Kaori Tsutsumi

Involvement of inorganic polyphosphate in expression of SOS genes.

Inorganic polyphosphate (poly(P)) is a linear polymer that has been found in every organism so far examined. To elucidate the functions of poly(P) in the regulation of gene expression, the level of cellular poly(P) in Escherichia coli was reduced to a barely detectable concentration by overproduction of exopolyphosphatase (exopoly(P)ase) with a plasmid encoding yeast exopoly(P)ase (Shiba et al., Proc. Natl. Acad. Sci. USA 94 (1997) 11210-11215). It was found that exopoly(P)ase-overproducing cells were more sensitive to UV or mitomycin C (MMC) than were control cells. Poly(P) accumulation was observed after treatment with MMC, whereas the poly(P) level was below the detectable level in cells that overproduced exopoly(P)ase. When exopoly(P)ase-overproducing cells were transformed again by a multiple copy number plasmid that carries the polyphosphate kinase gene (ppk), the cells accumulated a great amount of poly(P) and restored the UV and MMC sensitivities to the level of control cells. In exopoly(P)ase-overproducing cells, the expression of recA and umuDC were not induced by MMC. In addition, a strain containing multiple copies of ppk accumulated not only a large amount of poly(P) but also recA mRNA. Since recA expression was induced in a recA-deletion strain harboring a plasmid with the ppk gene, poly(P) could be necessary for regulating the expression of SOS genes without depending on the RecA-LexA regulatory network.

Morphogenetic Study on the Maturation of Osteoblastic Cell as Induced by Inorganic Polyphosphate

Since inorganic polyphosphates [poly(P)] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P) on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P) were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P). Ultrastructural analysis of cells cultured with poly(P) revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P), it implies that poly(P) can be an effective material for bone regeneration.

Visualization of Ras-PI3K interaction in the endosome using BiFC

Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidylinositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome.

Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model

The microdosimetric-kinetic (MK) model is one of the models that can describe the fraction of cells surviving after exposure to ionizing radiation. In the MK model, there are specific parameters, k and yD, where k is an inherent parameter to represent the number of potentially lethal lesions (PLLs) and yD indicates the dose-mean lineal energy in keV/μm. Assuming the PLLs to be DNA double-strand breaks (DSBs), the rate equations are derived for evaluating the DSB number in the cell nucleus. In this study, we estimated the ratio of DSBs for two types of photon irradiation (6 MV and 200 kVp X-rays) in Chinese hamster ovary (CHO-K1) cells and human non-small cell lung cancer (H1299) cells by observing the surviving fraction. The estimated ratio was then compared with the ratio of γ-H2AX foci using immunofluorescent staining. For making a comparison of the number of DSBs among a variety of radiation energy cases, we next utilized the survival data in the literature for both cells exposed to other photon types, such as 60Co γ-rays, 137Cs γ-rays and 100 kVp X-rays. The ratio of DSBs based on the MK model with conventional data was consistent with the ratio of γ-H2AX foci numbers, confirming that the γ-H2AX focus is indicative of DSBs. It was also shown that the larger yD is, the larger the DSB number is. These results suggest that k and yD represent the characteristics of the surviving fraction and the biological effects for photon irradiation.

A Simulation Study of the Radiation-Induced Bystander Effect : Modeling with Stochastically Defined Signal Reemission

The radiation-induced bystander effect (RIBE) has been experimentally observed for different types of radiation, cell types, and cell culture conditions. However, the behavior of signal transmission between unirradiated and irradiated cells is not well known. In this study, we have developed a new model for RIBE based on the diffusion of soluble factors in cell cultures using a Monte Carlo technique. The model involves the signal emission probability from bystander cells following Poisson statistics. Simulations with this model show that the spatial configuration of the bystander cells agrees well with that of corresponding experiments, where the optimal emission probability is estimated through a large number of simulation runs. It was suggested that the most likely probability falls within 0.63–0.92 for mean number of the emission signals ranging from 1.0 to 2.5.

Evaluation of the cell survival curve under radiation exposure based on the kinetics of lesions in relation to dose-delivery time

We have investigated the dose rate effects on cell damage caused by photon-beam irradiation. During a relatively long dose-delivery time with a low dose rate, lesions created in cells may undergo some reactions, such as DNA repair. In order to investigate these reactions quantitatively, we adopted the microdosimetric–kinetic (MK) model and deduced a cell surviving fraction (SF) formula for continuous irradiation. This model enabled us to estimate the SF from dose and dose rate. The parameters in the MK model were determined so as to generate the SF, and we attempted to evaluate the dose rate effects on the SF. To deduce the cell-specific parameters in the SF formula, including the dose rate, we performed a split-dose experiment and a single-dose experiment with a constant dose-delivery time (10 min) (to retain the condition for equivalent behavior of cell lesions) by means of a clonogenic assay. Then, using the MK model parameters, the SFs were reproduced for a variety of dose rates (1.0, 0.31, 0.18, 0.025 and 0.0031 Gy/min) and were compared with reported experimental data. The SF curves predicted by the MK model agreed well with the experimental data, suggesting that the dose rate effects appear in the kinetics of cell lesions during the dose-delivery time. From fitting the analysis of the model formula to the experimental data, it was shown that the MK model could illustrate the characteristics of log-SF in a rectilinear form at a high dose range with a relatively low dose rate.

