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Biomedical subjects

G M Hahn

Publications and source records attributed to G M Hahn.

189 records · Page 11Linked to original sources
PubMedDate not supplied

Adaptation to low pH modifies thermal and thermo-chemical responses of mammalian cells.

The thermal and thermochemical survival responses of mammalian cells growing routinely at pH 7.4 are affected by extracellular pH. The cells show an increase in sensitivity when heated at pH values below about pH 7.0. The possibility that this sensitivity at low pH can be modified by maintaining the cells at pH 6.8 ('EP-2' cells) or pH 6.5 ('EP-1' cells) was examined. When Chinese hamster cells (HA-1) were exposed to 42 degrees C at pH 6.5, 6.8 and 7.4, a dramatic increase in sensitivity was seen at pH 6.5. EP-2 cells similarly exposed showed considerably less difference at the three pH values. Variations in survival due to these pH changes were almost eliminated when the EP-1 cells were exposed to 42 degrees C. HA-1 cells tended to develop less thermotolerance rates and did so possibly at a reduced rate at pH 6.8 when compared to pH 7.4; the reduced pH had only a minor effect on the ability of EP-2 cells to develop thermotolerance. The effects of pH adaptation on the pH dependence of drug cytotoxicity at 37 degrees C or at 43 degrees C were also compared. Adaptation also modified drug cytotoxicity except that cell killing by BCNU (as a function of pH) was similar for both HA-1 and EP-2 cells at both temperatures. EP-2 cells were more sensitive to MMS treatment at 43 degrees C and more resistant at 37 degrees C than were HA-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMedDate not supplied

Effects of hyperthermia on the intracellular pH and membrane potential of Chinese hamster ovary cells.

The effects of hyperthermia (exposure to 41-47 degrees C) on the intracellular pH and membrane potential have been studied using Chinese hamster ovary HA-1 cells. Our goal was to determine whether intracellular pH changes or changes in membrane potential correlated with cell killing. The intracellular pH (pHi) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pHi reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for delta pH (pHi-pHo). The membrane potential difference (delta psi) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that delta psi falls to zero only upon prolonged exposure to temperatures above 43 degrees C. When the external pH was changed from normal values the drop in delta psi occurred more readily. Development of thermotolerance resulted in an increase in the time required to make delta psi change by half. The changes in delta psi were shown to be irreversible. When the proton electrochemical gradient (delta mu H+) was calculated using the measured values of delta psi and delta pH, the trends observed were the same as those seen for delta psi. The changes observed for pHi can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in delta psi and delta mu H+ may reflect the physical breakdown of the transmembrane H+ gradients, which may be the actual mechanical process of cell death. No correlation of cell survival with the measured parameters was observed.

Animals↗
PubMedDate not supplied

Changes in pH and blood flow induced by glucose, and their effects on hyperthermia with or without BCNU in RIF-1 tumours.

The changes in pH and blood flow of tumours caused by hyperglycaemia, and their effects on hyperthermia with BCNU, were investigated using RIF-1 tumours which grew intradermally in the thigh of C3H mice. The pH of the tumour was measured by inserting a microelectrode into the tumour. The blood flow in the tumour was estimated by flow cytometry from the uptake of the bisbenzamide fluorochrome Hoechst 33342 into the tumour. Administration of glucose resulted in a transient drop of both pH and blood flow of the tumour. The change in blood flow appeared earlier than that in pH. The response of the tumour to heat was enhanced by the administration of glucose in a time- and dose-dependent fashion, and closely correlated to the change in tumour pH. Glucose administration reduced the cytotoxicity of BCNU. This can be explained by the inhibitory effect of glucose on tumour blood flow. The effect of BCNU was enhanced by hyperthermia. This combined effect of heat and BCNU was further enhanced by the addition of glucose, but this effect was not dramatic. Because glucose inhibits blood flow in the RIF system at least, glucose is more effective for thermotherapy than for thermochemotherapy.

Animals↗
PubMedDate not supplied

Effects of hyperthermic temperatures and the synthesis of heat-shock proteins on the lateral diffusion of H-2Kk.

The effect of hyperthermia, fractionated heat and the synthesis of heat shock proteins on the lateral diffusion of H-2Kk was examined in RDM-4, Ch-1 and mouse L cells. Cells in suspension were examined immediately after heating, adaptation to growth at 40 degrees C for 72 h or 6 h after exposure to sodium arsenite (5 microM), iodoacetamide (10 microM) or a 20 min heat shock at 45 degrees C. No heat shock protein synthesis could be measured in CH-1 cells in response to either heat or chemical stress. Synthesis of these proteins was observed in both RDM-4 and mouse L cells in response to heat or chemical treatments, and the kinetics of synthesis were similar to those reported in the literature for other eukaryotic cell lines. The diffusion of fluorescein isothiocyanate-labelled monoclonal anti-H-2Kk bound to heat (or chemically) treated cells was measured by the technique of fluorescence recovery after pattern photobleaching. After a 30 min exposure to temperatures between 41 degrees C and 45 degrees C, a temperature-dependent decrease in recovery was observed. At 45 degrees C, 75-100% of the surface antigen was found to be immobile. The diffusion coefficients of the mobile fractions were not significantly changed at all the temperatures examined. Heat effects on the recovery patterns of all the three cell lines were qualitatively similar and unaffected by the presence or absence of heat or chemically induced heat shock proteins. After 48-60 h following a heat shock, the recovery patterns were similar to those of unheated control cells.

Animals↗
PubMedDate not supplied

An immunoassay for heat shock protein 73/72: use of the assay to correlate HSP73/72 levels in mammalian cells with heat response.

