Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells

AbstractThe effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10–100 μg ml−1 PI at different conductivities (0.8–14 mS cm−1) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl− and SO42−. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the ‘electroleaks’ during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm−1 in K+-free, NaCl-containing medium and about 0.9 kV cm−1 in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K+-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V

Identification of RNA markers associated with Parkinson's disease using multiplex gene expression analysis

Introduction. Parkinson's disease (PD) is a neurodegenerative disorder, and the development of biomarkers is essential due to complicated PD diagnosis and progression assessment. Objective. To identify PD RNA markers by multiplex expression profiling of 760 genes associated with the main neuropathological processes. Materials and methods. We studied the expression of 760 genes associated with the main neuropathological processes using Nanostring nCounter Human Neuropathology Panel in 29 blood samples obtained from PD patients, including 13 samples from those in the early stage and 16 samples from those in the advanced stage, and in 16 control blood samples. Results. The comparison of gene expression in the patients with early PD and in the controls demonstrated differential expression of genes CDKN1A and CPT1B. The comparison of gene expression in the patients with advanced PD and in the controls showed LRP1 upregulation in the advanced PD group. We also revealed СPT1B upregulation in advanced disease, with a positive correlation between СPT1B expression and PD duration. Discussion. The variably expressed genes may be relevant as PD biomarkers for diagnosis and progression assessment

Comparison of Chromatographic Methods for Determination of the Major Metabolites of Catecholamines and Serotonin

In the present review, we set out a new high-tech method for determining the major metabolites of catecholamines and serotonin in urine. We then discuss the effects of these substances and the value of their main metabolites in normal and various pathological conditions, and show the efficiency of applying this technique in the laboratory diagnosis of several diseases

FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy

Background Phosphorylated histone H2AX, also known as gamma H2AX, forms mu m-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, gamma H2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of gamma H2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of gamma H2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected. Results To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of gamma H2AX foci in z-image stacks acquired by confocal fluorescence microscopy. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and gamma H2AX foci, as well as evaluation of the total foci number per cell nucleus. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. We validated the FocAn algorithm using fluorescence-labeled gamma H2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to similar to 200 DSB/nucleus. Conclusions FocAn is freely available an open-source 3D foci analyzer. The user-friendly algorithm FocAn requires little supervision and can automatically count the amount of DNA-DSBs, i.e. fluorescence-labeled gamma H2AX foci, in 3D image stacks acquired by laser-scanning microscopes without additional nuclei staining

Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs

Background: Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. Methods: Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by gamma H2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (similar to 80% vs. similar to 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. Conclusions: The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism

Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells

Abstract Background Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells. Methods Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition. Results We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy. Conclusions Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered