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Biomedical subjects

E W Gerner

Publications and source records attributed to E W Gerner.

At least 91 records · Page 5Linked to original sources
PubMed1982

Effect of heating on lethality due to hyperthermia and selected chemotherapeutic drugs.

Because the rate of heating alters various cellular events associated with exposure to hyperthermia (including the rate of cell death), effect of this parameter on the cytotoxic interaction between selected anticancer drugs and hyperthermia was studied. The drugs cisplatin, 1,3-bis(2-chloroethyl)-1-nitrosourea, and adriamycin at 42.4 degrees C and bleomycin and methotrexate at 43 degrees C were tested in Chinese hamster ovary (CHO) cells in vitro. Heating times ranged from less than 3 minutes (immediate exposure) to 3 hours. For all drugs tested, synergistic cytotoxicity was not significantly altered by heating to peak temperature over 30 minutes as compared with immediate exposure. When heating to peak temperatures was prolonged to 3 hours, however, cell killing was markedly reduced, although significant sensitization to the drug remained. This was true despite the fact that, in CHO cells, heating to 42.4 degrees C over 3 hours produced no cell killing for up to 6 hours at that temperature. These results suggested that the mechanism(s) responsible for induced thermotolerance are probably different from those that cause cellular resistance to the cytotoxic effects of the drugs tested. These results may also partially explain the relative lack of clinical success with whole-body hyperthermia and chemotherapeutic drugs an anticancer treatment, because heating to therapeutic temperatures often requires 2-3 hours.

Animals↗
PubMed1982

Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture.

The weight ratio of either cholesterol or phospholipid to protein contents in 7 different cell lines, growing exponentially at 37 degrees, correlates positively with increasing resistance of the cells to subsequent hyperthermic cell killing. The relative heat resistance of each cell line is derived from survival curves obtained when the different cell lines are exposed to 43 degrees. Cholesterol and phospholipid amounts in the particulate fraction correlate with survival sensitivity to 43 degrees when the values are expressed per mg protein but not when expressed per cell number. Also, cholesterol:phospholipid molar ratios and the amount of protein in the particulate fraction do not display linear correlations with sensitivity of the respective cell lines to 43 degrees-induced cell killing. The relative degree of fatty acid saturation at 37 degrees also is independent of whether cells show a higher degree of heat resistance. These data suggest that lipid (both cholesterol and phospholipid):protein weight ratios correlate with increasing resistance of cells to an elevation in temperature. The major implication of these data is that major membrane components can influence and perhaps predict cellular survival to hyperthermia.

Animals↗
PubMed1982

Interstitial thermoradiotherapy.

The results of a phase I clinical trial in which heat was combined with interstitial (low dose rate) radiation are described with emphasis on response and technical and physical aspects of heating. We treated 25 patients (27 lesions) using interstitial implants to locally recurrent, accessible tumors heated by radiofrequency currents to 43 degrees -45 degrees C for 30 minutes, with needle guides as electrodes. They were subsequently irradiated with either 192Ir or 226Ra. All patients had failed previous conventional treatments including surgery, chemotherapy, and, in most, near tolerance doses of irradiation. In all but one, a single hyperthermic treatment was given and the average dose of low dose irradiation was under 3,000 rad over 60 hours. No patient failed to respond, 63% achieved a complete disappearance of tumor in the treated volume, and 37% had partial response (50% less than volume reduction less than 100%). Duration of response was from 2 to 30 months, and no patient showed regrowth at the site treated. Normal tissue complications were minimal. Interstitial thermoradiotherapy is both a safe and an effective means of treatment in advanced or recurrent accessible disease.

Adenocarcinoma↗
PubMed1981

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.

Animals↗
PubMed1981

Rate of heating as a determinant of hyperthermic cytotoxicity.

In Chinese hamster ovary cells and in normal and transformed rat embryonic fibroblasts, survival as a function of time at 42.4 degrees was dependent upon the rate of heating from 37 degrees to 42.4 degrees. Unexpectedly, the untransformed rat fibroblasts were more heat sensitive than were the transformed cells, and the protective effect of slow rates of heating upon survival at 42.4 degrees was also more pronounced in the normal cells than in the transformed cells. In Chinese hamster ovary cells, total cellular cholesterol content and cell volume were found to change significantly with time at 42.4 degrees when cells were heated immediately (37 to 42.4 degrees within 3 min) but did not vary significantly during 6 hr at 42.4 degrees in cells heated from 37 to 42.4 degrees over 3 hr. Chinese hamster ovary cells heated immediately to 42.4 degrees also showed a significant drop in the protein content of the particulate fraction with time at 42.4 degrees. In contrast, cells heated over 3 hr showed a significant increase in the protein content of the particulate fraction with time at 42.4 degrees. These data suggest that, if cells are heated to hyperthermic temperatures over sufficiently long intervals, mechanisms have time to develop which protect the cell membrane against changes associated with cell death in rapidly heated cells. The protective effect of slow rates of heating may partially explain the relative lack of success thus far observed with the use of whole-body hyperthermia in which heating from 37 degrees to 42 degrees often requires 2 to 3 hr.

Animals↗
PubMed1981

Reversal of resistance to methotrexate by hyperthermia in Chinese hamster ovary cells.

