Cell Survival Data Research Articles

Systematic analysis of RBE and related quantities using a database of cell survival experiments with ion beam irradiation

For tumor therapy with light ions and for experimental aspects in particle radiobiology the relative biological effectiveness (RBE) is an important quantity to describe the increased effectiveness of particle radiation. By establishing and analysing a database of ion and photon cell survival data, some remarkable properties of RBE-related quantities were observed. The database consists of 855 in vitro cell survival experiments after ion and photon irradiation. The experiments comprise curves obtained in different labs, using different ion species, different irradiation modalities, the whole range of accessible energies and linear energy transfers (LETs) and various cell types. Each survival curve has been parameterized using the linear-quadratic (LQ) model. The photon parameters, α and β, appear to be slightly anti-correlated, which might point toward an underlying biological mechanism. The RBE values derived from the survival curves support the known dependence of RBE on LET, on particle species and dose. A positive correlation of RBE with the ratio α/β of the photon LQ parameters is found at low doses, which unexpectedly changes to a negative correlation at high doses. Furthermore, we investigated the course of the β coefficient of the LQ model with increasing LET, finding typically a slight initial increase and a final falloff to zero. The observed fluctuations in RBE values of comparable experiments resemble overall RBE uncertainties, which is of relevance for treatment planning. The database can also be used for extensive testing of RBE models. We thus compare simulations with the local effect model to achieve this goal.

The specificity of UVA-induced DNA damage in human melanocytes

Exposure to solar UV radiation is the origin of most skin cancers, including deadly melanomas. Melanomas are quite different from keratinocyte-derived tumours and exhibit a different mutation spectrum in the activated oncogenes, possibly arising from a different class of DNA damage. In addition, some data suggest a role for UVA radiation in melanomagenesis. To get further insight into the molecular mechanisms underlying induction of melanoma, we quantified a series of UV-induced DNA damage in primary cultures of normal human melanocytes. The results were compared with those obtained in keratinocytes from the same donors. In the UVB range, the frequency and the distribution of pyrimidine dimers was the same in melanocytes and keratinocytes. UVA was also found to produce thymine cyclobutane dimer as the major DNA lesion with an equal efficiency in both cell types. In contrast, following UVA-irradiation a large difference was found for the yield of 8-oxo-7,8-dihydroguanine; the level of this product was 2.2-fold higher in melanocytes than in keratinocytes. The comet assay showed that the induction of strand breaks was equally efficient in both cell types but that the yield of Fpg-sensitive sites was larger in melanocytes. Our data show that, upon UVA irradiation, oxidative lesions contribute to a larger extent to DNA damage in melanocytes than in keratinocytes. We also observed that the basal level of oxidative lesions was higher in the melanocytes, in agreement with a higher oxidative stress that may be due to the production of melanin. The bulk of these results, combined with qPCR and cell survival data, may explain some of the differences in mutation spectrum and target genes between melanomas and carcinomas arising from keratinocytes.

Compatibility of the repairable-conditionally repairable, multi-target and linear-quadratic models in converting hypofractionated radiation doses to single doses

We investigated the applicability of the repairable-conditionally repairable (RCR) model and the multi-target (MT) model to dose conversion in high-dose-per-fraction radiotherapy in comparison with the linear-quadratic (LQ) model. Cell survival data of V79 and EMT6 single cells receiving single doses of 2–12 Gy or 2 or 3 fractions of 4 or 5 Gy each, and that of V79 spheroids receiving single doses of 5–26 Gy or 2–5 fractions of 5–12 Gy, were analyzed. Single and fractionated doses to actually reduce cell survival to the same level were determined by a colony assay. Single doses used in the experiments and surviving fractions at the doses were substituted into equations of the RCR, MT and LQ models in the calculation software Mathematica, and each parameter coefficient was computed. Thereafter, using the coefficients and the three models, equivalent single doses for the hypofractionated doses were calculated. They were then compared with actually-determined equivalent single doses for the hypofractionated doses. The equivalent single doses calculated using the RCR, MT and LQ models tended to be lower than the actually determined equivalent single doses. The LQ model seemed to fit relatively well at doses of 5 Gy or less. At 6 Gy or higher doses, the RCR and MT models seemed to be more reliable than the LQ model. In hypofractionated stereotactic radiotherapy, the LQ model should not be used, and conversion models incorporating the concept of the RCR or MT models, such as the generalized linear-quadratic models, appear to be more suitable.

Cyclosporine A and bromocriptine attenuate cell death mediated by intracellular calcium mobilization

To identify the novel inhibitors of endoplasmic reticulum stress-induced cell death, we performed a high throughput assay with a chemical library containing a total of 3280 bioactive small molecules. Cyclosporine A and bromocriptine were identified as potent inhibitors of thapsigargiin-induced cell death (cut-off at 4σ standard score) . However, U74389G, the potent inhibitor of lipid peroxidation had lower activity in inhibiting cell death. The inhibition effect of cyclosporine A and bromocriptine was specific for only thapsigargin-induced cell death. The mechanism of inhibition by these compounds was identified as modification of the expression of glucose regulated protein-78 (GRP-78/Bip) and inhibition of phosphorylation of p38 mitogen activated protein kinase (MAPK). However, these compounds did not inhibit the same events triggered by tunicamycin, which was in agreement with the cell survival data. We suggest that the induction of protective unfolded protein response by these compounds confers resistance to cell death. In summary, we identified compounds that may provide insights on cell death mechanisms stimulated by ER stress.

A model for the relative biological effectiveness of protons: The tissue specific parameter α/β of photons is a predictor for the sensitivity to LET changes

Background. The biological effects of particles are often expressed in relation to that of photons through the concept of relative biological effectiveness, RBE. In proton radiotherapy, a constant RBE of 1.1 is usually assumed. However, there is experimental evidence that RBE depends on various factors. The aim of this study is to develop a model to predict the RBE based on linear energy transfer (LET), dose, and the tissue specific parameter α/β of the linear-quadratic model for the reference radiation. Moreover, the model should capture the basic features of the RBE using a minimum of assumptions, each supported by experimental data. Material and methods. The α and β parameters for protons were studied with respect to their dependence on LET. An RBE model was proposed where the dependence of LET is affected by the (α/β)phot ratio of photons. Published cell survival data with a range of well-defined LETs and cell types were selected for model evaluation rendering a total of 10 cell lines and 24 RBE values. Results and Conclusion. A statistically significant relation was found between α for protons and LET. Moreover, the strength of that relation varied significantly with (α/β)phot. In contrast, no significant relation between β and LET was found. On the whole, the resulting RBE model provided a significantly improved fit (p-value < 0.01) to the experimental data compared to the standard constant RBE. By accounting for the α/β ratio of photons, clearer trends between RBE and LET of protons were found, and our results suggest that late responding tissues are more sensitive to LET changes than early responding tissues and most tumors. An advantage with the proposed RBE model in optimization and evaluation of treatment plans is that it only requires dose, LET, and (α/β)phot as input parameters. Hence, no proton specific biological parameters are needed.

Radiobiological description of the LET dependence of the cell survival of oxic and anoxic cells irradiated by carbon ions

Light-ion radiation therapy against hypoxic tumors is highly curative due to reduced dependence on the presence of oxygen in the tumor at elevated linear energy transfer (LET) towards the Bragg peak. Clinical ion beams using spread-out Bragg peak (SOBP) are characterized by a wide spectrum of LET values. Accurate treatment optimization requires a method that can account for influence of the variation in response for a broad range of tumor hypoxia, absorbed doses and LETs. This paper presents a parameterization of the Repairable Conditionally-Repairable (RCR) cell survival model that can describe the survival of oxic and hypoxic cells over a wide range of LET values, and investigates the relationship between hypoxic radiation resistance and LET. The biological response model was tested by fitting cell survival data under oxic and anoxic conditions for V79 cells irradiated with LETs within the range of 30–500 keV/µm. The model provides good agreement with experimental cell survival data for the range of LET investigated, confirming the robustness of the parameterization method. This new version of the RCR model is suitable for describing the biological response of mixed populations of oxic and hypoxic cells and at the same time taking into account the distribution of doses and LETs in the incident beam and its variation with depth in tissue. The model offers a versatile tool for the selection of LET and dose required in the optimization of the therapeutic effect, without severely affecting normal tissue in realistic tumors presenting highly heterogeneous oxic and hypoxic regions.

Applying a Hypoxia-Incorporating TCP Model to Experimental Data on Rat Sarcoma

To verify whether a tumor control probability (TCP) model which mechanistically incorporates acute and chronic hypoxia is able to describe animal in vivo dose-response data, exhibiting tumor reoxygenation. The investigated TCP model accounts for tumor repopulation, reoxygenation of chronic hypoxia, and fluctuating oxygenation of acute hypoxia. Using the maximum likelihood method, the model is fitted to Fischer-Moulder data on Wag/Rij rats, inoculated with rat rhabdomyosarcoma BA1112, and irradiated in vivo using different fractionation schemes. This data set is chosen because two of the experimental dose-response curves exhibit an inverse dose behavior, which is interpreted as due to reoxygenation. The tested TCP model is complex, and therefore, in vivo cell survival data on the same BA1112 cell line from Reinhold were added to Fischer-Moulder data and fitted simultaneously with a corresponding cell survival function. The obtained fit to the combined Fischer-Moulder-Reinhold data was statistically acceptable. The best-fit values of the model parameters for which information exists were in the range of published values. The cell survival curves of well-oxygenated and hypoxic cells, computed using the best-fit values of the radiosensitivities and the initial number of clonogens, were in good agreement with the corresponding in vitro and in situ experiments of Reinhold. The best-fit values of most of the hypoxia-related parameters were used to recompute the TCP for non-small cell lung cancer patients as a function of the number of fractions, TCP(n). The investigated TCP model adequately describes animal in vivo data exhibiting tumor reoxygenation. The TCP(n) curve computed for non-small cell lung cancer patients with the best-fit values of most of the hypoxia-related parameters confirms previously obtained abrupt reduction in TCP for n < 10, thus warning against the adoption of severely hypofractionated schedules.

SU-E-T-646: Feasibility Study of Radiobiological Effectiveness-Based Treatment Plan Optimization for Spot-Scanned Proton Therapy Beams

Purpose: To develop novel methodologies for determination of the relative biological effectiveness (RBE) in treatment plans created with a feasibility search optimization scheme for the spot scanned proton pencil beams at the Proton Therapy Center in Houston. Methods: We explored methods with clinical practicality to calculate related physical and RBE‐weighted doses for spot scanned proton beams. We studied a test case in which an organ‐at‐risk (OAR) was surrounded by the PTV. Results were demonstrated with simulated proton therapy data. We optimized the dose distribution for a planning treatment volume (PTV) within a water phantom with a feasibility search method, used previously to design conventional photon and electron beamradiation therapy plans and applied here for the first time to proton therapy planning. Prospective and retrospective intercomparison scenarios were run with computed RBEs using linear quadratic model parameter values taken from the in vitro measured survival data of Chinese hamster V79 cells. Results: We designed methods to carry out intercomparisons between treatment plans with different modalities. We found a higher RBE‐ weighted dose in the OAR than that expected with a constant RBE value of 1.1, which is currently used in clinical practice for range modulated proton beams. The TCP (tumor control probability)/NTCP (normal tissue complication probability) dose‐response analysis showed that our test planned irradiation would not have been acceptable if the OAR structure connection were highly serial, such as in the spinal cord. Conclusion: This exploratory study has provided a framework which should be helpful in the ongoing search to include detailed RBE effects in intensity modulated proton therapytreatment planning.

SU‐E‐T‐05: Biophysical Modeling Intercomparison of Proton Radiation Effectiveness

Purpose: Evaluate the power of three biophysical models to predict proton relative biological effectiveness (RBE) using cell survival data for 60CoMethods: The Repair‐Misrepair‐Fixation (RMF), Linear Scaling (LSM) and Total‐isoeffective Dose Ratio (TiDR) models were compared to experimental data for V79 cells irradiated in vitro using gamma‐rays from 60Co and at selected depths in a proton spread out Bragg peak (SOBP). Dose, Linear Energy Transfer (LET) and fluence were computed for this SOBP using Monte Carlo (GEANT4) simulation. Biological parameters for the LSM and TiDR were obtained from published survival data for protons, whereas parameters for the RMF were determined using only the V79 survival data for 60CoResults: The models predict similar trends in cell survival with dose and depth within the SOBP. Some small differences in model predictions for lower and higher doses are attributable to uncertainties in the experimental data and to assumptions for the LET dependence of beta. For 2 Gy, the predicted proton RBE ranges from 1.06 to 1.12 at a depth of 17.2 mm, and from 1.18 to 1.3 on the distal edge of the SOBP (depth=155.8 mm). In the dose fall‐off region of the SOBP, the RBE may be as large as 1.5 to 2.4. Estimates of RBE from all three models are in good agreement with the measured data for V79 cellsConclusions: The current study provides evidence for substantial variations in proton RBE within the SOBP. The three models considered in the study mainly differ in their use of physical parameters (e.g., dose and LET) and the way intrinsic radiation sensitivity is parameterized. Still, all three models appear surprisingly robust in their ability to predict proton RBE from data for other radiations in the same cell line (RMF) or from published data for other cell lines (TiDR and LSM)

