Biomedical subjects

P M Corry

Publications and source records attributed to P M Corry.

At least 55 records · Page 3Linked to original sources

PubMed1993

Homoharringtonine induces heat protection and facilitates dissociation of heat shock transcription factor and heat shock element complex.

We investigated the effects of combined treatment with homoharringtonine (HHT) and hyperthermia on cytotoxicity and transcriptional regulation of heat shock genes in human colon carcinoma (HT-29) cells. The drug (100 ng/ml) which inhibited protein synthesis by 93% protected cells from killing at 43 degrees C. For example, treatment with HHT 2 hr before and during heating produced a 9-fold increase in survival from 3.7 x 10(-2) to 3.2 x 10(-1) after 10 hr at 43 degrees C. Little or no protection was observed if the drug was added only during heating. Interestingly, adding the drug (100 ng/ml) 2 hr before and during heat facilitated the dissociation of heat shock transcription factor-heat shock element (HSF-HSE) complex during continuous heating at 43 degrees C. These findings related to the literature suggest that the free pool of HSC70 is increased by inhibiting protein synthesis. An increase in the level of free HSC70 may more effectively protect or repair thermolabile targets and consequently affect regulation of heat shock response.

Antineoplastic Agents, Phytogenic↗

PubMed1993

Sensitivity of human cells to mild hyperthermia.

The cytotoxic effects of short duration, high temperature, and long duration, low temperature hyperthermia were determined in human cells growing in culture. The human tumor cell lines A549 (lung carcinoma), WiDr (colon carcinoma), and U87MG (glioblastoma) were used. In addition, a normal human lung fibroblast cell type 18Lu was used. Sensitivity to direct cell killing was measured at 41, 43, and 45 degrees C. Heat induced perturbations of cell cycle and proliferation were also analyzed. The results obtained on sensitivity of the above human cell lines at 43 and 45 degrees C are similar to those of the previous work of others in that the human cell lines were observed to be relatively resistant to thermal killing at 43 or 45 degrees C, when compared to heat sensitive rodent cell lines. The comparison is important because most prior hyperthermic research has been performed with rodent cells and clinical protocols have been designed with the use of rodent data. In contrast to the 43 degrees C response, most of the human cells we tested were killed by 41 degrees C heating to an extent greater than that observed for rodent cells. The heat sensitivities of the four different human cell lines varied widely. This appeared to be due to differences in both intrinsic heat sensitivity and tolerance development. During 41 degrees C heating, human cells did not proliferate and cell cycle perturbations developed but did not correlate with sensitivity to killing. Our heat sensitivity measurements point out the shortcomings of using data derived from rodent systems to predict clinical outcome of hyperthermia therapy.

Animals↗

PubMed1993

Synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29 cells are not mediated by alteration of induced protein levels.

In this study, we investigated the mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29. When cells were heated at 42 degrees C in the presence of recombinant human tumor necrosis factor (rhTNF-alpha), recombinant interferon-gamma (rhIFN-gamma), or in a combination of both, a synergistic increase in the cytotoxic effects of the respective drugs was observed. We hypothesized that alteration of cytokine or heat-induced polypeptides synthesis was responsible for a synergistic interaction between heat and cytokine. Five heat shock proteins (HSPs, M(r) 110,000, 100,000, 90,000, 70,000, and 28,000) were preferentially synthesized during chronic heating at 42 degrees C. In contrast, the synthesis of two proteins (M(r) 60,000 and 29,000) was induced by treatment with rhIFN-gamma (1,000 U/ml). Although the combination of chronic hyperthermia (42 degrees C) with TNF-alpha, IFN-gamma, or TNF-alpha + IFN-gamma increased cytotoxicity, alteration/induction of polypeptides was not correlated with the synergistical cytotoxic effects of cytokine and heat. Thus, the synergistic effects of cytokine and hyperthermia are not mediated through an induction of polypeptides.

Antineoplastic Agents↗

PubMed1993

Effect of thermotolerance on heat-induced excess nuclear-associated proteins.

Earlier studies reported that thermotolerance had two effects on the heat-induced increase in nuclear-associated proteins (NAPs); reduction in NAP levels immediately following hyperthermia and facilitation of NAP recovery to control levels. It has also been demonstrated that there are two phases of thermotolerance; one that requires newly synthesized proteins (protein synthesis dependent thermotolerance; PSDT), and another that does not (protein synthesis independent thermotolerance; PSIT). This study was designed to determine if these two phases of thermotolerance affected NAP binding in a similar or different manner. The results demonstrated that protein synthesis during thermotolerance development was not required to reduce NAP levels measured immediately following hyperthermia, but was required to facilitate NAP recovery to control levels following hyperthermia. Reducing NAP levels was the predominant mechanism by which thermotolerance protected cells from this lesion at 43.0 degrees C while facilitated NAP recovery predominated in protecting against exposure to 45.5 degrees C. The facilitated recovery of NAPs required only proteins synthesized following thermotolerance induction and prior to the second heat challenge. Proteins synthesized following the second heat challenge were not requisite. Finally, the processes that facilitate NAP recovery were inhibited at 3 degrees C, suggesting that they are enzymatically mediated.

