Metabolic network analysis of DB1 melanoma cells: how much energy is derived from aerobic glycolysis?

A network model has been developed for analysis of tumor glucose metabolism from (13)C MRS isotope exchange kinetic data. Data were obtained from DB1 melanoma cells grown on polystyrene microcarrier beads contained in a 20-mm diameter perfusion chamber in a 9.4 T Varian NMR spectrometer; the cells were perfused with 26 mM [1,6-(13)C(2)]glucose under normoxic conditions and 37°C and monitored by (13)C NMR spectroscopy for 6 h. The model consists of ∼150 differential equations in the cumomer formalism describing glucose and lactate transport, glycolysis, TCA cycle, pyruvate cycling, the pentose shunt, lactate dehydrogenase, the malate-aspartate and glycerophosphate shuttles, and various anaplerotic pathways. The rate of oxygen consumption (CMRO(2)) was measured polarographically by monitoring differences in pO(2). The model was validated by excellent agreement between model predicted and experimentally measured values of CMRO(2) and glutamate pool size. Assuming a P/O ratio of 2.5 for NADH and 1.5 for FADH2, ATP production was estimated as 46% glycolytic and 54% mitochondrial based on average values of CMRO(2) and glycolytic flux (two experiments).

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

Feasibility of lung cancer hyperthermia using breathable perfluorochemical (PFC) liquids. Part I: Convective hyperthermia.

Clinical studies have shown that hyperthermia in combination with radiotherapy and/or chemotherapy may be effective in the treatment of advanced cancer. No method of lung hyperthermia, however, has been accepted as standard or superior. This investigation sought to demonstrate in animals the thermal and physiologic feasibility of lung hyperthermia induced using heated breathable perfluorochemical (PFC) liquids, a method termed liquid-filled lung convective hyperthermia (LCHT). The ability to use LCHT is rooted in the development of both PFC liquid ventilation, now in clinical development with the PFC perflubron (LiquiVent), and a PFC blood substitute also in late Phase III trials (Oxygent). As LCHT background, the PFC technologies and biology are first reviewed. The physical properties of a variety of PFCs were evaluated for LCHT and it was concluded that more than one liquid is suitable based on such properties. Using total liquid ventilation type devices, LCHT was shown to deliver successfully localized (lobar) lung heating in sheep, and bilateral whole lung heating and whole-body hyperthermia in rabbits, cats and lambs. During LCHT, lung parenchymal temperatures were uniform (<1 degree C) across heated regions. In addition, based on patterns relating lung tissue temperatures to inspiratory and expiratory PFC liquid temperatures in the endotracheal tube, LCHT may minimize invasive thermometry requirements in the lung. Based on acute experiments, it was concluded that LCHT appears feasible and may simplify lung hyperthermia. It was recommended that potentially synergistic combinations of LCHT with other whole-body hyperthermia or local heating modalities, and with chemotherapeutic lung drug delivery, also be explored in the future.

Feasibility of lung cancer hyperthermia using breathable perfluorochemical (PFC) liquids. Part II: Ultrasound hyperthermia.

Enhanced local control of disease in lung cancer has been shown to improve survival, and controlled clinical trials of hyperthermia adjunctive to radiotherapy in other cancers have shown improved disease control and survival over radiotherapy alone. The challenge of lung hyperthermia, however, persists. This investigation sought to demonstrate the feasibility of localized lung hyperthermia at depth via therapeutic ultrasound. The method is based on using breathable perfluorochemical liquids as acoustic coupling media in the lung, liquids that have also been shown to enable controlled liquid-filled lung convective hyperthermia (LCHT). The ability to use both lung convective hyperthermia and liquid-filled lung ultrasound hyperthermia (LUHT) provides potential flexibility in heating patterns for the hyperthermic treatment of lung cancer with concurrent radiotherapy and/or chemotherapy. Using custom ultrasound transducers designed and built for these studies, the acoustic properties of three candidate perfluorochemicals were characterized over a range of temperatures, gas contents and ultrasound frequencies and acoustic intensities. Both sound speed and attenuation were measured in the neat liquids and in isolated lungs filled with the perfluorochemicals. Successful ultrasound hyperthermia at depth was demonstrated in vivo in sheep lung lobes in intraoperative conditions. In addition, the use of ultrasound diagnostic imaging was explored as a tool for use in conjunction with lung ultrasound hyperthermia.

Acute extracellular acidification reduces intracellular pH, 42 degrees C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Quercetin sensitizes cells in a tumour-like low pH environment to hyperthermia.

Quercetin has been shown to act as a hyperthermia sensitizer by inhibiting the synthesis of heat shock protein 70 (HSP70) in a variety of tumour cell lines. It is most effective under conditions of low pH. This study was designed to test the hypothesis that quercetin suppresses thermotolerance development in cells adapted to growth at low pH and renders them as responsive as acutely acidified cells to hyperthermia-induced cytotoxicity. Chinese hamster ovarian carcinoma cells (OvCa) were exposed to 42 degrees C hyperthermia and/or quercetin (50-200 mm) at their growth pH of either 7.3 or 6.7 or after acute acidification from 7.3 to 6.7. Thermotolerance development was measured by colony survival. HSP70 synthesis and total protein synthesis were measured by radioactive precursor pulse labelling techniques. Quercetin, in a concentration-dependent manner, reduced the rate of total protein synthesis and increased cytotoxicity equally after acute acidification to pH 6.7 or growth at pH 6.7 at 37 degrees C, and to a greater extent than it did in cells at pH 7.3. At 42 degrees C, 100 mm quercetin inhibited total protein synthesis, HSP70 synthesis and thermotolerance development to a similar extent in cells grown at pH 6.7 or acutely acidified to pH 6.7. In contrast, quercetin reduced but did not completely inhibit HSP70 synthesis and thermotolerance development in cells grown and heated at pH 7.3. These results support the hypothesis that quercetin can specifically reduce thermotolerance development in tumour cells adapted to growth at pHe 6.7 so that they respond similarly to acutely acidified cells. Since many tumours are adapted to growth at low pH and may resist a wide variety of therapeutic modalities, inhibition of thermotolerance expression by quercetin may not only enhance the response to hyperthermia but the response to commonly used therapies such as chemotherapy and radiation.

Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing.

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42 degrees C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2 h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42 degrees C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42 degrees C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42 degrees C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.

Non-invasive magnetic resonance thermometry using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA(-)).

Non-invasive thermometry is pivotal to the future advances of regional hyperthermia as a cancer treatment modality. Current magnetic resonance (MR) thermometry methods suffer from poor thermal resolution due to relatively weak dependence of chemical shift of the (1)H water signal on temperature. This study evaluated the feasibility of using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA(-)) for MR thermometry. TmDOTA(-) is non-toxic and the gadolinium complex of DOTA(4-) is widely used as a MR contrast agent. The results demonstrate that the temperature dependence of the TmDOTA(-) proton shifts are about two orders of magnitudes higher than the water proton and, thus, provide excellent accuracy and resolution. In addition, TmDOTA(-) proton shifts are insensitive to the paramagnetic complex concentration, pH, Ca(2+) or presence of plasma macromolecules and ions. Because hyperthermia is known to produce changes in tissue pH and other physiological parameters, these properties of TmDOTA(-) greatly simplify the procedures for using the lanthanide complex for MR thermometry. Application of TmDOTA(-) for measurement of temperature in a subcutaneously implanted human melanoma xenograft is demonstrated. Finally, the feasibility of imaging one of the (1)H resonances of the lanthanide complex is demonstrated in phantom experiments. Overall, TmDOTA(-) appears to be a promising probe for MR thermometry in vivo.

Betulinic acid sensitization of low pH adapted human melanoma cells to hyperthermia.