Empirical model estimation of relative biological effectiveness for proton beam therapy

A simple model for proton relative biological effectiveness (RBE) is proposed. It describes the RBE as a function of proton depth, the dose and the linear energy transfer (LET) when proton passes through tissue-like materials. Radiobiological parameters were first obtained by fitting the published experimental cell survival data. The dose-averaged LET values were calculated for 250-MeV proton beam in a water phantom by using GEANT4 Monte Carlo simulation code and were then used as input values to calculate the values of RBE as function of depths. The model was also applied to proton spread-out Bragg peak, where the increasing RBE with depth causes an extended RBE-weighted dose in the distal fall-off region. This model was found to be able to reproduce the measured RBE values as a function of LET, depth and dose for a specific cell line.

Modelling of the oxygen enhancement ratio for ion beam radiation therapy

The poor treatment prognosis for tumours with oxygen-deficient areas is usually attributed to the increased radioresistance of hypoxic cells. It can be expressed by the oxygen enhancement ratio (OER), which decreases with increasing linear energy transfer (LET) suggesting a potential clinical advantage of high-LET radiotherapy with heavy ion beams compared to low-LET photon or proton irradiation. The aim of this work is to review the experimental cell survival data from the literature and, based on them, to develop a simple OER model to estimate the clinical impact of OER variations. For this purpose, the standard linear-quadratic model and the Alper–Howard-Flanders model are used. According to our calculations for a carbon ion spread-out Bragg peak at clinically relevant intermediate oxygen levels (0.5–20 mmHg), the advantage of carbon ions might be relatively moderate, with OER values about 1%–15% smaller than for protons. Furthermore, the variations of OER with LET are much smaller in vivo than in vitro due to different oxygen partial pressures used in cell experiments or measured inside tumours. The proposed OER model is a simple tool to quantify the oxygen effect in a practical way and provides the possibility to do hypoxia-based biological optimization in treatment planning.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Temporal characterization and in vitro comparison of cell survival following the delivery of 3D-conformal, intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT)

A phantom was designed and implemented for the delivery of treatment plans to cells in vitro. Single beam, 3D-conformal radiotherapy (3D-CRT) plans, inverse planned five-field intensity-modulated radiation therapy (IMRT), nine-field IMRT, single-arc volumetric modulated arc therapy (VMAT) and dual-arc VMAT plans were created on a CT scan of the phantom to deliver 3 Gy to the cell layer and verified using a Farmer chamber, 2D ionization chamber array and gafchromic film. Each plan was delivered to a 2D ionization chamber array to assess the temporal characteristics of the plan including delivery time and ‘cell's eye view’ for the central ionization chamber. The effective fraction time, defined as the percentage of the fraction time where any dose is delivered to each point examined, was also assessed across 120 ionization chambers. Each plan was delivered to human prostate cancer DU-145 cells and normal primary AGO-1522b fibroblast cells. Uniform beams were delivered to each cell line with the delivery time varying from 0.5 to 20.54 min. Effective fraction time was found to increase with a decreasing number of beams or arcs. For a uniform beam delivery, AGO-1552b cells exhibited a statistically significant trend towards increased survival with increased delivery time. This trend was not repeated when the different modulated clinical delivery methods were used. Less sensitive DU-145 cells did not exhibit a significant trend towards increased survival with increased delivery time for either the uniform or clinical deliveries. These results confirm that dose rate effects are most prevalent in more radiosensitive cells. Cell survival data generated from uniform beam deliveries over a range of dose rates and delivery times may not always be accurate in predicting response to more complex delivery techniques, such as IMRT and VMAT.

Relation between Lineal Energy Distribution and Relative Biological Effectiveness for Photon Beams according to the Microdosimetric Kinetic Model

Our cell survival data showed the obvious dependence of RBE on photon energy: The RBE value for 200 kV X-rays was approximately 10% greater than those for mega-voltage photon beams. In radiation therapy using mega-voltage photon beams, the photon energy distribution outside the field is different with that in the radiation field because of a large number of low energy scattering photons. Hence, the RBE values outside the field become greater. To evaluate the increase in RBE, the method of deriving the RBE using the Microdosimetric Kinetic model (MK model) was proposed in this study. The MK model has two kinds of the parameters, tissue-specific parameters and the dose-mean lineal energy derived from the lineal energy distributions measured with a Tissue-Equivalent Proportional Counter (TEPC). The lineal energy distributions with the same geometries of the cell irradiations for 200 kV X-rays, (60)Co γ-rays, and 6 MV X-rays were obtained with the TEPC and Monte Carlo code GEANT4. The measured lineal energy distribution for 200 kV X-rays was quite different from those for mega-voltage photon beams. The dose-mean lineal energy of 200 kV X-rays showed the greatest value, 4.51 keV/µm, comparing with 2.34 and 2.36 keV/µm for (60)Co γ-rays and 6 MV X-rays, respectively. By using the results of the TEPC and cell irradiations, the tissue-specific parameters in the MK model were determined. As a result, the RBE of the photon beams (y(D): 2~5 keV/µm) in arbitrary conditions can be derived by the measurements only or the calculations only of the dose-mean lineal energy.

Analytical Description of the LET Dependence of Cell Survival Using the Repairable-Conditionally Repairable Damage Model

In light-ion radiation therapy, both the dose and the local energy spectrum, which is often characterized with the linear energy transfer (LET), must be considered. In treatment optimization, it is advantageous to use a radiobiological model that analytically accounts for both dose and LET for the ion type of interest. With such a model the biological effect can also be estimated for dose and LET combinations for which there are no observations in the underlying experimental data. In this study, the repairable-conditionally repairable (RCR) damage model was extended by expressing its parameters as functions of LET to provide a radiobiological model that accounts for both the dose and the LET for a given ion type and cell line. This LET-parameterized RCR model was fitted to published cell survival data for HSG and V79 cells irradiated with carbon ions and for T1 cells irradiated with helium ions. To test the robustness of the model, fittings to only a subset of the data were performed. Good agreement with the cell survival data was obtained, including survival data for LET values not used for model fitting, opening up the possibility of using the model in treatment planning for light ions.

MO-E-BRB-07: Relative Biological Effectiveness of Proton - An Empirical Model Prediction

Purpose: To propose a simple Relative Biological Effective (RBE) model for proton that describes the RBE as a function of proton depth, dose, and the linear energy transfer (LET). Methods and Materials: Radiobiological parameters α and β were obtained by fitting the published experimental proton cell survival data where the surviving fraction (SF) as a function of dose (D) is given by SFSF(D) = exp(−αD − βD 2 ). The RBE defined as the ratio of photondose over protondose (D x /D p ) for the same SF, is given in the present model by, RBE = α x 2 − 4β x LnSF − α x α p 2 − 4β p LnSF − α p × β p β x where α p = α 0 + 1 − e −λ 1 * LET 2 λ 2 * LET and β p = β x . A software program gnuplot with the Levenberg‐Marquardt algorithm was used to fit the experimental data with the empirical formula mentioned above by minimizing χ2 with the selection of suitable parameters (λ1, λ2, α0). The dose average LET values were calculated for 250 MeV proton in water using GEANT4 (version 4.9.3) Monte Carlo simulation code. The model was then used to calculate the RBE values as a function of depths that includes the Bragg peak depths of 250 MeV protons, where the increasing RBE with depths causes an extended biological effective dose in the distal falloff range. Results: The best fit parameters obtained were α0=0.1 Gy−1, λ1=0.0013 (keV/μm)−2 and 2=0.045 (keV/μm)−1⋅Gy. The model was found to be able to reproduce the measured RBE values as a function of LET and dose. The biological effective dose (physical dose multiplied by RBE) as a function of depths for 250 MeV incident proton shows that the shift in the depth of distal edge at 90% level of physical dose is 1 mm. Conclusions: The new model is providing an improved understanding of the RBE as a function of depth, dose and LET for proton beam.

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase lambda (Pol lambda) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol lambda. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol lambda efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol lambda could modulate the cytotoxic effect of AraC.