Animals↗

PubMed1992

Absence of HSP28 synthesis and phosphorylation during the development of chronic thermotolerance in murine L929 cells.

We investigated the correlation between chronic thermotolerance development and phosphorylation, synthesis, or expression of the HSP28 family in murine L929 cells. Chronic thermotolerance developed during heating at 41.5 degrees C as indicated by a biphasic survival curve. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, we failed to detect HSP28 synthesis during chronic heating by using two-dimensional polyacrylamide gel electrophoresis. The lack of HSP28 synthesis was also confirmed in acute thermotolerance. Similar results were observed in NIH 3T3 cells. Although Southern blots clearly demonstrated the presence of the HSP28 gene in genomic DNA, Northern blots failed to demonstrate its expression. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, were stimulated during chronic heating at 41.5 degrees C in L929 cells. These results suggest that HSP28 synthesis and its phosphorylation are not required to develop both chronic and acute thermotolerance in L929 cells.

3T3 Cells↗

PubMed1992

Effect of pH on quercetin-induced suppression of heat shock gene expression and thermotolerance development in HT-29 cells.

When cells were heated for 15 min at 45 degrees C, they became thermotolerant to a second heat exposure at 45 degrees C. Thermotolerance developed rapidly, reached its maximum 6 hr after heat shock, and then gradually decayed. The development of thermotolerance was partially suppressed by treatment with various concentrations of quercetin (0.05-0.2 mM) at pH 7.4 after the initial heat treatment. In contrast, the drug markedly inhibited thermotolerance development at pH 6.5. Furthermore, a combination of low pH and quercetin treatment distinctively altered the expression of HSP70 gene compared with that of HSP28 or HSP90 gene. These results demonstrate a good correlation between the amount of HSP70 gene expression and development of thermotolerance.

Blotting, Northern↗

PubMed1992

Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.

Animals↗

PubMed1992

Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.

Animals↗

PubMed1992

Constitutive HSP70: oligomerization and its dependence on ATP binding.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.

Adenosine Triphosphate↗

PubMed1992

Elimination of dose-rate effects by mild hyperthermia.

PURPOSE: Preferential amplification of low dose-rate irradiation toxicity in tumor cells is one way of improving presently applied brachytherapy. Low temperature hyperthermia applied to a tumor volume during irradiation is one candidate for reaching this goal. The ranges of relevant temperatures and dose-rate have been determined in a tissue culture system. In addition, the role of inhibition of sublethal damage repair in inhibition of dose-rate sparing has been investigated. METHODS AND MATERIALS: Dose-rate modification by long duration, mild hyperthermia was investigated in rat 9L gliosarcoma cells at dose-rates between 0.0833 and 132 Gy/hr. Enhancement of toxicity was measured using the colony formation technique. RESULTS: A biphasic dose-rate effect curve was observed when cells were irradiated at 37 degrees C. The dose required to kill 99% of cells irradiated at 37 degrees C increased sharply between 20 and 5 Gy/hr and also below 1 Gy/hr. When cells were irradiated at 41 degrees C, dose-rate sparing disappeared from 132 to 0.0833 Gy/hr. Elimination of dose-rate sparing appeared to be caused by both inhibition of sublethal damage repair and blockage of cell proliferation. The temperature threshold for sensitizing low dose-rate irradiation was determined at temperatures between 24 degrees C and 41 degrees C during 0.5 Gy/hr irradiation. Temperature dependent sensitization occurred above 39 degrees C. The mechanisms by which low temperature hyperthermia sensitizes low dose-rate irradiation was investigated using split dose experiments. Survival curve shoulder recovery was inhibited when cells were incubated at 41 degrees C between acute irradiations. This effect appeared to be caused by both inhibition of recovery from sublethal damage from the first radiation and preheating sensitization of the second irradiation. In single acute dose experiments, sensitization from preheating at 41 degrees C increased gradually over a 6 hr period. CONCLUSIONS: The mechanism by which 41 degrees C hyperthermia sensitizes low dose-rate irradiation is inhibition of radiation repair at medium dose rates and inhibition of repair and proliferation at very low dose rates. If low temperature hyperthermia is able to sensitize human tumor cells to brachytherapy similar to what has been described with 9L gliosarcoma cells, the addition of this modality could potentially greatly improve presently applied therapy.

Animals↗

PubMed1992

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.

Adaptation, Physiological↗

PubMed1991

Sensitization of rat 9L gliosarcoma cells to low dose rate irradiation by long duration 41 degrees C hyperthermia.

