DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Wnt Signaling in Skeletal Muscle Dynamics: Myogenesis,Neuromuscular Synapse and Fibrosis

The signaling pathways activated by Wnt ligands are related to a wide range of critical cell functions, such as cell division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the activation of stem cells of the adult muscles. We also discuss how Wnt signaling regulates the precise formation of neuromuscular synapses, by modulating the differentiation of presynaptic and postsynaptic components, particularly regarding the clustering of acetylcholine receptors on the muscle membrane. In addition, based on previous evidence showing that Wnt pathways are linked to several diseases, such as Alzheimer's and cancer, we address recent studies indicating that Wnt signaling plays a key role in skeletal muscle fibrosis, a disease characterized by an increase in the extracellular matrix components leading to failure in muscle regeneration, tissue disorganization and loss of muscle activity. In this context, we also discuss the possible cross-talk between the Wnt/β-catenin pathway with two other critical profibrotic pathways, transforming growth factor β and connective tissue growth factor, which are potent stimulators of the accumulation of connective tissue, an effect characteristic of the fibrotic condition. As it has emerged in other pathological conditions, we suggests that muscle fibrosis may be a consequence of alterations of Wnt signaling activity.     

Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor

Conclusion Experimental evidence has accumulated over the past few years to suggest that the GPI protein anchor may play a broad role in the regulation of membrane protein function. The significant changes in the biophysical properties of proteins that are membrane-anchored through GPI in lieu of a hydrophobic transmembrane peptide indicates a variety phobic transmembrane peptide indicates a variety of potential new functions served by the anchor structure itself. Moreover, the number of structural variations within the family of GPI molecules indicates a further opportunity for subspecialization of such anchored proteins, especially regarding cellular localization, mobility, metabolism and susceptibility to enzymatically-induced release. It is likely that further exploration of the structure and function of the GPI anchor may reveal additional roles for this unusual mechanism of membrane-protein attachment.     

Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America

Species can respond to environmental pressures through genetic and epigenetic changes and through phenotypic plasticity, but few studies have evaluated the relationships between genetic differentiation and phenotypic plasticity of plant species along changing environmental conditions throughout wide latitudinal ranges. We studied inter‐ and intrapopulation genetic diversity (using simple sequence repeats and chloroplast DNA sequencing) and inter‐ and intrapopulation phenotypic variability of 33 plant traits (using field and common‐garden measurements) for five populations of the invasive cordgrass Spartina densiflora Brongn. along the Pacific coast of North America from San Francisco Bay to Vancouver Island. Studied populations showed very low genetic diversity, high levels of phenotypic variability when growing in contrasted environments and high intrapopulation phenotypic variability for many plant traits. This intrapopulation phenotypic variability was especially high, irrespective of environmental conditions, for those traits showing also high phenotypic plasticity. Within‐population variation represented 84% of the total genetic variation coinciding with certain individual plants keeping consistent responses for three plant traits (chlorophyll b and carotenoid contents, and dead shoot biomass) in the field and in common‐garden conditions. These populations have most likely undergone genetic bottleneck since their introduction from South America; multiple introductions are unknown but possible as the population from Vancouver Island was the most recent and one of the most genetically diverse. S. densiflora appears as a species that would not be very affected itself by climate change and sea‐level rise as it can disperse, establish, and acclimate to contrasted environments along wide latitudinal ranges.     

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein

Journal of Physiology and Biochemistry - The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical...     

Inherited fungal symbionts enhance establishment of an invasive annual grass across successional habitats

Plants infected with vertically transmitted fungal endophytes carry their microbial symbionts with them during dispersal into new areas. Yet, whether seed-borne endophytes enhance the host plant’s ability to overcome colonisation barriers and to regenerate within invaded sites remains poorly understood. We examined how symbiosis with asexual endophytic fungi (Neotyphodium) affected establishment and seed loss to predators in the invasive annual grass Lolium multiflorum (Italian ryegrass) across contrasting successional plots. Italian ryegrass seeds with high and low endophyte incidence were sown into three communities: a 1-year-old fallow field, a 15-year-old grassland, and a 24-year-old forest, which conformed to an old-field chronosequence in the eastern Inland Pampa, Argentina. We found that endophyte infection consistently increased host population recruitment and reproductive output. Endophyte presence also enhanced aerial biomass production of ryegrass in a low recruitment year but not in a high recruitment year, suggesting that symbiotic effects on growth performance are density dependent. Endophyte presence reduced seed removal by rodents, although differential predation may not account for the increased success of infected grass populations. Overall, there was no statistical evidence for an endophyte-by-site interaction, indicating that the fungal endosymbiont benefitted host establishment regardless of large differences in biotic and abiotic environment among communities. Our results imply that hereditary endophytes may increase the chances for host grass species to pass various ecological filters associated with invasion resistance across a broad range of successional habitats.     

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies

Background

Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.

Results

Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.

Conclusions

Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1450-3) contains supplementary material, which is available to authorized users.     

Protein kinase C nu/protein kinase D3 nuclear localization,catalytic activation,and intracellular redistribution in response to G protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC micro/PKD, PKD2, and PKCnu/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.