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1.  Response of irradiated and bystander cells to charged particles in two-dimensional and three-dimensional colon models 
Journal of Radiation Research  2014;55(Suppl 1):i48-i49.
The radiation-induced bystander effect, wherein unirradiated cells near to or sharing medium with irradiated cells express biological responses, most often has been studied in two-dimensional monolayer cultures, although some studies with three-dimensional models and in vivo have also shown bystander signaling. We have shown previously that DNA damage, measured as foci of the DNA repair-related protein 53BP1, occurs in unirradiated bystander cells in a three-dimensional skin epithelium model irradiated with protons or iron ions (Lumpkins et al., submitted). In the current work, we extend the studies to a second epithelial model, colon, with studies in both two-dimensional monolayer and a three-dimensional tissue model using Caco2 human colon epithelial cancer cells and AG01522 human fibroblasts.
For the monolayer studies, Caco2 cells in exponential growth were irradiated then co-cultured, sharing medium in an insert system, with unirradiated cells. Cells were irradiated with 250 kVp X-rays at Massachusetts General Hospital or with 1 GeV/amu protons, silicon ions or iron ions at the National Space Radiation Laboratory at Brookhaven National Laboratory. At varying times after irradiation, cell damage was assayed as micronuclei (MN) induction or formation of 53BP1 foci in both irradiated and bystander cells. For the three-dimensional studies, AG01522 fibroblasts were embedded in a collagen gel, then Caco2 cells were grown on the top of the gel. Each three-dimensional construct was cut in half prior to irradiation, with one half irradiated then immediately placed in contact with the other, bystander, half for co-culture. At selected times after irradiation, irradiated and bystander construct halves were fixed and sectioned, and 53BP1 foci were counted.
In monolayer culture, irradiated Caco2 cells showed a dose-dependent increased fraction of cells with MN after exposure to X-rays, protons, iron ions or silicon ions. Bystander Caco2 cells sharing medium with the irradiated cells also showed an increased fraction of cells with MN, reaching similar levels of ∼16% cells with MN, about a threefold increase over controls, after 1 Gy of all types of radiation. The fraction of cells with 53BP1 foci depended on radiation type and time of assay after irradiation, with the induction of foci generally greatest 5 h after irradiation and increasing with radiation dose. In bystander Caco2 cells, the appearance of foci generally was delayed, with the maximal fraction of cells showing foci at 12 h. In three-dimensional culture, after X-ray or proton exposure, cells showed similar trends to those seen in two-dimensional growth, i.e. with both the Caco2 and the AG01522 cells, the fraction of irradiated cells having 53BP1 foci reached a maximum at 5 h, but with bystander cells, the maximum occurred at 12 h after irradiation. This delay in the appearance of foci in bystander cells compared with irradiated cells is similar to our findings in the three-dimensional skin model composed of keratinocytes and fibroblasts.
In summary, our data now show in two different epithelial tissue models in both two-dimensional and three-dimensional models, radiation-stimulated intercellular signaling results in substantial levels of DNA damage in unirradiated cells. Because Caco2 cells are a carcinoma cell line, the studies are now being extended to a three-dimensional colon model using normal human colonic epithelial cells.
PMCID: PMC3941552
bystander effects; heavy ions; three-dimensional epithelial model; colon cancer
2.  Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM and ATR 
Cancer research  2013;73(8):2574-2586.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.
PMCID: PMC3760004  PMID: 23436801
mTOR; ATM; ATR; lung cancer; kinase inhibitors
3.  Technical approach to individualized respiratory-gated carbon-ion therapy for mobile organs 
We propose a strategy of individualized image acquisitions and treatment planning for respiratory-gated carbon-ion therapy. We implemented it in clinical treatments for diseases of mobile organs such as lung cancers at the Gunma University Heavy Ion Medical Center in June 2010. Gated computed tomography (CT) scans were used for treatment planning, and four-dimensional (4D) CT scans were used to evaluate motion errors within the gating window to help define the internal margins (IMs) and planning target volume for each patient. The smearing technique or internal gross tumor volume (IGTV = GTV + IM), where the stopping power ratio was replaced with the tumor value, was used for range compensation of moving targets. Dose distributions were obtained using the gated CT images for the treatment plans. The influence of respiratory motion on the dose distribution was verified with the planned beam settings using 4D CT images at some phases within the gating window before the adoption of the plan. A total of 14 lung cancer patients were treated in the first year. The planned margins with the proposed method were verified with clinical X-ray set-up images by deriving setup and internal motion errors. The planned margins were considered to be reasonable compared with the errors, except for large errors observed in some cases.
