bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells

Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease.     

Autocrine TNF is critical for the survival of human dendritic cells by regulating BAK, BCL-2, and FLIPL

The life span of dendritic cells (DCs) is determined by the balance of pro- and antiapoptotic proteins. In this study, we report that serum-free cultured human monocyte-derived DCs after TLR stimulation with polyinosinic acid-polycytidylic acid or LPS underwent apoptosis, which was correlated with low TNF production. Apoptosis was prevented by the addition of exogenous TNF or by concomitant stimulation with R-848, which strongly amplified endogenous TNF production. Neutralization of TNF confirmed that DC survival was mediated by autocrine TNF induced either by stimulation with R-848 or by ligation of CD40. DCs stimulated by polyinosinic acid-polycytidylic acid or IFN-β, another known inducer of DC apoptosis, were characterized by high levels and activation of the proapoptotic protein BAK. The ratio of antiapoptotic BCL-2 to BAK correlated best with the survival of activated DCs. Addition of TNF increased this ratio but had little effect on BAX and XIAP. Knockdown experiments using small interfering RNAs confirmed that the survival of activated and also of immature DCs was regulated by BAK and showed that TNF was protective only in the presence of FLIP(L). Together, our data demonstrate that the survival of DCs during differentiation and activation depends on autocrine TNF and that the inhibition of BAK plays an important role in this process.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Genome sequence comparison reveals independent inactivation of the caspase-15 gene in different evolutionary lineages of mammals

We have recently demonstrated that placental mammalian species such as pig and dog express a novel proapoptotic protease, caspase-15, whereas mouse and humans lack this enzyme. Here we investigated the evolutionary fate of the caspase-15 gene in different mammalian lineages by analyzing whole-genome shotgun sequences of 30 mammalian species for the presence of caspase-15 orthologs. Caspase-15 gene sequences were found in representatives of all major mammalian clades except for the superorders Afrotheria (tenrec, rock hyrax, and elephant) and Euarchontoglires (rodents, rabbit, tree shrew, and primates), which either lacked any caspase-15-like sequences or contained mutated remnants of the caspase-15 gene. Polymerase chain reaction screenings confirmed the results of the database searches and showed that the caspase-15 gene is expressed not only in various placental mammals but also in the marsupial, Monodelphis domestica. The observed species distribution implies that caspase-15 has originated in an early ancestor of modern mammals and has been conserved, over more than 180 Myr, in marsupials and many placental mammals, whereas it was independently lost in 2 phylogenetically distant clades of placental mammals, that is, Afrotheria and Euarchontoglires. Our data suggest that the inactivation of the caspase-15 gene was not counteracted by, and may even have been driven by, evolutionary constraints in these clades, and therefore, caution against the uncritical use of gene absence for the inference of phylogenetic relationships.     

HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Delta disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to <10% of its normal cellular concentration. Other than Acc1p, the N-terminal sequence of Hfa1p comprises a canonical mitochondrial targeting signal together with a matrix protease cleavage site. Accordingly, the HFA1-encoded protein was specifically assigned by Western blotting of appropriate cell fractions to the mitochondrial compartment. Removal of the mitochondrial targeting sequence abolished the competence of HFA1 DNA to complement hfal null mutants. Conversely and in contrast to the intact HFA1 sequence, the signal sequence-free HFA1 gene complemented the mutational loss of cytoplasmic acetyl-CoA carboxylase. Expression of HFA1 under the control of the ACC1 promoter restored cellular ACC activity in ACC1-defective yeast mutants to wild type levels. From this finding, it is concluded that HFA1 encodes a specific mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for intraorganellar fatty acid and, in particular, lipoic acid synthesis.     

Concurrent increase of cholesterol,sphingomyelin and glucosylceramide in the spleen from non-neurologic Niemann-Pick type C patients but also patients possibly affected with other lipid trafficking disorders

Niemann-Pick type C disease (NPC) is a neurovisceral (or, extremely rarely, only visceral) lipidosis caused by mutations in the NPC1 gene or, in a few patients, the HE1 gene, which encode sterol regulating proteins. NPC is characterised by a complex lipid anomaly including a disturbed cellular trafficking of cholesterol but also multi-lipid storage in visceral organs and brain. Lipids were studied using conventional methods in enlarged spleens that had been removed from five patients for different therapeutic and diagnostic reasons and found to have microscopic signs of lysosomal storage disease not suspected clinically. The spleen lipid findings with a concurrent accumulation of cholesterol, sphingomyelin and glucosylceramide (Acc-CSG) allowed us to suggest NPC diagnoses for these patients, who were free of neurologic symptoms. From two patients no material for confirmatory studies was available, but in two other patients NPC diagnoses could be confirmed with the filipin cytochemical cholesterol assay and NPC1 gene analysis, respectively. However, these tests and also HE1 gene analysis were negative in a third patient. Since the Acc-CSG lipid pattern seems to indicate a multi-lipid trafficking defect rather than being highly specific for NPC, this patient, if not affected with very atypical NPC, may be a candidate for a different lipid trafficking disorder. The Acc-CSG pattern was considered to be similar to the lipid pattern known for the lipid rafts, these functional cell structures being probably disorganised and accumulated in late endosomes and lysosomes of NPC cells.     

Long-term follow-up of childhood-onset hypopituitarism in patients with the PROP-1 gene mutation

OBJECTIVE: The PROP-1 gene mutation is a rare disorder leading to combined pituitary hormone deficiencies over time. The aim was to analyze the clinical picture of 40 years of an almost untreated PROP-1 gene mutation. METHODS: We describe the clinical and hormonal data of 2 brothers from childhood to adulthood as well as imaging procedures (MRI of the pituitary gland, bone mineral density by QCT and DPX). The PROP-1 gene mutation (301-302delAG) was confirmed by DNA sequencing. RESULTS: Although long-standing untreated hypopituitarism was present, there was normal physical and professional activity. Bone mineral density was low only in 1 patient. Adrenocortical deficiency occurred late at 45 and 39 years. CONCLUSIONS: The biological evolution of the PROP-1 gene mutation illustrates the importance of continuous care for these patients. Hormonal deficiencies do not necessarily lead to the same phenotype as is obvious in differences of bone age and bone mineral density.