KCa3.1-dependent uptake of the cytotoxic DNA-binding dye Hoechst 33258 into cancerous but not healthy cervical cells.

The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.

A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution.

Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q2PISH allows for the analysis of both qualitative and quantitative (q2) gene expression on cultured cells for quality control measures with single cell resolution. q2PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q2PISH serves as an accurate quality control with single cell resolution for cultured cells.