Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation

The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.     

The effect of caffeine on post-replication repair and survival in two L5178Y cell lines with different sensitivities to UV irradiation

2 Strains of murine lymphoma L5178Y cells that varied from the point of view of sensitivity to UV irradiation (mean lethal doses: 3.6 and 8.5 J/m2 for L5178Y-R and L5178Y-S cells, respectively) also differed with respect to sensitization by caffeine. L5178Y-S cells were sensitized to UV irradiation by 0.75 mM caffeine, whereas in the same conditions L5178Y-R cells were not sensitized. Sedimentation analysis of the newly synthesized DNA indicated UV-induced gap formation in L5178Y-S cells only. The subsequent gap filling was inhibited by caffeine. Exposure to UV irradiation induced no gaps in L5178Y-R cells. However, when caffeine was added immediately after irradiation, DNA with reduced molecular weight was found in irradiated cells of both strains after a 2-h chase. On the other hand, caffeine inhibited elongation of undamaged DNA strands in neither of the 2 cell strains.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). II. Differential effects of caffeine.

Two strains of L5178Y murine lymphoma, inversely cross-sensitive to X-rays and UV light, were shown previously to respond to treatment with an antitumour platinum complex, cis-dichlorobis(cyclopentylamine)-platinum(II) (cis-PAD), in a similar manner as to UV. Enhancement of chromosomal damage and potentiation of lethal effect of cis-PAD by 0.75 mM caffeine were found in cis-PAD and UV light-resistant L5178Y-S strain but not in cis-PAD and UV light-sensitive L5178Y-R strain. These results suggest that the extreme sensitivity of L5178Y-R strain to cis-PAD and UV light is caused to some extent by deficiency in a caffeine-sensitive post-replication repair system.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

Heat-induced inhibition of DNA synthesis in L5178Y-S cells is reversed by caffeine

Heating L5178Y cells for 15 min at 43 degrees C caused a decrease in [3H]thymidine incorporation, which could be reversed by post-treatment with 0.75 mM caffeine in an L5178Y-S (radiation-sensitive, heat-resistant) but not in an L5178Y-R (radiation-resistant, heat-sensitive) strain. The reversal was accompanied by a sparing effect of the treatment: survival of L5178Y-S cells increased by a factor of 1.5. The effect of combined (heat + caffeine) treatment of L5178Y-R cells was cumulative.     

Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.     

Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). I. Cross-sensitivity to cis-PAD and UV light.

The response to cis-dichlorobis(cyclopentylamine)platinum(II) (cis-PAD) an antitumour platinum complex, was studied in two strains of murine lymphoma L5178Y cross-sensitive to X-rays and UV light. Dose-survival relationship, DNA synthesis formation of chromatid aberrations, progression through the cell cycle, and growth and viability changes after 1 h cis-PA; treatment at 37 degree C were examined and compared with the effects of X-rays and UV light. In both strains, cis-PAD caused immediate inhibition of progression through the cell cycle, reduced rate of DNA synthesis, delayed appearance of chromatid aberrations, and delayed death; however, there is a marked difference in sensitivity to cis-PAD between L5178Y-S strain (D0 approx. 5.8 microgram/ml) and L5178Y-R strain (D0 approx. 2.5 microgram/ml). In both strains a close resemblance was found between dose-survival relationships after cis-PAD and UV light treatment, respectively.     

Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster

Cells with mutated autophosphorylation sites in the ABCDE cluster of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are defective in the repair of ionising radiation-induced DSB, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. Nevertheless, the mutated DNA-PK is able to undergo ATP-dependent autophosphorylation and inactivation. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in DSB repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line. The purpose of this work was to examine the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells. Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences coding for the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PK autophosphorylation sites (ABCDE casette).     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Recovery from potentially lethal damage of irradiated L5178Y-R and L5178Y-S cells: the effect of temperature and benzamide

Mouse lymphoma L5178 Y-S and Y-R cells differing in radiosensitivity by 1.5 times were treated with benzamide, an inhibitor of poly(ADP-ribosylation), for 24 h before and 18 h after X-irradiation, and incubated after irradiation at 25 degrees C and 37 degrees C. Clonogenic capacity of LY-S cells incubated at 25 degrees C exceeded that of the same cells incubated at 37 degrees C; the clonogenic capacity of LY-R cells did not vary with the postirradiation incubation temperature. Benzamide increased equally the radiosensitivity of LY-R cells incubated at both temperatures, whereas that of LY-S cells was only increased at 37 degrees C. Repair of potentially lethal damages to LY-S cells incubated at 25 degrees C was independent of the effectiveness of poly(ADP-ribosylation).     

Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells

Three classes of TFTr variants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells can be identified--large colony (lambda), small colony (sigma), and tiny colony (tau). The sigma and lambda mutants are detectable in the routine mutagenesis assay using soft agar cloning. The tau mutants are extremely slow growing and are quantitated only in suspension cloning in microwells. Variants of all three classes have been analyzed in the process of evaluating the usefulness of the thymidine kinase locus in L5178Y/TK+/- mouse lymphoma cells for detecting induced mutational damage. 150 of 152 variants from mutagen treated cultures and 163 of 168 spontaneous mutants were TFTr when rechallenged approximately 1 week after isolation (3 weeks after induction). All of the 41 mutants assayed for enzyme activity were TK-deficient. The sigma and tau phenotypes were found to correlate with slow cellular growth rates (doubling time greater than 12 h), rather than from effects of the TFT selection or mutagen toxicity. Cytogenetic analysis of sigma mutants approximately 3 weeks after induction shows an association between the sigma phenotype and readily observable (at the 230-300 band level) chromosomal abnormalities (primarily translocations involving that chromosome 11 carrying the functional TK gene) in 30 of 51 induced mutants studied. Using an early clonal analysis of mutants (approximately 2 weeks after induction) 28 of 30 sigma mutants showed chromosome 11 rearrangements. All lambda mutants studied (17 of 17 evaluated 3 weeks after induction and 8 of 8 evaluated 2 weeks after induction) showed normal karyotypes (at the 230-300 band resolution level), including the chromosome 11s. These observations support the hypothesis that sigma (and likely tau) mutants represent chromosomal mutations and lambda mutants represent less extensive mutations affecting the TK locus. The inclusion of sigma mutants in the total induced mutant frequency, as well as distinguishing them as a separate subpopulation of TK-deficient mutants, is, therefore, essential in obtaining maximum utility of the information provided by the L5178Y/TK+/- mouse lymphoma assay.     

High-resolution cytogenetic characterization of the L5178Y TK+/- mouse lymphoma cell line

High-resolution chromosome preparations from L5178Y TK+/- 3.7.2C mouse lymphoma cells were obtained using acridine orange in the cell harvest procedure. With this technique it is possible to visualize over 500 bands in elongated mouse lymphoma cell chromosomes as compared to the approximately 230 bands visualized in metaphase preparations. High-resolution lymphoma cell chromosomes are described, and chromosome rearrangements carried in the cell line are characterized by ideograms representing the position, number, size, and relative staining intensity of the G-band patterns. Use of elongated chromosomes of mouse lymphoma TK+/- mutants should facilitate analysis of the cytogenetic effects associated with TK+/- ----TK-/- mutagenesis.     

Spindle poisons induce allelic loss in mouse lymphoma cells through mitotic non-disjunction

Aneuploidy is an important contributor to reproductive failure and tumor development. It arises spontaneously or as a result of exposure to aneugenic agents through non-disjunction. Two spindle poisons, colchicine (COL) and vinblastine (VBL) are mutagenic in the mouse lymphoma assay (MLA), a gene mutation assay that targets the heterozygous thymidine kinase (tk) gene on chromosome 11 in mouse lymphoma L5178Y tk+/- 3.7.2c cells. To investigate the mechanisms of spindle poison mutagenesis, we analyzed the COL- and VBL-induced TK mutants at the molecular and cytogenetic level. Loss of heterozygosity (LOH) analysis employing a microsatellite region within the tk locus revealed that almost all mutants had lost the functional tk allele. To determine the extent of the LOH, we further examined LOH mutants for heterozygosity at nine microsatellite loci spanning the entire chromosome 11. Interestingly, every microsatellite marker showed LOH in all COL- and VBL-induced LOH mutants, suggesting that these mutants were generated by loss of the whole chromosome 11 through mitotic non-disjunction. Chromosome painting analysis supported this hypothesis; there were no mutants showing structural changes such as deletions or translocations involving chromosome 11. In contrast, spontaneous TK mutants followed from point mutations, deletions and recombinational events as well as whole chromosome loss. Our present study indicates that spindle poisons induce mutations through mitotic non-disjunction without structural DNA changes and supports a possible mechanism in which a recessive mutation mediated by aneuploidy may develop tumors.     

T cell immunity induced by live, necrotic, and apoptotic tumor cells

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.