Biomedical subjects

R Skryma

Publications and source records attributed to R Skryma.

At least 19 recordsLinked to original sources

PubMed2007

Differential role of TRP channels in prostate cancer.

A major clinical problem with PC (prostate cancer) is the cell's ability to survive and proliferate upon androgen withdrawal. Indeed, deregulated cell differentiation and proliferation, together with the suppression of apoptosis, provides the condition for abnormal tissue growth. Here, we examine the differential role of TRP (transient receptor potential) channels in the control of Ca(2+) homoeostasis and growth of PC cells.

Cell Differentiation↗

PubMed2005

Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl- current associated with neuroendocrine differentiation of prostate cancer epithelial cells.

Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant, swelling-activated Cl- current (I(Cl,swell)), associated with NE differentiation of androgen-dependent LNCaP prostate cancer epithelial cells. NE-differentiating regimens, namely, chronic cAMP elevation or androgen deprivation, resulted in generally augmented I(Cl,swell) and enhanced RVD. This occurred as a result of both the increased endogenous expression of ClC-3, which is a volume-sensitive Cl- channel involved, as we show, in I(Cl,swell) in LNCaP (lymph-node carcinoma of the prostate) cells and the weaker negative I(Cl,swell) control from Ca2+ entering via store-dependent pathways. The changes in the RVD of NE-differentiated cells generally mimicked those reported for Bcl-2-conferred apoptotic resistance. Our results suggest that strengthening the mechanism that helps to maintain volume constancy may contribute to better survival rates of apoptosis-resistant NE cells.

Androgens↗

PubMed2004

2-APB inhibits volume-regulated anion channels independently from intracellular calcium signaling modulation.

It has previously been suggested that volume-regulated anion channels (VRACs) and store-operated channels (SOCs) interact with each other according to their expected colocalization in the plasma membrane of LNCaP cells. In order to study interactions between these two channels, we used 2-aminoethoxydiphenyl borate (2-APB) as a regular SOC inhibitor. Surprisingly 2-APB reduced VRAC activity in a dose-dependent manner (IC(50)=122.8 microM), but not 2,2-diphenyltetrahydrofuran (a structural analog of 2-APB). This effect was also present in keratinocytes. We conclude that 2-APB is an inhibitor of the VRAC family, and is also a potent tool to study the SOC-VRAC interaction in LNCaP cells.

Boron Compounds↗

PubMed2004

Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells.

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.

Apoptosis↗

PubMed2004

[Calcium-dependent programmed cell death in prostate cancer].

In the present review we describe the major molecular determinants of calcium homeostasis in prostate cancer cells and establish their role in the transformation to apoptosis-resistant cell phenotypes typical of advanced androgen-independent prostate cancer. We show that the hallmark of such transformation is complete loss of apoptosis pathway associated with endoplasmic reticulum calcium store depletion.

Animals↗

PubMed2003

Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells.

Although the prostate gland is a rich source of alpha1-adreno- (alpha1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP3-sensitive Ca2+ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations (PK(25) > PCs(4.6) > PLi(1.4) > PNa(1.0)), and the following permeability sequence for divalent cations: PCa(1.0) > PMg(0.74) > PBa(0.6) > PSr(0.36) > PMn(0.3). They are 4.3 times more permeable to Ca2+ than Na+ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, alpha1-ARs and m1-AChRs are functionally coupled to Ca2+-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.

Base Sequence↗

PubMed2003

A new human gene KCNRG encoding potassium channel regulating protein is a cancer suppressor gene candidate located in 13q14.3.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K+ channels. Using the patch-clamp technique we determined that KCNRG suppresses K+ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K+ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

Amino Acid Sequence↗

PubMed2002

Direct modulation of volume-regulated anion channels by Ca(2+) chelating agents.

Ca(2+) chelating agents are widely used in biological research for Ca(2+) buffering. Here we report that BAPTA, EDTA and HEDTA produce fast, reversible, voltage-dependent inhibition of swelling-activated Cl(-) current (I(Cl,swell)) in LNCaP prostate cancer epithelial cells that is unrelated to their Ca(2+) binding. BAPTA was the most effective (maximal blockade 67%, IC(50)=70 microM, at +100 mV) followed by EDTA and HEDTA. I(Cl,swell) blockade by EDTA was pH-dependent. BAPTA blocked I(Cl,swell) also in other cell types. We conclude that Ca(2+) chelating agents block I(Cl,swell) by acting directly on the underlying channel, and that the negative charge of the free chelator form is critical for the blockade.

Calcium↗

PubMed2002

[The influence of extracellular pH on volume-activated chloride current in the prostate cancer epithelial cells].

The influence of extracellular pH on characteristics of volume-activated chloride current, ICl,swell, in the human prostate cancer epithelial cell line, LNCaP, was studied using the patch-clamp technique. Acidification of the extracellular hypotonic solution used to develop the current shortened the temporal parameters of ICl,swell development and reduced its maximal density. Sudden shifts of the pH towards acidification caused fast, transient potentiation of the current followed by its sustained inhibition. Voltage-dependent inactivation of ICl,swell was the most pronounced in the narrow range of pH = 6-7. Based on the analysis of our data we hypothesize that volume-regulated anion channels underlying ICl,swell possess in their structure at least two pH-sensitive molecular groups with similar pK = 6, titration of which modulates the current amplitude and two additional proton-sensitive groups that determine channel's inactivation.

Chloride Channels↗

PubMed2001

Verapamil inhibits proliferation of LNCaP human prostate cancer cells influencing K+ channel gating.

The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K+ currents (I(K)) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type) inactivation. With 2 mM [Mg(2+)](o), I(K) activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC(50) = 11 microM) rapidly (12 s) inhibited I(K) in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 microM) did not influence whole-cell I(K), although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 microM) had no influence on I(K). Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I(K) were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 +/- 0.5 to 3.2 +/- 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 microM) reduced an open-channel probability (from 0.45 +/- 0.1 in control to 0.1 +/- 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.

Antineoplastic Agents↗

PubMed2000

Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis.

1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

Androgens↗

PubMed2000

Evidence of functional ryanodine receptor involved in apoptosis of prostate cancer (LNCaP) cells.

BACKGROUND: Very little is known about the functional expression and the physiological role of ryanodine receptors in nonexcitable cells, and in prostate cancer cells in particular. Nonetheless, different studies have demonstrated that calcium is a major factor involved in apoptosis. Therefore, the calcium-regulatory mechanisms, such as ryanodine-mediated calcium release, may play a substantial role in the regulation of apoptosis. METHODS: We assessed the presence of such functional receptors in LNCaP prostate cancer cells, using fluorimetric measurements of intracellular calcium and expression assays of mRNA encoding ryanodine receptors. RESULTS: We show here that LNCaP cells responded to caffeine, a ryanodine receptor agonist, by mobilizing calcium. Another ryanodine receptor agonist, 4-chloro-m-cresol, had a similar effect and promoted calcium release. These effects were inhibited by pretreatment with ryanodine or thapsigargin. In addition to a calcium release, caffeine was able to produce a calcium entry blocked by nickel. We used a reverse transcription-polymerase chain reaction assay to investigate the expression of ryanodine receptors in LNCaP cells. Two types of ryanodine receptor mRNAs were expressed in LNCaP cells: RyR1 and RyR2 mRNAs. Finally, we show that ryanodine receptor activation by caffeine slightly stimulates apoptosis of prostate cancer cells, and that the inhibition of these receptors by ryanodine protects the cells against apoptosis. CONCLUSIONS: The combination of results showed that LNCaP cells, derived from a human prostate cancer, express functional RyRs able to mobilize Ca(2+) from intracellular stores and which might control apoptosis.

Apoptosis↗

PubMed2000

Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line.

Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl(-) was the primary charge carrier (termed I(Cl,swell)). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br(-) approximately I(-) > Cl(-) > F(-) > methanesulfonate >> glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC conductance was determined at 9.6 +/- 1.8 pS. Conventional Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) and DIDS (100 microM) inhibited whole cell I(Cl,swell) in a voltage-dependent manner, with the block decreasing from 39.6 +/- 9.7% and 71.0 +/- 11. 0% at +50 mV to 26.2 +/- 7.2% and 14.5 +/- 6.6% at -100 mV, respectively. Verapamil (50 microM), a standard Ca(2+) antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I(Cl,swell) (genistein with an IC(50) of 2.6 microM and lavendustin A by 60 +/- 14% at 1 microM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 microM) stimulated I(Cl,swell) by 54 +/- 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.

Adenocarcinoma↗

PubMed1998

Prolactin induces an inward current through voltage-independent Ca2+ channels in Chinese hamster ovary cells stably expressing prolactin receptor.

There is still only limited understanding of the early steps of prolactin (PRL) signal transduction in target cells. Recent studies have identified some of the essential first steps: these include the rapid association of the PRL receptor with JAK tyrosine kinases and tyrosine phosphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the other hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channels involved have not been clearly established. This work was undertaken to characterise the channels responsible for calcium influx and to obtain an insight into their activation processes. Using the patch-clamp technique in the cell-attached configuration, single Ca2+ channel currents were recorded following PRL application (10 nM) in Chinese hamster ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statistical analysis showed that the recorded currents were voltage-independent, with a slope conductance of 16 pS. Although these channels were present in excised patches, the fact that PRL was unable to activate them suggested that a soluble cytoplasmic component may be required. Application of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 microM), to the inside of the excised patch membrane activated the voltage-independent 16 pS Ca2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,4,5)P5 and Ins(1,4,5)P3 did not affect channel activity while InsP6 (20 microM) had some effect, although less marked than that of Ins(1,3,4,5)P4. Using the anion-exchange HPLC technique, we then studied the effects of PRL (10 nM) on the turnover of inositol phosphates (InsPs) in CHO-E32. Our studies showed that PRL induces rapid increases in the production of Ins(1,3,4,5)P4 (207% at 30 s), InsP5 (171% at 30 s), and InsP6 (241% at 30 s). Conversely, Ins(1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomitant increase in Ins(1,3,4,5)P4, suggesting that the former could be transiently phosphorylated to produce the latter. Comparison of the production kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)P4, InsP5, and InsP6 indicated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca2+ entry through voltage-independent Ca2+ channels that may be activated by Ins(1,3,4,5)P4 and InsP6.

