Enzyme-linked immunosorbent assay for pharmacological studies targeting hypoxia-inducible factor 1alpha.
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-1alpha and HIF-1beta. The only analytical methods available for measuring HIF-1alpha levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1alpha-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-1alpha levels in cultured tumor cell lines in vitro. HIF-1alpha 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-1alpha under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1alpha protein levels and should be useful in preclinical pharmacological studies targeting HIF-1alpha.
Formento
J L
JL
Oncopharmacology Unit Centre, Antoine Lacassagne 33, Avenue de Valombrose, 06189 Nice Cedex 2, France. jean-louis.formento@nice.fnclcc.fr
Berra
E
E
Ferrua
B
B
Magné
N
N
Simos
G
G
Brahimi-Horn
C
C
Pouysségur
J
J
Milano
G
G
D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009363:Neoplasm Proteins / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000032:analysis*
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors