Expression of thymidine kinase is essential to low dose radiation resistance of rat glioma cells.

We have found that thymidine kinase expression is a major radioresponse determinant in rat glioma cells. Cells that lack thymidine kinase expression are significantly more radiosensitive relative to the wild-type cells. The degree of sensitization is large, particularly at the dose levels used in fractionated radiotherapy. The difference in low dose survival can be accounted for by a marked difference in the ability of the cells to undergo repair of sublethal damage. When herpes thymidine kinase was introduced into the thymidine kinase-deficient mutant cells, radioresistance was partially restored, and sublethal damage repair was also enhanced. All other radiobiological responses, including DNA double-strand break repair, potentially lethal damage repair, G2 arrest, and cell cycle distribution, appeared similar among the cell lines. These data suggest that the thymidine kinase enzyme or its cellular gene may be an excellent therapeutic target to increase radiosensitivity and thereby, to enhance the radiocurability of malignant brain gliomas.

Sigma 2 receptors as potential biomarkers of proliferation in breast cancer.

sigma 1 and sigma 2 receptors have been shown to exist in a number of rodent and human tumor cell lines. Although their expression is heterogeneous and their function is unknown, sigma receptors have been proposed as potential targets for diagnostic tumor-imaging agents. In this study, the density of sigma 2 receptors in proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66, was examined. Scatchard analyses of sigma 2 receptors were performed on membrane preparations of 66 P cells from 3-day cultures and 66 Q cells from 7-, 10-, and 12-day cultures. The Scatchard studies revealed that 66 P cells had approximately 10 times more sigma 2 receptors/cell than the 66 Q cells from 10-day cultures. Although > 97% of the cells were quiescent after 7 days in culture, the maximum differential in the sigma 2 expression between 66 P and 66 Q cells was not attained until these cells had been in culture for 10 days. These data suggest that ligands labeled with positron-emitting or single photon-emitting radionuclides, which selectively bind sigma 2 receptors, have the potential to noninvasively assess the proliferative status of human breast tumors.

Temperature dependence of radiation-induced DNA-protein crosslinks formed under hypoxic conditions.

Recently, we demonstrated that the oxygen dependence of the formation of DNA-protein crosslinks (DPCs) in irradiated mammalian cells measured by the alkaline elution technique is the mirror image of the oxygen dependence of radiation-induced cell killing. Consequently, these radiation-induced DPCs could be used to detect hypoxic cells or estimate the hypoxic fraction of cells in solid tumors. Although several techniques, including alkaline elution, gas chromatography/mass spectrometry (GC/MS) and nitrocellulose filter binding, have been used to measure radiation-induced DPCs, the published reports suggest that the characteristics of these DPCs may depend on both the type of sample irradiated (cellular compared to model systems, oxygenated compared to hypoxic, etc.) and the technique used to measure these radiation-induced DPCs. In the present study, the radiation-induced DPCs measured by our alkaline elution technique with and without proteinase K in the lysis solution were characterized by studying the dependence of their formation on temperature in hypoxic rat 9L brain tumor cells. Exponentially growing 9L rat brain tumor cells were rendered hypoxic at 4 degrees C or at 37 degrees C and then irradiated with either 7.5 Gy or 15 Gy. The cells were trypsinized at 4 degrees C, either immediately after the irradiation or after one half-time of strand break repair at 37 degrees C. The results demonstrated that the radiation-induced DPCs produced in 9L cells under hypoxic conditions, measured by our alkaline elution technique after low to moderate radiation doses, required metabolism for their formation, unlike the radiation-induced DPCs reported by others using the GC/MS or nitrocellulose filter binding technique.

Radiation-induced DNA damage in tumors and normal tissues: V. Influence of pH and nutrient depletion on the formation of DNA-protein crosslinks in irradiated partially and fully hypoxic tumor cells.

It has been demonstrated that the yield of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) appears to depend predominantly on the oxygen concentration in the microenvironment around the DNA of mammalian cells (Radiat. Res. 142, 163-168, 1995). Consequently, these DNA lesions have the potential to be used to detect hypoxic cells or estimate the hypoxic fraction of solid tumors. Although it has been demonstrated that physiological factors (e.g. pH, temperature, nutrient depletion, etc.) have no influence on the induction and repair of both DNA strand breaks and DPCs in irradiated oxygenated cells (Radiat. Res. 140, 321-326, 1994), there are no data to suggest how these physiological factors influence the induction and repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic cells. In the present study, the influence of pH (6.6-7.3) and nutrient depletion on the formation and/or repair of radiation-induced DNA strand breaks and DPCs in partially hypoxic (O2 concentration in the gas phase of 2.0-8.0%) or fully hypoxic (O2 concentration in the gas phase of 0.3-0.4%) populations of exponential (day 3) and plateau-phase (day 5) 9L rat brain tumor cells in culture was determined. Each population of cells was irradiated at 37 degrees C with 15 Gy and trypsinized at 4 degrees C after one half-time of strand break repair at 37 degrees C, and the DNA damage was measured using our alkaline elution technique, with or without proteinase K (PK) in the lysis solution. An analysis of the DNA-protein crosslink factor as a function of the oxygen concentration and a statistical comparison of the single-strand scission factor measured at eluted fraction 7 (SSSF7), with or without PK in the lysis solution, indicate that pH and nutrient depletion do not significantly influence the formation and/or repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic 9L cells. The data also demonstrate that the DNA lesions produced in irradiated partially hypoxic cells will cause the hypoxic fraction or fractional hypoxic volume to be overestimated by this DNA damage assay, not underestimated. However, this potential overestimation of the hypoxic fraction or fractional hypoxic volume should not limit the usefulness of this DNA damage assay in most laboratory studies, because it will take the contribution of a large cohort of partially hypoxic cells to overcome the dominance of the signal from a relatively small cohort of fully hypoxic cells.

Radiation-induced DNA damage in tumors and normal tissues. VI. Estimation of the hypoxic fraction of experimental tumors.

For several years, we have concentrated our efforts on validating the use of radiation-induced DNA strand breaks and DNA-protein crosslinks to assess the oxygenation status of tumors and normal tissues. We have demonstrated that (1) the oxygen dependence of strand break formation is identical to that of radiation-induced cell killing; (2) the oxygen dependence of DNA-protein crosslink formation is the mirror image of that of radiation-induced cell killing; and (3) the formation of these radiation-induced DNA lesions is predominantly dependent on the oxygen concentration near the DNA and is independent of the cell type, metabolic status, proliferative status, pH of the surrounding environment, and composition or properties of the proteins tightly associated with the DNA. In the present study, the hypoxic fraction of three experimental tumors was estimated using our assay of radiation-induced DNA damage. The average hypoxic fraction of a large number of tumors estimated with this assay of radiation-induced DNA damage for (1) WiDR human colorectal carcinoma xenografts (40.8 +/- 4.2%), (2) 66 mouse mammary adenocarcinoma tumors (41.8 +/- 3.1%), and (3) subcutaneous tumors grown from 9L rat brain tumor cells (95% CI =-8.2-4.2%) was not statistically different from that of a large number of tumors measured for each of these tumor models by the paired survival curve method (38.3 +/- 6. 3%, 28.9 +/- 5.5%, 95% CI = 2.2-4.4%, respectively). When the hypoxic fraction measured by the alkaline elution method on one half of an individual tumor was compared to that measured by the paired survival curve method on the other half of the same tumor, no statistical correlation was found for either 66 or WiDR tumors. Although this assay of radiation-induced DNA damage can be used effectively in the laboratory to answer a number of important questions about the oxygenation status of animal tumors and normal tissues, failure to reliably estimate the hypoxic fraction of individual tumors and technical considerations make it unlikely that the assay can be used in the clinic to estimate the hypoxic fraction of human tumors.

Photosensitization of MCF-7 cells with diaziquone using visible light: correlation with DNA strand breaks and free radical production.

The ability of visible light to enhance the activity of diaziquone (AZQ) was evaluated in MCF-7 human breast cancer cells. MCF-7 cells were sensitive to AZQ, while visible light illumination had no appreciable effect on cell survival. In the presence of AZQ, visible light potentiated AZQ's cytotoxicity. This potentiation of AZQ activity correlated with a 2-2.5-fold increase in the formation of free radicals (hydroxyl radicals and AZQ semiquinone) and with the production of DNA strand breaks as measured by electron paramagnetic resonance and gel electrophoresis respectively. These results support the hypothesis that free radical formation is part of the mechanism of action of AZQ.

Effect of L-buthionine sulfoximine on the radiation response of human renal carcinoma cell lines.

BACKGROUND: Renal cell carcinoma (RCC) is a progressive and relatively radioresistant disease. Currently, no data are available on the in vitro radiobiologic characterization of renal tumor cells to the authors' knowledge. METHODS: Two RCC were cultured from specimens from previously untreated patients after either surgical resection of the primary tumor or from the malignant ascites. These two cell lines were characterized with respect to cytogenetic abnormalities, gamma radiation survival response, intracellular levels of glutathione and its related detoxification enzymes, and the effect of glutathione depletion on radiation toxicity. RESULTS: The two RCC grew as adherent monolayer cultures with a median doubling time of 29 hours and 37 hours, respectively. Histopathologic analysis of the tumor cells grown in the renal capsule of the athymic mice confirmed their epithelial neoplastic growth. Both cell lines were aneuploid (range, 65-100 chromosomes) and had several marker chromosomes, including those derived from chromosomes 3, 7, and 11. In vitro radiation survival analysis indicated the relative radioresistance (RR; Do, 2.35 Gy) and relative radiosensitivity (RS; Do, 1.42 Gy), respectively, of these tumor cell lines. The levels of intracellular glutathione (GSH) were higher in the RR cells compared with the RS cells. The enzymatic activities of GSH S-transferase, GSH reductase, and the levels of GSH peroxidase and superoxide dismutase were elevated in the RS cells compared with the RR cells. L-Buthionine sulfoximine (BSO) treatment (concentration, 20 microM, applied for 17 hours) resulted in 77% and 63% GSH depletion compared with the untreated RR and RS cells, respectively. Pretreatment with higher concentration of BSO (50 microM for 17 hours) caused a modest radiosensitization of the RR cells (Do, 1.78 Gy). CONCLUSIONS: RCC have a differential pattern of radiosensitivity. BSO treatment causes moderate radiosensitization of the relatively radioresistant renal tumor cells.

Effect of ploidy, recruitment, environmental factors, and tamoxifen treatment on the expression of sigma-2 receptors in proliferating and quiescent tumour cells.

Recently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation (Mach et al (1997) Cancer Res 57: 156-161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors.