Consistency of endometrial receptivity array and histologic dating of spatially distinct endometrial samplings: a prospective, blinded study.

Objective: To compare the consistency of endometrial receptivity array (ERA) and histologic dating among 3 spatially distinct endometrial samples obtained during a cycle of exogenous estrogen and progesterone. Design: Prospective blinded study. Setting: University practice. Patients: Twelve patients undergoing a mock frozen embryo transfer cycle. Intervention: Endometrial biopsy was performed in a manner that provided a spatially organized endometrial specimen, corresponding to the fundus, middle, and lower segment. Each of these 3 sections was further divided into immediately adjacent specimens for ERA and histology. Main Outcome Measure: Consistency of the ERA and histology results among fundal, mid, and lower endometrial biopsy specimens. Results: The ERA showed variability in outcome among different patients but dated all specimens originating from the same patient identically. Histologic dating showed variability between patients as well as between different locations within the uterus. When comparing average dating results for each patient, we saw a positive correlation between histologic and ERA dating (Spearman Rho = 0.45); however, this did not reach statistical significance. The ERA results from upper, mid, and lower uterine biopsy specimens were identical for each autologous biopsy, whereas histologic dating showed variability with an average standard deviation of 0.71 days. Conclusions: The increased heterogeneity of histologic dating is likely to be attributed to the subjectivity of the test. Furthermore, we did not observe a consistent lag or advancement in histologic or ERA dating between the fundal or lower uterine biopsies. Overall, clinicians should be reassured that endometrial tissue will return consistent ERA results independent of the location within the uterus in which it was obtained.

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research.

Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological "normoxic" limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard "normoxic" oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen's sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed "consumptive oxygen depletion" (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.

Minimally invasive pancreatectomy for cancer: a critical review of the current literature.

Minimally invasive surgery (MIS) has transformed operative practices by offering patients procedures with reduced hospital stay and recovery compared to that of open operations. In spite of the advantages of a MIS approach, the application to pancreatectomy has only recently emerged. This review aims to analyze and discuss available comparative studies as they relate to resection techniques for treatment of malignant disease. A PubMed search was used to obtain original studies and meta-analyses relating to MIS pancreatectomy from 2008 to 2013. Several studies were identified that reported on the application of MIS specifically to the treatment of cancer, many of which were retrospective, single-institution studies. Notwithstanding an inherent selection bias, several studies suggest that MIS can provide equivalent R0 resection rates, number of lymph nodes harvested, and survival to that of open resection. Furthermore, parameters such as blood loss and length of stay are significantly reduced in patients treated with MIS. The current literature supports the conclusion that MIS is safe and effective as a treatment for cancer in well-selected patients in the hands of experienced surgeons. However, the published studies to date are observational in nature and therefore higher quality studies will be needed to support the application and generalizability of MIS in the treatment of pancreatic malignancies.

Prolyl-4-hydroxylase 3 (PHD3) expression is downregulated during epithelial-to-mesenchymal transition.

Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O2. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O2 and the migratory and metastatic potential of tumor cells.

Prolyl-hydroxylase 3: Evolving Roles for an Ancient Signaling Protein.

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD 1-3). These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that proline-4-hydroxylation controls much more than HIF signaling, with PHD3 emerging as an exceptionally unique and functionally diverse PHD isoform. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This review will examine the evolution of oxygen sensing by the HIF-family of PHD enzymes, with a specific focus on complex nature of PHD3 expression and function in mammalian cells.

Aberrant promoter CpG methylation is a mechanism for impaired PHD3 expression in a diverse set of malignant cells.

BACKGROUND: The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. METHODOLOGY/PRINCIPAL FINDINGS: Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2'-deoxycytidine significantly increased the expression of PHD3. CONCLUSIONS/SIGNIFICANCE: We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Cbl controls EGFR fate by regulating early endosome fusion.

Amino acid residues 1 to 434 of the E3 ubiquitin ligase Cbl control signaling of the epidermal growth factor receptor (EGFR) by enhancing its ubiquitination, down-regulation, and lysosomal degradation. This region of Cbl comprises a tyrosine kinase-binding domain, a linker region, a really interesting new gene finger (RF), and a subset of the residues of the RF tail. In experiments with full-length alanine substitution mutants, we demonstrated that the RF tail of Cbl regulated biochemically distinct checkpoints in the endocytosis of EGFR. The Cbl- and ubiquitin-dependent degradation of the regulator of internalization hSprouty2 was compromised by the Val(431)--> Ala mutation, whereas the Cbl- and EGFR-dependent dephosphorylation or degradation of the endosomal trafficking regulator Hrs was compromised by the Phe(434)--> Ala mutation. Deregulated phosphorylation of Hrs correlated with inhibition of the fusion of early endosomes and of the degradation of EGFR. This study provides the first evidence that Cbl regulates receptor fate by controlling the fusion of sorting endosomes. We postulate that it does so by modulating the abundance of tyrosine-phosphorylated Hrs.

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate.

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.