Our Chinese hamster ovary cells are extremely resistant to methotrexate (MTX) (100% survival after 500 microgram/ml for 13 hr). However, exposure to 43 degrees (but not 41 degrees or 42 degrees) for 1 hr sensitizes the cells to MTX so that a 50% cell kill in excess of that due to hyperthermia occurs. Treatment of cells at 43 degrees increases net MTX uptake by about 30% at 30 min but causes a substantial reduction after 1 hr. This negative effect is greater in cells continually heated at 43 degrees than in those exposed for only 1 hr. Treatment at 43 degrees for 1 hr also markedly increases efflux of MTX out of cells over the that 2 hr. Dihydrofolate reductase activity was found to decrease to about 50% of control values by 4 to 5 hr after exposure to 43 degrees. The biological half-life of dihydrofolate reductase in Chinese hamster ovary cells was determined to be about 4.5 hr, indicating that hyperthermia-induced cessation of protein synthesis may explain both the decrease in dihydrofolate reductase activity and the sensitization to MTX observed with heat exposure. In scheduling experiments, lethality due to exposure to 43 degrees for 1 hr in conjunction with MTX was maximum when 1-hr drug exposure began just at the end of heat treatment.

Animals↗
PubMed1980

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells.

Ornithine decarboxylase activity increases at least 4-5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G(1) and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G(1)-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.

Animals↗
PubMed1980

Cholesterol levels inversely reflect the thermal sensitivity of mammalian cells in culture.

Cholesterol is a primary component of the mammalian cell plasma membrane. Although its function is unknown, it may be of major importance in maintaining membrane fluidity and rigidity. In artificial membrane systems, the addition of cholesterol results in a condensing effect--thickening the bilayer and inducing higher order in the acyl chains of the phospholipids. Permeability profiles indicate that the addition of cholesterol into egg-lecithin bilayers increases the half-time of solute transport. In addition, decreased amounts of sterol in the membrane increase glucose permeability, and, in L cells, increase the transport of rubidium. These studies suggest a role for cholesterol in changing the physical characteristics of the membrane resulting in the alteration of membrane permeability. We now provide evidence that cholesterol may act, presumably via changes in physical membrane properties, with yet another biological consequence; regulating the survival sensitivity of mammalian cells to hyperthermic temperatures.

Animals↗
PubMed1979

Separation of normal human bone marrow cells by counterflow centrifugal elutriation. I. Morphological analysis and subfractionation of neutrophilic granulocytes.

We have used counterflow centrifugal elutriation to separate the nucleated cells of normal human bone marrow into their major morphologically identifiable cellular components. Human bone marrow cells were separated simultaneously into the major cell types--lymphocytes, monocytes, granulocytes, and erythroid precursors. Significant concentration of bone marrow cell types could be achieved, including myeloblasts (x 25), promyelocytes (x 43), early myelocytes ( x 22), late myelocytes (x 17), metamyelocytes (x 6), eosinophils (x 40), basophils (x 55), erythroblasts (x 35), monocytes (x 40), and plasma cells (x 35). Cell loss is minimal (85% recovery of input) and random, and cell viability, as measured by trypan blue staining, is high (greater than 95%). Factors interfering with separation are also described: a low loading flow rate, the presence of an excess of mature red cells, and the absence of fetal calf serum resulted in poor cell recovery and viability. The use of heparin as the anticoagulant resulted in cell loss, decrease in cell viability, and an ineffective separation. Separation of cells from normal bone marrow by centrifugal elutriation should facilitate the study of bone marrow cell interactions and the purification of individual types of bone marrow cells.

Bone Marrow↗
PubMed1978

Cyclic AMP levels and types I and II cyclic AMP-dependent protein kinase activity in synchronized cells and in quiescent cultures stimulated to proliferate.

Cyclic AMP as well as the specific activity of cyclic AMP-dependent protein kinase decreased from the first two hours after Chinese hamster ovary cells in plateau phase were stimulated to proliferate by tripsinization of confluent cultures and dilution in fresh media. From two to five hours after this stimulation, the cyclic AMP level and the specific activity of cyclic AMP-dependent protein kinase increased two-fold. There was a 40--50% increase in the degree of activation of cyclic AMP-dependent protein kinase during this same time interval. In plateau cultures prior to being stimulated to proliferate, type I cyclic AMP-dependent protein kinase was the predominant soluble form of these enzymes. At five hours after release from plateau, the predominant type of cyclic AMP-dependent protein kinase was type II. However, there was also a significant amount of type I present at this time. Types I and II cyclic AMP-dependent protein kinases were differentially detectable during the cell cycle of Chinese hamster ovary cells synchronized by mechanical selection of metaphase cells following colcemid treatment. During mitosis, type I kinase was predominant with only a small amount of type II activity detectable. The amount of activity of type I then progressively decreased as cells entered G1. During early G1, there was no detectable activity of type II kinase, but its activity increased from mid to late G1 and then decreased during the S phase. These data show a tight temporal relationship between the levels of cyclic AMP, the total cellular pool of type I and II cyclic AMP-dependent protein kinases, and the degree of activation of these kinases as cells traversed G1 toward S phase. These data suggest that the expression of each type of kinase may be important for the regulation of substrate phosphorylation during the cell cycle.

Animals↗