Abstract 5373: Modulation of temozolomide-mediated DNA damage in glioblastoma multiforme by the HDM2 antagonist, nutlin3

Abstract Development of efficacious strategies for eradication of glioblastoma multiforme (GBM) remains a significant challenge. While treatment of GBM with radiation and temozolomide (TMZ) has improved clinical outcome, lack of long-term efficacy can be due to the ability of GBM to acquire resistance to therapy by modulation of signaling pathways that control DNA repair and cell survival. The HDM2 antagonist, Nutlin3, blocks interactions of HDM2 with key signaling molecules such as p53, p73α, and HIF1α, and modulates their downstream effector function. A panel of human GBM cell lines is currently being used to monitor the extent to which Nutlin3 can sensitize cells to TMZ. In vitro survival assays indicate that Nutlin3 potentiates TMZ-mediated cell death; analysis of cell survival data using Dose Effect CalcuSyn software indicates that the combination index value was &amp;lt; 1 and indicative of a synergistic effect between Nutlin3 and TMZ. Western analyses of p53 wild-type GBM indicated increased p53 stabilization and activation of down-stream targets in cells treated with the combination of Nutlin3/TMZ compared to vehicle, TMZ, or Nutlin3. In addition, time-course studies indicated that U87-MG cells treated with Nutlin3/TMZ in comparison to single agent resulted in increased γ-H2AX and DNA-strand breaks. Efficacy studies using ectopic U87-MG xenografts indicated that 2 cycles of 5-consecutive days of Nutlin3/TMZ led to a significant decrease in tumor growth compared to vehicle or single agent (p &amp;lt; 0.001) with no signs of off-target toxicity. Regulation of DNA repair was further analyzed using the primary GBM.10 line that when grown as an ectopic xenograft, expresses the direct-reversal repair protein, O6-methylguanine DNA methyltransferase (MGMT) and proteins involved in base-excision repair (BER). In vivo target-validation studies using GBM.10 xenografts confirmed that repair of TMZ-mediated DNA damage is modulated in the presence of Nutlin3. GBM.10 xenografts were treated with vehicle, TMZ, nutlin3, or TMZ/Nutlin3 and modulation of critical targets determined by Western analyses. Increases in total p53, p53 phosphorylated at residue serine16, p73α, and substantial decreases in MGMT and the BER protein, APE-1, were observed following treatment with Nutlin3/TMZ compared to vehicle or single-agent exposure. Busso et al (Oncogene 2009) recently reported that HDM2 can monoubiquitinate APE-1 leading to proteosomal-mediated degradation. In addition, recombinant activity assays in our laboratory indicate that HDM2 directly monoubiquitinates recombinant MGMT and may play a role in its degradation. These data suggest that DNA repair proteins required for repair of TMZ-mediated DNA damage can be downregulated by altering HDM2-mediated signaling. Combination therapy that targets the p53-HDM2 E3 ligase network and DNA repair represents a novel approach towards improving treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5373.

Cellular apoptosis by nanosecond, high-intensity electric pulses: Model evaluation of the pulsing threshold and extrinsic pathway

A simple, bistable rate-equation based model is used to predict trends of cellular apoptosis following electric pulsing. The caspase-8 extrinsic pathway with inherent delays in its activation, cytochrome c release, and an internal feedback mechanism between caspase-3 and cleavage of Bid are incorporated. Results obtained were roughly in keeping with the experimental cell-survival data and include an electrical pulse-number threshold followed by a near-exponential fall-off. The extrinsic caspase-8 mechanism is predicted to be more sensitive than the mitochondrial intrinsic pathway for electric pulse induced cell apoptosis. Also, delays of about an hour are predicted for detectable molecular concentration increases following electrical pulsing. Finally, our results suggest that multi-needle electrode systems with adjustable field orientations would likely enhance apoptosis in the context of pulsed voltage-induced inactivation of tumor cells.

RBE estimation of proton radiation fields using a telescope

A new monolithic silicon DeltaE-E telescope was evaluated in unmodulated and modulated 100 MeV proton beams used for hadron therapy. Compared to a classical microdosimetry detector, which provides one-dimensional information on lineal energy of charged particles, this detector system provides two-dimensional information on lineal energy and particle energy based on energy depositions, collected in coincidence, within the DeltaE and E stages of the detector. The authors investigated the possibility to use the information obtained with the DeltaE-E telescope to determine the relative biological effectiveness (RBE) at defined locations within the proton Bragg peak and spread-out Bragg peak (SOBP). An RBE matrix based on the established in vitro V79 cell survival data was developed to link the output of the device directly to RBE(alpha), the RBE in the low-dose limit, at various depths in a homogeneous polystyrene phantom. In the SOBP of a 100 MeV proton beam, the RBE(alpha) increased from 4.04 proximal to the SOBP to a maximum value of 5.4 at the distal edge. The DeltaE-E telescope, with its high spatial resolution, has potential applications to biologically weighted hadron treatment planning as it provides a compact and portable means for estimating the RBE in rapidly changing hadron radiation fields within phantoms.