Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression.

The association between chronic inflammation and the development and progression of malignancy is exemplified in the biliary tract where persistent inflammation strongly predisposes to cholangiocarcinoma. The inflammatory cytokine interleukin-6 (IL-6) enhances tumor growth in cholangiocarcinoma by altered gene expression via autocrine mechanisms. IL-6 can regulate the activity of DNA methyltransferases, and moreover, aberrant DNA methylation can contribute to carcinogenesis. We therefore investigated the effect of chronic exposure to IL-6 on methylation-dependent gene expression and transformed cell growth in human cholangiocarcinoma. The relationship between autocrine IL-6 pathways, DNA methylation, and transformed cell growth was assessed using malignant cholangiocytes stably transfected to overexpress IL-6. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine decreased cell proliferation, growth in soft agar, and methylcytosine content of malignant cholangiocytes. However, this effect was not observed in IL-6-overexpressing cells. IL-6 overexpression resulted in the altered expression and promoter methylation of several genes, including the epidermal growth factor receptor (EGFR). EGFR promoter methylation was decreased and gene and protein expression was increased by IL-6. Thus, epigenetic regulation of gene expression by IL-6 can contribute to tumor progression by altering promoter methylation and gene expression of growth-regulatory pathways, such as those involving EGFR. Moreover, enhanced IL-6 expression may decrease the sensitivity of tumor cells to therapeutic treatments using methylation inhibitors. These observations have important implications for cancer treatment and provide a mechanism by which persistent cytokine stimulation can promote tumor growth.

Analysis of the CaMKIIα and ß splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation.

Four CaMKII isoforms are encoded by distinct genes, and alternative splicing within the variable linker-region generates additional diversity. The α and ß isoforms are largely brain-specific, where they mediate synaptic functions underlying learning, memory and cognition. Here, we determined the α and ß splice-variant distribution among different mouse brain regions. Surprisingly, the nuclear variant αB was detected in all regions, and even dominated in hypothalamus and brain stem. For CaMKIIß, the full-length variant dominated in most regions (with higher amounts of minor variants again seen in hypothalamus and brain stem). The mammalian but not fish CaMKIIß gene lacks exon v3N that encodes the nuclear localization signal in αB, but contains three exons not found in the CaMKIIα gene (exons v1, v4, v5). While skipping of exons v1 and/or v5 generated the minor splice-variants ß', ße and ße', essentially all transcripts contained exon v4. However, we instead detected another minor splice-variant (now termed ßH), which lacks part of the hub domain that mediates formation of CaMKII holoenzymes. Surprisingly, in an optogenetic cellular assay of protein interactions, CaMKIIßH was impaired for binding to the ß hub domain, but still bound CaMKIIα. This provides the first indication for isoform-specific differences in holoenzyme formation.

IGF-I stimulates Rab7-RILP interaction during neuronal autophagy.

Restoration of autophagy represents a potential therapeutic target for neurodegenerative disorders, but factors that regulate autophagic flux are largely unknown. When deprived of trophic factors, cultured Purkinje neurons die by an autophagy associated cell death mechanism. The accumulation of autophagic vesicles and cell death of Purkinje neurons is inhibited by insulin-like growth factor, by a mechanism that enhances autophagic vesicle turnover. In this report, we identify Rab7 as an IGF-I regulated target during neuronal autophagy. Purkinje neurons transfected with EGFP-Rab7-WT and constitutively active EGFP-Rab7-Q67L contained few RFP-LC3 positive autophagosomes and little co-localization with GFP-Rab7 under control conditions. Upon induction of autophagy, RFP-LC3 positive autophagosomes increased and co-localized with GFP-Rab7. Conversely, expression of the dominant negative mutant EGFP-Rab7-T22N increased the accumulation of autophagosomes under control conditions, which accumulated even further during trophic factor withdrawal. There was no vesicular co-localization between Rab7-T22N and RFP-LC3 under control or trophic factor withdrawal conditions. During prolonged trophic factor withdrawal, a condition that leads to the accumulation of autophagic vesicles and cell death, Rab7 activity decreased significantly. IGF-I, added at the time of trophic factor withdrawal, prevented the deactivation of Rab7 and increased the interaction of Rab7 with its interacting protein (RILP), restoring autophagic flux. These results provide a novel mechanism by which IGF-I regulates autophagic flux during neuronal stress.

Anti-Parkinson botanical Mucuna pruriens prevents levodopa induced plasmid and genomic DNA damage.

Levodopa is considered the 'gold standard' for the treatment of Parkinson's disease. However, a serious concern is dyskinesia and motor fluctuation that occurs after several years of use. In vitro experiments have shown that in the presence of divalent copper ions, levodopa may induce intense DNA damage. Mucuna pruriens cotyledon powder (MPCP) has shown anti-parkinson and neuroprotective effects in animal models of Parkinson's disease that is superior to synthetic levodopa. In the present study two different doses of MPCP protected both plasmid DNA and genomic DNA against levodopa and divalent copper-induced DNA strand scission and damage. It exhibited chelation of divalent copper ions in a dose-dependent manner. The copper chelating property may be one of the mechanisms by which MPCP exerts its protective effects on DNA.