Yoshio Hosoi

Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse

Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

A PHOSPHATIDYLINOSITOL 3-KINASE INHIBITOR WORTMANNIN INDUCES RADIORESISTANT DNA SYNTHESIS AND SENSITIZES CELLS TO BLEOMYCIN AND IONIZING RADIATION

ATM and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3‐kinase (PI3‐kinase). In order to determine the contribution of ATM and DNA‐PKcs to the increased sensitivity of cells to DNA‐damaging agents observed in the presence of PI3‐kinase inhibitors, we examined the effects of a PI3‐kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X‐irradiation and ultraviolet (UV)‐irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X‐irradiation, but not to MMC or UV‐irradiation. Hypersensitivity to BLM and X‐irradiation and normal sensitivity to MMC and UV‐irradiation are characteristic phenotypes of both AT and scid mice. DNA‐dependent protein kinase (DNA‐PK) activity was suppressed by wortmannin to 45–65% of the control values in all of the cells except SCF, in which DNA‐PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X‐irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA‐PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy. Int. J. Cancer 78:642–647, 1998.

Decreased Incidence of Thymic Lymphoma in AKR Mice as a Result of Chronic, Fractionated Low-Dose Total-Body X Irradiation

We have investigated whether low-dose total-body X irradiation could suppress the development of lymphoma in AKR mice. Male mice were irradiated with 5 cGy three times a week or 15 cGy two times a week from 11 weeks of age for 40 weeks. The incidences of lymphoma were 80.5% in sham-irradiated mice, 67.5% in mice irradiated with 5 cGy three times a week and 48.6% in mice irradiated with 15 cGy twice a week. Incidence of lymphoma was significantly reduced by irradiation with 15 cGy twice a week (P = 0.006). The mean survival time was significantly prolonged from 283 +/- 3 days in control mice to 316 +/- 10 days in mice irradiated with 15 cGy twice a week (P = 0.008) and to 309 +/- 14 days in mice irradiated with 5 cGy three times a week (P = 0.040). Because about 80% of male AKR mice die of lymphoma, it is likely that the prolonged life span observed in the irradiated mice is due to the reduced incidence of lymphoma.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT‐4 or U937 cells following X‐irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac‐DEVD‐CHO. p35 was also produced in vitro by treating MOLT‐4 cell lysate with recombinant caspases, suggesting that p35 was a caspase‐cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA‐PKcs. However, p60 was found in ataxia‐telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA‐PKcs but not ataxia‐telangiectasia mutated (ATM).

Suppressive effect of low dose total body irradiation on lung metastasis: dose dependency and effective period

The dose dependency and effective period of suppressive effects by low dose total body irradiation (TBI) in both artificial and spontaneous lung metastasis were investigated using murine squamous cell carcinoma. In the artificial lung metastasis of the tumour cells injected through a tail vein, the TBI of 0.15-0.20 Gy resulted in significant net suppression of lung colony formation. With this dose range, the effective period lasted for 12 h ranging from 9 h before to 3 h after the tumour cell injection. Similar suppressive effect was also observed in the spontaneous lung metastasis. These data suggest that careful assessment of effective dose and duration of low dose TBI on human metastasis could provide a new therapy to suppress clinical metastasis.

Induction of interleukin-1β and interleukin-6 mRNA by low doses of ionizing radiation in macrophages

We have previously reported the antimetastatic effects and augmentation of immune responses, which would be a mechanism of the antimetastatic effects, of 0.1 to 0.2 Gy total body irradiation. To elucidate the cellular mechanisms of the augmentation of immune response, we investigated the effects of low‐dose irradiation on gene expression of interleukin‐1β (IL‐1β) and IL‐6 using mouse peritoneal macrophages in vitro. Absolute mRNA quantification was carried out using competitive polymerase chain reaction. Gene expression of IL‐1β and IL‐6 was increased 1 to 2 hr after 2.0 Gy irradiation and then decreased to below the basal expression level 4 hr after irradiation. Irradiation with 0.1 Gy increased IL‐6 expression 2 hr after irradiation, but it did not affect IL‐1β expression. Downregulation of IL‐1β and IL‐6 observed 4 hr after 2.0 Gy irradiation was not observed with 0.1 Gy irradiation. The protein kinase C (PKC) inhibitor H7 and the phosphatidylinositol 3‐kinase (PI3‐kinase) inhibitor wortmannin inhibited induction of IL‐1β and IL‐6 expression, which suggests that radiation‐induced IL‐1β and IL‐6 expression is achieved by PKC‐ and PI3‐kinase‐mediated signaling.

