To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl− channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl− currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56–80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl− channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 μM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl− channel blocker, 4-acetamido-4′-isothiocyanostilbene at 100 μM. Glibenclamide (500 μM) and arachidonic acid (100 μM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl− channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd3+, a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl− currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach ∼13 μM by a biosensor technique using P2X2 receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl− channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 μM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl− channel; and third, the ATP release is not a prerequisite to activation of the Cl− channel.
Purpose: The aim of this study is to find a laboratory marker for overall survival in advanced cancer patients who were vaccinated with peptides based on pre-existing, peptide-specific CTL precursors in the circulation.Experimental Design: A group of 113 patients with advanced cancer (28 colorectal, 22 prostate, 15 lung, 14 gastric, and 34 other cancers) was enrolled in a Phase I clinical study of peptide vaccination in which peptidespecific CTL precursors of prevaccination peripheral blood mononuclear cells were measured, followed by vaccination with these peptides (maximum of four). For cellular responses, pre and postvaccination (sixth) peripheral blood mononuclear cells were provided for measurement of both peptide-specific CTL precursors by IFN-␥ release assay and tumor reactivity by 51 Cr release assay. Delayed type hypersensitivity was also measured. For humoral response, pre and postvaccination (sixth) sera were provided for measurement of peptide-reactive IgG by an ELISA.Results: The median survival time and 1-year survival rate of the total cases were 346 ؎ 64.9 days and 44.6%, respectively, and those of patients vaccinated more than six times (n ؍ 91) were 409 ؎ 15 days and 54.4%, respectively. In these 91 patients, the overall survival of patients whose sera showed increased levels of peptide-reactive IgG (n ؍ 60) was significantly more prolonged (P ؍ 0.0003) than that of patients whose sera did not (n ؍ 31), whereas none of cellular responses correlated with overall survival.Conclusions: Peptide-specific IgG in postvaccination sera could be a suitable laboratory maker for the prediction of prolonged survival in advanced cancer patients vaccinated with peptides based on pre-existing CTL precursors.
A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin‐luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling‐induced ATP release by 10–20 times under hypotonic conditions (<= 80 % osmolality). Glibenclamide failed to suppress swelling‐induced ATP release from C127/CFTR cells. In contrast, whole‐cell patch‐clamp recordings showed that both the cAMP‐activated ohmic Cl− currents and volume‐sensitive outwardly rectifying (VSOR) Cl− currents were prominently suppressed by glibenclamide. Gd3+ markedly blocked swelling‐induced ATP release but failed to suppress both cAMP‐ and swelling‐activated Cl− currents in the CFTR‐expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl− currents were activated normally. The continuous presence of an ATP‐hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl− currents in C127/CFTR cells. The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells. On balance, the following conclusions can be deduced. First, swelling‐induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl− channel. Second, swelling‐induced ATP release is not mediated by the VSOR Cl− channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl− channels in C127/CFTR cells.
1 Di erences in the acetylcholine (ACh)-induced endothelium-dependent relaxation and hyperpolarization of the mesenteric arteries of Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. 2 Relaxation was impaired in preparations from SHRSP and tendency to reverse the relaxation was observed at high concentrations of ACh in these preparations. 3 Relaxation was partly blocked by N G -nitro-L-arginine (L-NOARG, 100 mM) and, in the presence of L-NOARG, tendency to reverse the relaxation was observed in response to higher concentrations of ACh, even in preparations from WKY. The relaxation remaining in the presence of L-NOARG was also smaller in preparations from SHRSP. 4 The tendency to reverse the relaxation observed at higher concentrations of ACh in preparations from SHRSP or WKY in the presence of L-NOARG were abolished by indomethacin (10 mM). 5 Elevating the K + concentration of the incubation medium decreased relaxation in the presence of both indomethacin and L-NOARG. 6 Relaxation in the presence of L-NOARG and indomethacin was reduced by the application of both apamin (5 mM) and charybdotoxin (0.1 mM). This suggests that the relaxation induced by ACh is brought about by both endothelium-derived relaxing factor (EDRF, nitric oxide (NO)) and hyperpolarizing factor (EDHF), which activates Ca 2+ -sensitive K + channels. 7 Electrophysiological measurement revealed that ACh induced endothelium-dependent hyperpolarization of the smooth muscle of both preparations in the presence of L-NOARG and indomethacin; the hyperpolarization being smaller in the preparation from SHRSP than that from WKY. 8 These results suggest that the release of both NO and EDHF is reduced in preparations from SHRSP. In addition, indomethacin-sensitive endothelium-derived contracting factor (EDCF) is released from both preparations; the release being increased in preparations from SHRSP.
Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.
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