Milton B. Yatvin

The Influence of Membrane Lipid Composition and Procaine on Hyperthermic Death of Cells

The mechanism of hyperthermic killing, a component of some cancer therapy, is not known. Cell-survival curves during hyperthermic exposure can be used to elucidate the effects of membrane modifying procedures on cell death. Experiments were designed to test whether procedures that were reported to increase membrane fluidity also resulted in increased killing on hyperthermic exposure. An E. coli K12 mutant, K1060, is used to predictably alter the degree and amount of unsaturated fatty acids incorporated into membranes. Changing from an 18:1 to an 18:3 unsaturated fatty acid increases killing. Decreasing the amount of unsaturated fatty acid cells incorporated by increasing growth temperature decreases killing. Procaine, a drug known to decrease membrane viscosity, increases heat killing. These data are most simply explained by the hypothesis that membrane disorganization occurs as a result of temperature increasing to a point where a lipid transition causes a membrane structural change, which results in cell-death.

Correlation of Hyperthermic Sensitivity and Membrane Microviscosity in E. Coli K1060

We have demonstrated a positive correlation between membrane microviscosity and the temperature required to kill E. coli. Batches of cells with differing unsaturated fatty acid (u.f.a.) compositions were prepared from the u.f.a.-requiring E. coli K12 mutant K1060. The membrane microviscosity of these cells is estimated from the extent of fluorescence polarization of the probe molecule 1,6-diphenyl-1,3-5,-hexatriene dissolved in the membrane. For the same growth temperature, cells grown in oleic acid (18:1) have a greater microviscosity and u.f.a. content than linolenic acid (18:3) grown cells. the rate of decrease in microviscosity with increasing temperature is correlated with the amount of u.f.a. present in the membrane. From survival curves determined at several hyperthermic exposures, one can interpolate the hyperthermic temperature required to kill 90 per cent of the cells in three hours. These equivalent kill temperatures are directly related to the cell microviscosity. These data support the hypothesis that cell membrane microviscosity plays a critical role in hyperthermic killing.

Influence of Unsaturated Fatty Acids, Membrane Fluidity and Oxygenation on the Survival of an E. Coli Fatty Acid Auxotroph Following γ-irradiation

Escherichia coli K1060, a fatty acid auxotroph unable to either synthesize or degrade unsaturated fatty acids (uFAs), was used to study the effect of membrane fluidity on survival after exposure to ionizing radiation. Using this strain of E. coli, significant alterations in the fatty acid composition of the membrane have been produced and verified by gas chromatography. Linolenic, oleic, elaidic and palmitelaidic acids were the uFAs used. Survival above the transition temperature (Tt) (liquid crystal in equilibrium gel) was comparable for these fatty-acid-supplemented membranes after exposure to gamma-irradiation, whereas gamma-irradiation below Tt resulted ina significant decrease in survival. An oxygen enhancement effect was observed for each experimental condition employed.

Radiation Killing of E. Coli K1060: Role of Membrane Fluidity, Hypothermia and Local Anaesthetics

The enhancement of killing by gamma-irradiation, which is seen when E. coli K1060 are cooled below the transition temperature of their membrane lipids, is blocked by procaine-HCl. These data are consistent with the hypothesis that increased killing associated with irradiation at 0 degree C is the result of membrane microviscosity increases, since procaine is known to fluidize membranes. A cooling enhancement ratio (c.e.r.) is defined as the ratio of radiation D0 at 22 degrees C to its value at 0 degree C. The c.e.r. for oxygen-bubbled cells is 1.5 and for nitrogen-bubbled cells is 2.1. In the presence of 25 mM procaine the respective c.e.r. values are 1.08 and 1.29. The oxygen enhancement ratio (o.e.r.) at 22 degree C is 3.43 and at 0 degree C is 2.45. The addition of procaine does not change the o.e.r. Thus, the temperature effect on o.e.r. does not appear to be related to membrane fluidity.

“Repair” of plasma membrane injury and DNA single strand breaks in γ-irradiated Escherichia coliBr and Bs−1

Abstract Treatment of Escherichia coli spheroplasts with a series of hypotonic saline solutions produces cell lysis and by plotting the percent of remaining spheroplasts against osmolality a typical sigmoid curve is obtained. From this curve the 50 % level of osmotic lysis, a convenient measure of plasma membrane fragility, can be derived. When E. coli B r or Bs-1 are irradiated with γ-rays (18–54 krad) prior to being placed in the hypotonic environment, there is a marked increase in spheroplast fragility. Reincubation of irradiated B r cells results in less fragile spheroplasts. Irradiated E. coli Bs−1 act in a similar fashion with the spheroplasts becoming more fragile. However, in contrast to E. coli B r , reincubation of E. coli Bs-1 cells does not result in a decrease in spheroplast fragility. The “repair” of membrane damage after reincubation of E. coli B r cells is prevented by the presence of either quinicrine, acriflavin or chloramphenicol or the absence of glucose in the media during reincubation. E. coli B r cells which separate into three zones on linear Renografin gradients (45–70 %) after being irradiated and reincubated, show differential rates of osmotic fragility for the various zones. In addition, after irradiated E. coli B r cells have been reincubated for 40 min, the percent of cells in each zone varied with dose. The third zone contained 4 % of the total cell population after 18 krad and had over 20 % after 54 krad. Alkaline sucrose gradient analysis of single-strand DNA from control, irradiated and irradiated-reincubated E. coli B r cells, as well as irradiated-reincubated cells separated on Renografin gradients was performed. The data were plotted in two ways: normalized to 104 counts and on the basis of counts per 107 cells. Renografin Zones 1 and 2, after 18 krad and 40 min reincubation, had a pattern similar in shape to the control. In contrast, cells from Zone 3 showed little similarity to the normal DNA profile, even when plotted on a percent basis. After the reincubation of cells exposed to 54 krad, none of the cell zones, regardless of how the data were plotted, yielded normal-appearing alkaline sucrose gradient profiles. It is possible that only little rejoining of radiation-caused single strand breaks takes place during reincubation, but that degradation of DNA strands broken by irradiation occurs and when plotted on a percent of recovered radioactivity basis an overestimate of repair is obtained. An alternative hypothesis to the occurrence of any major amount of single strand breaks in irradiated reincubated E, coli B r is presented and discussed.

Hyperthermic Sensitivity and Growth Stage in Escherichia coli

Hyperthermic sensitivities of Escherichia coli B/r and Bs-1 were determined for lag-, midlog-, and stationary-phase cells at 47, 48, and 49 degrees C. In both strains midlog-phase cells were strikingly more heat sensitive (100-fold greater killing after 4 h at 48 degrees C) than stationary-phase cells, with intermediate sensitivity for lag-phase cells. In contrast to the reported difference in the radiation sensitivity between these two strains, very little difference in heat sensitivity was seen. Patterns of fatty acid composition of both strains were very similar at each phase of growth. From midlog to stationary phase, 16:1 and 18:1 unsaturated fatty acids decrease from 16 and 30% to 0.5 and 3%, respectively, while the C17 and C19 cyclopropane fatty acids increase from 7 and 3% to 22 and 25%, respectively. Concomitant with these changes in fatty acid composition, substantially higher membrane microviscosity values were recorded for stationary-phase cells. Total membrane microviscosity was positively associated with the C17 and C19 cyclopropane fatty acid composition and with cell survival following hyperthermia. In contrast to hyperthermic sensitivity, radiation survival differences between B/r and Bs-1 are little affected by growth stage. We propose that these results are consistent with a critical influence of membrane lipids on cellular hyperthermic sensitivity and further that the target sites for radiation and hyperthermia are different in these cells.

Potentiation of differential hyperthermic sensitivity of AKR leukemia and normal bone marrow cells by lidocaine or thiopental.

