Early ionic events associated with phorbol ester induced differentiation and inhibition of cell growth in hematopoietic tumor cell lines

The effects of 12-O-tetradecanoylphorbol-13 acetate (TPA) on DNA synthesis, phenotypic expression, cytoplasmic Ca2+ (Ca2+i), intracellular pH (pHi) and membrane potential were studied in the monoblastic U-937 and the erythroleukemic K-562 cells. In both cell lines DNA synthesis was inhibited and in the U-937 cells this was accompanied by the appearance of macrophage differentiation markers. The erythroid characteristics of K-562 cells, on the other hand, were markedly suppressed. Intracellular pH (pHi) was increased by TPA treatment; however, while the alkalinization of K-562 cells was dependent on the presence of extracellular Na+, the response of U-937 cells was unaffected by the removal of this cation. In each cell type the protein kinase C (PKC) inhibitor H-7 largely attenuated the TPA induced increase of pHi. Moreover, the alpha-stereoisomer of TPA, which does not activate PKC, had no effects. TPA caused a dose-dependent decrease in Ca2+i which was more pronounced in U-937 cells. Measurements of membrane potential revealed a marked TPA depolarization of the K-562 cells, but no such effects were observed in the U-937 cell line. The depolarizing response of K-562 cells could be abolished by substituting extracellular Na+ with choline+, indicating the presence of a TPA sensitive Na+ permeability. The results show that the phorbol ester TPA, which inhibits proliferation and causes phenotypic modulation, also induced a number of early, apparently PKC dependent and cell type specific, changes of intracellular ion activities. The possible role of intracellular ion fluxes in the regulation of cell growth and differentiation is discussed.     

Reduction of intracellular pH as a possible mechanism for killing cells in acidic regions of solid tumors: effects of carbonylcyanide-3-chlorophenylhydrazone

The environment of cells within solid tumors is known to be acidic relative to that in normal tissue, and the viability of tumor cells may depend on mechanisms which maintain intracellular pH (pHi) above the extracellular pH (pHe). We have assessed therefore the toxicity in vitro of the proton ionophore carbonylcyanide-3-chlorophenylhydrazone (CCCP), since this agent has been reported to be capable of transporting H+ equivalent through artificial lipid bilayers and mitochondrial membranes. CCCP was toxic to the human bladder carcinoma cell line MGHU1 and to the murine mammary sarcoma cell line EMT-6 only at pH, less than 6.5. CCCP transported H+ equivalents through cell membranes at physiological (7.35) and low pHc (6.20). Cell lines were found to have steady-state pHi values approximately 0.1 to 0.2 pH units above pHc at pHc less than 6.50. Addition of CCCP led to a decrease in steady-state pHi values as compared to untreated cells at pHc less than 6.50, whereas there was no apparent effect of CCCP on steady-state pHi values at pHc greater than 6.50. The CCCP-induced reduction in steady-state pHi combined with the uncoupling of oxidative phosphorylation by CCCP appeared to be the major mechanisms leading to cell death at pHc less than 6.50. The toxicity of CCCP under acidic conditions was enhanced by amiloride and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid, agents which are known to inhibit membrane-based ion exchange mechanisms which regulate pHi under acidic conditions. When both agents were combined with CCCP, cell killing was observed at pHc less than 7.30. Our results suggest that mechanisms which regulate pHi under acidic conditions which occur in solid tumors may represent targets for new forms of tumor-specific therapy.     

Heat-induced changes in intracellular Na+, pH and bioenergetic status in superfused RIF-1 tumour cells determined by 23Na and 31P magnetic resonance spectroscopy.

The acute effects of hyperthermia on intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) were investigated in superfused Radiation Induced Fibrosarcoma-1 (RIF-1) tumour cells using shift-reagent-aided 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy. Hyperthermia at 45 degrees C for 30 min produced a 50% increase in Na, a 0.42 unit decrease in pHi and a 40-45% decrease in NTP/P(i). During post-hyperthermia superfusion at 37 degrees C, pHi and NTP/P(i) recovered to the baseline value, but Na initially decreased and then increased to the hyperthermic level 60 min after heating. Hyperthermia at 42 degrees C caused only a 15-20% increase in Nai+. In the presence of 3 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, the increase in Nai+ during 45 degrees C hyperthermia was attenuated, suggesting that the heat-induced increase in Nai+ was mainly due to an increase in Na+/H+ anti-porter activity. EIPA did not prevent hyperthermia-induced acidification. This suggests that pHi is controlled by other ion exchange mechanisms in addition to the Na+/H+ exchanger. EIPA increased the thermo-sensitivity of the RIF-1 tumour cells only slightly as measured by cell viability and clonogenic assays. The hyperthermia-induced irreversible increase in Nai+ suggests that changes in transmembrane ion gradients play an important role in cell damage induced by hyperthermia.     

