Natsuko Kondo

DNA damage induced by alkylating agents and repair pathways.

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine.

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation.

Purpose : Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. Materials and methods : The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with γ-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). Results : Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.

Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation

Purpose : To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. Materials and methods : The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with γ-rays at 1.5 Gy (0.001Gy min -1 for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min -1 for 3 min). Results : When the wild-type mouse was previously exposed to chronic irradiation (1.5Gy) at a low dose-rate (0.001 Gy min -1) , apoptosis induced by acute irradiation (3.0 Gy, 1.0Gy min -1) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. Conclusions : These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage.

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.

p53- dependent hyperthermic enhancement of tumour growth inhibition by X-ray or carbon-ion beam irradiation

To elucidate p53 -dependency on combined treatment with radiation and hyperthermia, growth inhibition and apoptosis were analysed using transplantable human tumour. Human head and neck squamous cell carcinoma (HNSCC) cells carrying different p53 genes were transplanted into the thigh of nude mice. When the mean diameter of tumour reached 5-6 mm, the tumours were exposed to X-rays (2 Gy) or Carbon-ion (C-) beams (1 Gy) followed by heating at 42°C for 20 min. Tumour growth inhibition was evaluated by measuring the diameters of tumour. The induction of apoptosis and accumulation of apoptosis-related proteins were also analysed by immunohistochemical staining. Synergistic enhancement of tumour growth inhibition by hyperthermia was observed in wild-type p53 tumours treated with X-rays or C-beams but not in mutant p53 tumours. The incidence of apoptotic cells and activated-caspase-3-positive cells after combined treatment with them were significantly high in wild-type p53 tumours compared with that in mutant p53 tumours. The hyperthermic enhancement of tumour growth inhibition by X-ray- or C-beam-irradiation was p53 -dependent, suggesting that it might be highly correlated with p53 -dependent apoptosis.

The acceleration of boron neutron capture therapy using multi-linked mercaptoundecahydrododecaborate (BSH) fused cell-penetrating peptide.

New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting.

DNA ligase IV as a new molecular target for temozolomide

Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.

Boron Neutron Capture Therapy for Malignant Brain Tumors

Boron neutron capture therapy (BNCT) is a biochemically targeted radiotherapy based on the nuclear capture and fission reactions that occur when non-radioactive boron-10, which is a constituent of natural elemental boron, is irradiated with low energy thermal neutrons to yield high linear energy transfer alpha particles and recoiling lithium-7 nuclei. Therefore, BNCT enables the application of a high dose of particle radiation selectively to tumor cells in which boron-10 compound has been accumulated. We applied BNCT using nuclear reactors for 167 cases of malignant brain tumors, including recurrent malignant gliomas, newly diagnosed malignant gliomas, and recurrent high-grade meningiomas from January 2002 to May 2014. Here, we review the principle and history of BNCT. In addition, we introduce fluoride-18-labeled boronophenylalanine positron emission tomography and the clinical results of BNCT for the above-mentioned malignant brain tumors. Finally, we discuss the recent development of accelerators producing epithermal neutron beams. This development could provide an alternative to the current use of specially modified nuclear reactors as a neutron source, and could allow BNCT to be performed in a hospital setting.

DNA ligase IV is a potential molecular target in ACNU sensitivity

Nimustine (ACNU) is a chloroethylating agent which was the most active chemotherapy agent used for patients with high‐grade gliomas until the introduction of temozolomide, which became the standard of care for patients with newly diagnosed glioblastomas in Japan. Since temozolomide was established as the standard first‐line therapy for glioblastoma multiforme (GBM), ACNU has been employed as a salvage chemotherapy agent for recurrent GBM in combination with other drugs. The acting molecular mechanism in ACNU has yet to be elucidated. ACNU is a cross‐linking agent which induces DNA double‐strand breaks (DSBs). The work described here was intended to clarify details in repair pathways which are active in the repair of DNA DSBs induced by ACNU. DSBs are repaired through the homologous recombination (HR) and non‐homologous end‐joining (NHEJ) pathways. Cultured mouse embryonic fibroblasts were used which have deficiencies in DNA DSB repair genes which are involved in HR repair (X‐ray repair cross‐complementing group 2 [XRCC2] and radiation sensitive mutant 54 [Rad54]), and in NHEJ repair (DNA ligase IV [Lig4]). Cellular sensitivity to ACNU treatment was evaluated with colony forming assays. The most effective molecular target which correlated with ACNU cell sensitivity was Lig4. In addition, it was found that Lig4 small‐interference RNA (siRNA) efficiently enhanced cell lethality which was induced by ACNU in human glioblastoma A172 cells. These findings suggest that the down‐regulation of Lig4 might provide a useful tool which can be used to increase cell sensitivity in response to ACNU chemotherapy. (Cancer Sci 2010)

Pilot clinical study of boron neutron capture therapy for recurrent hepatic cancer involving the intra-arterial injection of a 10BSH-containing WOW emulsion

A 63-year-old man with multiple HCC in his left liver lobe was enrolled as the first patient in a pilot study of boron neutron capture therapy (BNCT) involving the selective intra-arterial infusion of a (10)BSH-containing water-in-oil-in-water emulsion ((10)BSH-WOW). The size of the tumorous region remained stable during the 3 months after the BNCT. No adverse effects of the BNCT were observed. The present results show that (10)BSH-WOW can be used as novel intra-arterial boron carriers during BNCT for HCC.