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Biomedical subjects

G M Hahn

Publications and source records attributed to G M Hahn.

At least 73 records · Page 4Linked to original sources
PubMed1986

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock.

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.

Animals↗
PubMed1986

Glucocorticoid-induced heat resistance in mammalian cells.

Chinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10(-6)M developed after a lag of 2-3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100-fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.

Animals↗
PubMed1986

Dansyl lysine, a new probe for assaying heat-induced cell killing and thermotolerance in vitro and in vivo.

Heat induces an increase in the fraction of cells staining with the nontoxic fluorescent membrane dye dansyl lysine (DL). The fraction of cells excluding DL can, under certain circumstances, be closely correlated to the fraction of cells surviving a heat treatment. Dansyl lysine has previously been shown to select for cholesterol-free membrane domains. We now describe the use of DL to provide rapid estimates of the kinetics of thermotolerance development and decay in vitro and in vivo. Following a 45 degree C-10-min heat shock, Chinese hamster ovary cells develop resistance to the lethal effects of a second heat treatment. This thermotolerance, as measured by both clonogenicity and resistance to DL staining, is maximal at approximately 12 h and gradually decays with a t1/2 of 2 to 3 days. DL staining also has utility in predicting the heat survival response in vivo. Radiation-induced fibrosarcomas grown in C3H mice were heated to 43 degrees C for 30 min. From 1 to 3 days later the tumors were excised and a single cell suspension was prepared. Tumor cells were then heated in vitro and assayed for survival or scored microscopically for DL staining. The two assays again yielded similar results showing maximal resistance by 19 h which decreased toward control heat sensitivity by 49 h. The evaluation of intrinsic heat sensitivity and induced thermotolerance is critical to rational treatment design in clinical hyperthermia. DL staining is rapidly quantitated by flow cytometry and can be applied to biopsy samples to provide estimates of heat sensitivity within hours.

Animals↗
PubMed1986

Tumor responses following multiple hyperthermia and X-ray treatments: role of thermotolerance at the cellular level.

We have investigated the effect of increasing numbers of hyperthermia fractions given at 7-day intervals, with or without fractionated radiotherapy, on tumor cure, tumor growth, and cell survival after in vivo or in vitro heat. The murine RIF tumor was treated by capacitive radiofrequency hyperthermia at 44.0 degrees C for 20 min for one to five treatments at weekly intervals (1-5 wk D1). Single treatments (1 wk D1) induced cure in 5% of tumors. Additional treatments (2-5 wk D1) induced similar rates of cure (0-16%, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). 1 wk D1 resulted in marked growth delay compared to controls. Mean tumor diameter doubling times increased from 13.2 days to 27.5 days (P less than or equal to 0.01). 2-5 wk D1 induced little additional growth delay (doubling times, 27.8-32.3 days, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). Fractionated radiotherapy of 3200 rads (400 rads given twice each week) significantly prolonged mean tumor doubling time to 26.2 days. The addition of one hyperthermia session to the fractionated radiotherapy (1 wk D1 + XRT) further increased doubling time to 34.2 days (P less than or equal to 0.01). Additional treatments (2-5 wk D1 + XRT) only modestly increased doubling times (36.0-39.5 days, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). In vitro assay of cells dissociated from tumors 5, 10, or 15 days after 3 wk D1 showed increased survival to 44 degrees C compared to previously untreated controls, and this cellular thermoresistance proved to be transient and noninheritable (i.e., thermotolerance). These results indicate that tumors can develop a prolonged thermal resistance after multiple weekly treatments which significantly modifies the response to subsequent treatment and which is associated with cellular thermotolerance.

Animals↗
PubMed1986

Heat shock protein levels are not elevated in heat-resistant B16 melanoma cells.

Heat-resistant variants have been selected from B16 melanoma cells and from surface mutants previously derived from them. The aim of the present study was to explore the possible role of heat shock proteins in the manifestation of this heat resistance. The major heat shock proteins evident after heating have subunit molecular weights of 68, 70, 89, and 110K on sodium dodecyl sulfate-polyacrylamide gels. The 68-kDa protein is not evident in any of the unheated B16 cell lines while the levels of the other heat shock proteins are elevated after heating. The constitutive levels of the 70, 89, and 110-kDa heat shock proteins were assessed after gel electrophoretic separation of proteins in several of the heat-resistant variants. No major differences were found in the levels of these proteins between the heat-sensitive parent lines and the heat-resistant variants. We therefore conclude that heat shock proteins are not a determining factor in the heat-resistant phenotype of B16 melanoma cells.

