preparation-metabolic-assay-3-dimensional-spheroid-co-cultures

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Translational Genomics Research Institute

The yKu protein of Saccharomyces cerevisiae is important for genome stability by repressing recombination involving telomeric sequences. The mechanism of this repression is not known, but silent heterochromatin such as HML, HMR, and telomeres are compartmentalized at the nuclear periphery and yKu is proposed to interact with these regions and to play a role in telomeric silencing and tethering. We have utilized ChIP on chip, quantitative PCR, and quantitative recombination assays to analyze yKu binding and its effect on genome stability in wild-type and mutant backgrounds. Our data suggest that, although yKu binds to the TG1-3 repeats and other parts of the genome when needed, such as during nonhomologous end-joining, it specifically binds to core X sequences in addition to the mating-type loci, HML and HMR. Association with core X occurred in the absence of Sir proteins, and enhanced binding was observed at silenced ends compared to nonsilenced ends. In contrast, binding to HML and HMR was totally dependent on Sir2-4p and partially dependent on Sir1p with a stronger association at HML in both MATa and MATalpha strains. Using yku80 separation-of-function mutants, we show a direct correlation between core X binding and recombination rate. We believe our findings support our hypothesis that yKu and core X play a pivotal role in maintaining genome stability through nuclear architecture by mediating a defensive fold-back structure at yeast chromosome ends.

The association of yKu with subtelomeric core X sequences prevents recombination involving telomeric sequences.

Chromosome translocations are gross chromosomal rearrangements that have often been associated with cancer development in mammalian cells. The feasibility of drastically reshaping the genome with a single translocation event also gives this molecular event a powerful capacity to drive evolution. Despite these implications and their role in genome instability, very little is known about the molecular mechanisms that promote and accompany these events. Here, at the molecular level, we describe 10 morphologically and physiologically different translocants ensuing from the induction of the same bridge-induced translocation (BIT) event in the budding yeast Saccharomyces cerevisiae. We have demonstrated that, despite their common origin from the integration of the same linear DNA construct, all 10 translocation mutant strains have different phenotypes and the ability to sporulate and regulate gene expression and morphology. We also provide insights into how heterogeneous phenotypic variations originate from the same initial genomic event. Here we show eight different ways in which yeast cells have dealt with a single initial event inducing translocation. Our results are in agreement with the formation of complex rearrangements and abnormal karyotypes described in many leukemia patients, thus confirming the modellistic value of the yeast BIT system for mammalian cells.

Different aneuploidies arise from the same bridge-induced chromosomal translocation event in Saccharomyces cerevisiae.

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis.

Acute pancreatitis (AP) and chronic pancreatitis (CP) share etiologies, but AP can be more severe and is associated with a higher rate of mortality. We investigated features of CP that protect against severe disease. The amount of intrapancreatic fat (IPF) is increased in obese patients and fibrosis is increased in patients with CP, so we studied whether fibrosis or fat regulate severity of AP attacks in patients with CP.

Fibrosis reduces severity of acute-on-chronic pancreatitis in humans.

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

Plusieurs ph&eacute;nom&egrave;nes intra-acinar d&eacute;l&eacute;t&egrave;res sont d&eacute;clench&eacute;s simultan&eacute;ment portant ouverture d&#39;une pancr&eacute;atite aigu&euml;. Celles-ci aboutissent &agrave; blessure acineuse ou g&eacute;n&eacute;ration m&eacute;diateur inflammatoire in vitro et parenchymateuse endommager in vivo. C&eacute;rul&eacute;ine supraphysiologique est une tel initiateur qui active simultan&eacute;ment plusieurs voies de signalisation y compris non r&eacute;cepteurs tyrosine kinases comme de la famille Src. Elle provoque &eacute;galement une augmentation soutenue en calcium cytosolique-un joueur consid&eacute;r&eacute; comme crucial dans la r&eacute;gulation des ph&eacute;nom&egrave;nes d&eacute;l&eacute;t&egrave;res. Nous avons montr&eacute; la Src d&#39;&ecirc;tre impliqu&eacute;s dans le remodelage de l&#39;actine c&eacute;rul&eacute;ine induite, et c&eacute;rul&eacute;ine induit des changements dans l&#39;appareil de Golgi et le trafic de post-Golgi d&#39;&ecirc;tre impliqu&eacute;s dans l&#39;activation trypsinog&egrave;ne, qui lance des l&eacute;sions des cellules acineuses. Toutefois, on ignore si une augmentation de calcium cytosolique est n&eacute;cessaire d&#39;initier des blessures acineuses ou si blessure peut &ecirc;tre initi&eacute;e aux niveaux de calcium cytosolique basale par une autre voie. Pour &eacute;tudier l&#39;interaction entre la signalisation de la kinase de tyrosine et de calcium, nous avons trait&eacute; les cellules acineuses du pancr&eacute;as de souris avec le pervanadate inhibiteur de tyrosine phosphatase. Nous avons &eacute;tudi&eacute; l&#39;effet de l&#39;inhibiteur utilis&eacute; en clinique de la Src, Dasatinib (BMS-354825) sur pervanadate ou c&eacute;rul&eacute;ine induced changes in activation Src, activation du trypsinog&egrave;ne, l&eacute;sions des cellules, en amont de calcium cytosolique, actine et morphologie de l&#39;appareil de Golgi. Pervanadate, comme c&eacute;rul&eacute;ine supraphysiologique, induite par l&#39;activation Src, redistribution de l&#39;actine F de son emplacement normal dans la r&eacute;gion de sous-apicale aux r&eacute;gions basolat&eacute;rale et provoqu&eacute; la fragmentation de l&#39;appareil de Golgi ant&eacute;rograde. Ces changements, comme ceux induits par supraphysiologique c&eacute;rul&eacute;ine, ont &eacute;t&eacute; associ&eacute;s avec activation du trypsinog&egrave;ne et blessures acineuses, qui ont &eacute;t&eacute; emp&ecirc;ch&eacute;s par Dasatinib. Fait int&eacute;ressant, toutefois, pervanadate n&#39;a pas caus&eacute; une augmentation du calcium cytosolique, et l&#39;augmentation de calcium cytosolique c&eacute;rul&eacute;ine induite n&#39;a pas &eacute;t&eacute; affect&eacute;e par Dasatinib. Ces r&eacute;sultats sugg&egrave;rent qu&#39;intra-acinar ph&eacute;nom&egrave;nes d&eacute;l&eacute;t&egrave;res peuvent &ecirc;tre engag&eacute;es, ind&eacute;pendante de l&#39;augmentation de calcium cytosolique. Autres joueurs acineuses blessant ainsi que de la famille Src des tyrosine kinases restent &agrave; explorer.

