Radiosensitization of mammalian cells in vitro by nitroacridines

The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals.     

Radiosensitization of hypoxic mammalian cells in vitro by some 5-substituted-4-nitroimidazoles

The efficiencies of various 5-substituted-4-nitroimidazoles as radiation sensitizers have been determined in hypoxic Chinese hamster cells irradiated in vitro. Compared with published data on the sensitizing properties of substituted 2-nitro- and 5-nitroimidazoles, some of the 4-nitro derivatives show unusually high sensitizing efficiencies defined as the concentrations required to give an enhancement ratio of 1.6. The equilibrium one-electron reduction potentials of the compounds (E17) were measured by a pulse radiolysis technique and the results show that although sensitizing efficiencies are unexpectedly high, based on considerations of electron affinity, they still increase with increasing values of E17. Enhancement ratios were determined in two V79 cell lines for combinations of one of these compounds (a 4-nitroimidazole containing the group SO2.O.phenyl in the 5-position, NSC 38087) with various concentrations of misonidazole. The sensitization observed suggests that the two compounds may be operating by different mechanisms.     

Radiosensitization of cancer cells by hydroxychalcones

Radiation sensitization is significantly increased by proteotoxic stress, such as a heat shock. We undertook an investigation, seeking to identify natural products that induced proteotoxic stress and then determined if a compound exhibited radiosensitizing properties. The hydroxychalcones, 2′,5′-dihydroxychalcone (D-601) and 2,2′-dihydroxychalcone (D-501), were found to activate heat shock factor 1 (Hsf1) and exhibited radiation sensitization properties in colon and pancreatic cancer cells. The radiosensitization ability of D-601 was blocked by pretreatment with α-napthoflavone (ANF), a specific inhibitor of cytochrome P450 1A2 (CYP1A2), suggesting that the metabolite of D-601 is essential for radiosensitization. The study demonstrated the ability of hydroxychalcones to radiosensitize cancer cells and provides new leads for developing novel radiation sensitizers.     

Radiosensitization of mammalian cells by diamide.

The effect of diamide on the radiosensitivity of T-cells was investigated under oxic and anoxic conditions. The compound was found to sensitize the cells under both conditions. Under oxic conditions, exposure for 10 min before and during irradiation to 0.1, 0.5 and 1.0 mM diamide produced dose-modifying factors of 0.81, 0.60 and 0.55, respectively. Under anoxic conditions, exposure for 10 min before and during irradiation to 0.5 mM produced a dose-modifying factor of 0.34. When the cells in oxic conditions were exposed for just 20 min before irradiation, the sensitizing effect was smaller, but some sensitization effect was still apparent after a 120 min interval between diamide treatment and irradiation. Diamide also sensitized the cells after irradiation, but this effect was less than when it was present during irradiation. The presence of whole rat-blood in the incubation medium prevented sensitization. No sensitization could be detected in the whole animal. It is proposed that sensitization is due to lack of capacity for repair of radicals by hydrogen transfer and biochemical repair processes.     

Changes in the cytoskeleton pattern of tumor cells by cisplatin in vitro.

The influence of the antitumor drug cis-diamminedichloroplatinum(II) ('cisplatin') upon the structural pattern of the main cytoskeletal components, i.e. microtubules, intermediate filaments and microfilaments, was investigated in squamous carcinoma cells derived from the mouse stomach (G 22) or the human lung (L 266) and growing in vitro as monolayer cultures. The studies were performed by the indirect immunofluorescence technique using monoclonal antibodies against alpha-tubulin, type 19 cytokeratin and actin at the end of a 90-min exposure to 2.5 x 10(-6), 5 x 10(-6) or 10(-5) mol cisplatin/l and a subsequent 24-h recovery period. Under the influence of cisplatin, the cytoskeletal tubules and filaments, which were distributed in untreated cells as a finely organized network spreading through the whole cytoplasm like a spider's web, collapsed and aggregated to dense and circularly arranged bands of bright immunofluorescence around the nucleus or to cap-like structures apposing the nucleus. These phenomena developed in clear dependence upon the dose of cisplatin applied and were observable in a modified manner and to a different degree with the three structural elements of the cytoskeleton. During the subsequent 24-h interval, during which the cells were allowed to recover in drug-free growth medium, the before-mentioned collapse of the cytoskeletal network was only partially reversible following previous treatment with the medium (5 x 10(-6) mol/l) and the high (10(-5) mol/l) dose of cisplatin and restored totally to the normal structural pattern of untreated control cells when the low dose of 2.5 x 10(-6) mol cisplatin/l had been administered before. These results give evidence that the DNA cannot be the only cellular target for the antitumor drug cisplatin, but that it also effects other intracellular lesions which cause structural alterations of cellular organelles independently of the primary molecular attack at nuclear DNA strands. Probably, these additional interactions fortify the antiproliferative effect and contribute to the achievement of important biological and cytological effects of cisplatin such as growth inhibition or giant cell formation.     

Radiosensitization by inhibition of IkappaB-alpha phosphorylation in human glioma cells

To assess the role of nuclear factor kappaB (NFKB) in cellular radiosensitivity, three different IkappaB-alpha (also known as NFKBIA) expression plasmids, i.e., S-IkappaB (mutations at (32, 36)Ser), Y-IkappaB (a mutation at (42)Tyr), and SY-IkappaB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 ((15)Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IkappaB-alpha phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.     

Radiosensitization of cultured mammalian cells by 5-iodouridine.

