Role of Endonuclease G in Exogenous DNA Stability in HeLa Cells.

Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts.

[Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

Endonuclease G depletion may improve efficiency of first generation adenovirus vector DNA replication in HeLa cells.

First generation adenovirus (Ad5 ΔE1,E3) vectors are able to replicate their DNA in many tumour cells and can be used for oncotherapy. Highest rates of viral DNA replication occur in the G2/M transition of the cell cycle. In this study, we tried to increase the efficiency of Ad5 ΔE1,E3 DNA replication in the cervical carcinoma HeLa cells by using RNA interference (RNAi) to target endonuclease G (EndoG) whose depletion leads to an accumulation of cells in the G2/M transition. Targeting of EndoG by an shRNA encoded on an Ad5 ΔE1,E3 vector resulted in an early proliferation defect of cervical carcinoma HeLa cells. This effect coincided with enhanced DNA replication and encoded transgene expression of an Ad5 ΔE1,E3 vector. Applied in high concentrations, the EndoG-targeting Ad5 ΔE1,E3 vector showed enhanced HeLa cell killing ability relative to control Ad5 ΔE1,E3 vectors. These effects are most likely the result of EndoG depletion, which causes cells to accumulate in the G2/M transition of the cell cycle and extends favourable cellular conditions for Ad5 ΔE1,E3 DNA replication. Targeting of EndoG by RNAi may be a viable strategy for improving both the levels of transgene expression and the oncolytic properties of first generation adenovirus vectors.

SU-E-T-614: Dose-Reactive Methods in Adaptive Robust Radiation Therapy for Lung Cancer.

PURPOSE: To perform adaptive radiation therapy treatments for lung cancer using IMRT; to show that adjusting the target dose after each fraction, in order to'react' to errors in the dose delivered in prior fractions, can lead to significant changes in the daily tumor dose over the treatment. METHOD AND MATERIALS: Before the start of treatment, the beamlet intensities are optimized to deliver the current target dose distribution for a target set of breathing patterns at minimal healthy tissue dose. In each fraction, the current beamlet intensities are delivered, and the patient's breathing pattern is measured. The breathing pattern set is updated using the breathing pattern, and the target dose distribution is updated to account for dose errors realized in the previous fraction. The beamlet intensities are then re-optimized for the updated dose distribution and uncertainty set, to be used in the next fraction. This process continues until the end of the treatment. We consider three types of updates to the target dose distribution: the reactive± update, which responds to both under and overdose; the reactive- update, which responds only to underdose; and the reactive+ update, which responds only to overdose. RESULTS: On breathing pattern sequences obtained from real patients, dose-reactive methods result in final dose performance comparable to non-dose-reactive methods. However, as the treatment progresses, the reactive± update results in growing daily underdose and overdose, the reactive- update results in growing daily overdose, and the reactive+ update results in growing daily underdose. In contrast, non-reactive methods have stable or decreasing tumor underdose and overdose. CONCLUSIONS: By incorporating dose-reaction, the final tumor dose distribution can be made to conform closely to the target dose distribution, but at the cost of increasing tumor underdose and/or overdose. This increasing heterogeneity may have implications for the biological effectiveness of treatments obtained by dose-reaction.

Robotic system for prostate brachytherapy.

In contemporary brachytherapy procedures, needle placement at the desired target is challenging for a variety of reasons. A robot-assisted brachytherapy system can potentially improve needle placement and seed delivery, resulting in enhanced therapeutic outcome. In this paper we present a robotic system with 16 degrees of freedom (DOF) (9 DOF for the positioning module and 7 DOF for the surgery module) that has been developed and fabricated for prostate brachytherapy. Strategies to reduce needle deflection and target movement were incorporated after extensive experimental validation. Provision for needle motion and force feedback was included in the system to improve robot control and seed delivery. Preliminary experimental results reveal that the prototype system is sufficiently accurate in placing brachytherapy needles.

Efficacy of prostate stabilizing techniques during brachytherapy procedure.

During the prostate brachytherapy procedure, multiple needles are inserted into the prostate and radioactive seeds are deposited. Stabilizing needles are first inserted to provide some rigidity and support to the prostate, ideally this will provide better seed placement and an overall improved treatment. However, there is much speculation regarding the effectiveness of using regular brachytherapy needles as stabilizers. In this study, we explored the efficacy of (1) two types of needles--18 gauge brachytherapy needle vs. 18 gauge hooked needle; and (2) parallel vs. angulated needle configurations to stabilize the prostate. Prostate phantom movement and needle insertion progression were imaged using ultrasound (US). The recorded images were analyzed and prostate displacement was computed from images using implanted artifacts. Experimentation allowed us to further understand the mechanics behind prostate stabilization. We observed superior stabilization by the hooked needles compared to the regular brachytherapy needles (more than 40% for parallel stabilization). Prostate movement was also reduced significantly when regular brachytherapy needles were in an angulated configuration as compared to the parallel configuration (approximately 40%). When the hooked needles were angled for stabilization, further improvement in decreased displacement was observed. In general, for convenience of dosimetric planning, all needles are desired to be in parallel and in this case, hooked needles are better suited to improve stabilization of the prostate. On the other hand, both regular and hooked needles appear to be equally effective in reducing prostate movement when they are in angulated configurations, which will be useful in robotic permanent seed implantation (PSI).

Tumor Detection in vivo with Optical Spectroscopy.

Ultrasound induced blood stasis has been observed for a long time, but to date most experimental observations have been in vitro. In this paper we discuss a possible diagnostic use for this previously undesirable effect of ultrasound - tumor detection in vivo. We demonstrate that, using optical spectroscopy, effects of ultrasound can be used to differentiate tumor from non-tumor in murine tissue. Finally, we propose a novel diagnostic algorithm that quantitatively differentiates tumor from non-tumor with maximum specificity 0.83, maximum sensitivity 0.79, and area under ROC curve 0.90.

In vivo Optical Spectroscopy of Acoustically Induced Blood Stasis.

Ultrasound-induced blood stasis has been observed for more than thirty years. Most of the literature has been focused on the health risks associated with this phenomenon and methods employed to prevent stasis from occurring during ultrasound imaging. To date, experimental observations have been either in vitro or invasive. The current work demonstrates ultrasound- induced blood stasis in murine tumor and nontumor tissue, observed through noninvasive measurements of optical spectroscopy, and discusses possible diagnostic uses for this previously undesirable effect of ultrasound.