Radial Flow Perfusion Enables Real-Time Profiling of Cellular Metabolism at Low Oxygen Levels with Hyperpolarized 13C NMR Spectroscopy.

In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.

Predictive Value of Abnormal Findings on Covered Transjugular Intrahepatic Portosystemic Shunt Baseline Doppler Sonography.

Doppler ultrasound (DUS) is frequently performed as a screening and diagnostic modality to evaluate the transjugular intrahepatic portosystemic shunt (TIPS) for short- and intermediate-term complications of the procedure. However, the clinical significance of initial frequently observed abnormalities in flow velocities, gradient, and direction in patients with covered TIPS is less studied. A retrospective study was performed between January 2005 and December 2014, and all patients undergoing covered TIPS procedure for the management of portal hypertension were included. Abnormal DUS findings were defined as intrashunt peak systolic velocities (PSVs) less than 90 or greater than 190 cm/s, intrashunt gradient greater than 50 cm/s, and abnormal flow direction in the main, right, and/or left portal veins. A total of 283 patients with adequate clinical follow-up and baseline TIPS DUS were included in the study. Median follow-up was 18.2 months. During the follow-up period, portal hypertension symptoms recurred in 83 patients who underwent TIPS angiography and/or revision. Of the 83, 57 had an elevated portosystemic gradient (>12 mm Hg) requiring angioplasty/stenting. With regard to baseline ultrasound, low PSVs were present in 88 patients (31.1%), high PSVs in 44 patients (15.5%), and elevated gradient in 98 patients (34.6%). The rate of developing TIPS stenosis in the future was not higher in patients with abnormal DUS parameters. For example, 26 (19.7%) of 132 patients with abnormal TIPS velocities developed stenosis compared with 31 (20.5%) of 151 patients with normal velocities (P = 0.9). Based on these results, abnormal DUS findings observed on baseline TIPS ultrasound examination have low predictive value for future covered TIPS dysfunction and failure.

Near-Infrared Fluorescence Imaging of Matrix Metalloproteinase 2 Activity as a Biomarker of Vascular Remodeling in Hemodialysis Access.

PURPOSE: To establish the capability of near-infrared fluorescence (NIRF) imaging for the detection of matrix metalloproteinase 2 (MMP-2) activity as a biomarker of vascular remodeling (VR) in arteriovenous fistulae (AVFs) in vivo. MATERIALS AND METHODS: AVFs were created in the right groins of Wistar rats (n = 10), and sham procedures were performed in the contralateral groins. Fistulography via a left common carotid artery approach confirmed stenosis (> 50%) in a subset of animals (n = 5) 4 weeks after AVF creation. After administration of MMP-2-activated NIRF probe, near-infrared imaging was performed in vivo and ex vivo of both the AVF and the sham-treated vessels to measure radiant efficiency of MMP-2-activated NIRF signal over background. Histologic analyses of AVF and sham-treated vessels were performed to measure VR defined as inward growth of the vessel caused by intimal thickening. RESULTS: AVFs demonstrated a significantly higher percentage increase in radiant efficiency over background compared with sham vessels (45.5 ± 56% vs 16.1 ± 17.8%; P = .008). VR in AVFs was associated with increased thickness of neointima staining positively for MMP-2 (161.8 ± 45.5 µm vs 73.2 ± 36.7 µm; P = .01). A significant correlation was observed between MMP-2 activity as measured by relative increase in radiant efficiency for AVFs and thickness of neointima staining positively for MMP-2 (P = .039). CONCLUSIONS: NIRF imaging can detect increased MMP activity in remodeled AVFs compared with contralateral sham vessels. MMP-2-activated NIRF signal correlates with the severity of intimal thickening. These findings suggest NIRF imaging of MMP-2 may be used as a biomarker of the vascular remodeling underlying stenosis.

Differences between genders in colorectal morphology on CT colonography using a quantitative approach: a pilot study.

PURPOSE: To explore quantitative differences between genders in morphologic colonic metrics and determine metric reproducibility. METHODS: Quantitative colonic metrics from 20 male and 20 female CTC datasets were evaluated twice by two readers; all exams were performed after incomplete optical colonoscopy. Intra-/inter-reader reliability was measured with intraclass correlation coefficient (ICC) and concordance correlation coefficient (CCC). RESULTS: Women had overall decreased colonic volume, increased tortuosity and compactness and lower sigmoid apex height on CTC compared to men (p<0.0001,all). Quantitative measurements in colonic metrics were highly reproducible (ICC=0.9989 and 0.9970; CCC=0.9945). CONCLUSION: Quantitative morphologic differences between genders can be reproducibility measured.

Passive expansion of sub-maximally dilated transjugular intrahepatic portosystemic shunts and assessment of clinical outcomes.

AIM: To assess for passive expansion of sub-maximally dilated transjugular intrahepatic portosystemic shunts (TIPS) and compare outcomes with maximally dilated TIPS. METHODS: Polytetrafluoroethylene covered TIPS (Viatorr) from July 2002 to December 2013 were retrospectively reviewed at two hospitals in a single institution. Two hundred and thirty patients had TIPS maximally dilated to 10 mm (mTIPS), while 43 patients who were at increased risk for hepatic encephalopathy (HE), based on clinical evaluation or low pre-TIPS portosystemic gradient (PSG), had 10 mm TIPS sub-maximally dilated to 8 mm (smTIPS). Group characteristics (age, gender, Model for End-Stage Liver Disease score, post-TIPS PSG and clinical outcomes were compared between groups, including clinical success (ascites or varices), primary patency, primary assisted patency, and severe post-TIPS HE. A subset of fourteen patients with smTIPS underwent follow-up computed tomography imaging after TIPS creation, and were grouped based on time of imaging (< 6 mo and > 6 mo). Change in diameter and cross-sectional area were measured with 3D imaging software to evaluate for passive expansion. RESULTS: Patient characteristics were similar between the smTIPS and mTIPS groups, except for pre-TIPS portosystemic gradient, which was lower in the smTIPS group (19.4 mmHg ± 6.8 vs 22.4 mmHg ± 7.1, P = 0.01). Primary patency and primary assisted patency between smTIPS and mTIPS was not significantly different (P = 0.64 and 0.55, respectively). Four of the 55 patients (7%) with smTIPS required TIPS reduction for severe refractory HE, while this occurred in 6 of the 218 patients (3%) with mTIPS (P = 0.12). For the 14 patients with follow-up computed tomography (CT) imaging, the median imaging follow-up was 373 d. There was an increase in median TIPS diameter, median percent diameter change, median area, and median percent area change in patients with CT follow-up greater than 6 mo after TIPS placement compared to follow-up within 6 mo (8.45 mm, 5.58%, 56.04 mm2, and 11.48%, respectively, P = 0.01). CONCLUSION: Passive expansion of smTIPS does occur but clinical outcomes of smTIPS and mTIPS were similar. Sub-maximal dilation can prevent complications related to over-shunting in select patients.

Ischemia Induces Quiescence and Autophagy Dependence in Hepatocellular Carcinoma.

Purpose To characterize hepatocellular carcinoma (HCC) cells surviving ischemia with respect to cell cycle kinetics, chemosensitivity, and molecular dependencies that may be exploited to potentiate treatment with transarterial embolization (TAE). Materials and Methods Animal studies were performed according to institutionally approved protocols. The growth kinetics of HCC cells were studied in standard and ischemic conditions. Viability and cell cycle kinetics were measured by using flow cytometry. Cytotoxicity profiling was performed by using a colorimetric cell proliferation assay. Analyses of the Cancer Genome Atlas HCC RNA-sequencing data were performed by using Ingenuity Pathway Analysis software. Activation of molecular mediators of autophagy was measured with Western blot analysis and fluorescence microscopy. In vivo TAE was performed in a rat model of HCC with (n = 5) and without (n = 5) the autophagy inhibitor Lys05. Statistical analyses were performed by using GraphPad software. Results HCC cells survived ischemia with an up to 43% increase in the fraction of quiescent cells as compared with cells grown in standard conditions (P < .004). Neither doxorubicin nor mitomycin C potentiated the cytotoxic effects of ischemia. Gene-set analysis revealed an increase in mRNA expression of the mediators of autophagy (eg, CDKN2A, PPP2R2C, and TRAF2) in HCC as compared with normal liver. Cells surviving ischemia were autophagy dependent. Combination therapy coupling autophagy inhibition and TAE in a rat model of HCC resulted in a 21% increase in tumor necrosis compared with TAE alone (P = .044). Conclusion Ischemia induces quiescence in surviving HCC cells, resulting in a dependence on autophagy, providing a potential therapeutic target for combination therapy with TAE. © RSNA, 2017 Online supplemental material is available for this article.

