Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells.

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Apoptosis after gamma irradiation. Is it an important cell death modality?

Apoptosis and necrosis are two different forms of cell death that can be induced by cytotoxic stress, such as ionizing radiation. We have studied the importance of apoptotic death induced after treatment with 6 Gy of gamma-irradiation in a panel of eight human tumour cell lines of different radiosensitivities. Three different techniques based on the detection of DNA fragmentation have been used, a qualitative one--DNA ladder formation --and two quantitative approaches--in situ tailing and comet assay. No statistically significant relationship between the two quantitative assays was found (r= 0.327, P = 0.159) so these methods seem to show different aspects of the process of cell death. The presence of the DNA ladder related well to the end-labelling method in that the least amount of end labelling was seen in samples in which necrotic degradation rather than apoptotic ladders were seen. However, as the results obtained by the comet assay are not in agreement with the DNA ladder experiments, we suggest that the distinction between the degraded DNA produced by apoptosis and necrosis may be difficult by this technique. Finally, although apoptosis has been proposed to be dependent on p53 functionality, and this may explain differences in cellular radiosensitivity, no statistically significant relationship was found between these parameters and apoptosis in the eight cell lines studied.

Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroblasts.

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.

Glutathione manipulation and the radiosensitivity of human tumour and fibroblast cell lines.

We have studied the role of glutathione (GSH) in determining radiation response in five human tumour and one human fibroblast cell line. GSH concentration was measured using the Tietze assay and compared with clonogenic survival following gamma-irradiation. No relationship between GSH concentration and aerobic radiosensitivity was observed. The addition of 10 mM extracellular cysteamine produced protection factors in all cell lines, ranging from 1.6 to 2.1, but had little influence on cellular GSH concentration. Depletion of GSH by buthionine sulphoximine (0.1 mM for 18 h) had negligible effect on cell survival, though moderate radiosensitization resulted from extreme GSH depletion after 30-min treatment with 1 mM dimethylfumarate. The degree of aerobic sensitization did not correlate with GSH levels. Irradiation under hypoxia produced oxygen enhancement ratios varying from 1.6 to 2.6, with no relationship to GSH content.

The comet moment as a measure of DNA damage in the comet assay.

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination. Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant. We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis. These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Host cell reactivation of gamma-irradiated adenovirus 5 in human cell lines of varying radiosensitivity.

DNA repair processes play an important role in the determination of radiation response in both normal and tumour cells. We have investigated one aspect of DNA repair in a number of human cell lines of varying radiosensitivity using the adenovirus 5 host cell reactivation assay (HCR). In this technique, gamma-irradiated virions are used to infect cells and the ability of the cellular repair systems to process this damage is assayed by a convenient immunoperoxidase method recognising viral structural antigen expression on the cell membrane 48 h after infection. Reduced HCR was exhibited by radioresistant HeLa cells and by a radiosensitive neuroblastoma cell line, HX142. In contrast, an ataxia telangiectasia cell line, AT5 BIVA, did not show reduced HCR. On the basis of these results we can make no general conclusions about the relevance of HCR to cellular radiosensitivity. We have extended these studies to determine whether our cell lines exhibited enhanced viral reactivation (ER) following a small priming dose of gamma-radiation given to the cells before viral infection. No evidence for this phenomenon was found either in normal or tumour cell lines.

The intrinsic alpha/beta ratio for human tumour cells: is it a constant?

The radiation response of 15 mammalian cell lines comprising 11 human tumour, two human fibroblast and two murine lymphoma cell lines, has been analysed using the linear-quadratic equation. As well as using conventional analysis of acute dose-survival curves to derive values for alpha and beta (termed alpha ac and beta ac), low dose-rate and split-dose experiments have been used to derive independent values of alpha and beta (alpha 1dr and beta RR), respectively. alpha 1dr provides a measure of irrecoverable damage, the magnitude of which agreed well with the initial slope of the acute survival curve for most cell lines. beta RR derived from split-dose experiments represents a unique measure of recovery for each cell line. Large differences were found between individual values of beta ac and beta RR, especially in the radiosensitive cell lines. Since beta RR is a functional measure of recovery we suggest that this is the more relevant parameter in studies of dose sparing. The most striking result of this analysis was found in considering the alpha/beta ratios. No relationship was observed between alpha ac and beta ac resulting in values of alpha ac/beta ac ranging from 1 to 175. In contrast a positive correlation was observed between alpha 1dr beta RR in the 11 tumour cell lines, giving an alpha/beta ratio of 9.4 +/- 1.8 Gy. This observation of the relative constancy of the ratio for human tumour cells leads to an hypothesis about the role of initial damage as a determinant of radiosensitivity.