SRC d&eacute;pendant des blessures acineuses du pancr&eacute;as peuvent &ecirc;tre lanc&eacute; ind&eacute;pendamment de l&#39;augmentation de Calcium cytosolique.

Parenteral nutrition (PN) has been associated with a higher rate of adverse outcomes compared with enteral feeding in patients with acute pancreatitis (AP). However, PN may be necessary when feeding via the enteral route is poorly tolerated or impossible, and PN is recommended as a second-line nutrition therapy in AP. Intravenous (IV) lipids are commonly used as a part of PN in patients with AP. While the adverse outcomes related to the use of PN in AP have commonly been attributed to infectious complications, data suggest that the unsaturated fatty acids in the triglycerides used in IV lipids may contribute to the development of organ failure. We discuss the clinical and experimental data on this issue and the alternative lipid emulsions that are being studied.

Potential influence of intravenous lipids on the outcomes of acute pancreatitis.

Obese patients have worse outcomes during acute pancreatitis (AP). Previous animal models of AP have found worse outcomes in obese rodents who may have a baseline proinflammatory state. Our aim was to study the role of acute lipolytic generation of fatty acids on local severity and systemic complications of AP. Human postpancreatitis necrotic collections were analyzed for unsaturated fatty acids (UFAs) and saturated fatty acids. A model of biliary AP was designed to replicate the human variables by intraductal injection of the triglyceride glyceryl trilinoleate alone or with the chemically distinct lipase inhibitors orlistat or cetilistat. Parameters of AP etiology and outcomes of local and systemic severity were measured. Patients with postpancreatitis necrotic collections were obese, and 13 of 15 had biliary AP. Postpancreatitis necrotic collections were enriched in UFAs. Intraductal glyceryl trilinoleate with or without the lipase inhibitors resulted in oil red O-positive areas, resembling intrapancreatic fat. Both lipase inhibitors reduced the glyceryl trilinoleate-induced increase in serum lipase, UFAs, pancreatic necrosis, serum inflammatory markers, systemic injury, and mortality but not serum alanine aminotransferase, bilirubin, or amylase. We conclude that UFAs are enriched in human necrotic collections and acute UFA generation via lipolysis worsens pancreatic necrosis, systemic inflammation, and injury associated with severe AP. Inhibition of lipolysis reduces UFA generation and improves these outcomes of AP without interfering with its induction.

Acute lipotoxicity regulates severity of biliary acute pancreatitis without affecting its initiation.

Multiple deleterious signaling cascades are simultaneously activated in acute pancreatitis (AP), which may limit the success of pharmacologic approaches targeting a single step. We explored whether cooling acinar cells slows distinct steps initiated from a stimulus causing pancreatitis simultaneously, and the temperature range over which inhibition of such deleterious signaling occurs.

Hypothermia slows sequential and parallel steps initiated during caerulein pancreatitis.

Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators.

Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections.

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.

Lipolysis of visceral adipocyte triglyceride by pancreatic lipases converts mild acute pancreatitis to severe pancreatitis independent of necrosis and inflammation.

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis.

Studying the uptake of 2-deoxy glucose (2-DG) analogs such as 2-Deoxy-2-[18F] fluoroglucose (FDG) is a common approach to identify and monitor malignancies and more recently chronic inflammation. While pancreatitis is a common cause for false positive results in human studies on pancreatic cancer using FDG, the relevance of these findings to acute pancreatitis (AP) is unknown. FDG has a short half-life. Thus, with an aim to accurately characterize the metabolic demand of the pancreas during AP in real-time, we studied the uptake of the non-radioactive, near infrared fluorescence labelled 2-deoxyglucose analog, IRDye® 800CW 2-DG probe (NIR 2-DG; Li-Cor) during mild and severe biliary AP.

Pawan Noel

Molecular Medicine Division

Translational Genomics Research Institute

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Pawan Noel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular Medicine Division

Translational Genomics Research Institute

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Molecular Medicine Division