Controlling fluid-induced seismicity during a 6.1-km-deep geothermal stimulation in Finland.

We show that near-real-time seismic monitoring of fluid injection allowed control of induced earthquakes during the stimulation of a 6.1-km-deep geothermal well near Helsinki, Finland. A total of 18,160 m3 of fresh water was pumped into crystalline rocks over 49 days in June to July 2018. Seismic monitoring was performed with a 24-station borehole seismometer network. Using near-real-time information on induced-earthquake rates, locations, magnitudes, and evolution of seismic and hydraulic energy, pumping was either stopped or varied-in the latter case, between well-head pressures of 60 and 90 MPa and flow rates of 400 and 800 liters/min. This procedure avoided the nucleation of a project-stopping magnitude M W 2.0 induced earthquake, a limit set by local authorities. Our results suggest a possible physics-based approach to controlling stimulation-induced seismicity in geothermal projects.

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay.

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow® DNA Damage Kit-p53, γH2AX, Phospho-Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals' predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146-161, 2017. © 2017 Wiley Periodicals, Inc.

Interaction with ErbB4 promotes hypoxia-inducible factor-1α signaling.

The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.

Hypoxic conversion of SMAD7 function from an inhibitor into a promoter of cell invasion.

Smad7 is an inhibitor of the transforming growth factor-beta-activated signaling pathway. Under well-oxygenated conditions, Smad7 is a potent inhibitor of carcinoma cell invasion. Paradoxically, however, the expression of Smad7 is upregulated across several cancers and may promote cancer progression. Hypoxia, which is frequently met in solid tumors, is an enhancer of carcinoma cell invasion and cancer progression. Here, we report that hypoxia activates the expression of Smad7 in a hypoxia-inducible factor- and von Hippel-Lindau protein-dependent manner. As expected, in normoxia, the forced expression of Smad7 inhibited carcinoma cell invasion. In contrast with the normoxic condition, the inhibitory effect of Smad7 was lost under hypoxia. The block in carcinoma cell invasion by forced expression of Smad7 was released by hypoxia in two invasive carcinoma cell lines. Moreover, the noninvasive HaCaT keratinocytes become invasive upon simultaneous hypoxia and transforming growth factor-beta stimulus. The hypoxia-activated invasion was attenuated by inhibiting Smad7 expression by short interfering RNA. Finally, the increased Smad7 expression in human carcinomas correlated with hypoxic gene expression. The data provide evidence that hypoxia could convert Smad7 function from an invasion inhibitor into an activator of invasion. Furthermore, they might shed light as to why increased Smad7 expression is detected in cancers.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth.

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1alpha and -2alpha). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1alpha or -2alpha expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.

Hypoxia-activated Smad3-specific dephosphorylation by PP2A.

The transforming growth factor-beta (TGF-beta) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-beta promotes malignancy. TGF-beta activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-beta-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-beta receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-beta-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-beta on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses.

Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness.

PURPOSE: Hypoxia in tumors is associated with poor prognosis and resistance to treatment. The outcome of hypoxia is largely regulated by the hypoxia-inducible factors (HIF-1alpha and HIF-2alpha). HIFs in turn are negatively regulated by a family of prolyl hydroxylases (PHD1-3). The PHD2 isoform is the main down-regulator of HIFs in normoxia and mild hypoxia. This study was designed to analyze the correlation of the expression and subcellular localization of PHD2 with the pathologic features of human carcinomas and HIF-1alpha expression. EXPERIMENTAL DESIGN: The expression of PHD2 was studied from paraffin-embedded normal tissue (n = 21) and head and neck squamous cell carcinoma (HNSCC; n = 44) by immunohistochemistry. Further studies included PHD2 mRNA detection and HIF-1alpha immunohistochemistry from HNSCC specimens as well as PHD2 immunocytochemistry from HNSCC-derived cell lines. RESULTS: In noncancerous tissue, PHD2 is robustly expressed by endothelial cells. In epithelium, the basal proliferating layer also shows strong expression, whereas the more differentiated epithelium shows little or no PHD2 expression. In HNSCC, PHD2 shows strongly elevated expression both at the mRNA and protein level. Moreover, PHD2 expression increases in less differentiated phenotypes and partially relocalizes from the cytoplasm into the nucleus. Endogenously high nuclear PHD2 is seen in a subset of HNSCC-derived cell lines. Finally, although most of the tumor regions with high PHD2 expression show down-regulated HIF-1alpha, regions with simultaneous HIF-1alpha and PHD2 expression could be detected. CONCLUSIONS: Our results show that increased levels and nuclear translocation of the cellular oxygen sensor, PHD2, are associated with less differentiated and strongly proliferating tumors. Furthermore, they imply that even the elevated PHD2 levels are not sufficient to down-regulate HIF-1alpha in some tumors.

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases.

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.