Modification of survival by long duration, 41 degrees C hyperthermia in combination with low dose rate radiation (0.5 Gy/h) was determined in rat 9L gliosarcoma cells. Cells were exposed to radiation in a manner that simulated continuous irradiation at a dose rate relevant to clinical brachytherapy. High dose rate X-irradiation was fractionated in 1.0-Gy fractions at 2-h intervals (FLDRI). Previous studies had demonstrated that 9L cells exposed to FLDRI with these parameters have survival characteristics that are equivalent to continuous low dose rate irradiation. Cells exposed to 41 degrees C throughout FLDRI were sensitized significantly (thermal enhancement ratio of 2.07) compared with cells irradiated at 37 degrees C. Incubation for 24 h at 41 degrees C before and/or after FLDRI at either 37 degrees C or 41 degrees C did not increase the slope of the radiation survival curves but did reduce the shoulder. Similarly, heating at 43 degrees C for 30 or 60 min before and/or after irradiation at 0.5 Gy/h also did not enhance cell sensitivity. Survival of cells after irradiation at high dose rate (60 Gy/h) was independent of the temperature during irradiation. Preheat at 41 degrees C for 24 h did not sensitize cells to high dose rate irradiation by increasing the slope of the survival curve, although a loss of shoulder was observed. Sensitization of cells heated at 43 degrees C for 30 or 60 min before high dose rate irradiation was expressed as classical slope modification. Our results demonstrate that 41 degrees C heating during FLDRI greatly sensitizes cells to radiation-induced killing for exposure durations up to 36 h. Heating 9L cells at 41 degrees C or 43 degrees C adjacent to FLDRI at 0.5 Gy/h resulted in no additional enhancement of terminal sensitivity, although shoulder modification was observed. The sensitization by simultaneous heating described above occurred even though thermotolerance developed during extended incubation at 41 degrees C. These in vitro data demonstrate that simultaneous protracted heating at modest temperatures could greatly enhance the cytotoxic effects of low dose rate interstitial irradiation and could be of significance in clinical application.

Animals↗

PubMed1991

Mechanism(s) of heat killing: accumulation of nascent polypeptides in the nucleus?

To investigate the possibility that nascent polypeptides released from polysomes by heat shock accumulate in the nucleus, cells were pulse labeled with [35S]methionine for two minutes and heated immediately thereafter at 45.5 degrees C for 10 minutes. When isolated nuclei were subjected to gel electrophoresis and subsequently autoradiographed, heated nuclei exhibited an approximately 10-fold increase in radioactive polypeptides in comparison to nonheated controls. These nascent polypeptides were nonspecific molecules covering a wide range of molecular weights. It is plausible that the accumulation of polypeptides in the nucleus results in hyperthermic cytotoxicity. Therefore, we propose that a potential target for heat killing is within the nucleus, at sites where nascent polypeptides accumulate after heat shock.

Animals↗

PubMed1991

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells.

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.

Animals↗

PubMed1991

Effect of tunicamycin on glycosylation of a 50 kDa protein and thermotolerance development.

We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a heat treatment at 45.5 degrees C administered 12 hr later. The thermotolerance ratio at 10(-3) isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2-2 micrograms/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 micrograms/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 micrograms/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance development by treatment with a combination of 2 micrograms/ml tunicamycin and 10 micrograms/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis-independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein.

Animals↗

PubMed1991

Inhibition of protein synthesis and heat protection: histidinol-resistant mutant cell lines.

The mechanism of histidinol (HST)-induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST-resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125-fold increase in survival from 4 x 10(-3) to 5 x 10(-1) after 2 hr at 43 degrees C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 micrograms/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat protection.

Animals↗

PubMed1991

RTOG quality assurance guidelines for clinical trials using hyperthermia for deep-seated malignancy.

Quality assurance has been vague or lacking in many previous hyperthermia trials. Recent publications by the Hyperthermia Physics Center, the Center for Devices and Regulatory Health, and the Radiation Therapy Oncology Group have described general guidelines for quality assurance in equipment reliability and reproducibility, superficial applications, and microwave techniques. The present report details quality assurance factors that are believed to be important for hyperthermia of deep clinical sites, defined as extending at least 3 cm beyond the skin surface. This document will discuss patient and physician factors, as well as thermometric accuracy, assessment of specific absorption rates (SAR), assurance of adequate coverage of tumors by the energy deposition pattern of the treatment device, and recommended documentation of the location, quantity, and frequency of treatment, specifically oriented to deep hyperthermia. The recommendations are structured to facilitate compliance in multiinstitutional trials.

Clinical Protocols↗

PubMed1991

Differences in thermotolerance induced by heat or sodium arsenite: correlation between redistribution of a 26-kDa protein and development of protein synthesis-independent thermotolerance in CHO cells.

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.

Acclimatization↗