Hypoxic cell sensitization: low-dose intrinsic radiosensitivity is predictive for etanidazole efficacy in a panel of human tumour cell lines.

We used a cell sorter assay to evaluate the efficacy of the hypoxic cell sensitizer etanidazole over 3-4 logs of cell inactivation, with particular attention to the clinically relevant low-dose survival region. Analysis of the radiation responses in a panel of six human tumour cell lines under conditions of hypoxia and hypoxia with etanidazole revealed both a cell-line and radiation dose-dependence in the sensitizing ability of this drug. Fits of the linear-quadratic (LQ) model to the low-dose region of cell survival indicate that sensitization of hypoxic cells by etanidazole results primarily from a modification of the beta parameter. This results in selective sensitization of cell lines in which this parameter contributes significantly to cell kill (i.e. a low alpha/beta ratio) and implies that the efficacy of this drug may be tumour specific. Selective modification of beta also leads to a radiation dose-dependence of the sensitizing enhancement ratio (SER). Analysis of the alpha and beta parameters derived from fist to data at low doses of radiation, suggests that the dose-dependence of this sensitizer, and possibly others including oxygen, is cell-line dependent; cell lines exhibiting a low alpha/beta ratio (i.e. with a large shoulder) exhibit little or no SER dose dependence, while those with a high alpha/beta ratio (i.e. small shoulder) exhibit a reduced SER at low doses as compared to high doses. Furthermore, this analysis suggests that modelling of the low-dose radiation survival data under conditions of hypoxia, can be predictive for both the absolute sensitizing ability of etanidazole, and its dose dependence. Our results also indicate that measurement of the in vitro low-dose radiation survival response in a panel of human cell lines is a more effective assay for evaluating agents like etanidazole than simply high-dose measurements in rodent cell lines which have, in general, demonstrated more congruent survival responses.

Low-dose hypersensitivity and increased radioresistance in a panel of human tumor cell lines with different radiosensitivity.

It is well known that cells of human tumor cell lines display a wide range of sensitivity to radiation, at least a part of which can be attributed to different capacities to process and repair radiation damage correctly. We have examined the response to very low-dose radiation of cells of five human tumor cell lines that display varying sensitivity to radiation, using an improved assay for measurement of radiation survival. This assay improves on the precision of conventional techniques by accurately determining the numbers of cells at risk, and has allowed us to measure radiation survival to doses as low as 0.05 Gy. Because of the statistical limitations in measuring radiation survival at very low doses, extensive averaging of data was used to determine the survival response accurately. Our results show that the four most resistant cell lines exhibit a region of initial low-dose hypersensitivity. This hypersensitivity is followed by an increase in radioresistance over the dose range 0.3 to 0.7 Gy, beyond which the response is typical of that seen in most survival curves. Mathematical modeling of the responses suggests that this phenomenon is not due to a small subpopulation of sensitive cells (e.g. mitotic), but rather is a reflection of the induction of resistance in the whole cell population, or at least a significant proportion of the whole cell population. These results suggest that a dose-dependent alteration in the processing of DNA damage over the initial low-dose region of cell survival may contribute to radioresistance in some cell lines.

Physiological comparison of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone with benzodiazepine and barbiturate modulators of GABAA receptors.

The GABAA receptor/chloride ionophore (GABAR) is allosterically modulated by several classes of anticonvulsant agents, including benzodiazepines and barbiturates, and some alkyl-substituted butyrolactones. To test the hypothesis that the anticonvulsant butyrolactones act at a distinct positive-modulatory site on the GABAR, we examined the physiological effects of a butyrolactone, a benzodiazepine and a barbiturate on GABA-mediated currents in voltage-clamped neurons and cells transfected with various subunit combinations. The butyrolactone, alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha EMTBL), altered the EC50 for GABA and changed the apparent cooperativity of GABA responses. In contrast, the benzodiazepine chlordiazepoxide altered the EC50 for GABA with no effect on apparent cooperativity. The barbiturate phenobarbital altered both the EC50 and the amplitude of the maximal GABA response without altering apparent cooperativity. The GABA-mediated effect of the barbiturate, but not the benzodiazepine, added to the maximal effect of the butyrolactone, supporting the hypothesis that butyrolactones do not exert their effect at the barbiturate effector site. Both alpha EMTBL and phenobarbital potentiated GABA currents in transfected cells containing the alpha 1 beta 2 and alpha 1 gamma 2 subunit combinations, as well as alpha 1 subunits alone. Chlordiazepoxide had the minimum requirement of an alpha subunit and a gamma subunit. Specific GABARs lacking benzodiazepine or barbiturate modulation were tested for modulation by alpha EMTBL. The alpha 6 beta 2 gamma 2 combination was modulated by the butyrolactone but not chlordiazepoxide. However, GABARs comprising rho1 subunits were sensitive to both phenobarbital and alpha EMTBL. Although the molecular determinants for alpha EMTBL action appear similar to the barbiturates, our data support the conclusion that alpha EMTBL interacts with GABARs in a distinct manner from barbiturates and benzodiazepines.

