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Biomedical subjects

L E Gerweck

Publications and source records attributed to L E Gerweck.

At least 37 records · Page 2Linked to original sources
PubMed1991

The simultaneous determination of intracellular pH and cell energy status.

The relationship between the apparent equilibrium constant of creatine kinase and intracellular pH was evaluated in CHO and murine FSaII tumor cells. The apparent equilibrium constant, K' = [ATP][Cr]/[ADP][PCr], was determined from acid extracts at variable pH. Intracellular pH (pHi) was determined from the intracellular/extracellular distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. Over the intracellular pH range of 7.2 to 6.1, K' increased by a factor of approximately 10. Intracellular pH was related to the apparent equilibrium constant by the equation pHi = -log K' + log K, where the value of the constant log (log[K'/H+]) was 8.09. Over the same pH range, the concentration of phosphocreatine decreased with pH. Essentially identical results were obtained in CHO and FSaII tumor cells. The similar apparent equilibrium constants in CHO and FSaII cells suggest that assessment of the creatine kinase metabolites will be useful not only for determination of cell energy status but also for the determination of intracellular pH. This information may be useful for the design of therapeutic strategies which are influenced by pH or energy status such as hyperthermia, and drugs which are weak acids or bases, including hypoxic cell radiosensitizers.

Animals↗
PubMed1991

Regulation of pH in murine tumor and muscle.

The relationship between intracellular and extracellular pH was investigated in a murine tumor and normal tissue, prior to and following glucose injection. Isotransplants of the murine tumor FSa-II in the dorsum of the hind foot and leg muscle, were investigated in nonanesthetized mice. Extracellular pH was measured with a glass microelectrode, with a tip diameter of approximately 80 microns. Intracellular pH was evaluated by 31P-NMR spectroscopy, using a wide-bore NT-150 spectrometer operating at 60.75 MHz. Five grams per kilogram intraperitoneal glucose led to small changes in extracellular pH of muscle (-0.13 unit) measured with a microelectrode, and no change in intracellular pH measured by NMR. In contrast, tumor intracellular pH decreased by 0.34 units and tumor extracellular pH by 1.13 units. The differential effect of glucose on tumor vs normal tissue, and pronounced pH gradient which develops in tumor cells should markedly affect the intracellular:extracellular distribution of drugs which are weak acids or bases.

Animals↗
PubMed1990

31P NMR spectra of extremity sarcomas: diversity of metabolic profiles and changes in response to chemotherapy.

We have used 31P NMR spectroscopy to study 22 patients with suspected sarcomas prior to any treatment. The spectra are characterized by the same peaks noted in murine tumors. The mean pH was 7.14 +/- 0.08 and PCr/Pi was 1.18 +/- 0.83. Comparison of pH and PCr/Pi ratios in human and a murine tumor with a low hypoxic cell fraction revealed no significant differences. Six patients subsequently received chemotherapy and three responded to therapy (based on pathologic examination and/or tumor reduction greater than 50%). The three responding patients were noted to have significantly lower PDE/PME in their pretreatment spectra than the three nonresponding patients. The three responding patients with sarcomas also showed a rise of greater than 100% in PDE/PME during the first cycle of therapy. Two of the responding patients had an increase of 0.37 pH units during this interval, which was not detected in the nonresponding patients. These data suggest that 31P NMR spectroscopy may be a useful prognostic indicator in conjunction with other clinical parameters.

Animals↗
PubMed1990

Relationship of changes in pH and energy status to hypoxic cell fraction and hyperthermia sensitivity.

