complex that activates several target genes that may include those regulating cell cycle [21]. In mammals three prolyl hydroxylase isoforms termed PHD1, PHD2 and PHD3 (also called EGLN2, EGLN, EGLN3, respectively) have been characterized [22]. All of these isoforms have been shown to hydroxylate HIF-a in vitro and to have similar co-factor requirements. The most ubiquitous factor controlling the activity of these enzymes is oxygen concentration but they also require 2-oxoglutarate and iron as cofactors. Despite the similarities in their requirements, several differences in their function and characteristics exist [23]. Unlike the other two isoforms, PHD3 has been shown to cause apoptosis in neuronal cells as well as in cancer cells when expressed under normoxia at high levels [24,25,26,27]. PHD3 is also the isoform that shows most robust induction under hypoxia [28,29,30,31]. The elevated expression is likely to at least partially compensate for the reduced activity under hypoxia. In fact, PHD3 is known to retain much of its enzymatic activity at least under moderate hypoxia [32]. Here we have studied the role of PHD3 depletion under hypoxia in cancer cell lines. We show that PHD3 is upregulated in squamous cell carcinoma tumours. We show that PHD3 depletion under hypoxia leads to cell cycle arrest that occurs at the G1/S boundary. In line with this, the depletion of PHD3 reduces the amount of hyperphosphorylated retinoblastoma protein as well as the amount of cyclin D1. Moreover, PHD3 inhibition upregulates the expression of cyclin-dependent kinase inhibitor p27. Results Head and neck squamous cell carcinoma tumors overexpress PHD3 PHD3 has been reported to be activated by hypoxia in several cell types including diverse carcinoma cell lines [30,33,34]. In order to study hypoxic PHD3 expression in human head and neck squamous cell carcinoma (HNSCC) we used five different primary cell lines (UT-SCC) established from HNSCC patients at Turku university hospital [35]. The cells were exposed to normoxia or hypoxia for 6 and 48 hours. PHD3 was strongly induced at mRNA level in all cell lines tested with maximal induction seen after 48-hour hypoxia (Fig. 1A, Fig. S1A,B). In line with this, PHD3 was induced at protein level in three cell line tested (Fig. 1B). To study the PHD3 mRNA expression in human HNSCC, we analyzed tumor samples from 28 HNSCC patients and from anatomically matching samples in 11 healthy controls. The Q-RT- PCR analysis showed a 5-fold overexpression of PHD3 (p- value,0.0001) in the tumor samples as compared to control (Fig. 1C and D). To investigate whether the PHD3 expression associates with hypoxia, we analyzed the correlation between PHD3 and a widely used hypoxic marker glucose transporter Glut-1. Strong correlation between PHD3 and Glut-1 mRNA expression was detected (Spearman correlation 0.8, p-val- ue,0.0001) implying that PHD3 transcription is activated by hypoxia in HNSCC (Fig. 1D). To further study the clinical occurrence of PHD3 overexpres- sion in HNSCC we analyzed studies deposited in the Oncomine database [36]. From the five studies comparing PHD3 expression in HNSCC and normal tissue four showed overexpression of PHD3. In three studies the HNSCC overexpression was statistically significant (p-value,0,002 to 0,005) (Fig. 1E). More- over, one study demonstrated increased PHD3 mRNA level in different anatomical locations including tongue, mouth floor and tonsilla (Fig. 1F) [37]. PHD3 overexpression was not restricted to HNSCC (Table S1). Hypoxic Cell Cycle Arrest by PHD3 Inhibition PHD3 inhibition reduces hypoxic SCC growth As PHD3 was overexpressed in HNSCC we asked whether PHD3 provided growth advantage for the carcinoma cells. We used previously validated double stranded PHD3 siRNA (siPHD3) to knock down the expression of PHD3. As controls non-target (siScr) and PHD2-targeted siRNA (siPHD2) were used [38]. The siRNAs were transfected to UT-SCC2 cells followed by exposure to normoxia (21% O2) or hypoxia (1% O2) for 48 hours. The PHD3 siRNA was effective and specific as demonstrated by reduced hypoxic PHD3 expression in SCC2 cells (Fig. 2A, Fig. S1C). Noticeably, we detected that the PHD3 knock-down caused a marked reduction in cell number as compared to controls when grown under hypoxia (Fig. 2B). Quantification of the cell number in hypoxia demonstrated almost 70% reduction in surviving cells after 48-hour hypoxia with siPHD3 as compared to either control or siPHD2 transfected cells (Fig. 2B and C). In line with the fact that little PHD3 expression was seen under normoxia, siPHD3 had no marked effect in normoxia. In order to verify the specific effect of PHD3 siRNA we used a point-mutated double stranded siRNA (siPHD3mut) as a control. The mutant siRNA did not affect the PHD3 expression. Accordingly, siPHD3mut did not have marked effect on the cell number in hypoxia (Fig. 2E and F) further validating the specific effect of PHD3 knock-down in reducing the number of surviving cells under hypoxia. We further asked whether the reduction in cell number would be reflected as reduced proliferation rate in siPHD3 cells. PHD3 or control siRNAs were transfected to SCC2 cells followed by BrdU addition and exposure to normoxia or hypoxia for 48 hours. Indeed, BrdU incorporation was reduced in siPHD3 cells as compared to control indicating reduced cell growth under hypoxia and PHD3 inhibition (Fig. 2D). Finally, using a rescue experiment we studied whether the hydroxylase activity of PHD3 was required for supporting hypoxic cell survival. HeLa cells exposed to siPHD3 were transfected with PHD3-EGFP, PHD3(R206K)-EGFP (a hydroxylase-inactive mu- tant) [26] or EGFP as control followed by two day hypoxic exposure (Fig. 3). The transfection efficiency was controlled with confocal microscopy for EGFP expression (Fig. S2). Cells transfected with PHD3-EGFP demonstrated clearly increased survival as compared to control. Interestingly, PHD3(R206K)- EGFP displayed comparable enhancement in cell number suggesting that hydroxylase activity is not required for PHD3 supported cell survival. Inhibition of PHD3 does not activate significant apoptosis in SCC cells As hypoxia is a known inducer of apoptosis we asked whether the reduction in cell amount was due to enhancement of hypoxia- induced apoptosis [39]. SCC2 cells were transfected with control and PHD3 siRNA followed by hypoxia for 6 to 30 hours. The cells were stained for cleaved caspase-3 which is activated during apoptosis and widely used as an apoptotic marker. Activation of caspase-3 was detected in a subpopulation of siPHD3 transfected cells as compared to control cells after 24-hour hypoxia (Fig. 4A) while most cells remained caspase-3 negative. Quantification demonstrated increased activation of caspase-3 by siPHD3 at early hypoxia (24-h) that was lost at later time points (Fig. 4B). Caspase-3 activation in a subpopulation of siPHD3 cells could be suggestive for increased apoptosis. However, the increased caspase-3 cleavage does not necessarily lead to terminal apoptosis [40]. Therefore, we next used flow cytometry to further analyze the occurrence of apoptosis by studying the cell population in sub- PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27112