Cellular recovery in two sub-lines of the L5178Y murine leukaemic lymphoblast cell line differing in their sensitivity to ionizing radiation.

Cellular recovery was assessed in two sublines of L5178Y murine lymphoma cells of differing radiosensitivity (LY-S and LY-A4) using low dose-rate irradiation and split-dose experiments. No increase in cell survival was observed in the LY-S cell line until the dose-rate was reduced to 2 cGy/min, whereas in the LY-A4 cell line 20 cGy/min was low enough to detect changes in survival. The extent of this change, as assessed by dose reduction factors at 2 logs of cell kill, was greater in the LY-A4 cell line. Fitting these data with the incomplete repair model of Thames led to anomalous values for the half-time of repair. In split-dose experiments the maximum observed recovery ratio increased as a function of dose in a manner that is consistent with the linear-quadratic equation. As was found previously with radiosensitive human tumour cells, the LY-S cell line showed more split-dose recovery at any given dose than the LY-A4 cell line.

Lack of radiosensitization in Lewis lung carcinoma and a murine plasmacytoma by MTDQ and MTDQ-DA.

MTDQ, and its watersoluble derivative MTDQ-DA, have been tested in combination with radiation using the murine plasmacytoma X5563 and the Lewis lung carcinoma. Neither of the two compounds showed any effect on the single dose radiation response as measured by tumour growth delay. In addition no effect of MTDQ-DA on the response of the X5563 plasmacytoma to fractionated radiation was observed. As a control, studies with the established hypoxic cell sensitizer Misonidazole showed that both tumours could be sensitized to radiation by that drug, in the case of Lewis lung carcinoma the use of the excision cell survival assay showed that this was due to hypoxic cell sensitization.

A panel of human lung carcinoma lines: establishment, properties and common characteristics.

A panel of human lung carcinoma lines representing the four main histological types (squamous, small-cell, large-cell and adenocarcinoma), and derived from both primary and metastatic sites, has been established in xenograft and in tissue culture. The highest take rates were achieved when biopsy specimens were obtained from large tumour masses and cultured lines were most readily established after preliminary passages as xenografts. The established lines exhibited an overlapping spectrum of biochemical and morphological characteristics, and showed a tendency to change from one cell type to another, in keeping with the concept of a common endodermal cell of origin. Radiation resistance appeared to be related to the large-cell phenotype.

Split-dose and low dose-rate recovery in four experimental tumour systems.

The radiation dose-rate effect has been investigated in three murine tumour cell systems (MT carcinoma, Lewis lung tumour, B16 melanoma) and in the HX34 human melanoma xenograft taken directly from mice and irradiated in vitro. The four tumour types were remarkably similar in their radiation response characteristics, especially at low dose rate; the Lewis lung tumour tended to be the most radiosensitive at high dose rate. The data have been analysed using the Lethal-Potentially Lethal (LPL) model of Curtis and the Incomplete Repair (IR) model of Thames. The data are equally well fitted by both models. The most remarkable feature of these analyses is that both models lead to estimates for the half-time for recovery that are in the region of 0.1 h, considerably shorter than other published values. Split-dose experiments were also performed, taking care to keep the cells at 37 degrees C between exposures. In all cases the split-dose half times were longer than the values derived from dose-rate analysis and in the case of the Lewis lung and HX34 tumour lines the difference was by almost a factor of ten. The discrepancy between these estimates could be the result of biphasic cellular recovery.