Hypoxia-inducible factor (HIF) is a transcriptional regulator that plays a key role in many aspects of oxygen homeostasis. The heterodimeric HIF complex is regulated by proteolysis of its ␣-subunits, following oxygendependent hydroxylation of specific prolyl residues. Although three HIF prolyl hydroxylases, PHD1, PHD2, and PHD3, have been identified that have the potential to catalyze this reaction, the contribution of each isoform to the physiological regulation of HIF remains uncertain. Here we show using suppression by small interference RNA that each of the three PHD isoforms contributes in a non-redundant manner to the regulation of both HIF-1␣ and HIF-2␣ subunits and that the contribution of each PHD under particular culture conditions is strongly dependent on the abundance of the enzyme. Thus in different cell types, isoform-specific patterns of PHD induction by hypoxia and estrogen alter both the relative abundance of the PHDs and their relative contribution to the regulation of HIF. In addition, the PHDs manifest specificity for different prolyl hydroxylation sites within each HIF-␣ subunit, and a degree of selectively between HIF-1␣ and HIF-2␣ isoforms, indicating that differential PHD inhibition has the potential to selectively alter the characteristics of HIF activation.

The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed.

Summary Hypoxia inducible factors HIF1α and HIF2α are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1α and 2α protein levels, as a consequence of a redox-sensitive stabilization. The HIFαs enter the nucleus, heterodimerize with the HIF1β protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1α and HIF2α expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2α expression with HIF1α expression showed a significant association (P < 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P < 0.004), PD-ECGF (P < 0.003) and bFGF (P < 0.04) was noted. HIF1α correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2α was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2α protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2α expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1α expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2α expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1α and HIF2α overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting 881-890 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.2018, available online at http://www.idealibrary.com on http://www.bjcancer.comIn the present study we examined the expression patterns of HIF1α and of HIF2α in normal lung and in a series of non-smallcell lung carcinomas. The expression of HIFs was analysed in comparison with the microvessel density, with the expression of multiple angiogenic factors and receptors as well as with the expression of onco-proteins, previously shown to have a role in lung cancer. The prognostic role of HIF expression was also studied. MATERIALS AND METHODSWe examined 108 tumour samples from patients with early operable (T1,2-N0,1-M0 staged) non-small-cell lung cancer (72 squamous and 36 adenocarcinomas). 12 samples from normal lung obtained during thoracic surgery for reasons other than lung cancer were also examined. Histological diagnosis, grading and Nstage w...