Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs.

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress-induced autophagy-associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over-expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi-based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1-binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon.

NUPR1 works against the metabolic stress-induced autophagy-associated cell death in pancreatic cancer cells.

The incidence of pancreatic adenocarcinoma is increasing with more than 43,000 predicted new cases in the US and 65,000 in Europe this year. Pancreatic cancer patients have a short life expectancy with less than 3-4% 5-y survival, which results in an equivalent incidence and mortality rate. One of the major challenges in pancreatic cancer is the identification of pharmacological approaches that overcome the resistance of this cancer to therapy. Intensive research in the past decades has led to the classification of pancreatic cancers and the identification of the driver key genetic events. Despite the advances in understanding the molecular mechanisms responsible for pancreatic cancer pathogenesis, this knowledge had little impact on significantly improving the treatment for this dismal disease. In particular, we know today that the lack of therapeutic response in pancreatic cancer is due to the intrinsic high resistance of these tumors to chemotherapy and radiation, rather than to the inappropriate design of these therapeutic approaches. Thus, in order to ensure a better outcome for pancreatic cancer patients, there is a strong need for research focused on the mechanism that determines this resistant phenotype and the means that might drive enhanced response to therapy.

Nupr1-aurora kinase A pathway provides protection against metabolic stress-mediated autophagic-associated cell death.

PURPOSE: The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. EXPERIMENTAL DESIGN: Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle-associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. RESULTS: We showed that both Nupr1 and HIF1α are coexpressed in human pancreatic ductal adenocarcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage-dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. CONCLUSIONS: Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress.

Homotypic cell cannibalism, a cell-death process regulated by the nuclear protein 1, opposes to metastasis in pancreatic cancer.

Pancreatic adenocarcinoma (PDAC) is an extremely deadly disease for which all treatments available have failed to improve life expectancy significantly. This may be explained by the high metastatic potential of PDAC cells, which results from their dedifferentiation towards a mesenchymal phenotype. Some PDAC present cell-in-cell structures whose origin and significance are currently unknown. We show here that cell-in-cells form after homotypic cell cannibalism (HoCC). We found PDAC patients whose tumours display HoCC develop less metastasis than those without. In vitro, HoCC was promoted by inactivation of the nuclear protein 1 (Nupr1), and was enhanced by treatment with transforming growth factor ß. HoCC ends with death of PDAC cells, consistent with a metastasis suppressor role for this phenomenon. Hence, our data indicates a protective role for HoCC in PDAC and identifies Nupr1 as a molecular regulator of this process.

Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development.

BACKGROUND: In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasV12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. RESULTS: Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. CONCLUSIONS: Some of the genes identified in this work, whose expression is altered upon rasV12/E1A transformation of MEFs, could be new cancer therapeutic targets.

Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene.

BACKGROUND: Ras is an area of intensive biochemical and genetic studies and characterizing downstream components that relay ras-induced signals is clearly important. We used a systematic approach, based on DNA microarray technology to establish a first catalog of genes whose expression is altered by ras and, as such, potentially involved in the regulation of cell growth and transformation. RESULTS: We used DNA microarrays to analyze gene expression profiles of rasV12/E1A-transformed mouse embryonic fibroblasts. Among the approximately 12,000 genes and ESTs analyzed, 815 showed altered expression in rasV12/E1A-transformed fibroblasts, compared to control fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated and 410 were down-regulated. About one half of genes encoding transcription factors, signaling proteins, membrane proteins, channels or apoptosis-related proteins was up-regulated whereas the other half was down-regulated. Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, and cytosolic proteins were down-regulated whereas genes encoding DNA-associated proteins (involved in DNA replication and reparation) and cell growth-related proteins were up-regulated. These data may explain, at least in part, the behavior of transformed cells in that down-regulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with reversion of the phenotype of transformed cells towards a less differentiated phenotype, and up-regulation of cell growth-related proteins and DNA-associated proteins is consistent with their accelerated growth. Yet, we also found very unexpected results. For example, proteases and inhibitors of proteases as well as all 8 angiogenic factors present on the array were down-regulated in transformed fibroblasts although they are generally up-regulated in cancers. This observation suggests that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation. CONCLUSIONS: This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of post-transcriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation.

P53-dependent expression of the stress-induced protein (SIP).

The mouse stress-induced protein (SIP) mRNA is activated in the pancreas with acute pancreatitis and in several cell lines in response to various stress agents. The SIP gene is alternatively spliced, generating two proteins (SIP'8 and SIP27). Both proteins, located mainly in the nucleus, promote cell death when overexpressed in vitro. We show that induction by stress agents of the expression of SIP18 and SIP27 mRNAs, observed in human- and mouse-derived cell lines, is absent from cells with deleted, mutated or inactive p53, suggesting that regulation of SIP gene expression is dependent on p53. That hypothesis is consistent with the presence of a functional p53-response element within the promoter region of the mouse SIP gene and confirmed by the induction of SIP mRNA expression in mouse embryo fibroblasts upon activation of a p53-dependent pathway by transfection with rasV12 or rasV12/E1A. In conclusion, SIP being a proapoptotic gene induced through p53 activation could be a stress-induced gene with antitumour properties.

Cloning and expression of the rat vacuole membrane protein 1 (VMP1), a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation.

To characterize the emergency program set up by pancreatic cells in response to pancreatitis, we established the phenotype of the pancreatitis-affected pancreas by characterizing a large number of its transcripts. In this report, we describe the cloning, sequencing, and expression pattern of a new gene, named VMP1 (vacuole membrane protein 1). The VMP1 mRNA codes for a putative protein of 406 amino acids. In situ hybridization studies revealed that pancreatic expression of VMP1 mRNAs was restricted to the acinar cells. Interestingly, VMP1 mRNA was also overexpressed in kidney after transient ischemic injury. However, many healthy tissues express VMP1 mRNA. Structure analysis suggested that VMP1 is a transmembrane protein with six hydrophobic regions. VMP1/EGFP fusion protein was located to the Golgi apparatus and the endoplasmic reticulum area. Expression of this protein promoted the formation of intracytoplasmatic vacuoles and VMP1/EGFP was located to the membranes of these vacuoles. Cells overexpressing this protein died after 48 h. In conclusion, we have identified a new stress-induced gene which codes for a transmembrane protein that, when overexpressed, promotes formation of intracellular vacuoles followed by cell death.

Synthesis and contents of pancreatic exportable enzymes: effect of oleic acid intraduodenal administration

Ratas Sprague-Dawley fueron estimuladas intraduadernalmente con ácido oleico y sacrificadas 20, 40, 60 y 80 m luego de la instilación. En todos los grupos se administró, 10m antes del sacrifício, 50 uCi de 3H-fenilalanina (3H-F) intraperitoneal. Se cuantificaron los niveles intrapancreáticos de Am, Chtg, Tg y Li y la incorporación de 3H-F en las proteínas secretoras. A los 40 m de la administración de ácido oleico se registró la máxima estimulación de ácido oleico se registró la máxima estimulación para Chtg (45%), Tg (38%) y Li (23%) por sobre los valores controles, no modificándose la Am. Todos los valores enzimáticos cayeron por debajo de los controles a los 60 y 80 minutos. La incorporación 3H-F fue máxima a los 40 m decayendo a tiempos más prolongados. En el presente trabajo demostramos que la administración intraduodenal de ácido oleico genera un aumento no paralelo en el nivel intrapancreático de algunas enzimas exportables, y que dichos valores caen a partir de los 40 m post-instilación, siendo la síntesis proteica afectada de manera similar

Synthesis and contents of pancreatic exportable enzymes: effect of oleic acid intraduodenal administration