Modeling cell survival and change in amount of DNA during protracted irradiation

Abstract Hyper-radiosensitivity (HRS) is a well-known bioresponse under low-dose or low-dose-rate exposures. Although disorder of the DNA repair function, non-targeted effects and accumulation of cells in G2 have been experimentally observed, the mechanism for inducing HRS by long-term irradiation is still unclear. On the basis of biological experiments and a theoretical study, we have shown that change in the amount of DNA associated with accumulation of cells in G2 enhances radiosensitivity. To demonstrate continuous irradiation with 250 kVp X-rays, we adopted a fractionated regimen of 0.186 or 1.00 Gy per fraction at intervals of 1 h (i.e. 0.186 Gy/h, 1.00 Gy/h on average) to Chinese Hamster Ovary (CHO)-K1 cells. The change in the amount of DNA during irradiation was quantified by flow cytometric analysis with propidium iodide (PI). Concurrently, we attempted a theoretical evaluation of the DNA damage by using a microdosimetric-kinetic (MK) model that was modified to incorporate the change in the amount of DNA. Our experimental results showed that the fraction of the cells in G2/M phase increased by 6.7% with 0.186 Gy/h and by 22.1% with 1.00 Gy/h after the 12th irradiation. The MK model considering the change in amount of DNA during the irradiation exhibited a higher radiosensitivity at a high dose range, which could account for the experimental clonogenic survival. The theoretical results suggest that HRS in the high dose range is associated with an increase in the total amount of DNA during irradiation.

Matrixmetalloproteinases: up-regulated in subclones that survived 10-Gy irradiation

We report an interesting fi nding regarding the mRNA expression pattern of cell lines that survived 10-Gy irradiation. According to the linear quadratic model in a textbook, the survival curve shows a straight line at highdose areas, meaning that cells are killed at a constant rate. The model explains that there are two types of killing: α-killing, which is a function of dose D, and βkilling, which is a function of D 2 . This is a kind of modifi ed version of a hit model wherein some percentage of cells remain intact after irradiation. We used mouse fi brosarcoma cells (QRsP, p53 wild type); 1 × 10 4 cells were exposed to 10 Gy. At day 12, we harvested 6 of about 30 colonies and established them as QRsPIR-1 to QUsPIR-6. cDNA microarray analysis was performed for the parental QRsP and QRsPIR-1. The pattern was signifi cantly different between the two, suggesting that the cells that survived were not intact. Rather, they had a more aggressive nature than the parental QRsP: matrix metalloproteinase (MMP)13 (collagenase) and MMP3 were signifi cantly up-regulated (26-fold and 23-fold, respectively). These results encouraged us to perform an in vivo study in which 2 × 10 4 cells were injected subcutaneously into the fl anks of C57BL/6 mice (fi ve animals each). At day 28, a large tumor mass was palpated in each of the fi ve mice for QRSPIR-1. In contrast, only two mice developed a tumor mass of the parental QRsP. To form a tumor mass, degradation of surrounding tissue (mainly collagen) must be done. Our fi ndings are quite revealing in this regard.

Inorganic polyphosphate enhances radio-sensitivity in a human non–small cell lung cancer cell line, H1299:

Inorganic polyphosphate is a linear polymer containing tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. Polyphosphate has been recognized as a potent anti-metastasis reagent. However, the molecular mechanism underlying polyphosphate action on cancer cells is poorly understood. In this study, we investigated the involvement of polyphosphate in radio-sensitivity using a human non–small cell lung cancer cell line, H1299. We found that polyphosphate treatment decreases cellular adenosine triphosphate levels, suggesting a disruption of energy metabolism. We also found that the induction of DNA double-strand breaks was enhanced in polyphosphate-treated cells after X-ray irradiation and colony formation assay revealed that cell survival decreased compared with that of the control groups. These findings suggest that polyphosphate is a promising radio-sensitizer for cancer cells. Therefore, we hypothesized that polyphosphate treatment disrupts adenosine triphosphate–mediated energy transfer for cellular survival and DNA repair, thereby reducing the cellular capability to resist X-ray irradiation.

Tomosynthesis can facilitate accurate measurement of joint space width under the condition of the oblique incidence of X-rays in patients with rheumatoid arthritis

OBJECTIVE Accurate evaluation of joint space width (JSW) is important in the assessment of rheumatoid arthritis (RA). In clinical radiography of bilateral hands, the oblique incidence of X-rays is unavoidable, which may cause perceptional or measurement error of JSW. The objective of this study was to examine whether tomosynthesis, a recently developed modality, can facilitate a more accurate evaluation of JSW than radiography under the condition of oblique incidence of X-rays. METHODS We investigated quantitative errors derived from the oblique incidence of X-rays by imaging phantoms simulating various finger joint spaces using radiographs and tomosynthesis images. We then compared the qualitative results of the modified total Sharp score of a total of 320 joints from 20 patients with RA between these modalities. RESULTS A quantitative error was prominent when the location of the phantom was shifted along the JSW direction. Modified total Sharp scores of tomosynthesis images were significantly higher than those of radiography, that is to say JSW was regarded as narrower in tomosynthesis than in radiography when finger joints were located where the oblique incidence of X-rays is expected in the JSW direction. CONCLUSION Tomosynthesis can facilitate accurate evaluation of JSW in finger joints of patients with RA, even with oblique incidence of X-rays. ADVANCES IN KNOWLEDGE Accurate evaluation of JSW is necessary for the management of patients with RA. Through phantom and clinical studies, we demonstrate that tomosynthesis may achieve more accurate evaluation of JSW.