Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes.

Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR-1 and small LCL-HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman-Hodgkin-Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC4 (3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC4 (3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense.

A bisexually reproducing all-triploid vertebrate.

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community. The reproduction of naturally occurring triploids has been reported to occur only through parthenogenesis, gynogenesis or hybridogenesis. The bisexual reproduction of pure triploids has been considered to be impossible because of the problem of equally distributing three chromosome sets in meiosis. Here we report geographically isolated populations of green toads (Bufo viridis complex) that are all-triploid and reproduce bisexually.

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

The bis-barbituric acid oxonol, DiBAC(4)(3) is used as a standard potentiometric probe in human cells. However, its fluorescence depends not only on membrane potential but also varies with nonpotential related changes in the amount of intracellular free and bound dye. This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC(4)(3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC(4)(3), the logarithmic relation between cell fluorescence and dye concentration turned into a virtually linear function intersecting with zero. Consequently, calibration can then be confined to determination of the fluorescence of depolarized cells stained with the same concentration as used for the actual measurement of membrane potential. Unexpectedly, quenching of fluorescence occurred in totally depolarized cells at concentrations higher than 6,250 nM. Linearity and quenching could be confirmed by additional experiments on Chinese hamster ovary CHO-K1 and B lymphoblastoid LCL-HO cells.

Cultivated keratinocytes express N-methyl-D-aspartate receptors of the NMDAR2D type.

N-methyl-D-aspartate receptors (NMDAR) can regulate the intracellular calcium concentration of keratinocytes (KC) and seem to be important for their growth and differentiation. The objective of this study was to identify the subtype(s) of this receptor expressed by KC in vitro. The mRNA was isolated from primary cultures of KC as well as from a KC cell line (HaCaT) and expression of the NMDAR subtypes determined by using RT-PCR. At the mRNA level, we found expression of only the constant NMDAR1 as well as the subtype NMDAR2D. In contrast to the other subtypes of NMDAR, NMDAR2D is characterized by low influence of magnesium to the receptor function. This characteristic is consistent with previously published functional investigations in KC. The identification of the NMDAR2D subtype in KC may be of value for the development of new therapeutic approaches.

DNA image cytometry on sections compared with flow cytometry in human bone metastases.

The DNA analysis of tumor cells discloses characteristic features from which their biological behavior with respect to both the intensity and regulation of growth can be deduced. The objective of this study was to comparatively assess the results of image cytometry (ICM) and flow cytometry (FCM) in human bone metastases as standard methods of DNA analysis and evaluate their possible importance. The nuclear DNA content of surgically removed tumors of bone tissue was determined using ICM and FCM, and the percentage of tumor cells in various cell cycle phases and ploidy status in each case were determined based on the DNA distribution pattern. Comparable results were determined by ICM and FCM with respect to the ploidy status in about 58% of examined tumor samples. When tissue samples from various regions of a tumor were examined, it was found that DNA-euploid and -aneuploid tumor areas were present within the tumors. The DNA aneuploidy was detected in 90% of these tumors with ICM. The percentage cell-cycle phase distribution varied widely with ICM and FCM. Based on our results, the use of ICM in addition to FCM is mandatory under certain conditions for the assessment of the DNA analysis of bone metastases and necessary for the critical assessment of the obtained findings.

Intratumoral DNA stem-line heterogeneity in superficial spreading melanoma.

BACKGROUND: In primary melanomas, data on the degree of intratumoral heterogeneity to date have been lacking. OBJECTIVE: Our purpose was to investigate intratumoral DNA stem-line heterogeneity in superficial spreading melanoma (SSM). METHODS: Multiple measuring fields of 54 SSMs (tumor thickness median 1.60 mm) were studied by DNA image cytometry to obtain data on the number of DNA stem lines per tumor, their ploidy characteristics, and intratumoral distribution. Results were compared with standard histopathological criteria. RESULTS: Twenty-three of 54 SSMs were found to have two or three distinct proliferating tumor cell stem lines (1.46 +/- 0.57 per tumor). Stem lines appeared spatially separated in 22 of 23 SMMs. At least 3 measuring fields per tumor were necessary to identify all stem lines with a likelihood of 95%. DNA heterogeneity correlated with tumor thickness, but occurred in 5 of 19 cases of pT1 melanoma. CONCLUSIONS: Primary SSMs can be regarded as potentially clonally unstable with a tendency for spatial separation of tumor cell stem lines.

Relevance of reactive oxygen species in the induction of 8-oxo-2'-deoxyguanosine in HaCaT keratinocytes.

