From radioresistance to radiosensitivity: In vitro evolution of L5178Y lymphoma.

PURPOSE: To discuss the possible reasons for the loss of tumourigenicity and the acquisition of new phenotypic features (among them, sensitivity to X and UVC radiations) as a result of in vitro cultivation of L5178Y lymphoma cells. RESULTS: Ten years ago the phenotypic differences between LY-R (original L5178Y maintained in vivo and examined in vitro) and LY-S lines were reviewed in detail by the author. The loss of tumourigenicity of LY-R cells upon in vitro cultivation accompanying the acquirement of the LY-S phenotype had been described earlier by Beer et al. (1983). In spite of their common origin, the sublines were shown to differ in their relative sensitivity to a number of DNA damaging agents and in numerous other features. Here, selected differences between LY-R and LY-S lines are briefly reviewed. It is proposed that Wallace's concept (2010a) that mitochondria are the interface between environmental conditions and the genome may explain the LY-R-LY-S conversion under prolonged in vitro cultivation. CONCLUSION: The differences between the LY lines were probably of epigenetic rather than genetic character. The properties of LY-R cells changed as a result of exposure to an oxic in vitro milieu. The changes could be preconditioned by heteroplasmy and the selection of cells endowed with mitochondria best fitted to a high oxygen-low carbon dioxide environment.

Coffee-mediated protective effects against directly acting genotoxins and gamma-radiation in mouse lymphoma cells.

The cytokinesis-block micronucleus test was performed using L5178Y mouse lymphoma cells to ascertain whether or not standard (caffeinated) instant coffee, the commonly consumed polyphenolic beverage with antioxidant activity can protect against chromosomal damage induced by the directly acting agents N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), methyl methanesulfonate (MMS) and gamma radiation. Our results demonstrated significant reductions in the in vitro genotoxic effects of MNNG, MMC, and MMS following co-treatment of mouse lymphoma cells with standard instant coffee. Subsequently, the comet assay was carried out to assess the effect of coffee co-treatment on the level of DNA damage induced by MMS in mouse lymphoma cells. The results demonstrated a significant reduction in MMS-induced DNA damage following co-treatment with standard instant coffee. Protective effects were observed in mouse lymphoma cells which were treated with coffee immediately after exposure to gamma radiation (1 and 2 Gy). Another experiment showed protection when the mammalian cells were irradiated (0.5 and 1 Gy) midway (at 2 h) during a 4 h coffee treatment. However, the protective effect against the lower dose (0.5 Gy) was not significant. In addition we assessed the modulatory effect of coffee on MNNG-induced apoptotic frequency by flow cytometry. The results revealed only a minor influence of coffee on the frequency of apoptotic cells induced by the test compounds, rendering an increase in sensitivity for apoptosis as a reason for the reduced genomic damage an unlikely or at least incomplete explanation.

Action spectra of apoptosis induction and reproductive cell death in L5178Y cells in the UV-B region.

We investigated the action spectra for the induction of apoptosis and reproductive cell death in mouse lymphoma L5178Y cells using a high-performance spectroirradiator, the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki. L5178Y cells were exposed to monochromatic light at different wavelengths in the UV-B and UV-A regions. The frequencies of apoptosis induction and reproductive cell death were determined by counting cells with chromatin condensation and by a semi-solidified agarose colony formation assay, respectively. The measured action spectra for the two end-points were similar. The sensitivity decreased steeply with an increase of wavelength in the UV-B region, but showed no further decrement in the UV-A region. The action spectra were slightly steeper than that for the minimum erythematic dose (MED), and were similar to the light-absorption spectrum of DNA in the UV-B region. On the other hand, in the UV-A region, the spectra for both endpoints were close to the MED, but not to DNA absorption spectra. The difference between the measured spectra and that for MED may have been caused by the absorption of the light by the skin. Differences in the time course and morphological difference of apoptosis were found between the UV-B and UV-A region. These results suggest that although DNA damage induced by UV-B light can trigger apoptosis, or lead to reproductive cell death, other damage (membrane, protein and so on) may trigger those effects in the UV-A region.

Poly ADP-ribosylation in two L5178Y murine lymphoma sublines differentially sensitive to DNA-damaging agents.

PURPOSE: To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely related murine lymphoma sublines, L5178Y-R (LY-R) and L5178Y-S (LY-S), with differential sensitivity to various DNA damaging agents (UV-C and ionizing radiation, hydrogen peroxide). MATERIALS AND METHODS: LY cells were X-irradiated (2 Gy). NAD+ was determined in cell extracts by high-pressure liquid chromatography. ADP-ribose polymers were purified and analysed by densitometry after polyacrylamide gel electrophoresis. Nuclear matrix proteins were separated by SDS-polyacrylamide gel electrophoresis and processed for ADP-ribose polymer blots to estimate their ability to bind poly(ADP-ribose). RESULTS: In the radiosensitive LY-S cells, the constitutive levels of ADP-ribose polymers were twofold higher than in radioresistant LY-R cells, but unresponsive to a challenge with 2 Gy X-rays. The concentrations of NAD+ - the substrate for poly(ADP-ribose) synthesis - were identical in the two cell lines. X-rays (2 Gy) depleted NAD+ only in LY-S cells. These cells also produced shorter poly(ADP-ribose) molecules as compared with LY-R cells. Nuclear matrix preparations of LY-S cells exhibited lower poly(ADP-ribose)-binding capacity than those of LY-R cells. CONCLUSION: The results demonstrate disturbances in the poly ADP-ribosylation response of the radiosensitive LY-S cells and reduced poly(ADP-ribose)-binding affinity of the nuclear matrix of these cells.

Combined effect of cisplatin and 5-fluorouracil with irradiation on tumor cells in vitro.

The concomitant administration of chemotherapy and radiation is an alternative tool in cancer therapy. The antiproliferative and the radiosensitizing effect of Cisplatin and 5-Fluorouracil (5-FU) were studied on mouse lymphoma cells transfected with human MDR1 gene (mdr) and its parent cell line (par) combined with and without radiation. HEp2 radioresistant cell culture was used in our experiments as a model of radioresistance. The growth rate and antiproliferative effect was measured by the MTT method. Significant inhibition of tumor cell growth was observed at a low concentration of Cisplatin and 5-FU combined with radiation on the mouse lymphoma cell lines. However an extremely high dose of Cisplatin and 5-FU resulted in moderate growth inhibition in the case of the HEp2 cell line. We assume that the radiosensitizing effect of 5-FU and Cisplatin can be considered as a synergistic antitumor effect at low doses of chemotherapy and radiation in a radiosensitive cell line. In the case of a radio-and chemoresistant cell line, high doses of radiation and chemotherapeutic agent achieved moderate tumour growth inhibition without significant synergistic effect. In addition the simultaneous application of both treatments can result in remarkable toxicity.

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.

Nuclear translocation of the p65 subunit of NF-kappaB in L5178Y sublines differing in antioxidant defense.

We examined the induction of nuclear translocation of the p65 subunit of NF-kappaB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-kappaB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-kappaB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 microM, 24 h, 37 degrees C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-kappaB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells.

Increased resistance of the radiosensitive M10 mutant cells of the L5178Y mouse lymphoma cell line to heat-induced apoptosis.

M10 cells, which are deficient in the repair of DNA DSBs and are therefore radiosensitive, are about twofold more thermoresistant than their parental L5178Y cells. We found that, after heat shock at 43 degrees C under conditions resulting in 10% survival (D(10)), M10 cells did not undergo apoptosis, whereas L5178Y cells did undergo apoptosis. M10 cells, but not L5178Y cells, constitutively expressed Hsp72 protein. Moreover, the M10 cells accumulated higher amounts of the heat-inducible form of Hsp72. The patterns of activation of the DNA-binding activity of HSF (heat-shock factor) differed in M10 and L5178Y cells. In response to heat shock, M10 cells accumulated greater amounts of Trp53 protein (formerly known as p53) than the parental cells. Cdkn1a (formerly known as p21, Waf1) was constitutively expressed and further accumulated after heat shock only in M10 cells. We suggest that heat-inducible Hsp72 to a larger extent, and constitutive Hsp72 to a lesser extent, together with Cdkn1a may be involved in the protection of M10 cells against heat-induced apoptosis. Apoptosis in these cells is likely to occur in Trp53-dependent manner.

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 (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 microM) 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. x 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. x 5) were combined, the tumor control rate reached 90% at Day 30. Using the local tumor control assay (TCD50), 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.

Effects of topoisomerase I-targeted drugs on radiation response of L5178Y sublines differentially radiation and drug sensitive.

The murine L5178Y (LY) lymphoma sublines, LY-R (radiation resistant) and LY-S (radiation sensitive) display a difference in susceptibility to camptothecin (CPT): LY-S cells are less sensitive to killing by this inhibitor of topoisomerase I than LY-R cells. Post-treatment (CPT present until 3 h after irradiation) sensitizes only LY-S cells. In agreement with this, only in LY-S cells is the relative number of DNA-protein cross-links formed after treatment with CPT + X higher than expected for additivity of X-ray and CPT-induced damage. The pattern of changes in the labelling indices and cell cycle distribution in cells that underwent combined treatment is essentially like that seen for single-agent treatment: for LY-S cells like that for radiation, for LY-R cells like that for CPT. We found no direct relation between the patterns of cell cycle distributions and the enhancement of the lethal effect of X-irradiation by CPT post-treatment. The sublines are not markedly differentially sensitive to beta-lapachone, which modifies topoisomerase I activity, and not sensitized to X-rays by post-irradiation treatment with the drug.

Analysis of mitotic cell death caused by radiation in mouse leukaemia L5178Y cells: apoptosis is the ultimate form of cell death following mitotic failure.

The appearance of various abnormal cells after irradiation was investigated in growing mouse leukaemia L5178Y cells. Morphologically defined apoptotic cells started to emerge at 10 h after irradiation and the frequency reached a peak at around 48 h being similar to the frequency of other abnormal cells, i.e. micronucleated, multinucleated and giant cells. Necrotic cells were rarely seen. The frequency of apoptosis and other abnormal cells depended on the radiation dose. The typical DNA ladder pattern for apoptosis was observed in the agarose gel electrophoretic analysis of the cells at 24-96 h postirradiation. A decline in the frequency of apoptotic cells occurred with longer incubation, which was associated with a sharp increase in cloning efficiency. Changes in the growth rate of the irradiated cell population during the postirradiation period could be reasonably well described by a simple model using the frequencies of apoptosis and other abnormal cells. The results suggest that apoptosis is the ultimate form of cell death via mitotic failure caused by relatively small doses of radiation in L5178Y cells.

Non-nuclear damage and cell lysis are induced by UVA, but not UVB or UVC, radiation in three strains of L5178Y cells.

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340-400 nm, UVASUN 2000 lamp), (2) UVA+UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (G15T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1-15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m2 for the UVASUN lamp, 75 to 390 J/m2 for the FS20 lamp and 3.8 to 17.2 J/m2 for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible+IR components (> 400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.

[Does ionizing radiation induce an adaptive response in mouse L5178Y lymphoma cells?]. / Indutsiruet li ioniziruiushchee izluchenie adaptivnyi otvet v kletkakh limfomy L5178Y myshei?

Growth kinetics of LY-S and LY-R cells (radiosensitive and radioresistant sublines of murine lymphoma L5178Y) has been investigated after beta-irradiation at cumulative doses of 1.5 to 20 cGy and dose rates of 0.8-10 mGy/h. It has been found that after 48 h culture in a complete medium the number of cells differed 5 times, whereas after X- and gamma-irradiation, Do values differed 1.62 times. Using the growth rate as the end point in evaluating the combined effect of beta-irradiation (10 cGy) and subsequent X-irradiation with lethal doses, we observed an increased relative cell number, in comparison to that after X-irradiation alone (an "adaptive response", using this criterion), in LY-S cells irradiated with a dose of 2 Gy. In contrast, when reproductive death of LY-S and LY-R cells the end-point analyzed, the lethal effect of consecutive beta- and X-irradiation in LY-S cells was higher than that expected for X-radiation alone (the synergistic effect).

No radiosensitivity-related change in plasma membrane properties in X- or gamma-irradiated L5178Y-R and L5178Y-S cells.

