Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. P16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. P16-overexpressing and shRNA knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed. P16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci, and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.
11/6/20928activity against human colon tumor cell lines. Hopefully, through an understanding of the cytotoxic mechanisms of human KC, strategies to stimulate human KC to prevent or eradicate hepatic metastases can be developed rationally.T h e aim of this investigation is to determine if (1) human KC are cytotoxic against human colon carcinoma in vitro, (2) this cytotoxic activity can be stimulated, and (3) human KC mediate cytotoxicity through the secretion of T N F . MATERIAL AND METHODSIsolation of human KC. At the time of resection for colon cancer in six patients, a small (2 X 2 cm) wedge biopsy specimen of the liver was obtained. The specimen was placed in an enzyme solution (0.05% collagenase and deoxyribonuclease) and incubated in a shaking water bath at 37" C for 35 minutes. The cell suspension in Dulbecco's phosphate-buffered saline solution (DPBS) was placed on a 30% metrizamide gradient and centrifuged for 20 minutes (1400 g at 4" C). The KC were taken from the metrizamide-DPBS interface, washed, and placed in a 96-microwell plate at various effector concentrations (0.5 X lo', 1.0 X lo', and 2.5 X lo5 KC/well). The KC adhered for 2 hours and were washed twice with DPBS. This technique produced a suspension of >95% KC as determined by (1) macrophage morphology in a Wright-Giemsa-stained 400 SURGERY
574 Background: Neutropenic fever (NF) is a serious complication of the chemotherapies given to breast cancer patients and often limits their use. Hence, identifying which patients are at increased risk to develop NF is very important. The NBS1 gene product is important for the repair of double-strand DNA breaks and is activated by chemotherapy. The objective of this study was to determine if genetic variations of NBS1 polymorphisms predict the risk of chemotherapy-induced NF in breast cancer patients. Methods: Blood from 306 newly diagnosed breast cancer patients treated with chemotherapy were prospectively collected on a study approved by the institutional review board. The relationship of chemotherapy administration (e.g. dose, timing) and growth factor use were correlated with the absolute neutrophil count (ANC) and NF development. For each patient, we assessed three polymorphisms (924T>C, 8360G>C, and 30537G>C) of NBS1 gene using polymerase chain reaction-restriction fragment length polymorphism method. Two-sided Chi-square test was used for univariate analysis and a multivariable logistical regression analysis was used to calculate odds ratios. Results: In total, 167 (55%) patients experienced ANC less than 1,000 cells/microliter (CIN1000) and 30 (10%) patients developed NF. For 8360G>C polymorphism, 9.7% of patients had a 8360CC variant genotype and these patients had increased risk of NF than the other genotypes (NF in CC 20.7% vs. in others 8.1%; Odds Ratio [OR] = 3.0; 95% confidence interval [CI] = 1.1 - 8.0, p = 0.034). In multivariable logistic regression model, 8360CC genotype (OR = 5.0, 95% CI = 1.6 - 16.1, p = 0.007) and growth factor support (OR = 19.6, 95% CI = 4.4 - 87.6, p < 0.001) were significantly associated with NF development. No genotypes of 924T>C and 30537G>C polymorphisms increased the risk of NF and there was no statistical association between the three NBS1 gene polymorphisms and CIN1000. Conclusions: Breast cancer patients with 8360CC variant polymorphism in NBS1 gene have increased risk in developing NF with systemic chemotherapy. Analysis of polymorphisms of NBS1 and other DNA repair genes could potentially help identify who will develop chemotherapy-induced bone marrow toxicities. No significant financial relationships to disclose.
21043 Background: Only 25% of esophageal cancer patients achieve pathological complete response after standard chemoradiotherapy. Radiation dose escalation is associated with higher toxicity but no therapeutic improvement. In addition, esophageal cancer cells may develop radiation resistance (RR) after fractionated radiation exposure. Therefore, molecular targeting therapy for RR esophageal cancer is urgently needed. Methods: Six pairs of RR esophageal cancer cell lines were established by applying continuous 2 Gy fractionated irradiation. Ad/TRAIL-E1, an oncolytic adenoviral vector expressing both apoptotic TRAIL and viral E1A genes under the control of tumor specific human telomerase reverse transcriptase promoter, was constructed. Phosphate buffer solution and vectors expressing the TRAIL gene only, the GFP marker protein only, or the E1A gene only served as controls. Trans-gene expression, apoptosis activation, and the RR esophageal cancer cells targeted were evaluated in vitro and in vivo. A human esophageal RR cancer model was established and locally treated with Ad/TRAIL-E1 or controls. Results: After fractionated radiation exposure, esophageal cancer cell lines developed RR (up to 25-fold) that was associated with activation of the anti-apoptotic pathway. Ad/TRAIL-E1 activated an apoptotic cascade of caspases and selectively killed esophageal cancer cells but not normal cells. Ad/TRAIL-E1 preferentially targeted RR stem-like cancer cells with higher trans-gene expression and cell killing compared with parental cells. Overexpression (3 times) of Coxsackie's and adenoviral receptors in RR esophageal cancer cells compared with parental cells was noted. Ad/TRAIL-E1 therapy resulted in 40% tumor-free survival without the treatment- related toxicity found in human RR esophageal adenocarcinoma mouse models (p<0.05 as compared with controls). Conclusions: Esophageal cancer cells develop RR after fractionated radiation exposure. Ad/TRAIL-E1 preferentially targeted RR stem-like esophageal cancer cells, which resulted in a 40% cure rate. No significant financial relationships to disclose.
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