Hiroko Hama-Inaba

Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.

X-ray-sensitive mutant mouse cells with various sensitivities to chemical mutagens

Three X-ray-sensitive mutants (LX821, LX827 and LX830) have been isolated from mouse-lymphoma L5178Y cells. These mutants are much more sensitive to the lethal effects of ionizing radiation than the parental L5178Y cells but are as resistant to ultraviolet radiation as L5178Y cells. We have previously isolated a mutant M10 that is sensitive to methyl methanesulfonate (MMS) and cross sensitive to ionizing radiation and 4-nitroquinoline 1-oxide (4NQO). Unlike M10 cells, newly isolated mutants were not sensitive to MMS and were less sensitive to 4NQO. These results indicate that new mutants may be deficient in the repair of DNA damage specific to ionizing radiation. LX821 and LX827 cells were concomitantly resistant to 5-bromodeoxyuridine, whereas LX830 cells were not.

Involvement of Protein Kinase C-Related Anti-apoptosis Signaling in Radiation-Induced Apoptosis in Murine Thymic Lymphoma(3SBH5) Cells

Abstract Nakajima, T., Yukawa, O., Azuma, C., Ohyama, H., Wang, B., Kojima, S., Hayata, I. and Hama-Inaba, H. Involvement of Protein Kinase C-Related Anti-apoptosis Signaling in Radiation-Induced Apoptosis in Murine Thymic Lymphoma (3SBH5) Cells. Radiat. Res. 161, 528–534 (2004). Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (α and βI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (α, βI and βII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (α or β) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line

To analyze specific genes related to radiation-induced apoptosis, 12 apoptosis-resistant clones were isolated from cells of the radiosensitive mouse thymic lymphoma 3SB line after treatment with ethyl methanesulfonate. Five of 12 clonal cell lines were recloned and were examined for their susceptibility to X-ray-induced apoptosis. A cell survival assay showed that all five secondary cell lines were two to three times more resistant to X rays than 3SB cells. When 3SB cells were exposed to 5 Gy of X rays, the fraction of cells stained with erythrosin B increased quickly within 8 h of incubation after irradiation. However, no apoptosis occurred in these secondary mutant cells. In particular, the percentage of cells undergoing apoptosis in one clone, 1B1C4, was low even after incubation for 48 h. In contrast to X rays, after exposure to 20 J/m2 UV radiation, the proportion of apoptotic cells in these mutant cells increased and reached about 60 to 100% at 24 h, indicating a difference in the ability of X rays and UV radiation to induce apoptosis. A similar radioresistance was observed using agarose gel electrophoresis of DNA from cells of all X-irradiated secondary lines. Western blot analysis and a sequence-specific DNA-binding assay demonstrated that 1B1C4 cells had a functional defect in p53 protein, but the other four cell lines displayed wild-type p53 after X irradiation. Our results suggest the existence of separate radiation-specific p53-dependent and independent apoptosis in thymic lymphoma cells. Thus these apoptosis-resistant cell lines provide a useful tool to identify the genes involved in the signaling pathways leading to X-ray-specific apoptosis.

Survival and mutagenic responses of mitomycin C-sensitive mouse lymphoma cell mutants to other DNA cross-linking agents

Mitomycin C-sensitive mutants MCN 151 (complementation group I) and MCE 50 (complementation group II) derived from mouse lymphoma L5178Y cells were found to be also highly sensitive to the lethal effects of other DNA cross-linking agents, such as photoaddition of 8-methoxypsoralen (8-MOP) and cis-diamminedichloroplatinum II (cis-DDP). They were less sensitive to the monofunctional derivative 3-carbethoxypsoralen (3-CPs) and to trans-DDP to trans-DDP than their bifunctional counterparts. Incorporation levels of labeled 8-MOP or 3-CPs in wild-type cells and 2 mutants were almost the same, indicating that the sensitivity is not caused by differential incorporation of the agents. The rates of photoinduced mutations to 6-thioguanine resistance in the mutants, per unit dose of 8-MOP, were about 4 times higher for MCN 151 and 3 times higher for MCE 50 than that in L5178Y cells. However, the rates of induced mutations per viable cells in the mutants were nearly equal to those in wild-type cells. Cross-link repair was compared between mutants and wild-type cells by using the alkaline sucrose-gradient sedimentation technique. The results show that normal cells and both mutants are able to incise the cross-linked DNA, which is the first step of cross-link repair.

Repair of DNA single-strand breaks in radiation-sensitive mutants of mouse cells.

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.

Radio-sensitive murine thymoma cell line 3SB: characterization of its apoptosis-resistant variants induced by repeated X-irradiation

3SB, a mouse thymoma cell line, is one of the most radio-sensitive cells (D0 = 0.3 Gy), and its rapid apoptosis (4 h after 5 Gy irradiation, 90% apoptosis) seems to play a decisive role in enhancing the radiosensitivity. To understand the molecular mechanisms underlying extremely high radiosensitivity and rapid apoptosis, we attempted to isolate X-ray-resistant (XR) variants from 3SBH5, a stable subclone of 3SB, by repeating exposure of the cells to 2-5 Gy X-rays. Four independent stable XR variants, R111, R223, R316 and R429, were isolated by the repeated irradiation protocols. All XR cells possessed about 3 times higher D10 values than that of their parental 3SBH5. They were also resistant to apoptosis; only 10% cells underwent apoptosis 4 h after 5 Gy irradiation. The p53 protein was induced in all the cell lines after 5 Gy X-irradiation. These variants showed a cross resistance to a chemical reagent daunorubicin (DNR) that is known to be involved in the ceramide-mediated apoptosis. DNR, as well as C2-ceramide (5 muM) induced apoptosis in parental 3SBH5 cell, but not in two XR variants, R233 and R316 cells. Present result suggests that the induction of X-ray resistance by repeated X-irradiation might be achieved, at least partly, by the enhanced resistance to the ceramide-mediated apoptosis.

Inhibition and recovery of DNA synthesis after X-irradiation in radiosensitive mouse-cell mutants.

The mouse lymphoma L5178Y cell line and its radiosensitive variants M10 and LX830 were examined for DNA synthesis after X-irradiation. The dose-response curves show that the rates of DNA synthesis immediately after exposure are reduced in a dose-dependent fashion and that the extents of reduction in these 3 cell lines are similar to one another. But a difference was observed in the recovery of DNA synthesis with time of incubation. The recovered levels in M10 and LX830 cells were much higher than those in L5178Y cells at high doses of X-rays. These results are discussed in relation to radioresistant DNA synthesis in ataxia telangiectasia cells.

CHO · K1 cell mutants sensitive to active oxygen-generating agents. I. Isolation and genetic studies

Nine mutants isolated from CHO.K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation group, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis-diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

A temperature-sensitive mammalian cell mutant exhibiting micronucleation.

Abstract A temperature-sensitive mutant (ts 39) of murine leukemic cells was shown to undergo micronucleation upon exposure to the non-permissive temperature. The formation of micronucleate cells appeared to be preceded by an increase in the fraction of mitotic cells. Since this phenomenon resembles micronucleation induced by colcemid, it is possible that ts 39 cells may be defective in microtubule assembly.