Betulinic acid is a known inducer of apoptosis in human melanoma that is most effective under conditions of low pH. It was hypothesized that betulinic acid, in combination with acute acidification and/or hyperthermia, would induce higher levels of apoptosis and cytotoxicity in low pH-adapted human melanoma cells than in cells grown at pH 7.3. DB-1 human melanoma cells, adapted to a tumour-like growth pH of 6.7, were exposed to hyperthermia (2h at 42 degrees C) and/or betulinic acid (4-10 microg/ml) and compared with cells grown at a physiological pH of 7.3 or after acute acidification from pH 7.3-6.3 or pH 6.7-6.3. Betulinic acid induced higher levels of apoptosis and cytotoxicity in low pH-adapted cells than in cells grown at pH 7.3, as measured by the terminal deoxynucleotidyl transferase (TdT) DNA fragmentation assay (TUNEL), the MTS cell viability assay, and single cell survival. Acute acidification of low pH adapted cells rendered them more susceptible to betulinic acid-induced apoptosis and cytotoxicity. In the presence of hyperthermia at 42 degrees C for 2 h, cells grown at pH 7.3 were not sensitized to heat killing by betulinic acid, whereas cells grown at pH 7.3 and acutely acidified to pH 6.3, cells adapted to growth at pH 6.7 and cells adapted to growth at pH 6.7 and acutely acidified to pH 6.3 were all similarly sensitized to heat killing by betulinic acid, with survival values of 5, 9 and 2%, respectively. It is concluded that betulinic acid may be useful in potentiating the therapeutic efficacy of hyperthermia as a cytotoxic agent in acidotic areas of tumours with minimal effect in normal tissues growing at pH 7.3.

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Absence of Crabtree effect in human melanoma cells adapted to growth at low pH: reversal by respiratory inhibitors.

Because many tumors are acidic and hypoxic relative to normal tissues, glycolysis and oxygen consumption were investigated in early-passage human melanoma cells adapted to growth at pH 6.7. In the absence of glucose, the basal rate of oxygen consumption in low pH-adapted cells was 75% of that in cells grown at pH 7.3. The rate of lactic acid production in low pH-adapted cells was increased 4-fold by exposure to 16.7 mM glucose compared with a 10-fold increase in cells grown at pH 7.3. Furthermore, in low pH-adapted cells the rate of oxygen consumption was stimulated by the addition of glucose in contrast to the inhibition of oxygen consumption by elevated glucose in cells grown at pH 7.3 (i.e., the Crabtree effect). Both low pH-adapted cells and cells grown at pH 7.3 exposed to glucose plus 0.35 mM meta-iodo-benzylguanidine (MIBG), an inhibitor of mitochondrial respiration, had oxygen consumption reduced by approximately 60% and lactic acid production increased by approximately 65% relative to glucose alone. Although adaptation to growth at low pH was associated with a loss of the Crabtree effect and a higher ratio of oxygen consumption to lactic acid production, the rate of glycolysis was the same in both growth conditions in the presence of 0.1 mM dinitrophenol, an uncoupler of ATP synthesis. This indicates that the glycolytic capacity of low pH-adapted cells remains unchanged. Therefore, tumor acute acidification and oxygenation can be achieved by exposure to hyperglycemia combined with MIBG to improve therapeutic response.

Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport.

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level +/- standard error of the mean, 26 mmol/L +/- 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), alpha-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4.4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). MATERIALS AND METHODS: The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. RESULTS: In small tumors (< or = 8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the beta-nucleoside triphosphate (NTP)/inorganic phosphate (Pi) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P1 and beta-NTP/P1 ratios, respectively. The combination of hyperglycemia. MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. CONCLUSION: These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermnia or other pH-sensitive therapeutic modalities.

Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: a 31P magnetic resonance spectroscopy study.

In vivo 31P magnetic resonance spectroscopy demonstrates that human melanoma xenografts can be significantly acidified by induction of hyperglycemia combined with administration of m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. In melanoma xenografts (< or =8 mm diameter), intracellular pH (pHi, measured by the chemical shift of the Pi resonance) and extracellular pH (pHe, measured with 3-aminopropylphosphonate) was reduced by less than 0.2 unit during i.v. infusion of glucose for 40 min. Administration of MIBG (30 mg/kg) under hyperglycemic conditions (26 mM) reduced tumor pHi and pHe by approximately 0.4 (P < 0.001) and approximately 0.6 (P < 0.001) unit, respectively; coincidentally, the nucleoside triphosphates:Pi ratio decreased approximately 60% (P < 0.004) relative to the baseline level. Minimal changes in pHi and pHe and a small decrease in nucleoside triphosphates:Pi ratio (26%, P = 0.2) were observed in liver in response to MIBG plus hyperglycemia. These results suggest that under normoglycemic and hyperglycemic conditions, small human melanoma xenografts (< or =8 mm) may exhibit a relatively high level of oxidative phosphorylation that may be blocked by MIBG. The acidification may result from increased lactate production as a direct effect of MIBG inhibition of respiration in mitochondria of tumor cells, or through indirect systemic effects, which remain to be identified. The synergetic effects of MIBG and hyperglycemia result in significant acidification of the tumor and a decrease in tumor bioenergetic status, and the effects are largely selective for tumors in comparison with normal tissues.