Cytotoxicity of compounds that interfere with the regulation of intracellular pH: a potential new class of anticancer drugs

The extracellular pH (pHe) in many solid tumors is often lower than in normal tissues. Cells may survive conditions of acid pHe because antiports in their membrane exchange Na+ for H+, or HCO3- for Cl-, and thus regulate the intracellular pH (pHi). We have therefore assessed the effects of drugs which interfere with regulation of pHi on survival of Chinese hamster ovary and human bladder cancer MGH-U1 cells in tissue culture. Nigericin, an ionophore which acidifies the cytoplasm when cells are placed in medium at low pHe, was not toxic at pHe 6.5 or above but became very toxic as pHe was reduced below this value. Amiloride and 4,4'-diisothiocyanostilbene 2,2-disulfonic acid, inhibitors of the Na+/H+ and HCO3-/Cl- exchangers, respectively, decreased pHi in the presence of nigericin at low pHe. These drugs showed little or no toxicity in the pHe range of 6.0-7.0 but added greatly to the toxicity of nigericin. A combination of all three drugs led to toxicity in the pHe range of 6.5-6.8, well within the measured range of tumor pH, but not at pHe 7.0 or above. A combination of low pH and hypoxia, two conditions likely to be found in regions distant from tumor blood vessels, caused cell mortality in the absence of drugs, and this effect was increased by nigericin used alone or in combination with amiloride and 4,4'-diisothiocyanostilbene 2,2-disulfonic acid. These drugs may be regarded as prototypes for potential new anticancer agents that might achieve selective killing of tumor cells by interfering with the regulation of intracellular pH.     

Interdependent regulation of intracellular acidification and SHP-1 in apoptosis

The G protein-coupled receptor agonist somatostatin (SST)-induces apoptosis in MCF-7 human breast cancer cells. This is associated with induction of wild-type p53, Bax, and an acidic endonuclease. We have shown recently that its cytotoxic signaling is mediated via membrane-associated SHP-1 and is dependent on decrease in intracellular pH (pHi) to 6.5. Here we investigated the relationship between intracellular acidification and SHP-1 in cytotoxic signaling. Clamping of pHi at 7.25 by the proton-ionophore nigericin abolished SST-signaled apoptosis without affecting its ability to regulate SHP-1, p53, and Bax. Apoptosis could be induced by nigericin clamping of pHi to 6.5. Such acidification-induced apoptosis was not observed at pHi <6.0 or >6.7. pHi-dependent apoptosis was associated with the translocation of SHP-1 to the membrane, enhanced in cells overexpressing SHP-1, and was abolished by its inactive mutant SHP-1C455S. Acidification caused by inhibition of Na+/H+ exchanger and H+ ATPase (pHi = 6.55 and 6.65, respectively) also triggered apoptosis. The effect of concurrent inhibition of Na+/H+ exchanger and H(+)-ATPase on pHi and apoptosis was comparable with that of SST. Acidification-induced, SHP-1-dependent apoptosis occurred in breast cancer cell lines in which SST was cytotoxic (MCF-7 and T47D) or not (MDA-MB-231). We conclude that: (a) SST-induced SHP-1-dependent acidification occurs subsequent to or independent of the induction of p53 and Bax; (b) SST-induced intracellular acidification may arise due to inhibition of Na+/H+ exchanger and H(+)-ATPase; and (c) SHP-1 is necessary not only for agonist-induced acidification but also for the execution of acidification-dependent apoptosis. We suggest that combined targeting of SHP-1 and intracellular acidification may lead to a novel strategy of anticancer therapy bypassing the need for receptor-mediated signaling.     

