Increased Radiation Resistance Research Articles

Abstract PD09-09: The Akt inhibitor MK-2206 is an effective radio-sensitizer of p53 deficient triple negative breast cancer (TNBC) cells.

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive tumor with a higher locoregional recurrence compared to estrogen receptor (ER) positive breast cancer. Mutations in the PI3K/Akt/mTOR pathway leading to up-regulation of Akt occur with high frequency in TNBC. Hyperactive Akt is associated with increase radiation resistance, mediated through inhibition of apoptosis and up-regulation of DNA damage-induced G2 arrest. p53 plays a key role in mediating G1 cell cycle arrest following genotoxic stress such as radiation; p53-deficient cells however must rely on alternate mechanisms for cell cycle arrest in S- and G2-phases. Inhibition of Akt therefore would be expected to act in a synthetic lethal fashion to radiosensitize p53 deficient TNBC cells by decreasing their ability to arrest in G2. Given that p53 mutations occur in 44% of TNBC this may be an effective strategy for radiosensitizing TNBC. Methods: Experiments were conducted using MDA-MB-468, MDA-MB-231 and SUM 149 cells, all well established models for p53-deficient TNBC. Cells were treated with MK-2206, a potent allosteric Akt inhibitor, alone or in combination with increasing doses of radiation from 0–8 Gy. In vitro studies preformed included; cell survival assays, MTT assay, immunoblot analysis of key proteins, FACS analysis for cell cycle, apoptosis, and gH2AX staining. Results: We found that the combination of IR and Akt inhibition by MK-2206 is synergistic. MK-2206 inhibited both endogenous and radiation-induced Akt activation and phosphorylation of its down-stream targets. Decreased clonogenic survival was seen after 2 or 24 hours pretreatment with MK-2206 as well as decreased viability by MTT assay. Cell cycle analysis demonstrates MK-2206 decreases the proportion of cells in G2/M arrest following irradiation, as well as induced a greater proportion of apoptosis. Evaluation of the DNA damage response pathway demonstrated decreased levels of total DNA-PK, as well as a delayed DNA damage response. Conclusions: These studies demonstrate that targeted inhibition of Akt effectively radiosensitizes p53 deficient TNBC cells lines, possibly through a synthetic lethal effect on the DNA damage response. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-09.

Qualitative and quantitative differences in the absorption spectra of electron-irradiated reflective coatings based on mixtures of BaTio3 powder with ZrO2 micro- or nanopowder of different concentrations

The effect of the concentration and size (micro- or nanoparticles) of zirconium dioxide powders, heated in mixtures with barium titanate powder, on the radiation resistance of reflecting coatings on their basis is analyzed during accelerated electron irradiation. It is shown that nanoparticles provide increased radiation resistance of the coatings compared to microparticles.

Abstract B59: MET gene copy number related to radiation treatment response and prognosis in breast cancer

Abstract The hepatocyte growth factor receptor (HGFR), also known as the Met receptor, promotes cell survival and proliferation by activating signaling pathways such as the PI3K/Akt and RAS/MAPK pathway. HGFR, coded by MET, has been shown to be abundant in several cancers and in some cases it interferes with treatment response. In vitro, HGFR has been shown to be upregulated and activated after radiotherapy, increasing the protection from apoptosis and thus increasing radiation resistance. In this study, it was aimed to determine the number of MET gene copies in breast tumors and correlate these findings with different clinicopathological parameters that were available in this material. Tumors of 172 patients with primary breast cancer were analyzed for MET using qPCR. Increased MET copy number (three or more copies) was found in 34% of the tumors. Five or more gene copies were found in 4.7% of the patients and indicated a significantly higher risk of developing distant metastasis. For the patients that received radiotherapy as the only postoperative treatment, the number of MET copies was associated with increased risk of distant metastasis. In terms of locoregional recurrence, it was found that increased MET copy number was associated with worse response to postoperative radiation therapy compared with chemotherapy. These results suggest that radiotherapy in combination with a Met inhibitor might be an option for patients with increased MET gene copy number to overcome radiation resistance.

P3-01-06: NF-kB Inhibition Promotes Radiosensitivity of Breast Cancer Cells in Three-Dimensional Culture through Abating b1-Integrin Expression.

Abstract Therapy-associated tumor resistance, giving rise to recurrence and mortality, is a critical issue in cancer therapy. The molecular mechanisms causing tumor resistnace to the therapeutic radiation remain elusive. Nuclear factor-KB (NF-KB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling pathways. The cooperative function of NF-KB with other key stress elements in radioresistance remains to be elucidated. We have previously reported that induction of α5β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to high dose IR (ionizing radiation). Because a typical NF-KB binding site was located in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-KB. The aim of the present study was to reveal a connection between NF-KB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D ***lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-KB activity was correlated with enhanced clonogenic survival, and increased NF-KB heterodimer p50/p65 levels were associated with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-KB activation significantly reduced clonogenic survival with the inhibition of β1-integrin. These results indicate that NF-KB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of T4-2 colonies, formed at day 4, with NF-KB activation inhibitor in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Together, these results provide the first evidence that NF-KB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-KB/β1-integrin pathway may serve as an efficient drug target to re-sensitize radioresistant tumor cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-06.

Abstract C130: Inhibition of NF-κB activity promotes radiosensitivity of breast cancer cells in three-dimensional culture via a β1-integrin-mediated pathway.

Abstract Radioresistance markedly impairs the efficacy of tumor radiotherapy and involves prosurvival signal transduction pathways that prevent radiation-induced cell death. Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling in response to ionizing radiation (IR). The cooperative function of NF-κB with other key stress elements in radioresistance has not been clearly defined. We have previously reported that induction of β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to clinically relevant doses of IR. Because a typical NF-κB binding site was identified in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-κB. The aim of the present study was to reveal a connection between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-κB activity was correlated with enhanced clonogenic survival with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-κB activation significantly reduced clonogenic survival with the inhibition of β1-integrin, suggesting that NF-κB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of malignant human breast cell colonies, T4-2 with NF-κB activation inhibitor at Day 4 in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Using an ELISA-based DNA binding assay, we next determined that the binding of NF-κB to its target sequence on β1-integrin promoter region was significantly increased by IR. These data suggest that IR-induced increase in β1-integrin protein expression is mediated by NF-κB activation and subsequent binding of NF-κB to the β1-integrin promoter. Taken together, our results provide the first evidence that NF-κB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-κB/β1-integrin pathway could provide new insight into the development of new therapeutic modalities to re-sensitize radioresistant tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C130.

Repair of DNA Double-Strand Breaks Induced by Ionizing Radiation Damage Correlates with Upregulation of Homologous Recombination Genes in Sulfolobus solfataricus

The mechanisms used by members of the archaeal branch of life to repair DNA damage are not well understood. DNA damage responses have been of particular interest in hyperthermophilic archaea, since these microbes live under environmental conditions that constantly elevate the potential for DNA damage. The work described here focuses on the response of four Sulfolobus solfataricus strains to ionizing radiation (IR) damage. Cellular survival of three wild-type strains and a defined deletion mutant strain was examined following exposure to various IR doses. Using pulsed-field gel electrophoresis, we determined chromosomal DNA double-strand break persistence and repair rates. Among the strains, variable responses were observed, the most surprising of which occurred with the defined deletion mutant strain. This strain displayed higher chromosomal repair rates than the parent strain and was also found to have increased resistance to IR. Using quantitative real-time PCR, we found that transcript levels of homologous recombination-related genes were strongly upregulated following damage in all the strains. The mutant strain again had an enhanced response and dramatically upregulated expression of recombination genes above levels observed for the parent strain, suggesting that increased levels of recombinational repair could account for its increased radiation resistance phenotype. Our results demonstrate a transcriptional response to IR in S. solfataricus for the first time and describe a defined deletion mutant strain that may give the first insight into a damage-based archaeal control element.

The Phenotypic Radiation Resistance of CD44+/CD24−or low Breast Cancer Cells Is Mediated through the Enhanced Activation of ATM Signaling

Cancer initiating cells (CIC) are stem-like cells. CIC may contribute not only to the initiation of cancer but also to cancer recurrence because of the resistance of CIC both to chemotherapy and radiation therapy. From the MCF-7 and MDA-MB231 breast cancer cell lines and primary culture of patient breast cancer cells, we isolated by flow cytometry a CIC subset of cells with the CD44+/CD24−or low phenotype. The CD44+/CD24−or low subset showed increased sphere formation and resistance to radiation compared to the non- CD44+/CD24−or low subset. The increased radiation resistance was not dependent on the result of altered non-homologous end joining (NHEJ) DNA repair activity as both NHEJ activity and expression of the various proteins involved in NHEJ were not significantly different between the CD44+/CD24−or low and non- CD44+/CD24−or low subsets. However, activation of ATM signaling was significantly increased in CD44+/CD24−or low cells compared to non- CD44+/CD24−or low cells in both from breast cancer cell lines and primary human breast cancer cells. Application of an ATM inhibitor effectively decreased the radiation resistance of CD44+/CD24−or low subset, suggesting that targeting ATM signaling may provide a new tool to eradicate stem-like CIC and abolish the radiation resistance of breast cancer.

Abstract 2469: ATM inhibitor 55933 abolishes the radiation resistance of CD44+/CD24−or low subsets of breast cancer cells

Abstract CD44+/CD24− subsets of breast cancer cells are enriched in cancer stem-like cells (CSC) and are radiation resistant. We isolated ESA+/CD44+/CD24− or low subsets by flow cytometry from MCF7, MDA-MB231, and HCC1937 breast cancer cell lines and primary cultures of breast cancer cells from two patients. The isolated CD44+/CD24−orlow subsets showed a 50 to 70% increase of sphere formation by in suspension culture in serum-free medium compared to CD44−/CD24high subsets. CD44+/CD24−or low subsets of the cell lines and primary cultures showed a significantly increased radiation resistance compared to the CD44−/CD24high cell line subsets or the unsorted primary cells with > 40% increased survival at 2Gy. Post-radiation survival showed that the radiation resistance was dependent on the CD44+/CD24 −or low cell phenotype. The correlation of CD44/CD24 status with radiation resistance was demonstrated by culture of the subsets. After 2 weeks of culture the following transitions occurred: 1) CD44+/CD24−or low cells changed to CD44−/CD24high cells (MCF-7 and HCC1937) or to CD44+/CD24+ (MDA-MB-231); and 2) CD44−/CD24 high (MCF-7 and HCC1937) and CD44+/CD24+ (MDA-MB-231) cells became CD44+/CD24−or low cells. Cells that lost the CD44+/CD24 −or low phenotype were no longer radiation resistant. The mechanisms of increased radiation resistance in CD44+/CD24−orlow subsets was not a function of non-homologous end joining (NHEJ) as there was no in vivo and in vitro change in NHEJ activity in the resistant cells nor altered expression of the NHEJ related proteins Ku 80, Ku70, PARP-1, and DNA-PK. However, there were differences ATM activity between CD44+/CD24−or low and the CD44−/CD24 high (MCF-7 and primary breast cancer) or CD44+/CD24+ (MDA-MB-231) subsets. Depending on the cell type and radiation dose, ATM phsophorylation increased by 2-5 fold in radiated CD44+/CD24−or low subsets of breast cancer cell lines and primary cultured breast cancer cells compared to the control subsets. The ATM inhibitor 55933 in a dose-dependent manner increased radiation sensitivity of all subsets including significantly abolishing the radiation resistance of CD44+/CD24−or low subsets of cells. After 2Gy of radiation, the survival fractions of cells treated with10μM of 55933 were 8-10 fold lower than that of untreated cells and were now similar between CD44+/CD24−or low subsets and control subsets for the cell lines and primary cultures. In conclusion, ATM is a candidate target for eliminating the radiation resistance of CSC's of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2011-2469

Radiation-induced HIF-1α Upregulates Glycolysis and Exhibits Radioresistance in Glioblastoma

Hypoxic environment is known to mediate treatment resistance. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role to increase radiation resistance through the activations of several genes involved in the metabolism, angiogenesis, metastasis, and treatment resistance. Recent investigations have shown that radiation increases HIF-1α expression in several tumors in vivo and in vitro. However, there have been no reports that radiation-induced HIF-1α mediates radiation resistance through upregulation of glycolysis.