Isotope selection for permanent prostate implants? An evaluation of 103Pd versus 125I based on radiobiological effectiveness and dosimetry.

Transperineal interstitial permanent prostate brachytherapy (TIPPB) has become an increasingly popular treatment for early-stage/favorable-risk adenocarcinoma of prostate. Within TIPPB, permanent implants often use either (103)Pd (T(1/2) = 17 days) or (125)I (T(1/2) = 60 days). This review compares the radiobiological and treatment planning effectiveness of (103)Pd and (125)I implants by using the linear-quadratic model with recently published data regarding: prostate tumor cell doubling times, T(pot), alpha and alpha/beta, ratio. The tumor potential doubling times (T(pot)) were determined based on recently published proliferation constants (K(p)). The initial slope of the cell radiation dose survival curve, alpha, the terminal slope beta and the alpha/beta ratio were taken from recent published clinical and cellular results. The total dose delivered from each isotope was the dose used clinically, that is, 120 Gy for (103)Pd and 145 Gy for (125)I. Dale's modified linear-quadratic equation was used to estimate the biological effective dose, the cell-surviving fraction, the effective treatment time, and the wasted radiation dose for different values of T(pot). Treatment plans for peripherally loaded implants were compared. The T(pot) reported for organ-confined prostate carcinomas varied from 16 to 67 days. At short T(pot) both isotopes were less effective, but (103)Pd had much less dependence on T(pot) than (125)I. However, at long T(pot) both isotopes produced similar effects. The minimum surviving fraction for exposure to (103)Pd decreased from 1.40 x 10(-4) to 1.31 x 10(-5) as the T(pot) increased from 16 to 67 days. By contrast for exposure to (125)I, the minimum surviving fraction decreased from 3.98 x 10(-3) to 1.98 x 10(-5) over the same range of T(pot). A comparison of treatment plans revealed that (103)Pd plans required more needles and seeds; however, this was a function of seed strength. Both isotopes had similar dose-volume histograms for prostate, urethra, and rectum. The theoretical prediction of effectiveness using the linear quadratic equation for the common clinically prescribed total radiation doses indicated that (103)Pd should be more effective than (125)I because it had less dependence on T(pot). The greatest benefit of (103)Pd was shown to be with tumors with a short T(pot). Although the regrowth delay would be longer with (125)I, the benefit was inconsequential compared with the very slow doubling times of localized prostate cancer. Treatment planning with either isotope revealed no significant differences. These findings may explain why clinically there seemed to be no clear difference in treatment outcome with either isotope. Based on these predictions, we recommend a clinical trial to compare the efficacy of the two isotopes.

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line.

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.

The response of human tumour blood flow to a fractionated course of thermoradiotherapy.

The response of human tumour blood flow to a fractionated course of thermoradiotherapy was documented in four superficial but bulky tumours (three adenocarcinomas, one melanoma). Blood flow was measured 15, 30, 45, and 60 min after the onset of heating. These measurements were made at the same intra-tumour point during each heat fraction by use of a modified thermal clearance technique in which a correction was made for the heat dissipated by thermal conduction. This point was at least 2 cm beneath the surface in the central portion of the tumour. Extracellular pH was measured within 1 cm of this point prior to the first heat fraction and 2-3 weeks later. Hyperthermia was administered for 60 min, twice a week for 4 weeks by use of a 16-channel 915 MHz microwave applicator. Each patient also received a radiation dose of 40 Gy fractionated at 2 Gy/fx, five times a week (adenocarcinomas) or 4 Gy/fx, twice a week (melanoma). Blood flow remained relatively constant during heating after steady state conditions were attained. However, an overall decrease in tumour blood flow was observed in each patient over the course of thermoradiotherapy. In each case, a relatively small decrease in blood flow occurred between most heat fractions which resulted in an overall decrease which ranged from 50-100%. However, there was a tendency for blood flow to increase following the initial heat fraction at points where the steady state temperature was approximately 41 degrees C or less. Extracellular pH increased in two of three patients and decreased in the other.