Detailed quantitative assessment of colonic morphology at CT colonography using novel software: a feasibility and reproducibility study.

The aim of this study was to evaluate feasibility and reproducibility of quantitative assessment of colonic morphology on CT colonography (CTC). CTC datasets from 60 patients with optimal colonic distension were assessed using prototype software. Metrics potentially associated with poor endoscopic performance were calculated for the total colon and each segment including: length, volume, tortuosity (number of high curvature points <90°), and compactness (volume of box containing centerline divided by centerline length). Sigmoid apex height relative to the lumbosacral junction was also measured. Datasets were quantified twice each, and intra-reader reliability was evaluated using concordance correlation coefficient and Bland-Altman plot. Complete quantitative datasets including the five proposed metrics were generated from 58 of 60 (97 %) CTC examinations. The sigmoid and transverse segments were the longest (55.9 and 51.4 cm), had the largest volumes (0.410 and 0.609 L), and were the most tortuous (3.39 and 2.75 high curvature points) and least compact (3347 and 3595 mm2), noting high inter-patient variability for all metrics. Mean height of the sigmoid apex was 6.7 cm, also with high inter-patient variability (SD 6.8 cm). Intra-reader reliability was high for total and segmental lengths and sigmoid apex height (CCC = 0.9991) with excellent repeatability coefficient (CR = 3.0-3.3). There was low percent variance of metrics dependent upon length (median 5 %). Detailed automated quantitative assessment of colonic morphology on routine CTC datasets is feasible and reproducible, requiring minimal reader interaction.

Thermal Changes during Rheolytic Mechanical Thrombectomy.

PURPOSE: To characterize thermal changes induced by rheolytic thrombectomy (RT) within an ex vivo venous model and evaluate resultant changes of endothelial and vessel wall injury. MATERIALS AND METHODS: Patent human saphenous vein segments without thrombus were mounted in an ex vivo perfusion system with a temperature probe apposed to the adventitial surface. RT was performed over a guide wire to facilitate device centering. Continuous RT was performed for 4 minutes with temperature recorded every 10 seconds. Pulsed RT was performed for eight cycles of 30 seconds followed by 10 seconds of deactivation. Mean temperature increase, maximum temperature (Tmax), intimal/medial thickness, endothelial cell staining (CD31), and heat shock protein 90 (HSP90) expression were compared between untreated and RT-treated venous segments. RESULTS: Continuous RT produced a mean 7.6°C increase in temperature above baseline with mean Tmax of 44.1°C. Pulsed RT produced a mean 7.3°C increase in temperature and mean Tmax of 43.8°C. Differences in mean temperature increase (P = .66) and Tmax (P = .71) between the two groups were not statistically significant. RT-treated segments showed intima/media thinning (0.32 mm before RT and 0.18 mm after RT; P = .004) and reduction in intact endothelium (38.8% before RT and 13.8% after RT; P = .002). Staining for HSP90 showed a 3.1% increase in expression after RT (P = .31). CONCLUSIONS: RT in this venous model showed reproducible increases in vessel temperature and evidence of endothelial and vessel wall injury. Avoiding prolonged RT application to a focal vascular segment during clinical use may be beneficial.

Impact of hysterectomy on three-dimensional rectosigmoid morphology and endoscopy performance: a pilot study.

OBJECTIVES: Assess differences in three-dimensional colonic metrics on CTC in women with or without hysterectomy following incomplete endoscopy to determine if there is a correlation between colonic morphology and incomplete colonoscopy after hysterectomy. METHODS: Quantitative rectosigmoid metrics were derived from CTC datasets of 37 women with hysterectomy and 36 women without hysterectomy who underwent CTC for incomplete endoscopy. Evaluated metrics included colonic length, volume, tortuosity, and compactness and sigmoid apex height relative to the lumbosacral junction. Differences were measured using the Student's t test, and intra-reader reliability was assessed using ICC. The relative risk of incomplete rectosigmoid visualization was determined by reviewing the endoscopy reports. RESULTS: Women with hysterectomy had a lower sigmoid apex height (p = 0.002), as well as increased tortuosity (p = 0.012) and compactness (p = 0.001) and decreased length (p = 0.026) and volume (p = 0.016) of the rectosigmoid. Intra-reader reliability was high for centerline length (ICC = 0.9940) and sigmoid apex height (ICC = 0.9851). The relative risk of incomplete visualization of the rectosigmoid on endoscopy in women with hysterectomy was 2.068 (p = 0.043) compared to women without hysterectomy. CONCLUSION: Our pilot data show reproducible quantitative differences in three-dimensional metrics of the rectosigmoid in women with or without hysterectomy who underwent CTC for incomplete endoscopy and increased relative risk of incomplete endoscopic visualization of the rectosigmoid after hysterectomy. Our findings suggest that women with hysterectomy may benefit from CTC rather than endoscopy as the initial diagnostic test for evaluating the colon.

Portal Venous Interventions: State of the Art.

In recent decades, there have been numerous advances in the management of liver cancer, cirrhosis, and diabetes mellitus. Although these diseases are wide ranging in their clinical manifestations, each can potentially be treated by exploiting the blood flow dynamics within the portal venous system, and in some cases, adding cellular therapies. To aid in the management of these disease states, minimally invasive transcatheter portal venous interventions have been developed to improve the safety of major hepatic resection, to reduce the untoward effects of sequelae from end-stage liver disease, and to minimize the requirement of exogenously administered insulin for patients with diabetes mellitus. This state of the art review therefore provides an overview of the most recent data and strategies for utilization of preoperative portal vein embolization, transjugular intrahepatic portosystemic shunt placement, balloon retrograde transvenous obliteration, and islet cell transplantation.

Magnetic Resonance-Monitored Coaxial Electrochemical Ablation--Preliminary Evaluation of Technical Feasibility.

PURPOSE: To evaluate the technical feasibility of a coaxial electrode configuration to rapidly create a mechanically defined electrochemical ablation zone monitored by magnetic resonance (MR) imaging in real time. MATERIALS AND METHODS: A direct current generator supplied the nitinol cathode cage and central platinum anode for coaxial electrochemical ablation. Safety and efficacy were evaluated by measuring local pH, temperature, and current scatter in saline solutions. Ablation zone diameters of 3-6 cm (n = 72) were created on ex vivo bovine liver and verified by gross pathology. Feasibility of MR monitoring was evaluated using 8 swine livers to create ablations of 3 cm (n = 12), 4 cm (n = 4), and 5 cm (n = 4) verified by histology. RESULTS: Local pH was 3.2 at the anode and 13.8 at the cathode. Current scatter was negligible. Ablation progress increased relative to local ion concentration, and MR signal changes corresponded to histologic findings. In the ex vivo model, the times to achieve complete ablation were 15 minutes, 20 minutes, 35 minutes, and 40 minutes for diameters of 3 cm, 4 cm, 5 cm, and 6 cm, respectively. Ablation times for the in situ model were 15 minutes, 35 minutes, and 50 minutes for 3 cm, 4 cm, and 5 cm, respectively. CONCLUSIONS: The coaxial configuration mechanically defined the electrochemical ablation zone with times similar to comparably sized thermal ablations. MR compatibility allowed for real-time monitoring of ablation progress.

Long-Term Patency and Clinical Analysis of Expanded Polytetrafluoroethylene-Covered Transjugular Intrahepatic Portosystemic Shunt Stent Grafts.

PURPOSE: To evaluate long-term patency and symptomatic recurrence rates following transjugular intrahepatic portosystemic shunt (TIPS) creation with expanded polytetrafluoroethylene (ePTFE)-covered stent grafts and to determine the necessity of extended clinical follow-up beyond 2 years after TIPS creation. MATERIALS AND METHODS: A retrospective review including 262 TIPSs created with ePTFE-covered stent grafts between July 2002 and October 2012 was performed. Primary, primary assisted, and secondary patency rates were calculated. Assessment of clinical data included technical, hemodynamic, and clinical success rates, as well as mortality after TIPS creation. RESULTS: Primary patency rates at 2, 4, and 6 years were 74%, 62%, and 50%, respectively. Primary assisted patency rates at 2, 4, and 6 years were 93%, 85%, and 78%, respectively. Secondary patency rates at 2, 4, and 6 years were 99%, 91%, and 84%, respectively. Technical and hemodynamic success rates were 99% and 93%, respectively. Clinical success rates for refractory ascites were 66% (complete response) and 90% (partial response); clinical success rate for bleeding/varices was 90%. Mortality rates at 2, 4, and 6 years after TIPS creation were 27%, 38%, and 46%, respectively. At the median wait time until transplantation, patients had an 84% chance of being alive. TIPS dysfunction developed in 21% of patients; 30% of revisions occurred later than 2 years during follow-up. CONCLUSIONS: Beyond 2 years after TIPS creation, patency rates gradually decrease, mortality rates continue to increase, and the chance of recurrent ascites or bleeding remains present. Together, these findings suggest that continued clinical follow-up beyond 2 years is necessary in patients with a TIPS created with an ePTFE-covered stent graft.

Identification of genes expressed during hair follicle induction.

The hair follicle is one of the skin appendages that develops through reciprocal epithelial-mesenchymal interactions. Although a large number of studies have been made on the mechanisms of hair follicle development, the whole molecular mechanism that governs hair follicle development remains poorly defined. To further understand the molecular basis of hair follicle development, it is necessary to identify genes that drive hair morphogenesis. As an initial approach, we attempted to identify gene products associated with mouse hair follicle development. Genes upregulated in the vibrissal hair placodes were screened by polymerase chain reaction (PCR)-based cDNA subtraction. The genes thus isolated were evaluated for their hair development-associated induction and spatiotemporal expression by quantitative reverse-transcription-PCR analysis and whole-mount in situ hybridization, respectively. Finally, we identified four genes whose upregulation and spatiotemporal expression in developing hair follicles were confirmed. Successful identification of novel hair development-associated genes will be informative as clues for further characterization of hair follicle development at the molecular level.

Regulation of mesodermal differentiation of mouse embryonic stem cells by basement membranes.

Basement membranes (BMs) have been implicated in cell fate determination during development. Embryoid bodies (EBs) derived from mouse embryonic stem cells deficient in the laminin gamma1 chain are incapable of depositing a BM, resulting in failure of primitive ectoderm epithelialization. To elucidate the mechanisms involved in this phenomenon, we compared the gene expression profiles of EBs with or without a BM to identify the genes showing BM-dependent expression. We found that the expressions of marker genes for the epithelial-mesenchymal transition (EMT), including the transcription factor Snai2, were up-regulated in LAMC1(-/-) EBs, whereas restoration of a BM to LAMC1(-/-) EBs suppressed the up-regulation of these genes. Overexpression of Snai2 induced the EMT in control EBs by molecular and morphological criteria, suggesting that suppression of the EMT regulatory genes is involved in BM-dependent epithelialization of primitive ectoderm. Despite the failure of primitive ectoderm epithelialization in BM-deficient EBs, mesodermal differentiation was not compromised, but rather accelerated. Furthermore, at later stages of control EB differentiation, the BM was disrupted at the gastrulation site where mesodermal markers were strongly expressed only in cells that had lost contact with the BM. Taken together, these results indicate that the BM prevents the EMT and precocious differentiation of primitive ectoderm toward mesoderm in EBs, implying that BMs are important for the control of mammalian gastrulation.

Bortezomib sensitizes human head and neck carcinoma cells SQ20B to radiation.

The proteasome inhibitor bortezomib was tested in a cell screen as a single agent with good efficacy in multiple hematologic and solid cancer cell lines. Phase II/III studies have supported the use of bortezomib in hematologic malignancies. In solid tumors, however, the results have been poor. There is data that bortezomib can induce PTEN expression resulting in down-regulation of PI3K-Akt signaling. We and others have shown that down-regulation of Akt results in radiation sensitization. We therefore evaluated the use of bortezomib in the head and neck cancer cell line SQ20B as a radiation sensitizer. SQ20B have a constitutively active mutation in EGFR resulting in a robust Akt response. We found that 10 nM of bortezomib decreased Akt signaling to almost undetectable. This same concentration decreased the surviving fraction after 2 Gy (SF2) from 0.77 to 0.45. Given that radiation is usually given at 2 Gy increments daily for 30 or more treatments, the exponential difference in log kill could be as high as 7 logs. The dose of bortezomib is also 2 logs less as a sensitizer than that required for single agent efficacy. Further studies should be done to explore this model in vivo.