An enzyme-linked immunosorbent assay (ELISA) for measurement of levels of heat shock proteins 73 and 72 (HSP73/72) in cultured cells and tissues is described. The assay involves detection of HSP73/72 in cell homogenates in 96-well plates using a specific monoclonal antibody. The assay has been used to explore the relationship between the amount of HSP73/72 in a cell and its response to heat shock, both before and after the development of thermotolerance. Six mammalian cell lines with differing responses to heat were characterized with respect to their response to heat treatments at 44 degrees C and concentrations of HSP73/72. Contrary to the widely expressed idea that the amount of HSP73/72 dictates the degree of heat resistance, no positive correlation between levels of HSP73/72 and heat resistance was found for the six lines tested here: if one particular line, a mutant selected for heat resistance, was excluded from the analysis, there was a negative correlation between HSP73/72 levels and heat resistance. A different result was, however, obtained when thermotolerant (transiently resistant) cells were compared to control cells. Here, we found a good correlation between the extent of thermotolerance and the amount of HSP73/72, suggesting that an increase in HSP73/72 level is important for the development of thermotolerance. The validity of the ELISA technique was checked using a second method for quantifying levels of HSP73/72. This involved uniform radiolabeling of cellular proteins, separation on two-dimensional gels and radioscanning to quantify radioactivity in each protein. The second technique is more powerful in that different isoforms of HSP73/72 can be distinguished, but it is more difficult to perform, is more labour intensive and requires an expensive device for gel scanning. The results using the second technique agreed well with those from the immunoassay and indicated that the level of the highly inducible HSP72 correlated best with the extent of thermotolerance.

Animals↗
PubMedDate not supplied

Heat sensitivity of bleomycin-sensitive CHO derivatives is not due to improper initialization of heat shock response.

We have investigated whether differences in the heat shock response exist between CHO and three bleomycin-sensitive, heat-sensitive CHO derivatives. The binding of heat shock factor (HSF) in response to heat and varying concentrations of bleomycin in the four cell lines was examined using a gel shift assay and a synthetic heat shock element (HSE). Heat (45 degrees C, 10 min) and exposure to 1 micrograms/ml bleomycin for 1 h at 37 degrees C induced similar levels of HSF binding in all four cell lines. We also examined if bleomycin dose and the length of recovery from bleomycin treatment affected the induction of HSF binding. The level of activated HSF binding to HSE was higher in cells treated with low doses (1 ng/ml) of bleomycin than in cells treated with 1 or 25 micrograms/ml bleomycin. The amount of activated HSF was directly proportional to the time elapsed since bleomycin treatment. Our results therefore indicate no difference between CHO and its bleomycin-sensitive derivatives in the ability to initiate the heat shock response as determined by the production of activated HSF in response to either heat or bleomycin. We conclude that the intrinsic thermosensitivity of these cell lines is not related to the early response to heat shock, but either occurs later in the pathway or is unrelated to events after heating.

Animals↗
PubMedDate not supplied

Mammalian cell survival studies characterizing multiport negative pi-meson irradiation with the Stanford Medical Pion Generator (SMPG).

Radiobiological measurements have been made under various conditions of muliport pion irradiation using the Stanford Medical Pion Generator (SMPG). Chinese hamster cells (HA-1) were suspended in a tissue-equivalent 25% gelatin/medium solution. Hypoxic and aerobic HA-1 cells were irradiated simultaneously in a cylindrical water tank at either 4 or 16 degrees C. Irradiation at the focus of 60 converging pion beams, at a peak dose rate of 6 rads/min, gave relative biological effectiveness (RBEs) of 2.8, 1.8 and 1.4 at 50, 20 and 5% survival, respectively, and an oxygen enhancement ratio (OER) of 1.7. Plateau irradiation with crossing pion beams, at a peak dose-rate of 3 rads/min, resulted in survival values very close to those obtained with similar doses of 85 kV X-rays. Preliminary studies with large irradiation volumes in broadened pion stopping regions indicate RBEs significantly > 1 for survival > 50%. Supporting microdosimetric data with the SMPG are consistent with the radiobiological results.

Animals↗
PubMedDate not supplied

Analysis of HSF-1 phosphorylation in A549 cells treated with a variety of stresses.

In the absence of stress, heat shock transcription factor-1 (HSF-1) exists as a monomer. After the treatment of cells with variety of stresses, HSF-1 forms a trimer and binds to the heat shock element (HSE), a motif consisting of three consecutive NGAAN sequences located in an inverted orientation upstream of the heat shock genes. HSF-1 is then phosphorylated causing transactivation of heat shock mRNAs. Treatment of cells with some of the stresses has been shown to increase HSF binding to HSE without detectably increasing the synthesis of heat shock mRNAs. Here we used antibody specific to HSF-1 to detect its phosphorylation status following exposure of A549, a human lung carcinoma cell line to a variety of stresses in order to correlate HSF-1 phosphorylation with its transactivation ability. Our studies show that HSF-1 is phosphorylated following heat shock (43 degrees C for 1 h), hypoxia (5 h exposure to 0.02% oxygen), 8% ethanol (1 h exposure at 37 degrees C), or 200 microM sodium arsenite (1 h exposure at 37 degrees C). All such stresses have previously been shown to increase the synthesis of heat shock proteins (hsps). However, there are no detectable increases in HSF-1 phosphorylation after the treatment of cells with X-irradiation (2-8 Gy) or 100 microM canavanine, an amino acid analogue (1 h exposure at 37 degrees C). Treatment of cells with X-irradiation increases HSF binding to HSE without increasing the synthesis of hsps, while treatment of cells with canavanine has been shown to increase the synthesis of hsps.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