The relative concentrations of nucleotide triphosphates, creatine phosphate, inorganic phosphate, and pH have been evaluated as a function of tumor volume in a murine fibrosarcoma (FSaII) by 31P NMR spectroscopy. As the tumor volume increased from 60-1250 mm3, the ratio of phosphocreatine to inorganic phosphate systemically decreased. This decrease paralleled a decrease in the ratio of nucleotide triphosphate to inorganic phosphate in the same tumor volume range. The tumor pH as measured by 31P NMR decreased slightly with tumor growth. A pH of 7.17 +/- 0.07 (n = 17) was found for tumors between 60 and 150 mm3, whereas for tumors greater than 900 mm3, a pH of 7.05 +/- .03 (n = 6) was noted. Intermediate size tumors (151-900) had a pH of 7.12 +/- 0.09 (n = 18). The change in tumor energy status with tumor volume inversely paralleled the change in tumor radiobiologic hypoxic cell fraction and suggested a causal relationship between tumor nutrient status and energy status. Tumor thermal sensitivity also increased with tumor volume, suggesting a relationship between pH, energy status, and thermal sensitivity, as has been demonstrated under in vitro conditions. Each NMR parameter was found to correlate significantly with tumor volume independent of the other NMR parameters.

Adenosine Triphosphate↗
PubMed1989

Relationship between energy status, hypoxic cell fraction, and hyperthermic sensitivity in a murine fibrosarcoma.

The energy status, radiobiological hypoxic cell fraction, and hyperthermic sensitivity of a spontaneous murine fibrosarcoma, FSa-II, have been evaluated as a function of tumor size. Tumors were evaluated over the size range of 70 to 800 mm3. The concentration of the high-energy phosphate reservoir creatine phosphate progressively decreased by a factor of 5 with increasing tumor volume, and was matched by an increase in creatine. The concentration of ATP also decreased with increasing tumor size, although this decrease was substantially less pronounced. The sum of ATP, ADP, and AMP did not vary with tumor size, suggesting that the necrotic fraction remained constant. The decrease in energy status occurred in parallel with an increase in the size of the hypoxic cell fraction and with increasing thermal sensitivity. The results suggest that energy status may be an important modifier of hyperthermic sensitivity in vivo and reflect tissue oxygen concentration.

Adaptation, Physiological↗
PubMed1988

FSaII mouse tumor metabolic changes with different doses of glucose measured by 31P nuclear magnetic resonance.

Tumor acidosis and energy deprivation enhance thermal sensitivity. We have used in vivo 31P nuclear magnetic resonance spectroscopy to noninvasively monitor changes in pH and energy metabolism in FSaII mouse tumors after i.p. administration of glucose. A dose of 5 g/kg glucose induced a pH drop of 0.31 units without any statistically significant change in energy status. The pH changes resolved within 2 h. In contrast, administration of 10 g/kg glucose resulted in a severe acidosis (mean nadir pH of 6.19 corresponding to a mean pH drop of 0.96 units) and loss of energy, the latter most probably being due to an acidosis-induced inhibition of glycolysis during ischemic hypoxia. The resulting acidosis and energy loss were more persistent and resolved in 5.5-28 h. In contrast, after an identical dose of mannitol (10 g/kg), a pH drop of approximately only 0.1 units over 72 min was noted. The data suggest that both cleavage of glucose to lactic acid and blood flow inhibition are involved in glucose induced tumor acidosis. In vivo 31P nuclear magnetic resonance spectroscopy may be useful clinically to monitor therapeutic attempts at enhancing thermal sensitivity.

Animals↗
PubMed1988

Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity.

The effect of variable extracellular pH on intracellular pH, cell energy status, and thermal sensitivity was evaluated in CHO cells over the extracellular pH range of 6.0 to 8.6. Extracellular pH was adjusted with either lactic acid, HCl, or NaOH. Regardless of the method of pH adjustment, the results obtained were similar. The relationship between extracellular and intracellular pH was dependent upon the pH range examined. Intracellular pH was relatively resistant to a change in extracellular pH over the pHe range of 6.8 to 7.8 (i.e., delta pHi congruent to delta pHe X 0.33). Above and below this range, delta pHi congruent to delta pHe X 1.08 or X 0.76, respectively. Cellular survival after a 30-min heat treatment at 44 degrees C remained constant over the extracellular pH range of 7.0 to 8.4, but varied substantially over a similar intracellular pH range. The cellular concentration of the high energy phosphate reservoir, phosphocreatine, decreased with decreasing pH. However, the cellular concentrations of ATP, ADP, and AMP remained constant over the entire pH range examined. It is concluded that increased thermal sensitivity resulting from a change in extracellular pH is not due to cellular energy depletion. Furthermore, intracellular pH is a more accurate indicator of thermal sensitivity than is extracellular pH.