Radiation therapy for loco-regionally recurrent esophageal cancer after surgery

PURPOSE To evaluate the treatment outcome of radiation therapy for 33 loco-regionally recurrent esophageal cancer patients. METHODS Between 1988 and 1997, 33 patients with loco-regional recurrence of esophageal cancer after curative surgery received radiation therapy at an average total dose of 61 Gy. The site of recurrence was the supraclavicular region in 14 patients, the mediastinal region in 13 patients, and both the supraclavicular and mediastinal regions in six patients. If patients had ether distant metastasis or malignant pleural effusion, they were excluded from analysis. Patients who received prophylactic postoperative irradiation were also excluded from analysis. RESULTS The median survival period was 7 months. The survival rates at 1, 2, and 3 years were 33, 15, and 12%, respectively. In univariate analysis, patients with a short time interval between surgery and recurrence (P=0.0098) and patients with recurrence in both the supraclavicular and mediastinal regions (P=0.036) had a worse prognosis. In multivariate analysis, the time interval between surgery and recurrence (P<0.001) and age (worse prognosis in younger patients, P=0.019) were the significant prognostic factors. Complete or partial responses were observed in nine (27%) and 21 (64%) of the patients, respectively. Changes in clinical symptoms, such as dysphagia, chest pain and back pain, could be evaluated in 11 patients, and improvement in symptoms was obtained in eight (73%) patients. CONCLUSIONS The prognosis of patients who received radiation therapy for postoperative loco-regional recurrence of esophageal cancer is poor. However, there is symptomatic relief in a significant proportion of such patients, and long-term survival is possible in some patients.

Prostacyclin Production in Cultured Endothelial Cells is Highly Sensitive to Low Doses of Ionizing Radiation

In an attempt to detect if low doses of ionizing radiation affect the physiological function of blood vessels, the effect of prostacyclin production was examined using cultured endothelial cells. Irradiation with 5 cGy suppressed the production to 53% of the levels in unirradiated control cells. The PGI2 production decreased with an increase in the dose, showing a maximal decrease at 100 cGy without further decrease at 200 cGy. The suppressive effect lasted for 15 min after irradiation and disappeared later. Preincubation with alpha-tocopherol abrogated the suppressive effect completely, although preincubation with ascorbic acid showed little effect. These results suggest that low dose irradiation inhibits the prostacyclin production by changing the cellular membranes transiently.

Dysbindin-1, a Schizophrenia-Related Protein, Functionally Interacts with the DNA- Dependent Protein Kinase Complex in an Isoform-Dependent Manner

DTNBP1 has been recognized as a schizophrenia susceptible gene, and its protein product, dysbindin-1, is down-regulated in the brains of schizophrenic patients. However, little is known about the physiological role of dysbindin-1 in the central nervous system. We hypothesized that disruption of dysbindin-1 with unidentified proteins could contribute to pathogenesis and the symptoms of schizophrenia. GST pull-down from human neuroblastoma lysates showed an association of dysbindin-1 with the DNA-dependent protein kinase (DNA-PK) complex. The DNA-PK complex interacts only with splice isoforms A and B, but not with C. We found that isoforms A and B localized in nucleus, where the kinase complex exist, whereas the isoform C was found exclusively in cytosol. Furthermore, results of phosphorylation assay suggest that the DNA-PK complex phosphorylated dysbindin-1 isoforms A and B in cells. These observations suggest that DNA-PK regulates the dysbindin-1 isoforms A and B by phosphorylation in nucleus. Isoform C does not contain exons from 1 to 6. Since schizophrenia-related single nucleotide polymorphisms (SNPs) occur in these introns between exon 1 and exon 6, we suggest that these SNPs might affect splicing of DTNBP1, which leads to impairment of the functional interaction between dysbindin-1 and DNA-PK in schizophrenic patients.

Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis.