RESUMEN
Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.
Asunto(s)
Supervivencia Celular/genética , Neoplasias de la Próstata/radioterapia , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Supervivencia Celular/efectos de la radiación , Cromatina/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HeLa , Humanos , Masculino , Ratones , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de la radiaciónRESUMEN
Residual tumor cells remain beyond the margins of every glioblastoma (GBM) resection. Their resistance to postsurgical therapy is considered a major driving force of mortality, but their biology remains largely uncharacterized. In this study, residual tumor cells were derived via experimental biopsy of the resection margin after standard neurosurgery for direct comparison with samples from the routinely resected tumor tissue. In vitro analysis of proliferation, invasion, stem cell qualities, GBM-typical antigens, genotypes, and in vitro drug and irradiation challenge studies revealed these cells as unique entities. Our findings suggest a need for characterization of residual tumor cells to optimize diagnosis and treatment of GBM.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/metabolismo , Adulto , Anciano , Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Técnicas de Cultivo de Célula , Proliferación Celular , Separación Celular , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Tumorales CultivadasRESUMEN
PURPOSE: The various microenvironments that exist within the brain combined with the invasive nature of glioblastoma (GBM) creates the potential for a topographic influence on tumor cell radiosensitivity. The aim of this study was to determine whether specific brain microenvironments differentially influence tumor cell radioresponse. METHODS AND MATERIALS: GBM stem-like cells were implanted into the right striatum of nude mice. To measure radiosensitivity, proliferation status of individual tumor cells was determined according to the incorporation of 5-chloro-2'-deoxyuridine delivered at 4, 12, and 20 days after brain irradiation. As an additional measure of radiosensitivity, the percentage of human cells in the right hemisphere and the olfactory bulb were defined using digital droplet polymerase chain reaction. Targeted gene expression profiling was accomplished using NanoString analysis. RESULTS: Tumor cells were detected throughout the striatum, corpus callosum, and olfactory bulb. After an initial loss of proliferating tumor cells in the corpus callosum and striatum after irradiation, there was only a minor recovery by 20 days. In contrast, the proliferation of tumor cells located in the olfactory bulb began to recover at 4 days and returned to unirradiated levels by day 12 postirradiation. The percentage of human cells in the right hemisphere and the olfactory bulb after irradiation also suggested that the tumor cells in the olfactory bulb were relatively radioresistant. Gene expression profiling identified consistent differences between tumor cells residing in the olfactory bulb and those in the right hemisphere. CONCLUSIONS: These results suggest that the olfactory bulb provides a radioresistant niche for GBM cells.
Asunto(s)
Glioblastoma/patología , Bulbo Olfatorio/patología , Bulbo Olfatorio/efectos de la radiación , Tolerancia a Radiación , Nicho de Células Madre/efectos de la radiación , Animales , Ratones , Microambiente Tumoral/efectos de la radiaciónRESUMEN
A consequence of the intratumor heterogeneity (ITH) of glioblastoma (GBM) is the susceptibility to treatment-driven evolution. To determine the potential of radiotherapy to influence GBM evolution, we used orthotopic xenografts initiated from CD133+ GBM stem-like cells (GSC). Toward this end, orthotopic xenografts grown in nude mice were exposed to a fractionated radiation protocol, which resulted in a significant increase in animal survival. Brain tumors from control and irradiated mice were then collected at morbidity and compared in terms of growth pattern, clonal diversity, and genomic architecture. In mice that received fractionated radiation, tumors were less invasive, with more clearly demarcated borders and tumor core hypercellularity as compared with controls, suggesting a fundamental change in tumor biology. Viral integration site analysis indicated a reduction in clonal diversity in the irradiated tumors, implying a decrease in ITH. Changes in clonal diversity were not detected after irradiation of GSCs in vitro, suggesting that the radiation-induced reduction in ITH was dependent on the brain microenvironment. Whole-exome sequencing revealed differences in mutation patterns between control and irradiated tumors, which included modifications in the presence and clonality of driver mutations associated with GBM. Moreover, changes in the distribution of mutations as a function of subpopulation size between control and irradiated tumors were consistent with subclone expansion and contraction, that is, subpopulation evolution. Taken together, these results indicate that radiation drives the evolution of the GSC-initiated orthotopic xenografts and suggest that radiation-driven evolution may have therapeutic implications for recurrent GBM. SIGNIFICANCE: Radiation drives the evolution of glioblastoma orthotopic xenografts; when translated to the clinic, this may have therapeutic implications for recurrent tumors.