Integrin ß1, Osmosensing, and Chemoresistance in Mouse Ehrlich Carcinoma Cells.

BACKGROUND/AIMS: Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin ß1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines. METHODS: Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin ß1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis. RESULTS: We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and ß1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin ß1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin ß1 promoted cisplatin-induced caspase activity in adherent cells. CONCLUSION: Our data identifies integrin ß1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells.

Monovalent ions control proliferation of Ehrlich Lettre ascites cells.

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl(-) ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl(-) substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pH(i)) recovery after acid loading and increasing pH(i). Results using the potential sensitive dye DiBaC4(3) showed a reduced Cl(-) conductance in S compared with G(1) followed by transmembrane potential (E(m)) hyperpolarization in S. Cl(-) substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine-tuning of E(m) in S phase and altered Ca2+ signaling.

ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl- channels in adherent Ehrlich Lettré and NIH3T3 cells.

Addition of H(2)O(2) (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 +/- 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K(+), which was significant within 8 min, whereas no concomitant increase in the K(+) or Cl(-) conductances could be observed. The H(2)O(2)-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca(2+)-activated K(+) channels, respectively, and is unaffected by a raise in extracellular K(+) concentration to a value that eliminates the electrochemical driving force for K(+). On the other hand, the H(2)O(2)-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO(3)(-) for Cl(-), and when the driving force for KCl cotransport was omitted. It is suggested that H(2)O(2) activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K(+) and Cl(-) channels. Addition of H(2)O(2) to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K(+), whereas no additional increase in the K(+) and Cl(-) conductance was observed. The effect of H(2)O(2) on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H(2)O(2)-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H(2)O(2) to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K(+) and Cl(-) conductances after hypotonic cell swelling. Hence, H(2)O(2) induces KCl cotransport or K(+) and Cl(-) channels in nonadherent and adherent cells, respectively.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Single point mutations of aromatic residues in transmembrane helices 5 and -6 differentially affect TRPV4 activation by 4α-PDD and hypotonicity: implications for the role of the pore region in regulating TRPV4 activity.

The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure-function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5-6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Modulation of the transient receptor potential vanilloid channel TRPV4 by 4alpha-phorbol esters: a structure-activity study.

The mechanism of activation of the transient receptor potential vanilloid 4 (TRPV4) channel by 4alpha-phorbol esters was investigated by combining information from chemical modification of 4alpha-phorbol-didecanoate (4alpha-PDD, 2a), site-directed mutagenesis, Ca(2+) imaging, and electrophysiology. Binding of 4alpha-phorbol esters occurs in a loop in the TM3-TM4 domain of TRPV4 that is analogous to the capsaicin binding site of TRPV1, and the ester decoration of ring C and the A,B ring junction are critical for activity. The lipophilic ester groups on ring C serve mainly as a steering element, affecting the orientation of the diterpenoid core into the ligand binding pocket, while the nature of the A,B ring junction plays an essential role in the Ca(2+)-dependence of the TRPV4 response. Taken together, our results show that 4alpha-phorbol is a useful template to investigate the molecular details of TRPV4 activation by small molecules and obtain information for the rational design of structurally simpler ligands for this ion channel.

Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells.

Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current decreased in G1 and increased in early S phase, compared to that in G0. The isotonic steady-state current, which seems to be predominantly VRAC, also decreased in G1, and increased again in early S phase, to a level similar to that in G0. In contrast, the maximal CaCC current (500 nM free Ca2+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle-dependent changes in the capacity for conductive Cl- transport. It is suggested that in ELA cells, entrance into the S phase requires an increase in VRAC activity and/or an increased potential for regulatory volume decrease (RVD), and at the same time a decrease in CaCC magnitude.

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin.

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P(2)] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by approximately 20% in modestly, and approximately 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P(2) analogue or a PtdIns(4,5)P(2)-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P(2). It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.