ZDHHC8 knockdown enhances radiosensitivity and suppresses tumor growth in a mesothelioma mouse model.

Mesothelioma is an aggressive tumor caused by asbestos exposure, the incidence of which is predicted to increase globally. The prognosis of patients with mesothelioma undergoing conventional therapy is poor. Radiation therapy for mesothelioma is of limited use because of the intrinsic radioresistance of tumor cells compared with surrounding normal tissue. Thus, a novel molecular-targeted radiosensitizing agent that enhances the radiosensitivity of mesothelioma cells is required to improve the therapeutic efficacy of radiation therapy. ZDHHC8 knockdown reduces cell survival and induces an impaired G2/M checkpoint after X-irradiation in HEK293 cells. In the present study, we further analyzed the effect of the combination of ZDHHC8 knockdown and X-irradiation and assessed its therapeutic efficacy in mesothelioma models. SiRNA-induced ZDHHC8 knockdown in 211H and H2052 mesothelioma cells significantly reduced cell survival after X-irradiation. In 211H cells treated with ZDHHC8 siRNA and X-irradiation, the G2/M checkpoint was impaired and there was an increase in the number of cells with micronuclei, as well as apoptotic cells, in vitro. In 211H tumor-bearing mice, ZDHHC8 siRNA and X-irradiation significantly suppressed tumor growth, whereas ZDHHC8 siRNA alone did not. Immunohistochemical analysis showed decreased cell proliferation and induction of apoptosis in tumors treated with ZDHHC8 siRNA and X-irradiation, but not with ZDHHC8 siRNA alone. These results suggest that ZDHHC8 knockdown with X-irradiation induces chromosomal instability and apoptosis through the impaired G2/M checkpoint. In conclusion, the combination of ZDHHC8 siRNA and X-irradiation has the potential to improve the therapeutic efficacy of radiation therapy for malignant mesothelioma

A loss of function screen identifies nine new radiation susceptibility genes

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G(2)/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy

A Loss of Function Screening for Radiation Susceptibility Genes

　Carcinogenesis is thought to be a multistep process that occurs through the accumulation of mutations in multiple genes required for the maintenance of normal growth control. Genomic instability is considered the earliest cellular event in the process of carcinogenesis. Although genomic instability is induced by ionizing radiation, the molecular mechanisms underlying this process are poorly understood. Cell cycle checkpoints play a key role in cell survival following DNA damage. The failure of DNA repair and cell cycle regulation in response to DNA damage are thought to be important factors in the early stages of genomic instability. Several known genes are reportedly associated not only with DNA damage repair and cell cycle regulation but also with radiation susceptibility. Thus, the identification of novel radiation susceptibility genes will likely help elucidate the molecular mechanisms underlying DNA damage–induced genomic instability.　By the large-scale expression profiling of 15 human cell lines and three mouse strains having varying degrees of susceptibility to ionizing radiation, we identified 200 genes correlated with radiation susceptibility. We constructed the shRNA library of these 200 genes and screened this library using a 96-well format and measuring the cell survivals, as determined by a sulforhodamine B–based cell proliferation assay, after X-irradiation. We identified 12 genes involved in cellular proliferation after irradiation. Three genes, ATM, ATR and CDKN1A, were known to be radiation susceptibility genes and other nine genes have not been reported to be associated with radiation susceptibility. Eight of the 12 genes were reported to directly or potentially regulate cell cycle, and the remaining four genes have not been reported biological functions. We then performed cell cycle analysis of cells transfected with shRNA vectors against thee four genes following X-irradiation, and found that knockdown cells of one gene did not accumulate at G2/M phase. This result suggests that one gene is associated with G2/M checkpoint after DNA damage. Further study of these 12 genes would help elucidate the molecular mechanism for genomic instability and carcinogenesis, and develop novel drugs for effective tumor radiotherapy and early diagnosis.１３ｔｈ International Congress of Radiation Reserc

Down regulation of ZDHHC8 enhances radiosensitivity of human mesothelioma in vitro and in vivo

Malignant mesothelioma is highly lethal and the prognosis following therapy is very poor. The effect of radiation monotherapy for mesothelioma is reported to be minimal. To improve the efficacy of radiotherapy, a novel molecular-targeted radiosensitizing agent is needed to increase radiosensitivity of mesothelioma cells. We have previously identified ZDHHC8 as a novel radiation susceptibility gene based on the functional screening in human cells. In this study, we analyzed the effect of ZDHHC8 knockdown with radiation on mesothelioma cells and assessed the therapeutic efficacy in mouse model. In mesothelioma cells, knockdown of ZDHHC8 by siRNA significantly reduced cell survival after irradiation, induced the impairment of G2/M checkpoint, and increased the frequency of cells with micronuclei and apoptosis. In the mouse model, the treatment with ZDHHC8 siRNA and irradiation significantly suppressed tumor growth and increased apoptosis, whereas ZDHHC8 siRNA alone did not. These results suggest that ZDHHC8 knockdown with X-irradiation induced chromosomal instability and cell death including apoptosis through the defects of G2/M checkpoint, and the combination of ZDHHC8 depletion and irradiation has the potential to be effective therapy for malignant mesothelioma.14th International Congress of Radiation Research(ICRR’2011

18F-FDG PET for Semiquantitative Evaluation of Acute Allograft Rejection and Immunosuppressive Therapy Efficacy in Rat Models of Liver Transplantation