The potential of lactate and succinate to kill nutrient deprived tumor cells by intracellular acidification

We have investigated the ability of the weak acids, lactate, succinate, and the monomethylester of succinate, to cause intracellular acidification of EMT-6 and MGH-U1 cells. Each of the three substances caused a decrease of intracellular pH (pHi) when the cell lines were exposed at low extracellular pH (pHe) in the range 6.0-6.5. Only monomethylsuccinate caused intracellular acidification at neutral pHe. The fall in pHi increased with increasing dose of each agent and with decreasing pHe. The pHi recovered to almost normal values after exposure of 30 minutes to 50 mM lactate, but there was little or no recovery of pHi in the presence of succinate or monomethylsuccinate. Succinate and its methylester were toxic to cells at low pHe (less than 6.5), and cell killing increased with exposure time and with dose of the agents used. Lactate did not cause cell death at low pHe, and none of the three substances exhibited any cytotoxicity at neutral pHe. Solid tumors are known to have an acidic microenvironment, and pHe may be particularly low in regions of hypoxia. Succinate and its monomethylester may have the potential to kill cells in acidic regions of tumors and might therefore enhance the effect of radiation.     

Effects of amiloride on intracellular pH and thermosensitivity.

Amiloride, a diuretic drug, is a potent inhibitor of Na+/H+ exchange through the plasma membrane, and has been reported to enhance thermal damage in tumor cells in vitro. We investigated the possible relationship between changes in the thermal response of SCK mouse mammary tumor cells in vitro and changes in intracellular pH (pHi) due to amiloride in the present study. At a concentration of 0.5 mM, amiloride reduced the shoulder (Dq) without causing significant change in the slope (Do) of the survival curve of SCK cells heated once at 43 degrees C. On the other hand, 0.5 mM of amiloride sensitized thermotolerant cells to heat as shown by a reduction in Do. The presence of amiloride during the interval between the first and second heatings slightly reduced or inhibited the development of thermotolerance. The pHi was measured with the BCECF fluorescence method. The presence of 0.5 mM amiloride significantly reduced pHi in both pH 7.2 and 6.6 medium. Heating the SCK cells at 43 degrees C in pH 7.2 or 6.6 medium also reduced the pHi. The combined effect of heat and amiloride in reducing the pHi of SCK cells was additive. These results suggest that the effects of amiloride on the thermal response of SCK cells might be mediated in part by a decrease in pHi. The possibility that the effects of amiloride on the thermal response of tumor cells are mediated by other biochemical changes, such as inhibition of protein synthesis, however, could not be ruled out.     

Regulation of intracellular pH in tumor cell lines: influence of microenvironmental conditions.

The effect of microenvironmental factors on the regulation of intracellular pH (pHi) in MGH U1 cells and EMT-6 cells was studied using the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange were found to be present in both cell types. The activity of both exchangers was dependent on pHi, with low levels of activity at neutral pH and an increase in activity as pHi fell. The level of extracellular pH (pHe) also influenced the operation of the exchangers, with a fall in activity as pHe was reduced over the range 7.4-6.6. This effect was more marked for the Na(+)-dependent Cl-/HCO3- exchanger than for the Na+/H+ antiporter, suggesting that under conditions of reduced pHe the Na+/H+ antiporter is the major mechanism for regulation of pHi. Neither 6 h of radiobiological hypoxia nor variations in the extracellular [Ca2+] over the range 1-6 mM had an effect on the regulation of pHi, while extracellular lactate (5-10 mM) caused a small, concentration-dependent decrease in the combined activity of both exchangers. We conclude that under the microenvironmental conditions found in some regions of tumors, Na+/H+ exchange may be the major method of regulation of pHi.     

Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin

Multidrug resistant (MDR) tumor cells exhibit an altered pH gradient across different cell compartments, which favors a reduced intracellular accumulation of antineoplastic drugs and a decreased therapeutic effect. In our study, we have observed that the activity and expression of Na+/H+ exchanger (NHE), which is involved in the homeostasis of intracellular pH (pHi), are increased in doxorubicin-resistant (HT29-dx) human colon carcinoma cells in comparison with doxorubicin-sensitive HT29 cells. The pH(i) was significantly higher in HT29-dx cells, which accumulated less doxorubicin than HT29 cells. The NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) significantly reduced the pHi value and increased the intracellular accumulation of doxorubicin in both cell populations: in the presence of EIPA HT29-dx cells accumulated as much drug as control HT29 cells. On the other hand, monensin, a Na+/H+ ionophore mimicking NHE activation, and phorbol 12-myristate 13-acetate (PMA), which stimulates NHE, significantly increased the pHi and decreased the drug accumulation in HT29 cells to values similar to those observed in control HT29-dx cells. EIPA potentiated the cytotoxic effect of doxorubicin in HT29 cells, and made HT29-dx cells as sensitive to the cytotoxic effect of the drug as control HT29 cells. Instead, PMA and monensin made HT29 cells as insensitive to doxorubicin as HT29-dx cells. These results suggest that in MDR cells the higher cytosolic pH is likely to decrease drug accumulation, and that such resistance can be reverted by inhibiting the NHE activity. This result opens the possibility to revert MDR with the clinical use of NHE inhibitors.     

The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects.

Mean values of extracellular pH (pHe) in tumours tend to be about 0.5 pH units lower than in normal tissues, whereas values of intracellular pH (pHi) in tumours and normal tissues are similar. Previous studies have shown that drugs that acidify cells at lower pHe such as nigericin, used alone or with agents that inhibit the regulation of pHi, have toxicity to cultured cells at pHe < 6.5 in short-term exposure; these agents also lead to modest anti-tumour effects in mice when given acutely. To evaluate the long-term effects of these drugs at levels of pHe that might occur commonly in tumours, we exposed cells for up to 72h at pHe 6.8 or 7.2 in vitro. Nigericin (0.033 microM) caused time-dependent cell killing of murine KHT and EMT-6 cells at pHe 6.8 (but not at pHe 7.2) with a surviving fraction approximately 5 x 10(-3) after 72 h exposure. Cell killing was increased in the presence of 4,4-diisothiocyanstilbene 2,2-disulphonic acid (DIDS), an inhibitor of Na+-dependent HCO3-/CI- exchange, and to a lesser extent in the presence of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of Na+/H+ exchange. Cell killing was exquisitely sensitive to the level of pHe. Osmotic pumps were used to obtain a 72 h continuous infusion of nigericin in mice; this led to dose-dependent killing of cells in KHT tumours with surviving fraction of approximately 0.1 at maximum tolerated doses. Hydralazine, which may cause tumour hypoxia and lower pHi as well as pHe, caused cytotoxity when given alone by chronic infusion, and enhanced the cytotoxicity due to nigericin. The addition of DIDS and/or EIPA (using two pumps) further enhanced anti-tumour toxicity, with a surviving fraction of approximately 0.002 at tolerated doses of the four drugs used to treat KHT tumours. The experiments demonstrate the activity of drugs that inhibit the regulation of pHi against murine tumours when delivered by chronic infusion.     

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells.

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103

TAS-103 is a DNA intercalating indeno-quinoline derivative that stimulates DNA cleavage by topoisomerases. This synthetic drug has a broad spectrum of antitumor activity against many human solid tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic leukemia cells treated with TAS-103. The treatment of proliferating human leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the fluorochromes 3,3-dihexyloxacarbocyanine iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-Asp-Glu-Val-Asp-pNA, poly(ADP-ribose) polymerase, and pro-caspase-3 reveal unambiguously that caspase-3 is a key mediator of the apoptotic pathway induced by TAS-103. Caspase-8 is also cleaved, and the bcl-2 oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of TAS-103.     

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.     

The sensitivity of a malignant cell line to hyperthermia (42 degrees C) at low intracellular pH.