Two strains of murine lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S) differ in radiosensitivity (D0 ca 1 and 0.5, respectively), in Na+/K+ and Mg2+-ATPase activities and susceptibility to heat; their fatty acid composition is also slightly different. Nevertheless, neither Na+-dependent amino acid uptake, nor membrane fluidity change after X- or gamma-irradiation (10 Gy under aerobic conditions, 27 Gy under extreme hypoxia). Although in LY-S cells there is a decrease in partition coefficient in a two-phase system, which indicates a late (24 h after irradiation) change in surface charge, this change is not related to viability, membrane fluidity, amino acid transport and survival.

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells.

Effects of deuterium oxide (D2O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of gamma rays. Growth of irradiated and unirradiated cells was inhibited by 45% D2O but not by 3-aminobenzamide at 10 mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. The D2O did not inhibit the radiation-induced increase in poly(ADP-ribose) synthesis as measured by the incorporation of [14C]NAD into the acid insoluble fraction, contrary to 3-aminobenzamide. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of gamma rays. Possible mechanisms underlying the inhibition are discussed.

Radiation-induced DNA damage and cellular lethality in cultured mammalian cells.

X ray-induced DNA scissions and their repair were studied by an alkaline separation method. DNA damage in Chinese hamster V79 cells was assigned to one of three groups based on a repair profile previously used for mouse L5178Y cells: fast-reparable (T1/2 = 5 min), slow-reparable (T1/2 = 70 min), and nonreparable . The three kinds damage were investigated in relation to cellular lethality under conditions where radiosensitivity of cultured cells was modified: (1) different sensitivity in different cell lines, (2) cell cycle fluctuation of radiosensitivity, and (3) recovery after split-dose irradiation. Among the three types of lesions, only nonreparable damage or remaining lesions showed correlation to cell killing. The parallel relationship between nonreparable damage and cell killing implies that this type of damage could play an important role in radiation-induced cell death.

Murine L5178Y cells resulting in altered drug sensitivities from fractionated radiation exposure in vitro.

Exposure of murine L5178Y cells in vitro to fractionated X-irradiation (DXR) produced a subline that exhibited significantly altered responses to the cytotoxic effects of a range of antitumor drugs. A DXR schedule of 2 Gy per fraction for 10 fractions (total dose, 20 Gy) was used. Characterization of this subline revealed no marked differences when compared with the parent line in terms of growth rate, cloning efficiency, cell volume, chromosome number, macromolecular content, cell cycle distribution, or response to acute DXR exposure. Drug sensitivities were assessed following 24-hour exposures by colony formation in soft agar. Three distinct patterns of response were found: increased sensitivity of DXR-pretreated cells to bleomycin, cis-diamminedichloroplatinum (II), or dibromodulcitol; comparable responses in both lines to doxorubicin, 5-fluorouracil, or hydroxyurea; and decreased sensitivity of DXR-pretreated cells to vincristine or VP 16213.

Post-irradiation inhibition of scheduled DNA synthesis and stimulation of ADP-ribosylation in sensitive and resistant L5178Y murine lymphoma cells.

Post-irradiation changes in DNA synthesis and ADP-ribosyltransferase (ADPRT) activity in L5178YS and L5178YR, radiation sensitive and resistant murine lymphoma cells are described. DNA synthesis was inhibited to a greater extent in L5178YS than in L5178YR cells. The stimulation of ADPRT activity by irradiation was not significantly different between these two cell lines. These observations contribute to other evidence which has failed to confirm a general association of ADP-ribosylation with the DNA synthesis inhibition response. The contrast between the response of L5178Y cells and the corresponding behaviour of ataxia telangiectasia cells and normal human cells indicate that entirely different mechanisms are involved in determining the differences in radiosensitivity in these two systems.

Protection of cultured mammalian cells by S-2-(3-aminopropylamino)ethylphosphorothioic acid(WR-2721).

The ability of WR-2721 to protect cultured mammalian cells against radiation-induced killing was nearly the same as that of cysteamine when WR-2721 was activated by mouse liver extract. Without the liver extract, protection by WR-2721 required long incubations with the cells prior to irradiation. The protective activity increased in proportion to the cell concentration. The dose reduction factor at a concentration of 4 mM WR-2721 was 1.11 and 1.41 for 1.5 X 10(5) cells/ml and 15 X 10(5) cells/ml of cultured cells, respectively. A non-protein bound sulfhydryl group was detected in cell suspensions after incubation with WR-2721, but it was not a dephosphorylated product of WR-2721.