Animals↗

PubMed1994

Voltage-dependent Ca2+ channels in Chinese hamster ovary (CHO) cells.

Voltage-dependent Ca2+ channels were identified in CHO-K1 cells, currently used in molecular biology studies. Experimental data obtained at both macroscopic and single-channel levels using the patch-clamp technique show that the Ca2+ current in CHO cells is similar to the high-threshold L-type of Ca2+ current previously observed in excitable cells. It can be carried by Ca2+ or Ba2+ ions, blocked by both inorganic (Co2+) and organic (nifedipine, isradipine) Ca2+ channel blockers. The unitary Ca2+ channel activity was characterized by a conductance of 19 pS in 60 mM Ca2+, by single exponential distribution of open times (t = 0.84 ms) and biexponential distribution of closed times (tf = 1.67 ms, ts = 7.9 ms). However, the functional role of these Ca2+ channels in CHO cells remains unclear.

Animals↗

PubMed1994

Gonadotropin-releasing hormone induced Ca2+ influx in nonsecreting pituitary adenoma cells: role of voltage-dependent Ca2+ channels and protein kinase C.

The action mechanism of gonadotropin-releasing hormone (GnRH) on the cytosolic free calcium concentration ([Ca2+]i) and high-threshold voltage-dependent Ca2+ channel activity was studied in human nonsecreting (NS) pituitary adenoma cells. [Ca2+]i was monitored in individual cells by dual emission microspectrofluorimetry using indo 1 as intracellular fluorescent Ca2+ probe. The whole-cell recording patch-clamp technique was used to study Ca2+ channels. A short application of GnRH (1 to 100 nM) induced an increase in [Ca2+]i due to Ca2+ entry through plasma membrane voltage-sensitive L-type Ca2+ channels. Protein kinase C (PKC) depletion induced by a pretreatment with 1 microM PMA for 24 h abolished spontaneous Ca2+ transients and the action of GnRH on [Ca2+]i and Ca2+ channels. Phloretin (250 microM) and staurosporine (20 nM), two protein kinase C inhibitors, inhibited Ca2+ channel activity, thereby suppressing the effect of GnRH. On the other hand, activation of PKC by a short application of phorbol myristate acetate (10 nM) stimulated Ca2+ influx through Ca2+ channels. These findings demonstrate that, in human NS adenoma cells, GnRH (1 to 100 nM) induces an increase in [Ca2+]i, principally due to Ca2+ entry through L-type voltage-activated Ca2+ channels. PKC regulates this mechanism as well as basal ion channel activity, thus exerting both positive and negative control of [Ca2+]i in stimulated and unstimulated NS adenoma cells.

Adenoma↗

PubMed1994

Voltage-dependent ionic conductances in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are becoming a widely used biological material. A number of studies report membrane ion conductance changes after transfection of channels and receptors, but there are few data available on the properties of membrane ion conductances of CHO cells before transfection. In this work we studied voltage-dependent ionic conductances in cultures of CHO native (CHO-K1) cells. Three types of voltage-dependent ionic conductances were identified: 1) a K+ conductance showing sensitivity to Ca2+ and a unit conductance of approximately 210 pS in symmetrical 150 mM K+ outside-out patches (this conductance, which did not inactivate during a 160-ms pulse, was inhibited by 30 nM charybdotoxin but not by 30 mM extracellular tetraethylammonium); 2) a rapidly activating and inactivating tetrodotoxin (TTX)-sensitive inward current, peaking at about -10 to 0 mV (this current showed characteristics similar in many respects to Na+ current recorded in neurons); and 3) another voltage-dependent inward current, which had slow inactivation, was TTX insensitive but was blocked by Co2+ (current was also carried by Ba2+, peaked at approximately 0 to +10 mV, was identified as a Ca2+ conductance, and was inhibited by dihydropyridines but not by 10 microM omega-conotoxin). Cell-attached patch recordings of single Ca2+ channel currents demonstrated a unitary conductance of approximately 20 pS.

Animals↗