Use of novel monoclonal antibodies to determine the expression and distribution of the hypoxia regulatory factors PHD-1, PHD-2, PHD-3 and FIH in normal and neoplastic human tissues.
OBJECTIVE
The cellular response to hypoxia includes the hypoxia inducible factor (HIF)-induced transcription of genes involved in diverse processes such as glycolysis, angiogenesis and the growth of experimental tumours. Regulation of the level of hypoxia inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) is a primary determinant of HIF activity. Recent biochemical and candidate gene approach studies have led to the discovery of three HIF-regulatory prolyl hydroxylases, PHD-1, -2 and -3 and an asparaginyl hydroxylase, also known as FIH (factor inhibiting HIF). In this study, we raised and characterized monoclonal antibodies against PHD-1, PHD-2, PHD-3 and FIH.


RESULTS
Immunohistochemistry of normal tissues with these monoclonal antibodies demonstrated a wide distribution in epithelial cells, stromal cells and leucocytes, with cytoplasmic staining predominating over nuclear staining. A preliminary study of tumours showed variable staining in tumour, stromal and inflammatory cells. While all tumour types showed some positive staining with each antibody, the overall pattern suggested a slight decrease in the amount of staining seen with PHD-1, -2 and -3 and an increase in FIH staining in neoplasia compared with corresponding normal tissues.


CONCLUSIONS
These monoclonal antibodies will allow further larger scale studies to determine the significance of PHD and FIH expression in neoplasia.
Soilleux
E J
EJ
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK.
Turley
H
H
Tian
Y M
YM
Pugh
C W
CW
Gatter
K C
KC
Harris
A L
AL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D049308:Dioxygenases; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D006899:Mixed Function Oxygenases; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C439790:HIF1AN protein, human; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases