Induced Cell Lethality Research Articles

The potentiation of radiation response in human colon carcinoma cells in vitro and murine lymphoma in vivo by AG337 (thymitaq™), a novel thymidylate synthase inhibitor

Purpose: To determine whether the administration of Thymitaq TM (AG337), a selective inhibitor of thymidylate synthase (TS), enhances radiation-induced cytotoxicity in vitro and increases tumor control rate in vivo. Methods and Materials: In vitro studies were carried out with HT-29 human colon carcinoma cells. In vivo studies were carried out using L5178Y(TK −) murine lymphoma implanted in DBA/2 mice. Results: Pretreatment of HT-29 cells to nontoxic concentration of AG337 (<10 μM) for a short period of time (< 24 h) significantly enhanced the radiation induced cell lethality. The radiosensitizing enhancement ratio was 1.7. In contrast, there was no increased cell killing when the drug was exposed immediately after irradiation. In studies using L5178Y(TK −) tumors, the drug alone (50 mg/kg, i.p. × 5) had a minimal tumor growth delay, while a single dose of radiation (17 Gy) resulted in < 10% tumor control at day 30. When radiation and drug (17 Gy + AG337, 50 mg/kg, i.p. × 5) were combined, the tumor control rate reached 90% at Day 30. Using the local tumor control assay (TCD 50), the radiation dose modification factor after a single dose of radiation was 2.6. Conclusion: The concentration of drug shown to be of radiosensitizing value in the in vivo studies is achievable in humans. The results of the present study further supports the potential utility of AG337 in the treatment of human tumors by radiotherapy.

Intragenic Suppressors of Mutant DNA Topoisomerase I-induced Lethality Diminish Enzyme Binding of DNA

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of the antitumor drug camptothecin (Cpt). Mutation of several conserved residues in yeast top1 mutants is sufficient to induce cell lethality in the absence of camptothecin. Despite tremendous differences in catalytic activity, the mutant proteins Top1T722Ap and Top1R517Gp cause cell death via a mechanism similar to that of Cpt, i.e. stabilization of the covalent enzyme-DNA intermediate. To establish the interdomainal interactions required for the catalytic activity of Top1p and how alterations in enzyme structure contribute to the cytotoxic activity of Cpt or specific DNA topoisomerase I mutants, we initiated a genetic screen for intragenic suppressors of the top1T722A-lethal phenotype. Nine single amino acid substitutions were defined that map to the conserved central and C-terminal domains of Top1p as well as the nonconserved linker domain of the protein. All reduced the catalytic activity of the enzyme over 100-fold. However, detailed biochemical analyses of three suppressors, top1C273Y,T722A, top1G295V,T722A, and top1G369D,T722A, revealed this was accomplished via a mechanism of reduced affinity for the DNA substrate. The mechanistic implications of these results are discussed in the context of the known structures of yeast and human DNA topoisomerase I.

Expression of the pCloDF13 encoded bacteriocin release protein or its stable signal peptide causes early effects on protein biosynthesis and Mg2+ transport.

The effect of the pCloDF13 encoded bacteriocin release protein (BRP) on Escherichia coli cell lethality was studied. Induction of the BRP resulted in a strong inhibition of the incorporation of radioactive labeled amino acids and affected the transport of Mg2+ ions. Similar effects were obtained when the BRP stable signal peptide was expressed as a separate entity. Kinetic studies revealed that these effects occurred prior to quasi-lysis and release of cloacin DF13. The results indicated that the BRP induced cell lethality is caused by early effects on protein synthesis and Mg2+ transport, due to the accumulation of stable BRP signal peptides in the cytoplasmic membrane.

The spectrum of in vitro radiosensitivity in four human melanoma cell lines in not accounted for by differential induction or rejoining of DNA double strand breaks

Radioresistance is a significant clinical problem in advanced malignant melanoma and many melanoma cell lines show a radioresistant acute x-ray survival response in vitro. Given that the DNA double strand break is the lesion most closely correlated with x-ray induced cell lethality, differences in the induction and rejoining of these lesions may account for the radioresistance of some human melanoma cell lines. The above hypothesis was tested using pulsed field gel electrophoresis to measure x-ray induced DNA double strand break induction and rejoining in four human melanoma cell lines: MM138, MM170, MM96-L and HT 144. The MM138, MM170 and MM96-L cell lines were characterized in vitro by low alpha/beta ratios and broad x-ray survival curve shoulders. MM138 and MM170 were the most radioresistant and MM96-L had intermediate sensitivity. In contrast, HT144 was markedly x-ray sensitive, despite retaining a shoulder and like the other lines, having a low alpha/beta ratio. There were no significant differences in DNA double strand break induction between the cell lines, and thus no correlation existed between DNA double strand break induction and radiosensitivity. Consistent with the shoulders on the x-ray survival curves, all four cell lines showed efficient DNA double strand break rejoining. Highly efficient DNA double strand break rejoining could account for the radioresistance of one of the melanoma lines (MM138). For example, MM138 had rejoined 50% of the induced DNA double strand breaks by 5.5 min compared to 13-17 min for the other three cell lines. The development of postirradiation apoptosis was effectively excluded as the cause of the marked radiosensitivity of the HT144 cell line. Other factors (such as lesion repair fidelity or differential lesion tolerance) underlie the differences in the intrinsic radiosensitivity between these melanoma cell lines.

Effects of folinic acid on 5-fluorouracil induced cell lethality with or without cisplatin against head and neck laryngeal squamous carcinoma multicellular tumor spheroids.

We evaluated the efficacy of folinic acid (Leucovorin, LV) on cell lethality induced by 5-fluorouracil (FU) alone or in combination with cisplatin (DDP) by using the HEp-2 laryngeal squamous carcinoma multicellular tumor spheroids (MTS) system. For LV, non-toxic concentration of 10(-5) M was used. For cells in the monolayer, 6 and 24 h exposure to LV increased FU-induced cell lethality approximately 7- and 2-fold, respectively, whereas LV did not influence the effect of FU for MTS. LV's lack of effect on cells in MTS may be interpreted to mean that LV cannot penetrate the MTS. For the monolayer, simultaneous exposure to 3 drugs, DDP, FU and LV, produced synergistic interaction. However, sequential exposures were marginally synergistic or antagonistic, irrespective of sequence of DDP first or last. In contrast, DDP followed by FU plus LV was most synergistic for MTS. Simultaneous exposure was also synergistic, however, FU plus LV followed by DDP was antagonistic. These results suggest that LV is unable to penetrate into the MTS core to potentiate FU activity. DDP appears to have enhanced LV penetration into the MTS core. The exploration of means to overcome limited penetration of LV appears important for successful treatment of head and neck carcinoma.

A comparison of the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

One of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as GSH. Using the gas explosion technique it is possible to follow the kinetics of these fast (greater than 1 ms) reactions in intact cells. We have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to DNA single-strand and double-strand breaks, measured using filter elution techniques, with those leading to cell killing in V79 cells. The chemical repair rates for DNA dsb (670s-1 at pH 7.2 and 380s-1 at pH 9.6) and cell killing (530s-1) were similar. This is in agreement with the important role of DNA dsb in radiation induced cell lethality. The rate for DNA ssb precursors was significantly slower (210s-1). The difference in rate between DNA ssb and dsb precursors may be explained on the basis of a dsb free radical precursor consisting of a paired radical, one radical on each strand. The instantaneous probability of one or other of these radicals being chemically repaired and not proceeding to form a dsb will be twice that of ssb radical precursor. This agrees well with the concept of locally multiply damaged sites (LMDS) produced from clusters of ionizations in DNA (Ward 1985).

頭頚部へん平上皮癌におけるｃｉｓｐｌａｔｉｎと５‐ｆｌｕｏｒｏｕｒａｃｉｌの併用療法に対するｌｅｕｃｏｖｏｒｉｎ ｃａｌｃｉｕｍ投与の効果

頭頚部へん平上皮癌におけるｃｉｓｐｌａｔｉｎと５‐ｆｌｕｏｒｏｕｒａｃｉｌの併用療法に対するｌｅｕｃｏｖｏｒｉｎ ｃａｌｃｉｕｍ投与の効果

Effects of doxorubicin and cisplatin on multicellular tumor spheroids from human lung cancer.

We tested the sensitivity to doxorubicin (DXR) and cisplatin (DDP) of multicellular tumor spheroids (MTS) developed from 2 human lung cancer cell lines; PC-10 squamous cell carcinoma and PC-6 small cell carcinoma. DDP was able to maintain its efficacy in MTS: PC-10 MTS were only 3-fold more resistant to DDP than in monolayer and in PC-6 cells DDP induced cell lethality was essentially unchanged irrespective of cells being in a monolayer or MTS. Atomic absorption spectrometry revealed that DDP uptake was essentially identical, irrespective of cells being in monolayer or MTS. DDP's efficient cell kill effects in MTS seems to be explained by its good penetration into the MTS core. In contrast to DDP, these 2 types of cells responded differently to DXR. Thus, PC-10 MTS became progressively more resistant to DXR when their size increased, whereas the susceptibility of PC-6 MTS tended to increase when the MTS grew larger. Fluorescent microscopic study revealed that prominent DXR fluorescence was observed only at the outer layer of PC-10 MTS. In PC-6 MTS, however, DXR fluorescence was diffusely seen in the entire MTS at low concentrations; nevertheless, owing to PC-6 cells' high sensitivity DXR was able to exert cell lethality. The differences in distribution of DXR fluorescence between PC-10 and PC-6 MTS were corroborated by flow cytometric analysis.

Characterization of hydroxyl radical-induced damage after sparsely and densely ionizing irradiation.

The extent of hydroxyl radical mediated cell inactivation was measured for a variety of particle beams ranging from 8.5 Me V/u neon ions to 570 Me V/u argon ions. In general, the fraction of the total radiosensitivity caused by OH decreases from close to 60 per cent at low ionization density or low linear energy transfer (low LET) to close to 25 per cent at high LET for aerobically irradiated mammalian cells. The extent of OH induced cell lethality can be explained in terms of LET infinity only for low energy or low atomic number particles where fragmentations and complicated track structures do not contaminate the characteristic particle LET. For example, at a calculated LET infinity of 100 ke V/micron, the OH mediated fraction of the total radiation damage is about 25 per cent for low energy carbon but close to 40 per cent for high energy carbon ions. For low energy charged nuclei of approximately the same energy, as the 5.4-13.4 MeV/u He, Li, C and Ne ions in this report, there is a predictable diminution of the OH mediated effect with increasing LET infinity; however, the biological effect cannot be predicted accurately from calculated LET infinity values for high energy particle irradiation, nor indeed from a variety of low energy charged particles of quite different energies (incident velocities). This illustrates the unsuitability of using LET as a unifying parameter, except under specific circumstances. As more is learned about the energy deposition for energized charged particles in terms of track structure (core and penumbra), it may be possible to characterize the radiobiological data with a better physical parameter than LET infinity.

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minimum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion and mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle.

PHOTOTOXIC REACTION TO XANTHENE DYES INDUCED BY VISIBLE LIGHT

Many dyes, for instance methylene blue, rose bengal, and eosin, are known as photosensitizers, and in the presence of molecular oxygen they induce cell lethality and skin photosensitivity (1-4). Several dyes are used in cosmetic products, particularly in lipsticks. Human lip skin is therefore exposed to potential danger from dye-sensitized phototoxic reactions. Using an in vivo system of mammalian skin, such as the abdominal skin of rabbits, we established screening tests for the phototoxic potential of synthetic dyes in two ways: (a) intracutaneous injection; (b) topical application with and without damaging the barrier property of the stratum corneum. In the intracutaneous injection assay, distinct phototoxic reactions were induced by rose bengal, eosin Y.S., and dibromofluorescein. When these dyes were applied topically to intact skin, no phototoxic reactions were observed. Phototoxic reactions were, however, elicited when the dye solutions were applied to abraded or scratched skin. The intensity of phototoxic reaction was found to be influenced by the vehicle in which the dyes were suspended. Phototoxic reaction to the dyes was induced by artificial light as well as by sunlight. By using commercially available fluorescent lamps with different spectral emissions, the action spectra for the phototoxic reaction to these dyes were investigated and it was found that the maximum phototoxicities of the dyes were manifested by light within a spectral range of 400-600 nm. Further studies on action spectra, using a monochromatic irradiation system, revealed a high correlation between the action spectra of the dyes and their absorption spectra. Maximum effective wavelength for the phototoxic reaction of eosin Y.S. was 525 nm. This topical as well as intradermal assay for assesing phototoxic reaction to synthetic dyes in living skin will be a practical and useful measure for studying the phototoxicity of the dyes.

Effect of temperature on x-ray induced cell lethality and chromosomal aberrations.

Effect of temperature on x-ray induced cell lethality and chromosomal aberrations.