Apoptosis induced by persistent single-strand breaks in mitochondrial genome: critical role of EXOG (5'-EXO/endonuclease) in their repair.

Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5'-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5'-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5'-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the initial trigger for apoptotic signaling in mammalian cells.

Changes in Treatment Patterns and Overall Survival in Patients With Early-Stage Non-Small Cell Lung Cancer in the United States After the Incorporation of Stereotactic Ablative Radiation Therapy: A Population-based Analysis.

PURPOSE: Technologic developments have made radiation therapy (RT) more effective and have introduced new treatment options, such as stereotactic ablative radiation therapy (SABR). This study sought to determine changes in practice patterns for treatment of stage IA non-small cell lung cancer (NSCLC) after the introduction of SABR into the United States. This population-based study also examined changes in survival during this time period for all patients and specifically for patients treated with RT, surgery, or observation. METHODS: We included patients in the Surveillance, Epidemiology, and End Results database diagnosed with stage IA NSCLC diagnosed between 2004 and 2012. Changes in treatment patterns were assessed. Outcomes were compared across 2 time periods: 2004 to 2008 (pre-SABR) and 2009 to 2012 (post-SABR). Kaplan-Meier and Cox regression were performed to compare overall survival (OS) for patients treated with surgery, RT, or observation. RESULTS: A total of 32,249 patients met the specified criteria. Comparing patients diagnosed in 2004 to those diagnosed in 2012, RT use increased from 13% to 29% (P<0.001), surgery use decreased from 76% to 61% (P<0.001), and patients observed decreased from 11% to 10% (P=0.3). There was no significant OS improvement in all patients or those patients who were observed; there were significant improvements in OS for patients treated with RT (hazard ratio=0.768; 95% confidence interval, 0.711-0.829) and those patients treated with surgery (hazard ratio=0.9; 95% confidence interval, 0.855-0.962). CONCLUSIONS: There has been an increase in RT utilization and decrease in surgical utilization after the incorporation of SABR by radiation oncologists within the United States. In addition, there has been an improvement in OS for patients treated with definitive RT for early-stage NSCLC between 2004 and 2012 that may be associated with increased utilization of SABR.

Early outcomes of uveal melanoma treated with intraoperative ultrasound guided brachytherapy using custom built plaques.

PURPOSE: To report early outcomes of patients with uveal melanoma treated with Eye Physics iodine-125 episcleral plaque therapy using modern biopsy techniques and intraoperative ultrasound guidance at our institution. METHODS AND MATERIALS: A retrospective chart review was conducted for 48 consecutive patients with uveal melanoma who were treated with Eye Physics plaque brachytherapy performed by 1 ocular oncologist. All patients underwent intraoperative ultrasound for image guidance of plaque placement. A dose of 85 Gy was prescribed to the apical height of the tumor or 5 mm from the inner sclera, whichever was greater. Forty-five patients underwent biopsy. Visual acuity, complication data, and recurrence rates were recorded. RESULTS: Median age at presentation was 63.0 years (range, 19-86 years). Median follow-up was 21.6 months. Median tumor apical height was 3.3 mm (range, 1.8-11.5 mm). Median dose at apex for tumor height >5 mm was 85.0 Gy and 142.5 Gy for tumor height ≤5 mm. Mean percent decrease in tumor height from baseline at 12, 24, and 36 months was 39.6%, 51.8%, and 53.8%, respectively. At 24 months, 19/23 (82.6%) patients maintained vision within 3 lines of baseline visual acuity. Twelve patients developed radiation retinopathy, 6 of whom were treated with anti-vascular endothelial growth factor therapy in the context of a clinical trial. No patients to date have local failure. Three patients are alive with confirmed hepatic metastases. CONCLUSIONS: We reported 0% early local failure rate and steady reduction in tumor height in 48 patients with uveal melanoma, ranging from small to large size, who were treated with Eye Physics iodine-125 episcleral plaque therapy using intraoperative ultrasound guidance. This promising result emphasizes the importance of image guided brachytherapy with intraoperative ultrasound at the time of plaque placement.

Accelerated partial breast irradiation: Past, present, and future.

Accelerated partial breast irradiation (APBI) focuses higher doses of radiation during a shorter interval to the lumpectomy cavity, in the setting of breast conserving therapy for early stage breast cancer. The utilization of APBI has increased in the past decade because of the shorter treatment schedule and a growing body of outcome data showing positive cosmetic outcomes and high local control rates in selected patients undergoing breast conserving therapy. Technological advances in various APBI modalities, including intracavitary and interstitial brachytherapy, intraoperative radiation therapy, and external beam radiation therapy, have made APBI more accessible in the community. Results of early APBI trials served as the basis for the current consensus guidelines, and multiple prospective randomized clinical trials are currently ongoing. The pending long term results of these trials will help us identify optimal candidates that can benefit from ABPI. Here we provide an overview of the clinical and cosmetic outcomes of various APBI techniques and review the current guidelines for selecting suitable breast cancer patients. We also discuss the impact of APBI on the economics of cancer care and patient reported quality of life.

Changes in treatment patterns and impact of radiotherapy for early stage diffuse large B cell lymphoma after Rituximab: A population-based analysis.

PURPOSE: Use of Rituximab for diffuse large B cell lymphoma (DLBCL) has improved outcomes and led to further questions regarding the benefit of consolidative radiation therapy (RT). This study sought to determine changes in RT utilization following the incorporation of Rituximab for treatment of early stage DLBCL and to examine survival outcomes. MATERIALS/METHODS: We included patients in the Surveillance, Epidemiology, and End Results database, diagnosed with Stage I-II DLBCL between 1992 and 2011. Linear regression was performed to determine rate of RT utilization over time during the pre- and post-Rituximab eras (1992-2001 vs. 2002-2011). Kaplan-Meier and Cox Regression were performed to compare overall survival (OS) for patients treated with or without RT. Propensity-score matching was used to compare survival outcomes to account for indication bias. RESULTS: 34,680 patients met the specified criteria. RT utilization was 35.2% in the pre-Rituximab era and 29.9% in the post-Rituximab era (​P<0.001). Linear regression revealed that in the pre-Rituximab era the slope of the best fit line for RT utilization by year was positive (m=0.01, ​P=0.0046), while the slope was negative in the post-Rituximab era (m=-0.008, ​P=0.0102). RT use was associated with improved OS in both the pre-Rituximab era (hazard ratio [HR]=0.797; 95% confidence interval [CI] 0.756-0.841) and the post-Rituximab era (HR=0.745; 95% CI 0.702-0.789). Propensity-score matched analysis confirmed that RT use improved OS in the pre-Rituximab era (HR=0.844; 95% CI 0.793-0.897) and post-Rituximab era (0.754; 95% CI 0.703-0.809). CONCLUSION: RT utilization has decreased following incorporation of Rituximab for first line treatment of DLBCL. RT use is associated with improved OS in both pre- and post-Rituximab eras, suggesting that RT should continue to be used for management of early stage DLBCL, even in the era of Rituximab.

Age- and tissue-specific changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury.

In this study, we investigated age- and tissue-dependent changes in the DNA base excision repair (BER) of oxidative lesions in mitochondrial and nuclear extracts by measuring single-nucleotide (SN)- and long-patch (LP)-BER activities in five tissues isolated from 4-, 10- and 20-month-old mice. Age-dependent SN-BER and LP-BER activity was increased in the mitochondria of liver, kidney and heart, but generally decreased in skeletal muscles. In contrast, no significant changes in repair activity were observed in nuclear extracts of the same tissues, except for quadriceps, where the SN-BER activity was higher in the old animals. Moreover, the BER activities in both the nucleus and the mitochondria were significantly lower in skeletal muscles compared to liver or kidney of the same mice. The protein level of three antioxidant enzymes, Mn and Cu/Zn superoxide dismutases (SOD) and catalase, was also significantly lower in skeletal muscle compared to liver or kidney. In addition, we found higher levels of protein carbonylation in the mitochondria of skeletal muscle relative to other tissues. Thus, it appears likely that mouse skeletal muscle is highly susceptible to oxidative stress due to deficiency in both repair of oxidative DNA damage and antioxidant enzymes, contributing to age-dependent muscle loss.

Long patch base excision repair in mammalian mitochondrial genomes.

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice.

BACKGROUND: In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development. RESULTS: We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes. CONCLUSION: We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.