Acclimatization↗
PubMed1985

Cyclic AMP and the heat shock response in Chinese hamster ovary cells.

Heat shock leads to transient increases in cAMP levels in HA-1-CHO cells. Such pulses are correlated temporally with the induction of heat resistance (thermotolerance) and with heat shock protein synthesis. Although the kinetics of cAMP increase after heating suggest a role in thermotolerance induction, raising cAMP levels directly using dBcAMP did not produce full thermotolerance. The resistance induced by dBcAMP may thus be either a component of or different to heat-shock triggered resistance. Cells which had been made thermotolerant by heat shock did not produce a pulse in cAMP level on heating. The cAMP producing system thus seemed desensitized to heat in thermotolerant cells.

Animals↗
PubMed1985

Noninvasive thermometry using multiple-frequency-band radiometry: a feasibility study.

The potential use of multiple-frequency-band radiometry as a means of noninvasive sensing of one-dimensional temperature profiles is presented in this communication. The radiative energy transfer equation is solved numerically. Ideal-condition thermal noise spectra and distributions of received energy, associated with specific temperature-depth profiles, are presented. Performance characteristics are discussed.

Humans↗
PubMed1985

Investigation of adenylate energy charge, phosphorylation potential, and ATP concentration in cells stressed with starvation and heat.

We have attempted to determine the appropriate parameter of energy status associated with the survival of CHO fibroblasts under starvation conditions. Survival correlated well with adenylate energy charge (EC) but not so well with the phosphorylation potential or ATP concentration. Starved cells exhibited the capacity to resist (transiently) decreases in both EC and survival. A fall in EC was associated with decreased survival. Using this correlation, we subsequently investigated whether killing after thermal stress occurred by a mechanism analogous to starvation, perhaps due to inhibition of energy yielding pathways. This hypothesis proved to be false; over 99% of cells were killed before a decrease was observed in any of the parameters of energy status. Cells were, however, sensitized to heat under nutritionally deprived conditions, a finding which may be significant for tumor treatment by heat in vivo.

Adenosine Diphosphate↗
PubMed1985

The significance of thermotolerance after 41 degrees C hyperthermia: in vivo and in vitro tumor and normal tissue investigations.

We have investigated the development of thermotolerance in both tumors and normal tissues after 41 degrees C for durations as brief as 15 minutes. The murine RIF tumor, treated by both local radiofrequency and systemic methods, was assayed for thermotolerance by both tumor growth and cell survival analyses. The murine leg and ear, treated by conductive methods, were assayed using pre-defined tissue damage scoring systems. All of these treatments quickly induced substantial levels of thermotolerance. In the tumor studies using local heating, RIF mean diameter doubling time decreased from 17.8 days to a minimum of 13.0 days with a 9 hr interval between 41.0 degrees C for 15 minutes and 44.0 degrees C for 30 minutes (9 hr D1-D2); cell survival increased from 1.2 X 10(-2) to 3.4 X 10(-1) (same interval). Both assays showed some degree of tolerance present immediately after 41.0 degrees C for 15, 30 or 60 minutes (0 hr D1-D2). In the tumor studies using systemic heating, the kinetic pattern of the induced tolerance was similar to that observed after local heating. In the leg studies, 41.0 degrees for 30 minutes increased the time at 45 degrees C necessary to induce a specified level of tissue damage (mean score of 7) by a maximum of 1.8 times (24 hr D1-D2). The kinetic pattern was similar to that for the tumors. In the ear studies, 41.0 degrees C for 30 minutes increased the time at 45 degrees C necessary to induce ear necrosis in 50% of animals by a maximum of 3.5 times (48 hr D1-D2). The peak tolerance level occurred later for the ears, which have a lower normal temperature of 28-30 degrees C, than for the tumors or legs. These results indicate that: thermotolerance can begin to appear in tumors during treatment if hyperthermia sessions involve initial low thermal exposures (near 41 degrees C) for 15 minutes or longer; thermotolerance can develop in tumors after systemic heating and occurs with a kinetic pattern similar to that following local heating; and normal tissues also can develop high levels of thermotolerance after similar thermal exposures.

Animals↗
PubMed1985

Protein synthesis, thermotolerance and step down heating.