Adaptation, Physiological↗
PubMed1988

Lonidamine-induced, pH dependent inhibition of cellular oxygen utilization.

The inhibitory effect of lonidamine 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid on oxygen utilization by Chinese hamster ovary (CHO) and murine fibrosarcoma (FSa-II) cells was evaluated with a Clark oxygen electrode. The drug produced a small but statistically significant inhibition of oxygen uptake at normal pH (7.4) in CHO and FSa-II cells of 16 and 11%, respectively. However, at low pH (6.65) the inhibitory effect of lonidamine increased dramatically to 60% in both CHO and FSa-II cells. Because of the potential difference between tumor and normal tissue pH, lonidamine and similar drugs may be effective for selectively modifying oxygen utilization and concentration in tumor tissue which might lead to increased radiation and hyperthermic sensitization in tumors compared to normal tissue, resulting in an improvement in the therapeutic ratio.

Animals↗
PubMed1987

Variability in the kinetics of thermotolerance decay in three cell lines.

The magnitude of thermotolerance and rate of tolerance decay has been evaluated in three cell lines following isotime and isoeffect initial heat treatments at 44 degrees C. Following the development of maximal thermotolerance, all cell lines lost tolerance at an exponential rate. However, for the three cell lines examined, V-79, CHO, and A-7, differences were apparent in the time required for maximal tolerance development, magnitude of induced tolerance, and rate of tolerance decay. This variability in the thermotolerance response of cells suggests difficulties which might be encountered if in vivo fractionation protocols are to be developed to minimize damages to normal tissue and maximize damages to tumor tissue.

Adaptation, Physiological↗
PubMed1986

Cell proliferation, protein turnover, and the decay of thermotolerance in CHO cells.

The rates of cell proliferation, total protein and heat shock protein turnover, and thermotolerance decay were determined in exponential-phase CHO cells. Following a mild heat treatment of 44 degrees C for 10 min, the rate of total protein turnover slightly exceeded the rate of cell proliferation. Heated cells doubled approximately every 16 h and labeled total protein turned over with a half-time of 14 h. The turnover rate of heat shock proteins (10-h half-time) somewhat exceeded the total protein turnover rate and was similar to the thermotolerance decay rate. These data indicate that the turnover of total and heat shock proteins and thermotolerance occurs as a result of both cell division-dependent and division-independent processes.

Animals↗
PubMed1985

Temperature-dependent influence of thiols upon glutathione levels in Chinese hamster ovary cells at cytotoxic concentrations.

Chinese hamster ovary cells were exposed to the sulfhydryl compound cysteamine at different temperatures (5 degrees C, 37 degrees C, 44 degrees C) at concentrations known to generate activated oxygen species. At 37 degrees C, the cellular glutathione (GSH) content increased linearly over the time of drug exposure (2 h) as compared to untreated cells or to cells kept at 5 degrees C during drug treatment. The 2-4-fold increase in GSH induced by cysteamine was more rapid at 44 degrees C than at 37 degrees C and showed a saturation effect at the higher temperature. The elevation of GSH could be completely blocked by DL-buthionine-S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, or by incubation in a cystine-free medium during the period of drug treatment. The increased cellular GSH content induced by cysteamine alone at 37 degrees C or combined with heat at 44 degrees C decreased to the range of control values within 22 h after either treatment. Other thiols like cysteamine, namely cysteine, N-acetylcysteine, and dithiothreitol, were found to be similar in their potential to induce GSH elevation in Chinese hamster ovary cells. Cytotoxic effects of these sulfhydryl compounds were observed in the same concentration range as that for cysteamine (0-2 mM), but only if cells were plated at low densities (10(2)-10(4) cells/flask), and were completely blocked by the addition of catalase (50 micrograms/ml). In contrast, the elevation of GSH after thiol treatment (0.8 mM) was not modified by catalase. The data suggest that thiol treatment combined with hyperthermia leads to a rapid increase of GSH biosynthesis in Chinese hamster ovary cells which seems to be independent of the simultaneous generation of activated oxygen species by thiol autoxidation.