Acute allograft rejection remains a major complication after liver transplantation. We report a semiquantitative imaging method of detecting acute allograft rejection with 18F-FDG PET. Methods: Syngeneic and allogeneic transplanted rats, with or without immunosuppressive treatment, were subjected to serial PET. Autoradiography of the liver was conducted in both the syngeneic and the allogeneic rats. Results: A significant increment of 18F-FDG accumulation in liver allografts was observed byPET on day 2. The 18F-FDG signal was concentrated in the area where inflammatory cells around the vessels were detected by autoradiography. Allotransplanted rats treated with an immunosuppressive agent displayed a marked decrease in hepatic 18F-FDG uptake, compared with allotransplanted rats that were not treated. Conclusion: 18F-FDG PET may be a valid method for facilitating the development of protocols to diagnose graftrejection and to monitor the efficacy of immunosuppressive therapy

Tumor-specific imaging with radiolabeled human anti-transferrin receptor antibody in pancreatic cancer mouse model

We radiolabeled the antibody with 125I by the chloramine-T method, and with 67Ga or 89Zr using deferoxamine. We conducted in vitro cell binding and competitive inhibition assays using a human pancreatic carcinoma cell line, MIAPaCa-2, which highly expressed TfR and a mouse fibroblast cell line A4, which highly expressed human HER2 but not human TfR. We inoculated MIAPaCa-2 and A4 cells subcutaneously into each side of hind limb and conducted PET imaging from one to six days postinjection of the 89Zr-labeled antibody. The cell binding assay showed that radiolabeled antibodies specifically bound to MIAPaCa-2 cells, but not to A4 cells. The Bmax of 125I-, 67Ga- and 89Zr-labeled antibodies was 69, 86 and 64 %, respectively. The competitive inhibition assay showed that Kd of 125I-labeled antibodies was 2 nM, respectively. Temporal PET imaging with the 89Zr -labeled antibody showed that the MIAPaca-2 tumor was readily visualized on day 1 postinjection and became clearer thereafter, and the uptake in the MIAPaca-2 tumor increased with time and reached 24.8% of injected dose per gram on day 6, whereas that in the A4 tumor was low and decreased with time. In conclusion, the radiolabeled anti-TfR antibody could be applicable for TfR-specific PET imaging and help in selecting appropriate patients for TfR-targeted treatments.14th International Congress of Radiation Research(ICRR’2011

Construction of high-resolution and physical maps around a radiation susceptibility gene on rat chromosome 4 using LEC congenic lines

Even if the human genome has been sequenced completely, it is difficult to determine the genes associated with the complicated radiation-induced responses in human. Therefore, the genetic analysis of an appropriate animal model is a useful approach to gain understanding of radiation-induced responses. A LEC rat is known to be an animal model of highly susceptibility to X-ray irradiation characterized by intestinal death, bone marrow death and impaired DNA repair. In order to identify the radiation susceptibility gene in the LEC rat, congenic rat lines developed by introgressing a LEC chromosomal segment into a F344 background using a phenotype-driven breeding protocol, and we performed a genome-wide genotyping of congenic rats and their progeny for selection tests. We determined that the susceptibility gene for X-irradiation was located in the chromosomal region on rat chromosome 4. In order to construct a high-resolution map, we mapped 30 microsatellite markers, four known genes and 73 ESTs using rat radiation hybrid panel. Then, we constructed a BAC contig in the chromosomal region using hybridization and PCR, and we estimate that a physical distance could be approximately 1Mb using pulse-field gel electrophoresis. We performed the sequencing of four positional candidate genes, and we determined no mutation causing amino acid substitution in their coding sequences between LEC and F344 rats. In addition, we could not find other candidate genes in the conserved chromosomal regions on human chromosome 3q21 and mouse chromosome 6. These findings suggest that the radiation susceptibility gene in the LEC rat would be a novel gene. Therefore, the determination of the radiation susceptibility gene for the LEC rat will be helpful to understand the molecular mechanism of irradiation-induced response in human.The 12th International Congress of Radiation Research(ICRR

Preliminary PET Study of Carbon-11 Labeled RGD Peptide by Using Carbon-11 Formaldehyde

Positron emission tomography (PET), in particular, is a useful modality that enables in vivo biological information to be obtained in a noninvasive manner using a variety of PET radiopharmaceuticals. Short half-life positron emitters have generally been used for PET molecular imaging studies. It is possible to synthesize PET probes that possess the same chemical structures as the parent unlabeled molecules without altering their biological activity because, with the exception of 18F, the positron-emitting radionuclides described above can replace their stable analogues in biomolecules. However, 15O and 13N have only ever been used in simple compounds such as [15O]O2 and [13N]NH3, respectively, because of the limitations imposed by their short half-lives. Carbon is present in all organic molecules to such an extent that the introduction of carbon-11 to a molecule does not modify its properties.The development of procedures amenable to the synthesis of novel carbon-11 labeled agents for use as tracers in biomedical research is important for moving the PET imaging technique forward into the future. A procedure for the synthesis of a 11C-labeled oligopeptide containing [1-11C]1,2,3,4-tetrahydro--carboline-3-carboxylic acid from the corresponding Trp•HCl-containing oligopeptide has been developed involving a Pictet-Spengler reaction with [11C]formaldehyde [1]. Herein, we report the radiosynthesis and PET study of cyclic RGD ([11C]1) and cyclic RAD ([11C]2) peptide using [11C]formaldehyde. We attempted remote-controlled synthesis of [11C]1 and [11C]2 using an automatic production system for [11C]CH3I. Radiochemical yield of [11C]2 at end of synthesis was 4.3±1.5 % (n=3), and the total synthesis time was about 40 min. Further details about the tumor imaging studies by PET will be reported.第52回ペプチド討論