The heat sensitivity of mammalian cells is modified by the cells' previous thermal history. If CHO cells are exposed to 42 degrees C or lower, they show resistance to subsequent heating at higher temperatures ("thermotolerance," TT); if the sequence is reversed, then an increased sensitivity is seen ("step-down heating," SDH). There is considerable evidence that protein synthesis is required for the development of tolerance, but nothing is known about the molecular events leading to SDH. We now show that for HA1 cells, the rate of protein synthesis (rPS) is related to both TT and SDH. The rPS of TT cells is 30% higher than of unheated cells. There is only a transient reduction of rPS during the exposure of TT cells to temperatures up to 43 degrees C with recovery occurring during heating. At higher temperatures, the effect is more severe and no recovery is observed. No recovery is seen during heating at 43 degrees C in previously unheated cells. On the other hand, SDH sensitization occurs in unheated cells and only when there is a severe and prolonged inhibition of the rPS (less than 10% of the control value). TT cells do not show SDH and also only show transient rPS reductions. Our results indicate that proteins must be synthesized for the development of TT and that SDH develops primarily as a consequence of the inhibition of the development of TT.

Animals↗
PubMed1985

Differential effects of hyperthermia on the Na+,K+-ATPase of Chinese hamster ovary cells.

The effect of hyperthermia in the 41-50 degrees C range on the Na+,K+-ATPase of CHO cells has been investigated. Three separate activities of the enzyme, namely, ATP hydrolysis, K+ uptake, and binding of the specific inhibitor, ouabain, have been measured independently. The results can be summarized as follows: (1) The ouabain binding capacity of intact cells decreased with increasing temperature and with increasing time at any one temperature. (2) The loss of ouabain-sensitive K+ uptake after treating cells at 45 degrees C was very similar to the loss of ouabain binding capacity. In contrast, the ATP hydrolyzing activity was much more resistant to heat. (3) The ouabain binding capacity was more resistant to hyperthermia at 45 degrees C if the cells were given a prior exposure at 45 degrees C 12 hr earlier. In contrast, the ATP hydrolyzing activity did not display this tolerance to heat.

Adenosine Triphosphate↗
PubMed1985

Thermal sensitivity of endothelial cells.

Experimental work indicates that one of the mechanisms of tumor control by hyperthermia may be damage to blood vessels, resulting in decreased blood flow to the neoplasms. Among the various elements of the microvasculature, endothelial cells are the most important possible targets of thermal injury. Furthermore, neoplasms have a significantly higher proportion of proliferating endothelial cells than do normal tissues. Thus it is necessary to establish the thermal sensitivity of endothelial cells and to explore possible differences in response between resting and proliferating endothelium. We studied the in vitro thermal sensitivity of murine and human capillary endothelial cells compared to human fibroblasts by following cell survival and growth recovery. Nonstimulated endothelial cells are more sensitive than fibroblasts. Their sensitivity is dose dependent within the range of 42 to 45 degrees C/30 min. Stimulation to proliferate by endothelial cell growth factor (ECGF) renders these cells even more sensitive. Morphologic studies confirm these thermal effects in endothelial cells and fibroblasts. These findings support a direct effect of hyperthermia on endothelial cells, which appears to be more severe in proliferating cells. This may explain the reduced blood flow in heated tumors and may indicate a valuable therapeutic gain for hyperthermia.

Animals↗
PubMed1985

Thermal sensitivity to single and double heat treatments in normal canine liver.

The treatment of intrahepatic or perihepatic neoplasms by hyperthermia may be limited by the thermal sensitivity of normal liver tissue. To establish the temperature dependence of hepatic toxicity, eight canine liver lobes were exposed to a single 30-min dose of localized hyperthermia in the range of 43.0 degrees C-47.5 degrees C, induced by radiofrequency currents. Four additional liver lobes were conditioned with a pretreatment dose of 43.0 degrees C/30 min and challenged at either 44.5 degrees C/30 min or 47.5 degrees C/30 min, 4 h later. Temperature distributions were measured using implantable thermocouple sensors. Treated areas were sampled 28 days later, and liver damage was determined using histopathological criteria. Most treated sites showed only modest alterations. The parameters of tissue injury that correlated best with dose were: evidence of hepatocyte loss; focal fibrosis; and distortion of lobular architecture. Areas of necrosis were observed in several samples, but their presence or severity did not correlate with dose. Thermal damage to liver capsule, liver lobules, portal areas, and central veins did not exhibit monotonic dose-response relationships. The data do not demonstrate thermotolerance; in fact, they suggest, although do not prove, its absence. If thermotolerance did not develop, vascular effects might explain such a finding.

Animals↗
PubMed1985

Mechanistic implications of the induction of thermotolerance in Chinese hamster cells by organic solvents.