Effect of i.v. glucose versus combined i.v. plus oral glucose on human tumour extracellular pH for potential sensitization to thermoradiotherapy.

The purpose of this study was to determine whether intravenous or combined intravenous plus oral glucose administration was more effective inducing acute tumour acidification. Seventeen nondiabetic patients at the Henan Tumour Hospital with superficial tumour deposits of various histologies and size were administered, after fasting, either 50 g glucose intravenously (i.v., in 100 ml over 10 min) or 50 g i.v. glucose (in 100 ml over 10 min) combined with 100 g oral glucose (in 200 ml; i.v. + oral). Extracellular tumour pH (pHe) was determined with one or two indwelling needle combination pH microelectrodes. Blood glucose concentration was determined every 15-20 min by finger stick with Chem-Strips and a Glucometer. Ten patients received i.v. glucose, and seven patients received i.v. + oral glucose. Blood glucose rose to 430 +/- 15 mg/dL in both groups. However, the rate of clearance of blood glucose was greater for the i.v. glucose than for the i.v. + oral glucose group (p < 0.00002), and thus the blood glucose levels remained elevated longer after i.v. + oral than after i.v. glucose administration. Relative to the initial fasting blood glucose concentration, blood glucose was -2 +/- 7 mg/dL at 110 min after glucose administration by the i.v. route, whereas, blood glucose relative to initial values was 143 + 23 mg/dL by 110 min after glucose administration by the i.v. + oral route, p = 0.000004. The initial pHe values in the two groups of tumours were similar, 7.34 +/- 0.09 (6.78-7.71) and 7.35 +/- 0.08 (6.99-7.61), respectively. After i.v. glucose, tumour acidification occurred in nine of ten patients (-0.16 + 0.02 pH unit, range -0.24 to -0.05), and after i.v. + oral glucose tumour acidification occurred in six of seven patients (-0.19 +/- 0.07 pH unit, range -0.43 to -0.06). When the initial fasting blood glucose concentration was in excess of 82 mg/dL, all patients (12/12) exhibited tumour acidification during hyperglycaemia, whereas, only 3/5 patients exhibited tumour acidification when the initial blood glucose concentration was less than 82 mg/dL (p = 0.07). The time to maximum decrease in tumour pHe was significantly shorter after i.v. + oral glucose than after i.v. glucose (e.g., 67 +/- 11 versus 102 +/- 8 min, p = 0.02) and correlated with the rate of clearance of blood glucose (p = 0.02, r = 0.55). Larger tumours tended to exhibit a greater decrease in pHe (p = 0.08, r = 0.04). The only side effects of hyperglycaemia were transient nausea and increased urinary output. The effect of hyperglycaemia induced by administration of 200 g oral glucose was similar to i.v. administration in that 83% of tumours exhibited acidification of 0.14 +/- 0.02 pH unit by 91 +/- 7 min. We conclude that i.v. and i.v. + oral glucose administration are equally effective inducing tumour acute acidification, but no more effective than 200 g oral glucose, for investigation of hyperglycemic sensitization to thermoradiotherapy.

Temporal association between alterations in proton extrusion and low pH adaptation.

Cells which have been adapted to growth at low extracellular pH (pHe) typically develop both an upregulation of steady state intracellular pH (pHi) and an ability to develop thermotolerance to 42 degrees C hyperthermia. These properties were acquired at different times, however. Days were required at pHe = 6.70 for two cell lines to adapt to low pHe by the thermotolerance criterion, but both had elevated steady state pHi values after only 4 hours at pHe = 6.70. A better correlation with adaptation to low pHe (as defined by hyperthermia) was found with changes in proton extrusion and the rate of pHi recovery after cytosolic acidification.

Altered proton extrusion in cells adapted to growth at low extracellular pH.

Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/ H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl-/HCO(-3)exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification.

Effects of 42 degrees C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH.

PURPOSE: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi. METHODS AND MATERIALS: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7. RESULTS: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival. CONCLUSIONS: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.