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Evolución Molecular , Heterogeneidad Genética/efectos de la radiación , Glioblastoma/radioterapia , Células Madre Neoplásicas/efectos de la radiación , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Análisis Mutacional de ADN , Femenino , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Mutación/efectos de la radiación , Células Madre Neoplásicas/patología , Tolerancia a Radiación/genética , Microambiente Tumoral/genética , Microambiente Tumoral/efectos de la radiación , Secuenciación del Exoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The processes mediating the repair of DNA double-strand breaks (DSB) are critical determinants of radiosensitivity and provide a source of potential targets for tumor radiosensitization. Among the events required for efficient DSB repair are a variety of post-translational histone modifications, including methylation. Because trimethylation of histone H3 on lysine 27 (H3K27me3) has been associated with chromatin condensation, which can influence DSB repair, we determined the effects of radiation on H3K27me3 levels in tumor and normal cell lines. Irradiation of tumor cells resulted in a rapid loss of H3K27me3, which was prevented by the siRNA-mediated knockdown of the H3K27 demethylase UTX. Knockdown of UTX also enhanced the radiosensitivity of each tumor cell line. Treatment of tumor cells with the H3K27 demethylase inhibitor GSKJ4 immediately before irradiation prevented the radiation-induced decrease in H3K27me3 and enhanced radiosensitivity. As determined by neutral comet analysis and γH2AX expression, this GSKJ4 treatment protocol inhibited the repair of radiation-induced DSBs. Consistent with in vitro results, treatment of mice bearing leg tumor xenografts with GSKJ4 significantly enhance radiation-induce tumor growth delay. In contrast with results generated from tumor cell lines, radiation had no effect on H3K27me3 levels in normal fibroblast cell lines and GSKJ4 did not enhance their radiosensitivity. These data suggest that H3K27me3 demethylation contributes to DSB repair in tumor cells and that UTX, the demethylase responsible, provides a target for selective tumor cell radiosensitization. Mol Cancer Ther; 17(5); 1070-8. ©2018 AACR.
Asunto(s)
Histona Demetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Tolerancia a Radiación/efectos de la radiación , Células A549 , Benzazepinas/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , Humanos , Metilación/efectos de los fármacos , Metilación/efectos de la radiación , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Pirimidinas/farmacología , Interferencia de ARN , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genéticaRESUMEN
Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSC) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of clinically relevant XPO1 inhibitor selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. On the basis of γH2AX foci and neutral comet analyses, selinexor inhibited the repair of radiation-induced DNA double-strand breaks in GSCs, suggesting that the selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to selinexor and radiation. Treatment of mice bearing orthotopic xenografts with selinexor decreased tumor translational efficiency as determined from polysome profiles. Although selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves the global inhibition of gene translation. Mol Cancer Ther; 17(8); 1717-26. ©2018 AACR.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Hidrazinas/uso terapéutico , Tolerancia a Radiación/efectos de los fármacos , Triazoles/uso terapéutico , Animales , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/patología , Humanos , Hidrazinas/farmacología , Ratones , Ratones Desnudos , Triazoles/farmacologíaRESUMEN
Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood-brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM. Mol Cancer Ther; 17(6); 1207-16. ©2018 AACR.
Asunto(s)
Antineoplásicos/farmacología , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Glioblastoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Línea Celular Tumoral , Proteína Quinasa Activada por ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Glioblastoma/patología , Histonas/metabolismo , Humanos , Ratones , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."
Asunto(s)
Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/radioterapia , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Benzoxazoles/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Masculino , Ratones , Ratones Desnudos , Piperazinas/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Distribución Aleatoria , Transducción de Señal/efectos de la radiación , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078-87. ©2016 AACR.
Asunto(s)
Glioblastoma/patología , Aparato de Golgi/metabolismo , Mitocondrias/metabolismo , Células Madre Neoplásicas/patología , Polirribosomas/metabolismo , Biosíntesis de Proteínas/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Glioblastoma/genética , Glioblastoma/radioterapia , Aparato de Golgi/genética , Aparato de Golgi/efectos de la radiación , Humanos , Mitocondrias/genética , Mitocondrias/efectos de la radiación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Polirribosomas/genética , Polirribosomas/efectos de la radiación , ARN Mensajero/genética , Radiación Ionizante , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/efectos de la radiación , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Tolerancia a Radiación , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Análisis por Conglomerados , Técnicas de Cocultivo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Femenino , Perfilación de la Expresión Génica , Glioblastoma/genética , Histonas/metabolismo , Humanos , Ratones , Tolerancia a Radiación/genética , Factor de Transcripción STAT3/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs). METHODS: mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation. RESULTS: Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments. CONCLUSIONS: These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Morfolinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Células Madre Neoplásicas/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzamidas , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Femenino , Técnica del Anticuerpo Fluorescente , Glioblastoma/patología , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Células Tumorales Cultivadas , Terapia por Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines. EXPERIMENTAL DESIGN: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay. RESULTS: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation. CONCLUSION: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair.
Asunto(s)
Benzoxazoles/farmacología , Neoplasias Pancreáticas/radioterapia , Pirimidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Femenino , Humanos , Ratones , Ratones Desnudos , Biosíntesis de Proteínas/efectos de la radiación , Tolerancia a Radiación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133- counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133- cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133- cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Invasividad Neoplásica/patología , Células Madre Neoplásicas/patología , Antígeno AC133 , Antígenos CD/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Glioblastoma/metabolismo , Glicoproteínas/metabolismo , Humanos , Microscopía Electrónica de Transmisión de Rastreo , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Transcriptoma , Microambiente TumoralRESUMEN
Brain tumor xenografts initiated from glioblastoma (GBM) CD133(+) tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133(+) cells and their differentiated (CD133(-)) progeny. Under in vitro conditions, no difference was detected between CD133(+) and CD133(-) cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133(+) cells compared to their CD133(-) counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133(+) cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133(+) cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.
Asunto(s)
Antígenos CD/metabolismo , Neoplasias Encefálicas/patología , Encéfalo/patología , Glioblastoma/patología , Glicoproteínas/metabolismo , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Tolerancia a Radiación , Microambiente Tumoral , Antígeno AC133 , Animales , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Genes Reporteros , Glioblastoma/metabolismo , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de la radiación , Células Madre Neoplásicas/trasplante , Carga Tumoral/efectos de la radiación , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
PURPOSE: Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (TSC) simulate the biological characteristics of GBMs in situ. Therefore, to determine whether the brain microenvironment affects the intrinsic radiosensitivity of GBM cells, we compared the radioresponse of GBM TSCs grown in vitro and as brain tumor xenografts. EXPERIMENTAL DESIGN: As indicators of DNA double-strand breaks (DSB), γH2AX, and 53BP1 foci were defined after irradiation of 2 GBM TSC lines grown in vitro and as orthotopic xenografts in nude mice. Microarray analysis was conducted to compare gene expression patterns under each growth condition. RESULTS: Dispersal of radiation-induced γH2AX and 53BP1 foci was faster in the tumor cells grown as orthotopic xenografts compared with cells irradiated in vitro. In addition, cells irradiated in vivo were approximately 3-fold less susceptible to foci induction as compared with cells grown in vitro. Microarray analysis revealed a significant number of genes whose expression was commonly affected in the 2 GBM models by orthotopic growth conditions. Consistent with the decrease in sensitivity to foci induction, genes related to reactive oxygen species (ROS) metabolism were expressed at higher levels in the brain tumor xenografts. CONCLUSION: γH2AX and 53BP1 foci analyses indicate that GBM cells irradiated within orthotopic xenografts have a greater capacity to repair DSBs and are less susceptible to their induction than tumor cells irradiated under in vitro growth conditions. Because DSB induction and repair are critical determinants of radiosensitivity, these results imply that the brain microenvironment contributes to GBM radioresistance.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Tolerancia a Radiación , Microambiente Tumoral/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/genética , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Análisis por Micromatrices , Tolerancia a Radiación/genética , Tolerancia a Radiación/fisiología , Células Tumorales Cultivadas , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 +/- 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.
Asunto(s)
Rayos Láser , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Inmunohistoquímica , Teratoma/patologíaRESUMEN
The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.