Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

Klapperstück, Thomas; Glanz, Dagobert; Klapperstück, Manuela; Wohlrab, Johannes

doi:10.1002/cyto.a.21149

Cited by 12 publications

(42 citation statements)

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“…Five human MM cell lines-buf1280, C8161, IGR1, MeWo, and SK-Mel13-were obtained from the Medical Faculty of the University of Halle (generous gift from Dr B Seliger and Dr J Wohlrab) and cultured in the recommended medium (Seliger et al, 2001;Klapperstuck et al, 2009). Genomic DNA was extracted from frozen tissues and cultured cells by a standard proteinase K and phenol/chloroform procedure .…”

Section: Tissues and Cell Linesmentioning

confidence: 99%

RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi

Helmbold

Richter

Walesch

et al. 2012

Journal of Investigative Dermatology

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The Ras association domain family (RASSF) consists of several tumor suppressor genes, which are frequently silenced in human cancers. We analyzed the epigenetic inactivation of RASSF2 and RASSF10 in malignant melanoma (MM) of the skin, including 5 MM cell lines, 28 primary MM, 33 metastases of MM, 47 nevus cell nevi (NCN), and 22 control tissues. The RASSF2 promoter was epigenetically downregulated in two MM cell lines only, but not in any of the investigated tumor samples. In contrast, hypermethylation of the RASSF10 promoter was found in all investigated cell lines, 19/28 (68%) of the primary MM and 30/33 (91%) of the MM metastases, 2/18 (11%) of the dysplastic NCN, and 0/29 (0%) of the non-dysplastic NCN (difference between MM and all nevi, P<0.001). RASSF10 promoter hypermethylation correlated with a reduced RASSF10 mRNA expression in 3/4 MM cell lines, and treatment with a DNA methylation inhibitor reactivated RASSF10 transcription. Furthermore, immunohistological RASSF10 expression corresponds negatively to its promoter methylation state. In summary, RASSF10 proved to be a characteristically epigenetically silenced tumor suppressor in melanomagenesis, and analysis of RASSF10 methylation status represents a new candidate tool to assist in discrimination between MM and NCN.

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Section: Tissues and Cell Linesmentioning

confidence: 99%

RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi

Helmbold

Richter

Walesch

et al. 2012

Journal of Investigative Dermatology

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“…Treatment of the cells with the two polyphenolic compounds leads to a reduction of fluorescence intensity of the DiBAC 4 (3) labeled PBMCs, corresponding to a hyperpolarization effect [24]. The EGCG hyperpolarizing effect was stronger for both groups (5.80% for normoglycemia subjects and 9.58% for diabetes compared to Q that induced a 4.42% effect on normoglycemia subjects and a 5.99% effect on HG patients).…”

Section: Resultsmentioning

confidence: 99%

Quercetin and Epigallocatechin Gallate Induce in Vitro a Dose-Dependent Stiffening and Hyperpolarizing Effect on the Cell Membrane of Human Mononuclear Blood Cells

Margină

Ilie

Grădinaru

2012

IJMS

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The bioactivity of polyphenols is closely linked to their ability to interact with biological membranes. The study evaluates the in vitro effect of quercetin and epigallocatechin on the membrane anisotropy and transmembrane potential of peripheral blood mononuclear cells (PBMCs) isolated from 26 type 2 diabetes mellitus patients compared to 25 age matched controls. The in vitro assays were analyzed in correlation with the biochemical and inflammatory profile of the subjects and with insulin resistance parameters (HOMA-IR, plasma resistin) as well. For type 2 diabetes patients, the increase of HOMA-IR and resistin concentration was associated with a significant decrease of the PBMCs membrane anisotropy. The two tested polyphenols induced a dose-dependent hyperpolarizing effect and stiffening of the cell membranes for all tested subjects. Physiological levels of quercetin and epigallocatechin gallate had the tendency to normalize the PBMCs membrane anisotropy of the cells isolated from diabetes patients, bringing it to the level of cells isolated from normoglycemic ones. Epigallocatechin gallate induced higher effects compared to quercetin on the membranes isolated from subjects with higher cardiovascular risk. The decrease of membrane fluidity and the hyperpolarizing effect could explain the cardiovascular protective action of the tested compounds.

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“…Signals were amplified, acquired and stored on a personal computer using a self-designed Labview Ò -based data acquisition software tool (National Instruments, Austin, TX). At the end of each experiment, cells were fully depolarized with 30, 60 and 150 mM KCl to obtain maximum fluorescence (f max ) for calculation of the absolute membrane potential (Vm) using the Nernst equation [19]:…”

Section: Patch-clamp Experimentsmentioning

confidence: 99%

“…RINm5F cells were transfected with untagged SUR1D2 and subjected to fluorescent membrane potential measurements. Cells were loaded with 100 nM DiBAC4(3), a bis-oxonol, which is a membrane potential-sensitive dye that increases its fluorescence upon depolarization and strictly follows the Nernst equilibrium at this low concentration [19]. Following hyperpolarization of cells with 100 lM diazoxide, glibenclamide was added at 1, 3 or 6 nM (Fig.…”

Section: Effects Of Sur1d2 On Glibenclamide-induced Depolarizationmentioning

confidence: 99%

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action

Schmid

Stolzlechner

Sorgner

et al. 2011

Cell. Mol. Life Sci.

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An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K(ATP)) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K(ATP) channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K(ATP)-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.

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Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

Cited by 12 publications

References 0 publications

RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi

RASSF10 Promoter Hypermethylation Is Frequent in Malignant Melanoma of the Skin but Uncommon in Nevus Cell Nevi

Quercetin and Epigallocatechin Gallate Induce in Vitro a Dose-Dependent Stiffening and Hyperpolarizing Effect on the Cell Membrane of Human Mononuclear Blood Cells

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action

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