Radiosensitization of cultured mammalian cells was studied with halogenated pyrimidines, such as 5-iodouridine or 6-chloropurine, which have been shown to promote bacterial cell lethality when combined with gamma-irradiation. When Chinese hamster cells were exposed to gamma-rays to acidic pH values and the number of colonies was scored after 6 to 11 days of incubation, many more cells were inactivated in the presence of the drug than in its absence. This may be due to radiation-induced cytotoxic iodine radicals from the reagent in the case of 5-iodouridine, because the cells were inactivated efficiently only be contact with the previously-irradiated drug solution. The toxicity of the irradiated drug solution increased remarkably when the pH shifted to acidic side. The radiosensitization and the cytotoxic effects of gamma-irradiated drug solution were not found with 6-chloropurine. This may be the first observation on the lethal effect of chemical radicals on mammalian cells, and it is concluded that radiosensitization with 5-iodouridine does not require the drug incorporation into cellular DNA, at least under the conditions adopted in the present studies.     

Radiosensitization of a mouse tumour by Ro 03-8799: acute and protracted administration

The nitroimidazole Ro 03-8799 has been tested as a sensitizer of hypoxic tumour cells, using regrowth delay of a mouse mammary carcinoma. This drug has a short biological half-life and has previously proved to be less promising in tumour experiments than was predicted from in vitro studies and from artificially hypoxic skin. The postulate that this might result from the delay in penetrating to poorly vascularized tumour regions has been tested by maintaining constant blood and tumour levels for 2 hours before irradiation, using an infusion pump. For equivalent gross tumour concentrations at the time of irradiation there was no significant difference in the radiosensitization achieved with a single dose or with prolonged administration. This indicates that slow penetration of the drug is not a problem.     

Radiosensitization of yeast cells by inhibition of histone h4 acetylation

Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (gamma-H2A) induction after irradiation but a reduced rate of loss of gamma-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G1 and G2 checkpoints after exposure to ionizing radiation, suggesting that G1 and G2 checkpoint activation is independent of histone H4 acetylation.     

Metabolism of glutathione in tumour cells as evidenced by 1H MRS

1H MRS signals of glutathione and of free glutamate were examined in samples from cultured tumour cells, namely MCF-7 from mammary carcinoma and TG98 from malignant glioma, with the aim of relating signal intensities to aspects of GSH metabolism. Spectra of cells harvested at different cell densities suggest that GSH and glu signal intensities are related to cell density and proliferation and their ratio is dependent on the activity of the gamma-glutamyl cysteine synthetase. The hypothesis is confirmed by experiments performed on cells treated with buthionine sulfoximine that inhibits the enzyme activity.     

Radiosensitization of human T-cell leukemia by thymidine and 41.8 degrees C hyperthermia in vitro

This study tested whether thymidine, a naturally occurring nucleoside metabolite, could increase the sensitivity of human T-cell neoplasms to ionizing irradiation and whether adding 41.8 degrees C hyperthermia (X 1 h) to the combination of thymidine and irradiation would further enhance killing of cells by radiation. The magnitude of cytotoxicity induced directly by thymidine was also evaluated. Finally, the ability of 2'-deoxycytidine to prevent thymidine-induced cytotoxicity and radiosensitization was studied. Using JM and MOLT3, two human T-cell acute lymphoblastic leukemias, it was found that thymidine itself was cytotoxic and the toxicity appeared rapidly upon exposure to the drug (i.e., at 4 h). Thymidine caused significant radiosensitization, and thymidine plus 1 h of 41.8 degrees C hyperthermia enhanced radiation-induced killing significantly more than did thymidine or hyperthermia separately. The addition of 2'-deoxycytidine only partly reversed thymidine-induced killing and did not prevent thymidine-induced radiosensitization. The applicability of these results to the clinical management of T- and null-cell malignancies is discussed, as is the presumed mechanistic basis for these observations relative to deoxyribonucleoside metabolism, NAD metabolism, and inhibition of poly(ADP-ribose) polymerase.     

Mechanisms of cytostasis of tumours in vitro by syngeneic lymphoid cells of tumour bearers.

The cytostasis assay is an in vivo-in vitro radioactive technique which detects antitumour responses of the syngeneic tumour-bearing hosts. Examination and characterization of effector mechanisms at the cellular and humoral levels revealed that the cytostasis assay using Meth A (a 3-methylcholanthrene-induced) tumour was T cell independent. Furthermore, both B cells and macrophages were required. It was concluded that the mechanism involved complement-dependent antibody-mediated lysis of the tumour cells, with B cells producing antibody and macrophages producing the complement components during incubation. However, antibody-dependent cell-mediated cytotoxicity with or without complement could not be completely excluded. Although antibody was detected in vivo, specific antibody against Meth A tumour was produced in vitro by cultured lymphoid cells from the tumour-bearers. Antibody-coated Meth A cells caused regression of some tumours when inoculated into BALB/c mice. When these regressor mice were rechallenged with tumour, they were found to be permanently immune to the tumour. In the light of these findings, the role of antibody in the protection of tumours and its implications are discussed.     

Radiosensitization of human colorectal tumor cells by 4-nitro-5-sulfonatoimidazoles

Misonidazole, a clinically-effective 2-nitroimidazole hypoxic cell radiation sensitizer, and 12 4-nitro-5-sulfonatoimidazoles were tested in cultured human SW1116 colorectal adenocarcinoma cells for radiosensitizing efficiency. Octanol-water partition coefficients and HPLC capacity factors were determined for all agents as measurements of lipophilicity, and an excellent correlation was found between the two measurements. Cytotoxicity, in vitro glutathione reactivity, and one-electron reduction potential were also determined for each compound to evaluate potential utility as macromolecularly transported radiosensitizers. Ten members of the set were found to be 40 to 300 times more radiotoxic than misonidazole, but no correlation was found between their radiosensitizing efficiencies and the chemical and physical parameters.