Correction: Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of integrin-linked kinase in regulating the protein stability of the MUC1-C oncoprotein in pancreatic cancer cells.

MUC1-C overexpression has been associated with the progression of pancreatic tumors by promoting the aggressive and metastatic phenotypes. As MUC1 is a STAT3 target gene, STAT3 plays a major role in regulating MUC1-C expression. In this study, we report an alternative mechanism by which integrin-linked kinase (ILK) post-transcriptionally modulates the expression of MUC1-C by maintaining its protein stability in pancreatic cancer cells. We found that ILK acts in concert with STAT3 to facilitate IL-6-mediated upregulation of MUC1-C; ILK depletion was equally effective as STAT3 depletion in abolishing IL-6-induced MUC1-C overexpression without disturbing the phosphorylation or cellular distribution of STAT3. Conversely, ectopic expression of constitutively active ILK increased MUC1-C expression, though this increase was not noted with kinase-dead ILK. This finding suggests the requirement of the kinase activity of ILK in regulating MUC1-C stability, which was confirmed by using the ILK kinase inhibitor T315. Furthermore, our data suggest the involvement of protein kinase C (PKC)δ in mediating the suppressive effect of ILK inhibition on MUC1-C repression. For example, co-immunoprecipitation analysis indicated that ILK depletion-mediated MUC1-C phosphorylation was accompanied by increased phosphorylation of PKCδ at the activation loop Thr-507 and increased binding of PKCδ to MUC1-C. Conversely, ILK overexpression resulted in decreased PKCδ phosphorylation. From a mechanistic perspective, the present finding, together with our recent report that ILK controls the expression of oncogenic KRAS through a regulatory loop, underscores the pivotal role of ILK in promoting pancreatic cancer progression.

TGFß promotes mesenchymal phenotype of pancreatic cancer cells, in part, through epigenetic activation of VAV1.

The highly homeostasis-resistant nature of cancer cells leads to their escape from treatment and to liver metastasis, which in turn makes pancreatic ductal adenocarcinoma (PDAC) difficult to treat, especially the squamous/epithelial-to-mesenchymal transition (EMT)-like subtype. As the molecular mechanisms underlying tumour heterogeneity remain elusive, we investigated whether epigenetic regulation might explain inter-individual differences in the progression of specific subtypes. DNA methylation profiling performed on cancer tissues prior to chemo/radiotherapy identified one hypermethylated CpG site (CpG6882469) in the VAV1 gene body that was correlated with demethylation of two promoter CpGs (CpG6772370/CpG6772811) in both PDAC and peripheral blood. Transforming growth factor ß treatment induced gene-body hypermethylation, dissociation of DNMT1 from the promoter, and VAV1 expression via SMAD4 and mutant KrasG12D. Pharmacological inhibition of TGFß-VAV1 signalling decreased the squamous/EMT-like cancer cells, promoted nuclear VAV1 localization, and enhanced the efficacy of gemcitabine in prolonging the survival of KPfl/flC mice. Together, the three VAV1 CpGs serve as biomarkers for prognosis and early detection, and the TGFß-VAV1 axis represents a therapeutic target.

Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.

Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

PHRF1 promotes genome integrity by modulating non-homologous end-joining.

Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-ß cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.

Nanodomain induced anomalous magnetic and electronic transport properties of LaBaCo2O5.5+δ highly epitaxial thin films.

A giant magnetoresistance effect (∼46% at 20 K under 7 T) and anomalous magnetic properties were found in a highly epitaxial double perovskite LaBaCo2O5.5+δ (LBCO) thin film on (001) MgO. Aberration-corrected Electron Microscopy and related analytical techniques were employed to understand the nature of these unusual physical properties. The as-grown film is epitaxial with the c-axis of the LBCO structure lying in the film plane and with an interface relationship given by (100)LBCO || (001)MgO and [001]LBCO || [100]MgO or [010]MgO. Orderly oxygen vacancies were observed by line profile electron energy loss spectroscopy and by atomic resolution imaging. Especially, oxygen vacancy and nanodomain structures were found to have a crucial effect on the electronic transport and magnetic properties.

Does Time Constraint on Users Negate the Efficacy of Decision Support Systems?

Time constraint can impair decision performance: time-constrained decision makers process information faster, process less information, and use less rigorous decision strategies. On the other hand, properly designed decision support systems (DSSs) can induce decision makers to process more information and use more rigorous decision strategies, which can result in enhanced performance. In this study, we investigate, drawing on bounded rationality and cost-benefit theories of DSS use, whether these salutary effects of DSSs still hold in time-constrained environments. Our experimental results replicate past research regarding the effects of time constraint and DSSs taken separately and also show that the positive effects of DSSs are maintained when decision makers are under time constraint. That is, consistent with hypotheses, time-constrained participants processed information in a more compensatorylike manner when aided by DSSs than when not. Some of the results suggest that the negative effects of time constraint can be mitigated or even eliminated by the use of DSSs, but some participants did not take full advantage of the DSS to combat time constraint. Copyright 2001 Academic Press.

mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.

Reverse sequestration in a case of sickle crisis.

A patient with homozygous sickle cell disease presented with sickle crisis complicated by hepatic and pulmonary sequestration and required intensive therapy. During the recovery phrase she developed a rapid rise of haemoglobin concentration, unrelated to blood transfusion, followed by hypertension, congestive cardiac failure and catastrophic intracerebral haemorrhage. This serious haemodynamic disturbance was considered to be caused by a reversal of the sequestration process. Careful monitoring of a sickle cell patient's blood pressure, blood counts, haematocrits, haemoglobin S level and plasma viscosity, even after the end of a sequestration crisis, is recommended.

Morphine activates opioid receptors without causing their rapid internalization.

We have examined the endocytic trafficking of epitope-tagged delta and mu opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1/2 approximately 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either delta or mu opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.

Epidemiology of persistent proteinuria in type II diabetes mellitus. Population-based study in Rochester, Minnesota.

Clinical risk factors for nephropathy were assessed in a population-based study of Rochester, Minnesota, residents with diabetes mellitus initially diagnosed between 1945 and 1969 (incidence cohort). The 1031 Rochester residents with non-insulin-dependent diabetes mellitus (NIDDM) were followed through their complete medical records in the community to 1 January 1982. The prevalence of persistent proteinuria was 8.2% at the diagnosis of NIDDM. Among those initially free of persistent proteinuria, the subsequent incidence was 15.3/1000 person-yr. Twenty years after the diagnosis of diabetes, the cumulative incidence of persistent proteinuria was 24.6%. A proportional hazards model identified the following risk factors for persistent proteinuria in NIDDM: elevated initial fasting blood glucose (P less than .01); older age at onset of diabetes (P less than .01); male gender (P = .05); and presence of macrovascular disease (P = .05), diabetic retinopathy (P = .05), or glycosuria (P = .07) at the diagnosis of diabetes. Separate analyses controlling for attained age indicated no association between duration of NIDDM and the incidence of persistent proteinuria. Stratified analysis of the two most significant risk factors (fasting blood glucose and age) indicated that hyperglycemia was a stronger risk factor for proteinuria in younger diabetic subjects, perhaps because of a competing risk of death in the elderly diabetic patient. In contrast to a recently described decreasing secular trend of proteinuria in Danish insulin-dependent diabetes mellitus patients, there was no decrease over the past 40 yr in proteinuria risk in this NIDDM incidence cohort.

Alterations in physiologic functions and in brain monoamine content in streptozocin-diabetic rats.

In the present study, we used streptozocin (STZ) to induce diabetes in rats and observed alterations in several physiologic functions and in monoamine content of different brain regions. Rats with STZ diabetes displayed a thermoregulatory deficit in the cold. Both the body temperature and metabolic rate of the diabetic animals were reduced at ambient temperatures below 22 degrees C. These diabetic animals had a higher level of the spontaneous pain threshold, but displayed a reduced sensitivity of analgesic responses to morphine injection. In addition, these diabetic animals had a lower level of spontaneous motor activity, but displayed an increased sensitivity of locomotor stimulant responses to amphetamine administration. Biochemical examination revealed that the diabetic animals had a lower serotonin level in both the hypothalamus and the brainstem without changes in the serotonin levels of the corpus striatum. These diabetic animals also had a lower catecholamine level in the hypothalamus, but a higher catecholamine level in the corpus striatum. The alterations in brain monoamine content and in the above-mentioned physiologic parameters were reversed after insulin replacement therapy. The data suggest that alterations in various autonomic, somatosensory, and motor neural functions of untreated STZ-diabetic rats correlated with a reproducible pattern of monoamine content in various brain regions (a pattern that differed from that observed in healthy control rats), and that both the altered neural function and the altered brain monoamine pattern were reversed after insulin therapy.

Decreasing brain epinephrine levels with 2-cyclooctyl-2-hydroxyethylamine induces hyperglycemia in rats.

Intraperitoneal administration of 2-cyclooctyl-2-hydroxyethyl-amine (CONH; 25-75 mg/kg) produced dose-related reductions in the epinephrine (EPI) concentrations in both the hypothalamic and brainstem regions, but not in the adrenal glands. The reductions in the hypothalamic and brainstem EPI concentrations in rats anesthetized with pentobarbital sodium were accompanied by parallel increases in blood glucose. These data suggest that CONH induces its hyperglycemia action at least in part by altering central EPI biosynthesis. This hyperglycemic was antagonized by spinal transection or adrenalectomy, but not by vagotomy. This indicates that CONH increases the adrenal-sympathetic efferent activity and leads to hyperglycemia in rats.

Effects of TSH, TRH, LH and LHRH on thermoregulation and food and water intake in the rat.

The effects of administration of thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), luteinizing hormone (LH) or luteinizing hormone-releasing hormone (LHRH) into the lateral cerebral ventricle on thermoregulation and food and water intake were assessed in rats. Intracerebroventricular, but not intraperitoneal, injection of TSH, LH or LHRH produced hypothermia in rats at ambient temperatures of both 8 and 22 degrees C. The hypothermia in response to TSH injection was due to both decreased metabolic heat production and increased heat loss (cutaneous vasodilatation). The hypothermia in response to either LH or LHRH was due solely to decreased metabolic heat production. There was no change in respiratory evaporative heat loss in response to TSH, LH or LHRH injection. Furthermore, food but not water intake was greatly reduced following an intracerebroventricular injection of TSH or TRH in rats. On the other hand, intracerebroventricular administration of LH, but not LHRH, caused an increase in relative water intake (or water/food) in rats. However, intracerebroventricular administration of LH or LHRH had an insignificant effect on food intake. The data indicate that, in addition to their hormone actions, TSH, LH and their releasing hormones act through a central mechanism to influence some of the physiological or behavioral functions.