The postulate that low intracellular pH acts as a preconditioner for the destructuve effects of hyperthermia (42 degrees C) was examined, using a heat-sensitive line of malignant cells derived from rat mammary gland (SDB). Intracellular pH (pHi) was measured indirectly, from the distribution of the weak, non-metabolizable organic acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) between intra- and extra-cellular water. Respiration, aerobic and anaerobic and anaerobic glycolysis of the cells were studied at normal pHi (pH 7-0-7-4) or at low pHi (pH 6-2-6-6) and at 38 degrees C or 42 degrees C over 6 h in Warburg manometers; the ability of the cells to replicate in culture was examined after 3 h or 6 h incubation in the flasks. The relationship between pHi and extracellular pH (pHe) depended upon the buffer system used and the exact pH in question; no assumption regarding pHi based only on pHe measurement could be made. At 38 degrees C and low pHi, the Pasteur effect became negative due to a relatively greater inhibition of anaerobic than aerobic glycolysis. Respiration was unaffected and cell replicative ability unimpaired. At 42 degrees C and normal pHi, respiration was totally inhibited after 4 h and the Pasteur effect was decreased, in this case due to a compensatory increase in aerobic glycolysis without alteration in anaerobic CO2 production. Low pHi in the presence of hyperthermia enabled cell respiration to continue at a reduced level with no further change in glycolysis. There was delayed cell replication after 3 h at 42 degrees C and inability to multiply following 6 h hyperthermia: low pHi did not influence these results. It is concluded that with these cancer cells, pHi values maintained in the region of 1-0 pH unit below normal for 6 h had no deleterious effect on the cells. No sensitizing effect of the low pHi for the destructive effect of hyperthermia on the cells was observed.     

Effects of bafilomycin A1, a vacuolar type H+ ATPase inhibitor, on the thermosensitivity of a human pancreatic cancer cell line.

PURPOSE: It has been known that the thermosensitivity of tumour cells can be increased by lowering intra-cellular pH (pHi) by inhibiting pHi control mechanisms. The pHi is partially controlled by transport of H+ from cytoplasm into endocytic and secretary systems in the cells mediated by vacuolar type H+ATPase and also by transport of H+ through plasma membrane. METHODS: This study investigated the effects the bafilomycine A1, an inhibitor of the vacuolar type H+ATPase and the EIPA, an inhibitor of the Na+/H+ exchanger in plasma membrane, on thermosensitivity of AsPC-1 cells, a human pancreatic cancer cell line. It also investigated the effects of combination of bafilomycine A1 and EIPA. RESULTS: The treatment of cancer cells with bafilomycine A1 or EIPA individually slightly lowered pHi of the cells in vitro and increased the thermosensitivity of the cells. CONCLUSION: The combination of these two drugs significantly lowered pHi and increased thermosensitivity of cancer cells in vitro and enhanced the heat-induced the growth delay of AsPC-1 tumours grown s.c in the legs of BALB/cA nude mice.     

Effects of acute pH 6.6 and 42.0 degrees C heating on the intracellular pH of Chinese hamster ovary cells

Incubation of Chinese hamster ovary cells in pH 6.6 medium for 4 h prior to and during 42.0 degrees C heating enhanced thermal cell killing compared to cells heated under normal pH 7.3 conditions. We examined the relationship between the extracellular pH and intracellular pH (pHi) of Chinese hamster ovary cells using a flow cytometer with the pH-sensitive fluorescent molecule 2,3-dicyanohydroquinone. Using either normal (7.3) or low (6.6) pH conditions, the mean pHi and population pHi heterogeneity was studied as a function of time at 42.0 degrees C. Cells incubated at pH 6.6 for 4 h had a resting pHi 0.14 to 0.19 pH units lower than cells at normal pH 7.3, indicating the presence of an active pHi regulatory system. Heating 1 h at 42.0 degrees C at normal pH caused an increase in the pHi of 0.14 pH units. With further heating the cells gradually returned to the unheated (7.3) control levels. Similar pHi changes were observed with the cells incubated and heated at pH 6.6. However, the mean pHi was always more acidic than cells heated at normal pH. Active pHi regulation was still possible for a substantial (greater than 30%) number of cells even after 10 h of heating under low pH conditions. These results suggest that a breakdown in pHi regulation is not the mechanism of low pH-induced heat sensitization.     

Regulation of intracellular pH in subpopulations of cells derived from spheroids and solid tumours.