There is growing evidence that solar radiation-induced oxidative DNA damage may play an important role in carcinogenesis of the skin. One substantial modification in this context is the oxidation of the guanine base to 8-oxo-2'-deoxyguanosine. Using HaCaT keratinocytes, measurement of the 8-oxo-2'-deoxyguanosine content in this study was performed by flow cytometry on whole cells. Hydrogen peroxide and hydroxyl radicals seem not to be involved in the process of this DNA alteration. However, our results demonstrate that ultraviolet A can cause DNA damage at guanine sites primarily via photosensitized reactions. Although singlet oxygen can also lead to 8-oxo-2'-deoxyguanosine, the major mechanism seems to be based on formation of the guanylcation radical through excited riboflavin and can therefore proceed without the involvement of reactive oxygen species.

Evidence for a role of nitric oxide in the mediation of antiproliferative UVA effects in keratinocytes.

Using cultured human keratinocytes, the present study investigates the role of nitric oxide (NO) in the mediation of the antiproliferative effects of ultraviolet light A (UVA). UVA treatment of cells (3-21 J cm (-2)) caused a time- and dose-dependent increase in nitrite formation in a micromolar range. This effect was accompanied by a decrease in DNA synthesis by 53.5%. Moreover, UVA treatment slightly reduced cell viability by 23.8%. Preincubation of keratinocytes with the NO scavenger 4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO, 10-100 microM) or the NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA, 30-300 microM) significantly diminished the UVA-induced increase in nitrite. PTIO as well as l-NMMA partially protected keratinocytes from UVA-induced antiproliferative effects and increased DNA synthesis by 67 or 49% of the control. The co-application of UVA irradiation (10 J cm (-2)) and the essential cofactor of NO synthases tetrahydrobiopetrin (BH4, 500 microM) led to an overadditive increase in the release of nitrite as well as to a decrease in DNA synthesis. These results imply that NO is involved in the antiproliferative UVA effects in keratinocytes.

Effects of potassium and anion channel blockers on the cellular response of human osteoarthritic chondrocytes.

The aim of this article is to determine to what extent the proliferation, CD44 expression, and apoptosis behavior of cells can be influenced by the modulation of ion channel activity on the cell membrane of human osteoarthritic chondrocytes. The potassium channel blocker 4-aminopyridine (4-AP) and the chloride and anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS) were used as ion channel modulators. Assessment of the proliferation was done by incorporation of (3)H-thymidine. The detection of apoptotic cells and expression of the hyaluronic acid binding CD44-receptor were determined by flow cytometry. The results showed that 4-AP and SITS lead to a temporary increase in (3)H-thymidine incorporation, followed by a suppression of proliferation after a 12-day incubation. 4-AP causes considerable cytotoxic effects. SITS leads to necrotic cell damage. CD44 expression is increased up to 43% after incubation with 4-AP for 24 or 48 h, whereas prolonged incubation under SITS influence leads to a clear inhibition of CD44 expression. In conclusion, proliferation, CD44 expression, and apoptosis behavior of human chondrocytes can be influenced by modulation of ion channel activity. These results serve as a basis for further investigations to extend the therapeutic possibilities in the treatment of arthritis.

N-methyl-D-aspartate receptors influence the intracellular calcium concentration of keratinocytes.

In the present study, the distribution of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA)-receptor type was immunohistochemically demonstrated in healthy human skin (n = 22) and healthy buccal mucosa (n = 20). Moreover, the intracellular calcium concentration of HaCaT-cells and native human keratinocytes were studied under the influence of the selective agonist NMDA and the selective NMDA-antagonist MK-801. Immunohistochemical imaging of NMDA receptors in healthy epidermis showed a positive reaction in the stratum basale, spinosum and granulosum, whereby the greatest expression was observed in the granular layer. In the mucosal preparations, the distribution of NMDA receptors was observed to be equal in all cell layers. In the cell culture (HaCaT-cells), NMDA concentrations between 25 microM and 1 mM resulted in a significant increase in the number of cells showing elevated intracellular calcium concentration. This effect could be significantly reduced by prior application of MK-801 (100 micro M). In supplementary tests on HaCaT-keratinocytes, blockade of the keratinocytic NMDA receptors with MK-801 suppressed the differentiation of the cells (expression of cytokeratin 10). The proliferation of cells was not influenced by NMDA. The investigations showed that glutamate receptors of the NMDA type have an influence on keratinocytic calcium concentration. This appears especially important for the differentiation of keratinocytes.