Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

Resveratrol-induced limitation of dysfunction of mitochondria isolated from rat brain in an anoxia-reoxygenation model

Resveratrol protection on the main functions of purified rat brain mitochondria submitted to anoxia-reoxygenation was investigated. Resveratrol (<0.1 microM) reversed partly (23.3%) the respiratory control ratio (RCR) decrease by protecting both states 3 and 4. This effect was both observed when resveratrol was added before anoxia or reoxygenation. Resveratrol fully inhibited the release of cytochrome c in a concentration-dependent manner and significantly decreased the superoxide anion (O2(0-)) production at a concentration of 1 nM. The mitochondrial membranes damaged after the anoxia-reoxygenation were partly protected (about 70%) by resveratrol at 0.1 microM. The oxygen consumption of mitochondria in presence of NADH and cytochrome c was significantly inhibited by resveratrol with a low EC50 of 18.34 pM. Resveratrol inhibited the CCCP-induced uncoupling from about 20%. The effects of resveratrol on oxidative phosphorylation parameters were also investigated in rats after pretreatment (0.4, 2 and 10 mg/kg/day) for one week. After the isolation of brain mitochondria, the RCR was significantly less decreased in the resveratrol group compared to the control group. These results showed that resveratrol could preserve the mitochondrial functions with at least three mechanisms: antioxidant properties, action on complex III and a membrane stabilizing effect.     

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.     

Flow cytometric DNA ploidy, p53, PCNA, and c-erbB-2 protein expressions as predictors of survival in surgically resected gastric cancer patients

In order to determine retrospectively the impact of some cytometric and immunohistochemical parameters on the overall survival of gastric cancer patients treated with surgery alone, paraffin-embedded tumor samples from 137 gastric carcinoma patients undergoing curative resection from 1987-1993 were analyzed by flow cytometry (FCM) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). FCM-derived parameters were DNA ploidy and fraction of S-phase cells (SPF). Multiple regression analysis was applied to determine the prognostic significance of the conventional clinicopathologic findings together with the flow cytometric and immunohistochemical parameters on overall survival. When all parameters were entered simultaneously into the Cox regression model, stage and DNA ploidy (DNA index >1.35) clearly emerged as the only independent prognostic factors. When the stages were analysed separately, the independent prognostic factors resulted DNA ploidy in early stages (I-II) and grading in stage IIIA tumors. For stage IIIB tumors, no independent prognostic factor was found. These results indicate that the DNA ploidy pattern is a valuable predictor of survival in curatively resected gastric cancer patients, especially when less advanced tumors are taken into consideration.     

Deletion 2q31.3→2q33.3: gene dosage effect of ribulose 5-phosphate 3-epimerase

In a new case of interstitial del(2q), measurements of ribulose 5-phosphate 3-epimerase activity suggested that the locus for this enzyme might be localized to the subregion 2q32q33.3.     

Prefrontal cortex activity related to abstract response strategies

Many monkeys adopt abstract response strategies as they learn to map visual symbols to responses by trial and error. According to the repeat-stay strategy, if a symbol repeats from a previous, successful trial, the monkeys should stay with their most recent response choice. According to the change-shift strategy, if the symbol changes, the monkeys should shift to a different choice. We recorded the activity of prefrontal cortex neurons while monkeys chose responses according to these two strategies. Many neurons had activity selective for the strategy used. In a subsequent block of trials, the monkeys learned fixed stimulus-response mappings with the same stimuli. Some neurons had activity selective for choosing responses based on fixed mappings, others for choosing based on abstract strategies. These findings indicate that the prefrontal cortex contributes to the implementation of the abstract response strategies that monkeys use during trial-and-error learning.     

TWEAK signals through JAK–STAT to induce tumor cell apoptosis

The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK–STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK–STAT dependent, as JAK inhibitors block IFNγ?dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK–STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNβ, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNβ can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK–STAT pathway to induce IFNβ, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.