Solid tumours are known to develop regions of extracellular acidity and survival of tumour cells in such regions depends on membrane-based mechanisms which regulate intracellular pH (pHi). We have therefore developed a method, based on dual staining of cells and flow cytometry, to study the regulation of pHi in subpopulations of tumours and spheroids. The activity of membrane-based pHi regulating transporters was studied in EMT-6 and MGH U1 cells grown in monolayer culture, spheroids, and tumours. pHi was measured with the fluorescent pH probe 2''7''-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, and Hoechst 33342 was used to identify cells from different regions of tumours and spheroids. In monolayer culture, incubation of cells for 18 h at pHe 6.6 led to a 1.3-1.5-fold enhancement in the activity of both the Na+/H+ exchanger and the Na(+)-dependent Cl-@HCO3- exchanger. This effect was prevented by the protein synthesis inhibitor cycloheximide. Cells from the centre of EMT-6 spheroids had increased activity of the Na+/H+ exchanger compared to cells from the periphery, when spheroids were grown in medium at pH 6.6, but not at 7.4. By contrast, in MGH U1 spheroids, cells from the centre had increased activity of the Na+/H+ antiport under both sets of conditions. There was no significant difference in the activity of the Na+/H+ exchanger in cells derived from different subpopulations of EMT-6 tumours or MGH U1 xenografts in nude mice. Although upregulation of Na+/H+ exchange occurs after exposure to acidic conditions in vitro, the microenvironmental conditions found within solid tumours do not appear to cause this effect. Our results suggest the feasibility of pharmacological inhibition of Na+/H+ exchange activity as an approach to therapy directed against nutrient-deprived tumour cells.     

Rapid decrease of intracellular pH associated with inhibition of Na+/H+ exchanger precedes apoptotic events in the MNK45 and MNK74 gastric cancer cell lines treated with 2-aminophenoxazine-3-one

The effects of Phx-3 on changes in intracellular pH (pHi) in the MKN45 and MKN74 human gastric cancer cell lines were evaluated in order to determine the mechanism for the proapoptotic effects of 2-aminophenoxazine-3-one (Phx-3) on these cells. Phx-3 (100 μM) reduced pHi in MKN45 from 7.45 to 5.8, and in MKN74 from 7.5 to 6.2 within 1 min of engagement with these cells. Such a decrease of pHi was closely correlated with the dose of this phenoxazine and continued for 4 h. The activity of Na+/H+ exchanger isoform l (NHE1), which is involved in H+ extrusion from the cells, was dose-dependently suppressed by Phx-3 in these cells, and was greatly suppressed in the presence of 100 μM Phx-3. This result indicates that the decrease of pHi in MKN45 and MKN74 cells is closely associated with the inhibition of NHE1 in these cells. The morphology of these cells at 24 h after treatment with Phx-3 indicated shrinkage of the cells and condensation of the nuclear chromatin structure, which are characteristic of the apoptotic events in these gastric cancer cells. Cytotoxicity of Phx-3 against MKN45 and MKN74 cells was extensive because almost all MKN45 cells lost viability at 24 h in the presence of 20 μM Phx-3, and nearly 50% of the MKN74 cells lost viability in the presence of 50 μM Phx-3. These results suggest that rapid and extensive decrease of pHi in human gastric cancer MKN45 and MKN74 cells caused by Phx-3 might disturb intracellular homeostasis, leading to apoptotic and cytotoxic events in these cells. Phx-3 is a good candidate for therapeutics of gastric cancer that is intractable to conventional chemopreventive therapies.     

Reduction of intracellular pH inhibits the expression of VEGF in K562 cells after targeted inhibition of the Na+/H+ exchanger

To explore the effect of inhibition of Na(+)/H(+) exchanger isoform 1 (NHE1) on the expression of vascular endothelial growth factor (VEGF) mRNA and protein in human myeloid K562 cells. The expression of VEGFmRNA was detected by RT-PCR technique. The levels of VEGF protein were measured by Western blotting and immunocytochemistry assay. pHi values were measured with fluorescence spectrophotometer. The three RT-PCR products detected were VEGF121, VEGF165, and VEGF189, respectively. Treatment of K562 cells either with amiloride (an inhibitor of NHE1) or with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, a selective inhibitor of NHE1) resulted in significant decrease of VEGF mRNA and VEGF protein levels. Either amiloride or EIPA decreased intracellular pH (pHi) values in K562 cells. These data strongly suggested that the expression of VEGF mRNA and protein in K562 cells was inhibited accompanying its reduction in pHi value after targeted inhibition of NHE1.     

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.