Dual modulation of the gamma-aminobutyric acid type A receptor/ionophore by alkyl-substituted gamma-butyrolactones.

Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones exhibit convulsant or anticonvulsant activity, depending on the alkyl substituents. alpha-Substituted lactones with small alkyl substituents are anticonvulsant and potentiate gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas beta-substituted compounds are usually convulsant and block GABAA currents. We have now found that this distinction is not so clear-cut, in that some compounds can both block and augment GABAA currents, but with different time courses. For example, alpha,alpha-diisopropyl-GBL (alpha-DIGBL) potentiates exogenous GABA currents in cultured rat hippocampal neurons but diminishes GABA-mediated inhibitory postsynaptic currents. A more detailed analysis demonstrates a triphasic effect of alpha-DIGBL on GABA currents, with a rapid inhibitory phase, a slower potentiating phase, and then an "off response" when the GABA/alpha-DIGBL perfusion is stopped. Thus, alpha-DIGBL can inhibit and potentiate GABA currents with kinetically different time courses. Inhibition is more rapid, but at steady state potentiation dominates. Using a simplified model of the GABAA receptor/ionophore, we have simulated our experimental observations with alpha-DIGBL. Another lactone, beta-ethyl-beta-methyl-gamma-thiobutyrolactone, also has dual actions, with inhibition predominating at low concentrations and potentiation predominating at high concentrations. We propose two distinct GBL modulatory sites on the GABAA receptor, i.e., an inhibitory "picrotoxin" site and an enhancing "lactone site." New information on the structure of the GABAA receptor/ionophore may allow the molecular dissection of these two sites.

Developmental alteration in GABAA receptor structure and physiological properties in cultured cerebellar granule neurons.

Although we now have extensive knowledge about the GABAA receptor subunits determining benzodiazepine modulation of channel function, little is known about subunits influencing other modulatory sites on the GABAA receptor-chloride channel complex. We have identified a developmental change in subunit composition of the GABAA receptor in cultured cerebellar granule neurons that eliminates benzodiazepine-mediated enhancement of GABA responses and alters modulation by a substituted gamma-butyrolactone. Based on data from sequential PCR experiments, we mimicked the functional properties of early and mature receptors with heterologous expression of specific subunit combinations. This report describes one of the most extensive cell- and site-specific developmental changes for an ion channel seen to date.

Field-focusing hyperthermia and magnetic resonance imaging (MRI) with a grounded probe and a commercial MRI scanner.

A method for performing magnetic resonance imaging (MRI) and producing field-focusing hyperthermia sequentially in phantoms and rat tissues with a grounded hyperthermic probe and a commercial MRI scanner was demonstrated. In the treatment mode the MRI scanner was used as a radiofrequency (RF) power source, and an invasive, electrically grounded, tuned probe was used to produce hyperthermia in phantoms via induced eddy current convergence. Temperature increases of 4.5 degrees C/5 minutes in a dielectrically uniform phantom and 5.0 degrees C/6 minutes in the peritoneum of a rat were measured in the vicinity (3-5 mm) of the grounded probe with the transmitter of the MRI scanner working at 2 per cent duty cycle. The advantage of this combined diagnostic and therapeutic approach is that the position of the hyperthermic probe can be monitored before each treatment, with observation of the tumor during and after treatment, if desired. In addition, the total cost is significantly less than that of both an MRI scanner and an RF hyperthermia treatment system.

Focused hyperthermia with a magnetic resonance imaging (MRI) unit and an interstitial grounded probe.

The feasibility of using a commercial magnetic resonance imaging (MRI) scanner to do either imaging or hyperthermic treatment was demonstrated. Radiofrequency (RF) induced focal heating of phantoms and animal tissues was performed using a MRI scanner as the RF power source and a grounded interstitial probe as a device to produce hyperthermia via eddy current convergence. In the therapeutic mode, a pulse width of 900 microseconds and interval of 50 ms were used to give 2% duty cycle (closest simulation to continuous wave (CW) mode without bypassing imaging filters). Temperature in the vicinity of the grounded probe was measured with a field nonperturbing fluoroptic probe. Temperatures increased 4.5 degrees C in 5 minutes in a dielectrically uniform phantom, 3.1 degrees C in 6.7 minutes in rats' leg muscles, and 5.0 degrees C in 6.0 minutes in rats' peritoneum. The MRI of the phantom with the grounded probe and the fluoroptic probe was obtained using spin echo sequences. The potential advantage of this approach is visualization of deep-seated tumors and hyperthermic treatment with minimal modification of the MRI scanner.