Several organic solvents were shown to induce thermotolerance. These included: aliphatic alcohols (ethanol to octanol); local anesthetics; dimethyl sulfoxide; and dimethyl formamide. The minimum concentrations of the various alcohols required to induce tolerance were similar to the threshold doses for cytotoxicity. When only intramembrane concentrations were considered (by multiplying by the appropriate membrane:buffer partition coefficient), then the alcohols' threshold dose to induce tolerance was only mildly a function of carbon number. The efficiency increased almost linearly with a membrane dose from propanol to pentanol, but was slightly reduced for octanol. All alcohols induced tolerance rapidly (within a few hours after 30-min exposure), but the thermotolerance ratio decreased with increasing carbon number. Of the three anesthetics examined, lidocaine was a highly efficient inducer, procaine less so, and tetracaine did not induce tolerance even at a concentration mildly cytotoxic. Dimethyl sulfoxide and dimethyl formamide induced tolerance without cytotoxicity, but the kinetics of doing so was different from that seen with the other solvents. Only cells exposed to these water-soluble agents for 18 h or longer showed evidence of induced heat resistance. Our results indicate that membranes likely are the site for the initiation of solvent-induced cell killing and thermotolerance. The minimum dose of induction may be related to the degree of disorder induced by the individual alcohol, but the amount of tolerance induced is inversely related to this. The data on the effects of local anesthetics show that neither their common functional mode of inhibiting nerve transmission nor their similar level of cytotoxicity insures similar efficiency as tolerance inducers. Results with dimethyl sulfoxide and dimethyl formamide suggest that tolerance can be induced by more than one mechanism, or alternately, that a multiple-step mechanism can be activated at different points along this pathway. We also measured heat shock protein synthesis after several treatments; efficient inducers of thermotolerance also induced these proteins.

Alcohols↗
PubMed1985

Effects of proliferation on the decay of thermotolerance in Chinese hamster cells.

Development and decay of thermotolerance were observed in Chinese hamster HA-1 cells. The thermotolerance kinetics of exponentially growing and fed plateau-phase cells were compared. Following a 10-min heat exposure at 45 degrees C, cells in both growth states had similar rates of development of tolerance to a subsequent 45-min exposure at 45 degrees C. This thermotolerant state started to decay between 12 and 24 hr after the initial heat exposure. The decay appeared to initiate slightly sooner in the exponentially growing cells when compared to the fed plateau-phase cells. During the decay phase, the rate of thermotolerance decay was similar in the two growth conditions. In other experiments, cells were induced to divide at a slower rate by chronic growth (3 months) in a low concentration of fetal calf serum. Under these low serum conditions cells became more sensitive to heat and the rate of decay of thermotolerance remained the same for exponentially growing cells. Plateau-phase cells were also more sensitive, but thermotolerance decayed more rapidly in these cells. Although dramatic cell cycle perturbations were seen in the exponentially growing cells, these changes appeared not to be related to thermotolerance kinetics.

Acclimatization↗
PubMed1984

Inhibition of the recovery from potentially lethal damage by lonidamine.

Lonidamine [1-(2,4-dichlorobenzyl)-1-H-indazol-3-carboxylic acid] is shown to inhibit recovery from potentially lethal damage after exposure of cells to X-rays, methyl methane sulfonate, or bleomycin and heat (43 degrees C, 1h). Inhibition is most effective when the drug is present before and after exposure of 10 to 25 mg l-1, a concentration readily achievable in vivo.

Animals↗
PubMed1984

The polycation diethylaminoethyl dextran potentiates thermal cell killing.

The polycation diethylaminoethyl dextran (DEAE-dextran) causes marked potentiation of the effects of hyperthermia (45 degrees C) on HA-1 Chinese hamster ovary cells. The effects of the polycation appear to be mediated at the cell surface and are largely reversed by removal of DEAE-dextran with the polyanion dextran sulphate. Maximal potentiation is observed when polycation and heat are given simultaneously. The polycation also inhibits the capping of concanavalin-A acceptors when used singly and is additive with heat in inhibiting capping; reduction in con-A capping may indicate decreased mobility of cell surface glycoproteins. The mechanism of heat potentiation by DEAE-dextran is not fully understood. Effects on surface charge and cytoskeleton-membrane interactions may be involved. However, the polycation appears to have promise, both in probing mechanisms of heat killing and as an adjuvant to heat treatment; potentiation occurs at extremely low DEAE-dextran levels, with considerable potentiation at a concentration of 0.4 microM.

Animals↗