Previous work has utilized spleen colony formation to evaluate the fractional survival of AKR leukemia and normal bone marrow cells after in vitro heat exposure. An inherently greater sensitivity of neoplastic cells to thermal killing, as compared to normal syngeneic stem cells, has been established both at 41.8°C and 42.5°C. Normal bone marrow colony‐forming units were assayed in lethally irradiated (750 cGy) mice. Leukemic colony‐forming units were assayed in nonirradiated mice. Using this methodology, the authors demonstrated that the differential effect of hyperthermia on AKR murine leukemia and AKR bone marrow cells can be further enhanced by the addition of lidocaine or thiopental to incubation mixtures. These findings may have application to autologous bone marrow transplantation in humans.

Correlation of Bacterial Hyperthermic Survival with Anaesthetic Potency

We have evaluated several local anaesthetics and hypnotics for their relative ability to influence hyperthermic cell killing. Bacterial cell survival following exposure to heat and anaesthetic was used as the assay system. The E. coli bacterium used was the unsaturated fatty acid auxotroph, K1060. It was grown at 37 degrees C in medium supplemented with oleic acid and then exposed to 47 degrees C hyperthermia in the presence of an anaesthetic. The local anaesthetics tested were procaine, lidocaine, tetracaine, and benzocaine, and the general anaesthetics were barbital and pentobarbital. The dose response for each anaesthetic was determined over a five-hour heating period. The anaesthetic concentration required during heating to halve the time for cell killing found with heat alone is 5.9 mM for procaine, 0.8 mM for lidocaine, 0.12 mM for tetracaine, 2.0 mM for benzocaine, 6.7 mM for barbital and 1.2 mM for pentobarbital. There is a direct correlation between equivalent effect doses of the local anaesthetics and published data for the relative potency of the same anaesthetics as determined by respiratory arrest in mice and by myocardial contractile force in dogs. The assay we have described would be a convenient and easy test for the interaction of these drugs with hyperthermia. The use of this interaction with hyperthermia as an adjuvant in combined radiation-hyperthermia therapy should be tested.

Influence of membrane-lipid composition on translocation of nascent proteins in heated Escherichia coli

In studies using Escherichia coli we have shown that new protein species appear in the outer membrane fraction with concomitant losses of nascent proteins from the soluble and inner membrane fractions following heat exposure. Of the various explanations for this phenomenon, temperature-induced membrane disorganization appeared the most likely. It is suggested that heat mimics the action of the signal sequence of a protein on the lipid bilayer allowing non-signal-sequence-containing proteins to be translocated. To test this hypothesis we grew E. coli K1060 cells, an unsaturated fatty acid requiring auxotroph, supplemented during growth with fatty acids of varying chain length in an attempt to determine whether biological membranes of varying ability to maintain their bilayer configuration could be constructed. The rationale being that such membranes would allow us to determine whether differences in translocation would occur in cells grown at the same temperature supplemented with either 16:1 or 20:1 unsaturated fatty acids when the cells were subjected to a series of thermal insults. Protein translocation occurred to a greater extent and at lower temperatures in cells supplemented with the longer chain fatty acid. Treatment of outer membranes with either 1 M salt, 6 M urea or high pH and studies determining fluorescent polarization values by scanning up and down through a series of temperatures ranging from 15 to 49 degrees C indicate that the proteins translocated by heat to the outer membrane are integral. Protein translocation may represent an adaptive response to an altered environment enabling the cell to respond to stress by stabilizing its outer membrane.

Hyperthermia and surface morphology of p388 ascites tumour cells: effects of membrane modifications.

The quantitative distribution of cell surface alterations of heated P388 ascites tumour cells was determined by scanning electron microscopy. Cells harvested from host animals maintained on a standard rodent chow diet or one high in saturated fatty acids responded differently, to identical hyperthermic treatment in vitro, to cells obtained from animals on a highly unsaturated diet. The morphological response of cells from chow fed animals was modified by addition to the incubation medium, of procaine, a membrane-active drug. The pattern of response observed after these cells were heated in the presence of procaine resembled that seen following heat treatment of ascites cells obtained from animals fed diet high in unsaturated fatty acids. These data are consistent with the hypothesis that a cells response to hyperthermic insult is related to its membrane fluidity at the time of treatment.