A novel large-scale production system for modified basement membrane matrices using gene-swapped parietal endoderm cells.

Parietal endoderm-like cells, including Engelbreth-Holm-Swarm tumor and differentiated F9 embryonal carcinoma cells, produce huge amounts of basement membrane components, including laminin-1 (alpha1beta1gamma1). We employed a double-lox system-based gene-swapping strategy in F9 cells to replace the laminin alpha1 gene with a laminin alpha5 minigene. The gene-swapped F9 cells secreted laminin-10 (alpha5beta1gamma1) consisting of the exogenous alpha5 subunit and endogenous beta1 and gamma1 subunits on differentiation. The laminin-10 concentration in the conditioned medium exceeded 10 mg/l, which is 10-fold higher than the concentrations achieved by conventional recombinant expression systems. The gene-swapped F9 cells deposited basement membrane-like matrices containing laminin-10 on culture dishes, offering a novel microenvironment for in vitro cell manipulation.

Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle.

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.

Expression of MAEG, a novel basement membrane protein, in mouse hair follicle morphogenesis.

We screened for genes specifically expressed in the mesenchymes of developing hair follicles using representational differential analysis; one gene identified was MAEG, which encodes a protein consisting of five EGF-like repeats, a linker segment containing a cell-adhesive Arg-Gly-Asp (RGD) motif, and a MAM domain. Immunohistochemistry showed that MAEG protein was localized at the basement membrane of embryonic skin and developing hair follicles, while MAEG expression diminished at the tip of the hair bud. A recombinant MAEG fragment containing the RGD motif was active in mediating adhesion of keratinocytes to the substratum in an RGD-dependent manner. One of the adhesion receptors recognizing the RGD motif was found to be the alpha8beta1 integrin, the expression of which was detected in the placode close to MAEG-positive mesenchymal cells, but later became restricted to the tip of the developing hair bud. Given its localized expression at the basement membrane in developing hair follicles and the RGD-dependent cell-adhesive activity, MAEG may play a role as a mediator regulating epithelial-mesenchymal interaction through binding to RGD-binding integrins including alpha8beta1 during hair follicle development.

Sox7 plays crucial roles in parietal endoderm differentiation in F9 embryonal carcinoma cells through regulating Gata-4 and Gata-6 expression.

During early rodent development, the parietal endoderm appears from an inner cell mass and produces large amounts of basement membrane components, such as laminin-1 and collagen IV. To elucidate the regulatory network for gene expression during these procedures, we constructed a series of short interfering RNA expression vectors targeted to various transcription factors, transfected them into F9 embryonal carcinoma cells, and evaluated the effects of the gene silencing on the induction of parietal endoderm differentiation and basement membrane component production by treating F9 cells with all trans-retinoic acid and dibutyryl cyclic AMP. Among the transcription factors tested, silencing of Sox7 or combined silencing of Gata-4 and Gata-6 resulted in suppression of cell shape changes and laminin-1 production, which are the hallmarks of parietal endoderm differentiation. In cells silenced for Sox7, induction of Gata-4 and Gata-6 by retinoic acid and cyclic AMP treatment was inhibited, while induction of Sox7 was not affected in cells silenced for Gata-4 and Gata-6, indicating that Sox7 is an upstream regulatory factor for these Gata factors. Nevertheless, silencing of Sox7 did not totally cancel the action of retinoic acid, since upregulation of coup-tf2, keratin 19, and retinoic acid receptor beta2 was not abolished in Sox7-silenced F9 cells. Although overexpression of Sox7 alone was insufficient to induce parietal endoderm differentiation, overexpression of Gata-4 or Gata-6 in Sox7-silenced F9 cells restored the differentiation into parietal endoderm. Sox7 is therefore required for the induction of Gata-4 and Gata-6, and the interplay among these transcription factors plays a crucial role in parietal endoderm differentiation.

Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction.

PURPOSE: Locoregional recurrence is the dominant form of treatment failure in head and neck (H&N) cancer. The epidermal growth factor receptor (EGFR) is frequently amplified in this disease (