PMCID: PMC3709089  PMID: 23568337
Carbon-ion therapy; Respiratory motion; Respiratory gating; Treatment planning; 4D CT; Errors
4.  Risk factors for rectal bleeding associated with I-125 brachytherapy for prostate cancer 
Journal of Radiation Research  2012;53(6):923-929.
The purpose of this study was to determine the risk factors for rectal bleeding after prostate brachytherapy. Between April 2005 and September 2009, 89 patients with T1c-2cN0M0 prostate cancer were treated with permanent I-125 seed implantation alone. The prostate prescription dose was 145 Gy, and the grade of rectal bleeding was scored according to the Common Terminology Criteria for Adverse Events version 4.0. Post-treatment planning was performed with fusion images of computerized tomography and magnetic resonance imaging 4–5 weeks after brachytherapy. Patient characteristics and dosimetric parameters were evaluated to determine risk factors for bleeding. The calculated parameters included the rectal volume in cubic centimeters that received >50–200% of the prescribed dose (RV50–200) and the minimal doses received by 1–30% of the rectal volume (RD1–30). The median follow-up time was 42 months (ranging 18–73 months). Grade 1 rectal bleeding occurred in 24 (27.0%) patients, but no Grade 2 or severe bleeding was observed. Usage of anticoagulants had a significant correlation with the occurrence of bleeding (P = 0.007). The RV100–150 and RD1–10 were significantly higher in patients with rectal bleeding than in those without bleeding. The RV100 was identified as a possible threshold value; the 3-year rectal bleeding rate in patients with an RV100 > 1.0 cm3 was 36%, whereas that with an RV100 ≤ 1.0 cm3 was 14% (P < 0.05). Although no Grade 2 morbidity developed in this study, the RV100 should be kept below 1.0 cm3, especially in additional dose-escalated brachytherapy.
PMCID: PMC3483856  PMID: 22859567
prostate cancer; brachytherapy; rectal bleeding; dose-volume-histogram; anticoagulant
5.  Long-term results of curative intraluminal high dose rate brachytherapy for endobronchial carcinoma 
The treatment strategy of central lung tumors is not established. Intraluminal brachytherapy (ILBT) is widely used for palliative treatment of endobronchial tumors, however, it is also a promising option for curative treatment with limited data. This study evaluates the results after ILBT for endobronchial carcinoma.
Sixteen-endobronchial carcinoma of 13 patients treated with ILBT in curative intent for 2000 to 2008 were retrospectively reviewed. ILBT using high dose rate 192 iridium thin wire system was performed with 5 Gy/fraction at mucosal surface. The patient age ranged from 57 to 82 years old with median 75 years old. The 16 lesions consisted of 13 central endobronchial cancers including 7 roentgenographically occult lung cancers and 3 of tracheal cancers. Of them, 10 lesions were treated with ILBT of median 20 Gy combined with external beam radiation therapy of median 45 Gy and 6 lesions were treated with ILBT alone of median 25 Gy.
Median follow-up time was 32.5 months. Two-year survival rate and local control rate were 92.3% and 86.2%, respectively. Local recurrences were observed in 2 lesions. Three patients died due to lung cancer (1 patient) and intercurrent disease (2 patients). Complications greater than grade 2 were not observed except for one grade 3 dyspnea.
ILBT combined with or without EBRT might be a curative treatment option in inoperable endobronchial carcinoma patients with tolerable complication.
PMCID: PMC3411402  PMID: 22824158
Lung cancer; Radiation therapy; High dose rate; Intraluminal brachytherapy; Curative intent

Results 1-5 (5)