Abstract 1504: Inhibition of RNA-dependent protein kinase (PKR) enhances radiation sensitivity in lung cancer cells

Abstract We have previously demonstrated that radiation resistance in MEFs is mediated in part through RNA-dependent protein kinase (PKR) possibly through upregulation of AKT and NFk-B. The role of PKR in cancer cells has not been evaluated. In this study, we show that the transduction of MEFs and lung cancer cells with adenoviral wt PKR results in nuclear translocation of PKR, activation of AKT1/2 and increased radiation resistance. The transduction of lung cancer cells or MEFs with a dominant negative adenoviral PKR vector blocks nuclear translocation of PKR, inhibits AKT activation and leads to the reversal of radiation resistance. Plasmid transduction of MEFs and lung cancer cells with nuclear targeted wt PKR vectors also results in AKT1/2 activation and increased radiation resistance. This effect is selectively abrogated by plasmid transduction of dominant negative PKR vectors which inhibit AKT1/2 activation and restore radiation sensitivity. These findings suggest a novel role for PKR in lung cancer cells as a mediator of radiation resistance possibly through translocation of the protein product to the nucleus and activation of AKT1/2. 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 1504.

Electrically Small MNG ZOR Antenna With Multilayered Conductor

In this letter, an electrically small mu-negative (MNG) zeroth-order resonance (ZOR) antenna with multilayered conductor is presented. To improve the radiation efficiency, the structure of the MNG ZOR antenna is determined to increase radiation resistance by reducing the current cancellation between the conductor strip of the antenna. In particular, the multilayered conductor is employed to enhance the radiation efficiency by reducing ohmic loss. The MNG ZOR antenna using multilayered conductor (kr = 0.182) has a measured 3-dB fractional bandwidth of 0.846% and radiation efficiency of 18.66%.

Increased Radiation Resistance of Breast Cancer Cells by Endocrine Disrupting Chemical Bisphenol A.

Abstract Purpose: Bisphenol A (BPA) is used in the manufacture of plastics and belongs to a family of compounds called endocrine disrupting chemicals (EDCs). Because of extensive use of BPA in the manufacture of consumer goods and products, humans are exposed to levels of BPA which may cause adverse effects on endocrine-sensitive organs. Here, we evaluated the effects of BPA on the proliferation of breast cancer cells and the effect of BPA on tumor cell radioresponse.Materials/Methods: Proliferation was evaluated in estrogen-sensitive (MCF-7) and estrogen-insensitive (MDA-MB-231) human breast cancer lines that were treated for 72 hour with BPA (0.1µM; environmentally relevant concentration). The effect on radiation sensitivity was evaluated using clonogenic survival assays after exposure to various dose of BPA (0.1, 1, and 10 µM-) followed by irradiation (IR). DNA damage was evaluated using phosphorylated histone H2AX (γH2AX) and the neutral comet assay. BPA induced cell cycle changes were evaluated by staining of phosphor-H3 and flow cytomety. To investigate the mechanism of BPA±IR induced cell growth, we explored the activation of Akt and mitogen-activated protein kinase (MAPK) after treatments.Results: Exposure of BPA at low-dose (0.1µM) for 72 hour resulted in an increase survival rate in MCF-7 cancer cell (150 ± 19% of control) with more limited effects on MDA-MB-231 cancer cell (123 ± 17% of control). Exposure of MCF-7 to BPA for 72 hour before radiation resulted in an increase in radioresistance with dose-reduction factors at a surviving fraction of 0.1 of 1.23 and 1.31 with BPA 0.1µM, 10µM, respectively. BPA had no effect on the number of cells in each phase of the cell cycle nor on the activation of the G2 cell cycle checkpoint after IR. As a measure of DNA double strand breaks, γH2AX were determined as a function of time after the BPA±IR treatments. BPA also significantly decreased γH2AX expression at 24 hours after IR. Expression of phosphorylated Akt and phosphorylated mitogen-activated protein kinase (MAPK) increased with BPA± IR but decreased with IR alone at 24 hr.Conclusions: These results suggest that BPA can enhance phosphorylation of Akt and MAPK possibly leading to a resistance to radiation-induced cell death after IR. Further studies are needed to determine the clinical relevance of these findings. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6073.

The Suppression of Hypoxia-inducible Factor and Vascular Endothelial Growth Factor by siRNA Does not Affect the Radiation Sensitivity of Multicellular Tumor Spheroids

The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression. SQ5 human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Reversed transfection methods were employed for the spheroids. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay. Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. Hypoxic conditions elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions. We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions.

Moisture Degradation of Cyanate Ester/S2 Glass Composite Insulation Systems

The fusion devices currently being developed present several challenges for magnet designers. One challenge lies within the electrical insulation, which must be able to withstand extreme temperatures, large shear and compressive stresses, high operating voltages, and high levels of incident radiation. To address the need for better performing insulation systems, Composite Technology Development (CTD), Inc. has developed CTD-403, a cyanate ester resin with increased radiation resistance, ease of processing and fabrication, low moisture absorption characteristics, and high mechanical and electrical strength at cryogenic and elevated temperatures. The moisture absorption trends of CTD-403/S2 glass composite insulation were studied. The effects of humidity exposure on interlaminar shear strength (ILSS), compressive strength, dielectric strength, and glass transition temperature were also studied. The saturation level of the insulation was seen to increase with the relative humidity of the aging environment. Fickian behavior was seen at room temperatures below 97% relative humidity exposure. Non-Fickian behavior was seen at elevated temperatures. Saturation levels after 6 months exposure were seen to be below typical epoxy-based insulation systems, averaging 0.5% weight gain. Degradation of mechanical and electrical properties was seen with increased humidity exposure and moisture absorption. ILSS showed an average retention rate of 75% after 6 months exposure. The compressive strength showed no decrease after 6 months exposure at room temperature, and show retention rates greater than 90% at 75°C/79% RH. An average dielectric strength of 98.6 kV/mm was seen for all specimens at room temperature (above 90% retention) after 6 months exposure.

Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress

To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G 2–M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.

Acquisition of Stable Inducible Up-Regulation of Nuclear Factor-κB by Tumor Necrosis Factor Exposure Confers Increased Radiation Resistance without Increased Transformation in Breast Cancer Cells

High-grade breast cancers are better adapted to hypoxia and more resistant to chemotherapy and radiotherapy. Constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB) increases in breast tumors and in breast cancer cell lines, where it promotes chemoradiation resistance, in part by activation of antiapoptotic genes. The role for up-regulation of NF-kappaB in breast cancer progression is less clear. Here, we first show that whereas the constitutive activity of NF-kappaB is incrementally elevated from immortalized breast epithelial to frank transformed invasive ductal breast cancer cell lines (~3-fold, +/-0.1-fold, P < 0.05), inflammatory cytokine-inducible activity is further increased (up to 9-fold, +/-0.9-fold, P < 0.05). We then show that inhibition of NF-kappaB activity selectively sensitizes transformed but not immortalized cells to killing by ionizing radiation or low levels of tumor necrosis factor (TNF) by up to 10-fold (+/-1-fold, P < 0.05) but has little effect on hypoxia-mediated cell death. Prolonged cultivation of immortalized and partially transformed cells in TNF selected for cells displaying stable constitutive and strongly inducible overexpression of NF-kappaB even in the absence of TNF. Stable acquisition of increased NF-kappaB activity conferred resistance to ionizing radiation or inflammatory cytokines, which was dependent on elevated NF-kappaB activity, but had no effect on transformation potential measured by in vitro and in vivo parameters. Thus, TNF and possibly other inflammatory cytokines in the tumor-stroma matrix likely select for breast cancer cells that stably overexpress NF-kappaB, leading to greater cancer cell survival. Greater cell survival despite increased genomic injury may permit increased acquisition of malignant genetic alterations as well as resistance to chemoradiation therapy.

The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor κB and Akt Activation

Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

Intensity Modulating and Other Radiation Therapy Devices for Dose Painting

The introduction of intensity-modulated radiation therapy (IMRT) in the early 1990s created the possibility of generating dramatically improved dose distributions that could be tailored to fit a complex geometric arrangement of targets that push against or even surround healthy critical structures. IMRT is a new treatment paradigm that goes beyond the capabilities of the earlier technology called three-dimensional radiation therapy (3DCRT). IMRT took the older approach of using fields that conformed to the silhouette of the target to deliver a relatively homogeneous intensity of radiation and separated the conformal fields into many subfields so that intensity could be varied to better control the final dose distribution. This technique makes it possible to generate radiation dose clouds that have indentations in their surface. Initially, this technology was mainly used to avoid and thus control the dose delivered to critical structures so that they are not seriously damaged in the process of irradiating nearby targets to an appropriately high dose. Avoidance of critical structures allowed homogeneous dose escalation that led to improved local control for small tumors. However, the normal tissue component of large tumors often prohibits homogeneous dose escalation. A newer concept of dose-painting IMRT is aimed at exploiting inhomogeneous dose distributions adapted to tumor heterogeneity. Tumor regions of increased radiation resistance receive escalated dose levels, whereas radiation-sensitive regions receive conventional or even de-escalated dose levels. Dose painting relies on biologic imaging such as positron emission tomography, functional magnetic resonance imaging, and magnetic resonance spectroscopy. This review will describe the competing techologies for dose painting with an emphasis on their commonalities.