Animals↗
PubMed1984

Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection.

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoy's 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoy's 5A has been compared with that in Ham's F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoy's 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.

Catalase↗
PubMed1984

Persistence of thermotolerance in slowly proliferating plateau-phase cells.

Thermotolerance decay rates were determined under rapidly proliferating exponential growth conditions and under high-cell-density plateau-phase growth conditions. Thermotolerance was induced by a marginally toxic initial heat treatment of 10 min at 44 degrees C in Chinese hamster ovary cells. Second dose-response curves were obtained at 24-hr intervals for up to 96 hr after the initial heat treatment. Resistance to second heat treatment had disappeared by 72 hr after the initial heat treatment in proliferating cells, but was evident in plateau-phase cells at 96 hr. Regardless of the proliferative status, thermotolerance decayed in an exponential manner. The half-times of tolerance decay were approximately 13 hr in proliferating cells and 30 hr in plateau-phase cells.

Acclimatization↗
PubMed1984

Influence of nutrient and energy deprivation on cellular response to single and fractionated heat treatments.

The effect of hypoglycemia and hypoxia on cell survival following single and fractionated 44 degrees C heat treatments was investigated. These studies show that short-term glucose deprivation or oxygen deprivation does not increase sensitivity to single or fractionated heat treatments compared to normal nutrient conditions. However, simultaneous hypoglycemia and hypoxia leads to an increased sensitivity to single heat treatments, and to an even greater increase in sensitivity to fractionated heat treatments. Sensitivity to heat treatment under nutrient-deprived conditions correlates with intracellular ATP levels at the time of treatment. These data indicate that it is not nutrient concentration per se, but rather the effect of nutrient level on intracellular ATP concentration that affects thermal sensitivity.

Acclimatization↗
PubMed1984

Enhancement of thermal response of normal and malignant tissues by Corynebacterium parvum.

Further studies were carried out on the combined effects of Corynebacterium parvum and hyperthermia on animal tissues and cultured Chinese hamster ovary cells. Experimental animals were C3Hf/Sed mice derived from our defined flora mouse colony. Tumors were eighth-generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Hyperthermia was given by immersing the mouse foot or culture flasks in the constant temperature water bath. Present experiments include thermal enhancement of C. parvum at different temperatures, effect of the agent on the kinetics of thermal resistance, and the mechanism of the thermal enhancement. The thermal enhancement by C. parvum was independent of temperature in a range between 42.5 and 46.5 degrees, and it increased with decreasing temperature. The analysis of the Arrhenius plot suggested a comparable activation energy for combined treatments and for heat alone between 42.5 and 46.5 degrees. The thermal resistance developed very rapidly in both normal and tumor tissues. Systemic administration of C. parvum failed to modify the kinetics of thermal resistance. Several experiments were attempted in order to disclose the mechanism. A single injection of C. parvum-induced macrophages failed to enhance thermal response of the mouse foot, while 3 daily injections of the macrophages enhanced the response, indicating that the enhancement by C. parvum is at least partly attributed to the C. parvum-induced macrophages. Whole-body irradiation of 6 Gy and/or administration of anti-mouse T-cell serum and histamine failed to inhibit the C. parvum enhancement of thermal response. No thermal enhancement was observed for Chinese hamster ovary cells treated at 43.0 degrees in vitro with C. parvum or thiomersalate , a preservative supplemented in C. parvum, although cytotoxic effect was shown at a high concentration of thiomersalate .

Animals↗