TAp73 loss favors Smad-independent TGF-ß signaling that drives EMT in pancreatic ductal adenocarcinoma.

Advances made in pancreatic cancer therapy have been far from sufficient and have allowed only a slight improvement in global survival of patients with pancreatic ductal adenocarcinoma (PDA). Recent progresses in chemotherapy have offered some hope for an otherwise gloomy outlook, however, only a limited number of patients are eligible because of important cytotoxicity. In this context, enhancing our knowledge on PDA initiation and evolution is crucial to highlight certain weaknesses on which to specifically target therapy. We found that loss of transcriptionally active p73 (TAp73), a p53 family member, impacted PDA development. In two relevant and specific engineered pancreatic cancer mouse models, we observed that TAp73 deficiency reduced survival and enhanced epithelial-to-mesenchymal transition (EMT). Through proteomic analysis of conditioned media from TAp73 wild-type (WT) and deficient pancreatic tumor cells, we identified a secreted protein, biglycan (BGN), which is necessary and sufficient to mediate this pro-EMT effect. Interestingly, BGN is modulated by and modulates the transforming growth factor-ß (TGF-ß) pathway, a key regulator of the EMT process. We further examined this link and revealed that TAp73 impacts the TGF-ß pathway by direct regulation of BGN expression and Sma and Mad-related proteins (SMADs) expression/activity. Absence of TAp73 leads to activation of TGF-ß signaling through a SMAD-independent pathway, favoring oncogenic TGF-ß effects and EMT. Altogether, our data highlight the implication of TAp73 in the aggressiveness of pancreatic carcinogenesis through modulation of the TGF-ß signaling. By suggesting TAp73 as a predictive marker for response to TGF-ß inhibitors, our study could improve the classification of PDA patients with a view to offering combined therapy involving TGF-ß inhibitors.

Stromal SLIT2 impacts on pancreatic cancer-associated neural remodeling.

Pancreatic ductal adenocarcinoma (PDA) is a critical health issue in the field of cancer, with few therapeutic options. Evidence supports an implication of the intratumoral microenvironment (stroma) on PDA progression. However, its contribution to the role of neuroplastic changes within the pathophysiology and clinical course of PDA, through tumor recurrence and neuropathic pain, remains unknown, neglecting a putative, therapeutic window. Here, we report that the intratumoral microenvironment is a mediator of PDA-associated neural remodeling (PANR), and we highlight factors such as 'SLIT2' (an axon guidance molecule), which is expressed by cancer-associated fibroblasts (CAFs), that impact on neuroplastic changes in human PDA. We showed that 'CAF-secreted SLIT2' increases neurite outgrowth from dorsal root ganglia neurons as well as from Schwann cell migration/proliferation by modulating N-cadherin/ß-catenin signaling. Importantly, SLIT2/ROBO signaling inhibition disrupts this stromal/neural connection. Finally, we revealed that SLIT2 expression and CAFs are correlated with neural remodeling within human and mouse PDA. All together, our data demonstrate the implication of CAFs, through the secretion of axon guidance molecule, in PANR. Furthermore, it provides rationale to investigate the disruption of the stromal/neural compartment connection with SLIT2/ROBO inhibitors for the treatment of pancreatic cancer recurrence and pain.

TAp73 is required for macrophage-mediated innate immunity and the resolution of inflammatory responses.

The multiple isoforms of p73, a member of the p53 family, share the ability to modulate p53 activities but also have unique properties, leading to a complex and poorly understood functional network. In vivo, p73 isoforms have been implicated in tumor suppression (TAp73(-/-) mice), DNA damage (ΔNp73(-/-) mice) and development (p73(-/-) mice). In this study, we investigated whether TAp73 contributes to innate immunity and septic shock. In response to a lethal lipopolysaccharide (LPS) challenge, TAp73(-/-) mice showed higher blood levels of proinflammatory cytokines and greater mortality than their wild-type littermates. In vitro, TAp73(-/-) macrophages exhibited elevated production of tumor necrosis factor alpha , interleukin-6 and macrophage inflammatory protein-2 as well as prolonged survival, decreased phagocytosis and increased major histocompatibility complex class II expression. Mice depleted of endogenous macrophages and reconstituted with TAp73(-/-) macrophages showed increased sensitivity to LPS challenge. These results suggest that macrophage polarization is altered in the absence of TAp73 such that maintenance of the M1 effector phenotype is prolonged at the expense of the M2 phenotype, thus impairing resolution of the inflammatory response. Our data indicate that TAp73 has a role in macrophage polarization and innate immunity, enhancing the action field of this important regulatory molecule.

Consequences of DJ-1 upregulation following p53 loss and cell transformation.

p53 is a tumor suppressor that responds to various stress signals by initiating cell-cycle arrest, senescence and apoptosis. Mutations of the p53 gene are found in over 50% of human tumors, highlighting the importance of p53 in tumor suppression. Numerous studies have reported on the interactions between p53, IGF-1-AKT and mTOR pathways as potentially explaining some of the tumor suppressive activities of p53. To further understand the basis of these interactions, we analyzed the involvement of DJ-1, an oncogene known to drive AKT-mediated cell survival, in the p53-AKT axis. In this study, we show that DJ-1 and p53 are tightly 'linked': p53 prevents the accumulation of DJ-1 protein, whereas loss of p53 leads to stabilization and enhancement of DJ-1 expression. Interestingly, this increase in DJ-1 level is only observed when p53 loss is accompanied by transformation of cells. Moreover, DJ-1 seems to be required for the enhanced activation of AKT observed in p53-deficient cells. Such observation confers a new property to DJ-1 associated to transforming-process to its oncogenic ability to drive AKT activation. We also show that DJ-1 is necessary for p53 activation following oxidative stress, suggesting the existence of a finely regulated loop between these two proteins in transformed cells. Finally, we demonstrate that in the absence of p53, DJ-1 is stabilized by ROS accumulation, and surprisingly seems to be required for this high intracellular ROS production. These data offer new insights into the regulation of DJ-1 and suggest that DJ-1 is a target of p53. Importantly, our study highlights that during transformation, DJ-1 is having a key role in the p53-regulated AKT pathway and p53-driven oxidative-stress response.

Retinol-binding protein 4 levels in obese children and adolescents with glucose intolerance.

BACKGROUND: Retinol-binding protein 4 (RBP4) is known to be involved in obesity-associated insulin resistance. AIMS: To study the relationships between the degree of adiposity, insulin resistance indices, plasma lipids, inflammatory parameters, glucose intolerance (GI) status and plasma RBP4 levels in obese children and adolescents. PATIENTS AND METHODS: Prospective study comprising 199 obese patients (95 boys) aged 8-16 years (11.8 +/- 1.9). Fifty-three subjects (23 boys) of similar mean age, 11.3 +/- 2.1 years, served as controls. BMI, waist and hip circumferences, plasma lipids, and inflammatory parameters were measured and patients underwent an oral glucose tolerance test. Plasma RBP4 levels were determined by nephelometry. RESULTS: Plasma RBP4 levels (pg/ml) in obese patients with GI (n = 15) were higher (45.0 +/- 14.1) compared with those of obese patients without GI (35.9 +/- 11.7, p = 0.02; n = 184) and controls (31.5 +/- 12.3, p = 0.04) in a generalized linear model adjusted for age, sex, BMI and pubertal status. A negative correlation was found between the skeletal muscle insulin resistance index and RBP4; positive correlations were found between the RBP4 and BMI Z-score (r = 0.213, p < 0.001), waist circumferences (r = 0.135, p < 0.05), plasma triglycerides (r = 0.187, p = 0.005) and apolipoprotein B (0.187, p = 0.007). CONCLUSIONS: Our results suggest a direct relationship between circulating insulin and RBP4 levels, which indicates that this protein might contribute to the development of muscle insulin resistance.

Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.

We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.

Expression profiling in pancreas during the acute phase of pancreatitis using cDNA microarrays.

Most attacks of acute pancreatitis are self-limiting, suggesting that the pancreatic cells adapt their phenotype to prevent progression of the disease. Such phenotypic change must involve a coordinated modification in the expression of numerous genes. To identify differentially expressed genes, high-density mouse cDNA microarrays were hybridized with cDNA probes from both healthy pancreas and pancreas affected by acute pancreatitis. From the 7981 mouse genes analyzed, 239 showed significant changes in their expression during the acute phase of pancreatitis. Among them, 107 genes were up-regulated whereas 132 were down-regulated. They include genes whose function was not previously related to pancreatitis, suggesting that they are involved in some way into the acute pancreatic response. Finally, 40% of differentially expressed genes corresponded to ESTs. Demonstration that a large quantity of unexpected or yet uncharacterized genes showed altered expression during acute pancreatitis underscores the interest of a genome-based investigation. Some of these genes are certainly involved in the cellular defense against pancreatitis and, as such, deserve being studied further.

Cloning and expression of the mouse PIP49 (Pancreatitis Induced Protein 49) mRNA which encodes a new putative transmembrane protein activated in the pancreas with acute pancreatitis.

We have used a microarray-based strategy to characterize, at the molecular level, the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis. In this strategy, the phenotype of the pancreatitis-affected pancreas is established by characterization of a large number of its transcripts using a high-density mouse cDNA microarray. This method allows identification of transcripts differentially expressed during pancreatitis. We describe here the cloning, sequencing, and expression analysis of a new gene, named PIP49 (Pancreatitis Induced Protein 49). Its very strong expression is specific of acinar cells and occurs rapidly after initiation of the acute phase of pancreatitis. Analysis of its primary and secondary structures strongly suggests that PIP49 encodes a putative transmembrane protein.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Catecholaminergic development of fetal rat ventral mesencephalon: characterization by high-performance liquid chromatography with electrochemical detection and immunohistochemistry.

We determined dopamine (DA), noradrenaline (NA), and adrenaline (A), as well as immunohistochemically stained tyrosine hydroxylase (TH) and DA in dissected rat ventral mesencephalon (VM) tissue from Embryonic Day (ED) 14 to Postnatal Day (P) 17. Whole VM tissue DA, NA, and A contents increased with advancing age. VM DA/protein increased from ED15 to ED16, whereas NA/protein increased from ED15 to ED16 and from ED20 to P4. VM DA/NA ratio increased from ED14 to ED15 and decreased from ED18 to P4. VM cell suspensions exhibited higher DA/NA ratios than whole VM tissue. Washed cell suspensions had higher DA/NA than unwashed counterparts. We conclude that data from both VM immunohistochemistry and catecholamine assays relate to VM development. VM DA is contained mainly in cells, whereas VM NA is located in fibers that channel at the dorsal side of the VM. Determination of tissue catecholamine contents may be helpful for the biochemical characterization of tentatively identified VM grafts.

Lipoma intraneural / Intraneural lipoma

Se presenta un paciente de 22 anios con dos tumoraciones amarillentas localizadas en cara palmar del pulgar de la mano izquierda,de 1 cm de diametro cada una,asintomaticas. La histopatologia mostro proliferacion de tejido fibroadiposo de disposicion intra y perineural,haciendose el diagnostico de lipoma intraneural. No se implemento tratamiento

Lipoma intraneural / Intraneural lipoma

Se presenta un paciente de 22 anios con dos tumoraciones amarillentas localizadas en cara palmar del pulgar de la mano izquierda,de 1 cm de diametro cada una,asintomaticas. La histopatologia mostro proliferacion de tejido fibroadiposo de disposicion intra y perineural,haciendose el diagnostico de lipoma intraneural. No se implemento tratamiento

GABA-ergic component of rat embryonic ventral mesencephalic grafts: an in-vitro study.

In order to establish the number, the viability and the developmental potential of GABAergic neurons present in dopaminergic ventral mesencephalic (VM) grafts from embryonic rat, we have studied the survival and development of these neurons in culture. The GABAergic fraction demonstrated a highly disproportionate survival in culture in relation to other VM neurons resulting in a drastic change in the neuronal composition of the dissociated VM grafts. The occurrence of a similar gradual dominance of GABAergic neurons at the site of intracerebral implantation, may affect the development of grafted dopaminergic VM neurons and their interaction with host striatal cells.

Qualitative and quantitative examination of rat and human fetal dopaminergic grafts.

This study was carried out as a prelude to possible implantations of cultured human fetal dopaminergic grafts in parkinsonian patients. Examination of fetal rat ventral mesencephalon tissue for morphology, viability, and dopamine content showed an optimal gestational age for neural grafting in rat experiments of approximately 17 days. Moreover, fetal rat ventral mesencephalon tissue was cultured, and neural dopaminergic cells were observed in cell culture in 4 (11%) out of 36 ventral mesencephalon specimens derived from 15- to 21-day-old rat fetuses. Human fetal donor material from elective abortions was examined for morphology and cell culture possibilities. In 48 curettements, fetal tissue was seen in 34 (71%) of these, resulting in 17 (50%) cultures containing dopaminergic cells. Both fetal rat and human cell cultures were continued for approximately 8 weeks and appeared to remain positive upon immunocytochemical dopamine staining.

Interaction of phospholipid vesicles with rat hepatocytes: further characterization of vesicle-cell surface interaction; use of serum as a physiological modulator.

We investigated the interaction of small unilamellar phospholipid vesicle, containing the water-soluble fluorescent dye carboxyfluorescein (CF), with rat hepatocytes in vitro. Fetal calf serum (FCS) was previously shown to interfere with hepatocyte-vesicle interaction using egg-lecithin as a liposomal marker [Hoekstra, D., Scherphof, G (1979): Biochim, biophys, Acta 551, pp. 109-121.] We now demonstrate that FCS affects the binding of intact vesicles to the cell surface, as well as the transfer of individual lipid molecules between vesicles and cells. By contrast, transfer of the entrapped fluorophore from the vesicles to the interior of the cell is unaffected. These observations lead us to suggest that the sites on the surface of the hepatocyte at which stable adsorption, the transfer of vesicle contents, and the transfer of individual phospholipid molecules take place are, at least kinetically, not identical. The potential importance of the inhibitory effect of serum on defined steps in the process of vesicle-cell interaction is emphasized.

Interactions of phospholipid vesicles with rat hepatocytes in vitro. Influence of vesicle-incorporated glycolipids.

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either N-lignoceroyldihydrolactocerebroside or the monosialoganglioside, GM1, enhanced liposomal lipid uptake 4-5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin. In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [14C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%). The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryloleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicle is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.

Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture.

We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.