The hypoxic cell cycle arrest has been reported to be p53 and p21(Cip1/CDKN1A) independent. In line with this, the knock- down of PHD3 did not lead to upregulation of p21 under hypoxia (Fig. 6C). We further studied the expression of two other cyclin- dependent kinase inhibitors, p16(INK4a) and p27(Kip1/ CDKN1B) under hypoxic PHD3 inhibition. Similarly to p21, p16 levels remained unchanged under siPHD3 exposure. In striking contrast, the inhibition of PHD3 led to clear upregulation of p27, which is known to arrest cell cycle at G1-phase. This was evident both in normoxia and hypoxia (Fig. 6D). Interestingly, the p27 level was reduced by hypoxia at 24 hour time point but increased at 48 hours as compared to normoxia (Fig. 6E). Taken together the data indicate that PHD3 is required for cell cycle to progress from G1 to S-phase under hypoxia and that p27 may mediate the effect. Discussion PHD3 is upregulated by hypoxia and the elevated levels remain in prolonged hypoxia enabling long-term regulation of hypoxic cell fate [28,29,30]. Some PHD3 hydroxylation activity is preserved at least under moderate hypoxia (1% O2) where it has been suggested to desensitize and prevent excessive HIF-alpha activity [32,34]. Here we have studied the hypoxic function of PHD3 in carcinoma cells. First we demonstrated that PHD3 is elevated in human cancers of the head and neck region. The increase of PHD3 during carcinogenesis suggested that it may facilitate tumor progression. Supporting this, the inhibition of PHD3 expression in cells derived from SCC led to reduced cell survival under hypoxia. Importantly, we demonstrated that the inhibition of PHD3 causes cell cycle arrest of carcinoma cells in hypoxia. The arrest was pinpointed to the G1/S boundary and was accompanied by a block in Rb hyperphosphorylation as well as reduced expression of the cyclins D1 and B1. Together the data suggests that hypoxic upregulation of PHD3 in HNSCC is necessary for cell survival in hypoxia by allowing cell cycle to proceed from G1 to S-phase. Hypoxia is a well-characterized environmental factor that halts cell cycle and cell proliferation [5,6]. Since energy availability from oxidative phosphorylation is reduced in hypoxia, it is likely that cell cycle arrest is to prevent excessive cell death under reduced oxygen [42]. The hypoxic cell cycle arrest occurs at G1 preventing cells from proceeding to S-phase where they would be most susceptible to cell death. In keeping with this, hypoxia is known to result in Rb hypophosphorylation and reduced cyclin D1 expression [43,44]. However, during the growth of carcinomas a subpopulation of cells need to escape the cell cycle regulation in order to maintain cell growth also under low oxygen tension. The molecular mechanisms that underlie the hypoxia-induced cell cycle arrest or the mechanisms that can release the arrest in carcinoma cells are not fully understood. Our work directly links the primary oxygen sensors to the hypoxic cell cycle arrest as well as Rb hypophosphorylation and reduced cyclin D1 expression. Out of the three cyclin-dependent kinase inhibitors studied, p27 was the only one the expression of which displayed changes upon PHD3 inhibition. p27 has several functions in regulating cell cycle [45]. Expression of p27 at G1 phase inhibits cyclin E/A – CDK2 blocking the transcription of genes that are required for the G1 to S transition. In particular under adverse conditions p27 is also known to inhibit the activation of cyclin D – CDK4 complex and cause a block in S-phase transition. This is well in line with reduced cyclin D1 expression and G1 arrest seen by PHD3 inhibition. Therefore, it is feasible that p27 mediates the hypoxic effects of PHD3 on cell cycle. Whether PHD3 affects the Hypoxic Cell Cycle Arrest by PHD3 Inhibition transcription or protein degradation of p27 remains to be investigated. Moreover, it is not clear whether the hypoxic block requires p27 cell cycle inhibitor activity [10,11]. This may be context-dependent and also vary depending on the length of hypoxia. Our data indicate that p27 is first suppressed under hypoxia but reactivated upon longer exposure. Under hypoxia PHD3 expression is activated by HIF [28,30,31]. In turn, HIF-a is the main known enzymatic target of PHD3. Whether the downstream effects of PHD3 on cell survival are mediated by HIF remain elusive. Given the large body of evidence that HIF functions as a protective factor for hypoxic cells it is difficult to see how the knockdown of an HIF inhibitor PHD3 could cause increased cell death. However, one needs to keep in mind that PHD3 is likely to fine tune the HIF-a level in hypoxia where excessive HIF accumulation might shift the cell fate decision towards cell death. Experimentally it will be challenging to study the possible HIF-mediated effects on cell cycle under PHD3 inhibition, as the effect of PHD3 in hypoxia may be to retain HIF level within a narrow range that can support cell cycle progression. Reaching such level in a large cell population using simultaneous HIF and PHD3 inhibition will be challenging. Supporting the idea that the functions are not mediated by HIF, the rescue experiments suggested that PHD3 hydroxylase activity is not required for cell survival supporting function. This is not completely surprising since a number of hydroxylase activity independent functions have been ascribed for the PHD family members [23,46]. Interestingly also, the experiments suggested that even when only a fraction of the cells express PHD3, this is reflected in a survival benefit for a larger cell population, suggesting a bystander effect. Several studies have indicated that when overexpressed in normoxia, PHD3 can activate apoptotic cell death in neuronal cells [24,25,27,47]. Such cell death induced by PHD3 has also been reported in some carcinoma cells [26,48]. However, similarly to our work, more recent reports have also indicated that under hypoxia PHD3 may in fact protect cancer cells from cell death induced either by hypoxia or other apoptotic factors [49]. This implies that PHD3 has a dualistic function in regulating cell fate decisions that depend at least on cell-type and oxygen availability but probably also on other interacting factors. For example, a recent study suggests that inhibition of PHD3 in hypoxic carcinoma cells lacking PRP19 may reduce apoptosis [50]. In the present study we detected activation of caspase-3 in a subpopulation of siPHD3 transfected cells under hypoxia. Initially this could have been interpreted as an increase in the apoptosis rate. However, it has been well documented that caspase-3 activation does not necessarily lead to terminal apoptosis [40]. In keeping with this, we were unable to detect any significant increase of terminal apoptosis in siPHD3-exposed cells by flow cytometry. It needs to be underlined that these analyses are internally controlled by reduced S-phase entry. The data strongly argues against apoptosis as the main cause of reduced cell survival by PHD3 inhibition in hypoxic squamous carcinoma cells. PHD1 has recently also been shown to regulate cell cycle progression and cyclin D1 expression [51]. This was shown in estrogen-dependent breast carcinomas where loss of PHD1 led to decrease in cyclin D level and subsequently impaired cell proliferation. In the current work we focused on the hypoxia- induced PHD isoforms PHD2 and -3. In glioblastoma cells the inhibition of either PHD2 or PHD3 was protective against hypoxia and staurosporine-induced cell death [49]. In SCC cells however, PHD2 inhibition demonstrated far less cell death or effects on the cell cycle as compared to PHD3. Together the data suggests cell-type specific effects of the PHD isoforms on cell PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27112