to other tumors selectively. The expression of HIF-1 alpha in HeLa cells was induced by the hypoxia-mimetic DFO. Non-induced HeLa cells were used as 'genuine' negative controls in addition to routine ones. HeLa cells (both induced and not induced) were also examined by immunofluorescence and Western blotting. We found that the antibodies showed immunostaining patterns with remarkable qualitative and quantitative differences, an observation not emphasized in previous literature. Certain antibodies require careful application to avoid specificity issues, and others to avoid low sensitivity problems. Pairing certain antibodies can optimize evaluation of HIF-1 alpha expression. Most previous immunohistochemical studies of HIF-1 alpha have attempted to map hypoxic neoplastic tissues or to demonstrate hypoxia in studies of neoangiogenesis, rather than 'measuring' HIF-1 alpha expression or activation, because this requires a validated immunoassay. Our study thus allows for the development of a controlled and comparative HIF-1 alpha immunoassay, which could be valuable if HIF-1 alpha becomes a therapeutic target.