Prognostic Associations of Activated Mitogen-Activated Protein Kinase and Akt Pathways in Glioblastoma

Activation of mitogen-activated protein kinase (MAPK) and members of the Akt pathway have been shown to promote cell proliferation, survival, and resistance to radiation. This study was conducted to determine whether any of these markers are associated with survival time and response to radiation in glioblastoma. The expression of phosphorylated (p-)Akt, mammalian target of rapamycin (p-mTOR), p-p70S6K, and p-MAPK were assessed by immunohistochemical staining in 268 cases of newly diagnosed glioblastoma. YKL-40, a prognostic marker previously examined in these tumors, was also included in the analysis. Expression data were tested for correlations with response to radiation therapy in 131 subtotally resected cases and overall survival (in all cases). Results were validated in an analysis of 60 patients enrolled in clinical trials at a second institution. Elevated p-MAPK expression was most strongly associated with poor response to radiotherapy, a finding corroborated in the validation cohort. For survival, higher expressions of p-mTOR, p-p70S6K, and p-MAPK were associated with worse outcome (all P < 0.03). YKL-40 expression was associated with the expressions of p-MAPK, p-mTOR, and p-p70S6K (all P < 0.02), with a trend toward association with p-Akt expression (P = 0.095). When known clinical variables were added to a multivariate analysis, only age, Karnofsky performance score, and p-MAPK expression emerged as independent prognostic factors. p-MAPK and activated members of the Akt pathway are markers of outcome in glioblastoma. Elevated expression of p-MAPK is associated with increased radiation resistance and represents an independent prognostic factor in these tumors.

Thalidomide Sensitizes 32D cl 3 Hematopoietic Progenitor Cells to Ionizing Irradiation.

Thalidomide (TL) has been used in the treatment of multiple myeloma and due to its anti-angiogenic effects has been postulated to be a potential radiosensitizer of tumors in vivo. We first investigated the effect of TL on growth of IL-3 dependent murine hematopoietic 32D cl 3 cells in semisolid methylcellulose containing medium also supplemented with TL ranging from 0 to 200 μM. The cells were incubated at 37°C for 7 days at which time colonies of greater than 50 cells were counted. TL increased cell colony formation 2-fold over the dose range of 10 to 100 μM. In serially decreasing concentrations of IL-3, TL (50 μM) was growth stimulatory of 32D cl 3 colony formation in 15% IL-3 and 10% IL-3 conditioned medium (146.2 ± 7.2 and 89.3 ± 2.4 colonies/500 cells plated in TL vs. 110.8 ± 11.4 and 58.0 ± 2.1 without TL, p = 0.0304 or 0.0006). To determine whether thalidomide was a radiosensitizer, cells were placed in: 0, 50, or 150 μM TL in three protocols:1 hour before irradiation;1 hour before irradiation plus also in methylcellulose following irradiation; orwere placed in TL containing methylcellulose only following irradiation.Cells were irradiated to doses ranging from 0 to 800 cGy and were plated in methylcellulose containing medium. Seven days later, colonies of greater than 50 cells were counted and analyzed by single hit, multi-target or linear quadratic models. Cells grown in 50 μM TL which stimulated cell growth resulted in an increased radiation resistance compared to control irradiated cells (D0 = 1.87 ± 0.02 vs. 1.30 ± 0.09 Gy, respectively, p = 0.0366). However, 150 μM TL (which did not stimulate cell growth) increased radiation sensitivity compared to control irradiated cells (D0 = 1.22 ± 0.5 vs. 1.64 ± 0.11 Gy, respectively, p = 0.0245). To determine whether thalidomide radiosensitized tumor cells in the therapeutic 150 μM range, SSC-VII murine squamous cell carcinoma cells were grown in TL at concentrations ranging from 0 to 200 μM and were assayed for colony formation. In contrast, SSC-VII cells showed no detectable stimulation or inhibition of cell growth for unirradiated cells in any concentration of TL. However, at 150 μM TL, SSC-VII cells were radiosensitive when incubated in TL before irradiation compared to control irradiated cells (D0 = 2.02 vs. 2.55 Gy, respectively). These results demonstrate that thalidomide may be a clinically valuable radiosensitizer.