Ratas Sprague-Dawley fueron estimuladas intraduadernalmente con ácido oleico y sacrificadas 20, 40, 60 y 80 m luego de la instilación. En todos los grupos se administró, 10m antes del sacrifício, 50 uCi de 3H-fenilalanina (3H-F) intraperitoneal. Se cuantificaron los niveles intrapancreáticos de Am, Chtg, Tg y Li y la incorporación de 3H-F en las proteínas secretoras. A los 40 m de la administración de ácido oleico se registró la máxima estimulación de ácido oleico se registró la máxima estimulación para Chtg (45%), Tg (38%) y Li (23%) por sobre los valores controles, no modificándose la Am. Todos los valores enzimáticos cayeron por debajo de los controles a los 60 y 80 minutos. La incorporación 3H-F fue máxima a los 40 m decayendo a tiempos más prolongados. En el presente trabajo demostramos que la administración intraduodenal de ácido oleico genera un aumento no paralelo en el nivel intrapancreático de algunas enzimas exportables, y que dichos valores caen a partir de los 40 m post-instilación, siendo la síntesis proteica afectada de manera similar (AU)

Bethanechol-induced restricted stimulation of pancreatic juice secretion in mice

La liberación ("out-put") de enzimas y el volumen de secreción pancreática fueron estudiados "in vivo" en ratones, bajo estimulación con betanecol. La curva dosis-respuesta para ambos parámetros se ajustó a un patón bifásico, implicando que el flujo de jugo pancreático participa también en el fenómeno de estimulación restringida

Bethanechol-induced restricted stimulation of pancreatic juice secretion in mice

La liberación ("out-put") de enzimas y el volumen de secreción pancreática fueron estudiados "in vivo" en ratones, bajo estimulación con betanecol. La curva dosis-respuesta para ambos parámetros se ajustó a un patón bifásico, implicando que el flujo de jugo pancreático participa también en el fenómeno de estimulación restringida (AU)

Estudio in vivo de la secreción de calcio por el páncreas exocrino del raton: relación con la secreción enzimática / In vivo study of the calcium secretion by the exocrine pancreas of mice: relationship with enzymatic secretion

La secreción de calcio y su relación con la secreción de quimiotripsina (QT) por el páncreas del ratón, in vivo, fueron determinadas en jugo pancreático puro colectado bajo tres condiciones de estimulación: secretina 32 mU/gm; secretina 32 mU/mg más colecistocinina 16 mU/gm y secretina 32 mU/gm más betanecol 0.2 ug/gm. En las muestras obtenidas se determinó actividad de quimotripsina y calcio secretado en 30 minutos luego de la estimulación. La secreción de calcio y QT fue mayor en ratones tratados con secretina más CCK y secretina más betanecol. En estos dos últimos grupos, entre ambos parámetros se estableció una correlación positiva. Las rectas obtenidas no fueron diferentes de una recta única y la extrapolación al origen indica que existe calcio en el jugo pancreático aún ausencia de secreción enzimática. Los resultados obtenidos concuerdan con la existencia de al menos dos mecanismos diferentes de secreción de calcio por el páncreas exocrino del ratón (AU)

Estudio in vivo de la secreción de calcio por el páncreas exocrino del raton: relación con la secreción enzimática / In vivo study of the calcium secretion by the exocrine pancreas of mice: relationship with enzymatic secretion

La secreción de calcio y su relación con la secreción de quimiotripsina (QT) por el páncreas del ratón, in vivo, fueron determinadas en jugo pancreático puro colectado bajo tres condiciones de estimulación: secretina 32 mU/gm; secretina 32 mU/mg más colecistocinina 16 mU/gm y secretina 32 mU/gm más betanecol 0.2 ug/gm. En las muestras obtenidas se determinó actividad de quimotripsina y calcio secretado en 30 minutos luego de la estimulación. La secreción de calcio y QT fue mayor en ratones tratados con secretina más CCK y secretina más betanecol. En estos dos últimos grupos, entre ambos parámetros se estableció una correlación positiva. Las rectas obtenidas no fueron diferentes de una recta única y la extrapolación al origen indica que existe calcio en el jugo pancreático aún ausencia de secreción enzimática. Los resultados obtenidos concuerdan con la existencia de al menos dos mecanismos diferentes de secreción de calcio por el páncreas exocrino del ratón