Differential development of neuronal physiological responsiveness in two human neural stem cell lines

Background: Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to neurodegenerative disease. Overexpression of the myc family transcription factors in human primary cells from developing cortex and mesencephalon has produced two stable multipotential NSC lines (ReNcell VM and CX) that can be continuously expanded in monolayer culture.Results: In the undifferentiated state, both ReNcell VM and CX are nestin positive and have resting membrane potentials of around -60 mV but do not display any voltage-activated conductances. As initially hypothesized, using standard methods (stdD) for differentiation, both cell lines can form neurons, astrocytes and oligodendrocytes according to immunohistological characteristics. However it became clear that this was not true for electrophysiological features which designate neurons, such as the firing of action potentials. We have thus developed a new differentiation protocol, designated 'pre-aggregation differentiation' (preD) which appears to favor development of electrophysiologically functional neurons and to lead to an increase in dopaminergic neurons in the ReNcell VM line. In contrast, the protocol used had little effect on the differentiation of ReNcell CX in which dopaminergic differentiation was not observed. Moreover, after a week of differentiation with the preD protocol, 100% of ReNcell VM featured TTX-sensitive Na+-channels and fired action potentials, compared to 25% after stdD. Currents via other voltage-gated channels did not appear to depend on the differentiation protocol. ReNcell CX did not display the same electrophysiological properties as the VM line, generating voltage-dependant K+ currents but no Na+ currents or action potentials under either stdD or preD differentiation.Conclusion: These data demonstrate that overexpression of myc in NSCs can be used to generate electrophysiologically active neurons in culture. Development of a functional neuronal phenotype may be dependent on parameters of isolation and differentiation of the cell lines, indicating that not all human NSCs are functionally equivalent

Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells

<p>Abstract</p> <p>Background</p> <p>Pregnane X receptor (PXR) is a key transcription factor that regulates drug metabolizing enzymes such as cytochrome P450 (CYP) 3A4, and plays important roles in intestinal first-pass metabolism. Although there is a large inter-individual heterogeneity with intestinal CYP3A4 expression and activity, the mechanism driving these differences is not sufficiently explained by genetic variability of PXR or CYP3A4. We examined whether epigenetic mechanisms are involved in the regulation of PXR/CYP3A4 pathways in colon cancer cells.</p> <p>Methods</p> <p>mRNA levels of PXR, CYP3A4 and vitamin D receptor (VDR) were evaluated by quantitative real-time PCR on 6 colon cancer cell lines (Caco-2, HT29, HCT116, SW48, LS180, and LoVo). DNA methylation status was also examined by bisulfite sequencing of the 6 cell lines and 18 colorectal cancer tissue samples. DNA methylation was reversed by the treatment of these cell lines with 5-aza-2'-deoxycytidine (5-aza-dC).</p> <p>Results</p> <p>The 6 colon cancer cell lines were classified into two groups (high or low expression cells) based on the basal level of PXR/CYP3A4 mRNA. DNA methylation of the CpG-rich sequence of the <it>PXR </it>promoter was more densely detected in the low expression cells (Caco-2, HT29, HCT116, and SW48) than in the high expression cells (LS180 and LoVo). This methylation was reversed by treatment with 5-aza-dC, in association with re-expression of PXR and CYP3A4 mRNA, but not VDR mRNA. Therefore, PXR transcription was silenced by promoter methylation in the low expression cells, which most likely led to downregulation of CYP3A4 transactivation. Moreover, a lower level of <it>PXR </it>promoter methylation was observed in colorectal cancer tissues compared with adjacent normal mucosa, suggesting upregulation of the PXR/CYP3A4 mRNAs during carcinogenesis.</p> <p>Conclusions</p> <p><it>PXR </it>promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 mRNA expression and might be associated with the inter-individual variability of the drug responses of colon cancer cells.</p

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays

Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress