RESUMEN

Glial cells are crucial for the normal function of neurons and are intricately involved in the pathogenesis of neurodegenerative diseases as well as neurologic malignancies. A deeper understanding of the mechanisms by which glial cells influence the development of such pathologies will undoubtedly lead to new and improved therapeutic approaches. Commercially available human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), both of which can be differentiated into neural progenitors (NPs) and various neural cell lineages, have become widely used as sources for producing normal human central nervous system (CNS) cells. A better understanding of the DNA damage response (DDR) that occurs in these cells after therapeutic ionizing radiation (IR) and chemotherapy is essential for assessing the effects on healthy human brain.Neurodegenerative features associated with conditions such as ataxia telangiectasia and Nijmegen breakage syndrome highlight the importance of DNA double strand break (DSB) repair pathways in maintaining genomic integrity in cells of the CNS. Similarly, the development of brain tumors is also intricately linked to DNA repair. The importance of ATM and the other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, ATR and DNA-PKcs, is not fully defined in either CNS developmental or pathological states. While their roles are relatively well established in the DDR of proliferating cells, our recent work has demonstrated that these processes exhibit spatiotemporal evolution during cell differentiation. This chapter discusses and explores various laboratory techniques for investigating the role of ATM in hESCs and differentiated neural cells.

Asunto(s)

Daño del ADN/fisiología , Reparación del ADN/fisiología , Células Madre Embrionarias/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN/genética , Células Madre Embrionarias/citología , Humanos , Células Madre/citología , Células Madre/metabolismo

RESUMEN

PURPOSE: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12 to 15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of patients with GBM is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. Ataxia-telangiectasia mutated (ATM) is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro. EXPERIMENTAL DESIGN: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intracranially, and KU-60019 was administered intratumorally by convection-enhanced delivery or osmotic pump. RESULTS: Our results show that the combined effect of KU-60019 and radiation significantly increased survival of mice 2- to 3-fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. CONCLUSIONS: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada/genética , Neoplasias Encefálicas/terapia , Glioma/terapia , Morfolinas/administración & dosificación , Tioxantenos/administración & dosificación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/patología , Humanos , Ratones , Mutación , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Proteína p53 Supresora de Tumor/genética

RESUMEN

Essential oils from mint plants, including peppermint and pennyroyal oils, are used at low levels as flavoring agents in various foods and beverages. Pulegone is a component of these oils. In a 2-year bioassay, oral administration of pulegone slightly increased the urothelial tumor incidence in female rats. We hypothesized that its mode of action (MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately leading to tumors. Pulegone was administered by gavage at 0, 75, or 150 mg/kg body weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy and scanning electron microscopy (SEM) and bromodeoxyuridine (BrdU) labeling index. Pulegone and its metabolites, piperitenone, piperitone, menthofuran, and menthone, were tested for cytotoxicity in rat (MYP3) and human (1T1) urothelial cells by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. No abnormal urinary crystals were observed by light microscopy. Urine samples (18-h) showed the presence of pulegone, piperitone, piperitenone, and menthofuran in both treated groups. By SEM, bladders from treated rats showed superficial necrosis and exfoliation. There was a significant increase in the BrdU labeling index in the high-dose group. In vitro studies indicated that pulegone and its metabolites, especially piperitenone, are excreted and concentrated in the urine at cytotoxic levels when pulegone is administered at high doses to female rats. The present study supports the hypothesis that cytotoxicity followed by regenerative cell proliferation is the MOA for pulegone-induced urothelial tumors in female rats.

Asunto(s)

Monoterpenos/farmacología , Vejiga Urinaria/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica , Células Cultivadas , Monoterpenos Ciclohexánicos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Ratas , Ratas Endogámicas F344 , Vejiga Urinaria/ultraestructura

RESUMEN

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Glioblastoma/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tolerancia a Radiación , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular , Técnicas de Cocultivo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Activación Enzimática , Glioblastoma/enzimología , Glioblastoma/radioterapia , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Morfolinas/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Temozolomida , Tioxantenos/farmacología , Proteína 28 que Contiene Motivos Tripartito , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer.

Asunto(s)

Proteína BRCA1/genética , Mutación , Recombinación Genética , Secuencias de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Sitios de Unión , Neoplasias de la Mama/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , ADN/genética , ADN/metabolismo , Reparación del ADN , Endodesoxirribonucleasas , Femenino , Vectores Genéticos , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Unión Proteica , Estructura Terciaria de Proteína , Recombinasa Rad51/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación

RESUMEN

Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤ 2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (> 2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Bromodesoxiuridina/química , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Humanos , Fosforilación , Fotólisis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Rayos Ultravioleta

RESUMEN

We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Enzimas Reparadoras del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , ADN , Células Madre Embrionarias/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Astrocitos/citología , Astrocitos/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/citología , Humanos , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteína Quinasa C/fisiología

RESUMEN

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Astrocitos/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Linaje de la Célula , Células Cultivadas , Roturas del ADN de Doble Cadena/efectos de la radiación , Humanos , Recombinasa Rad51 , Radiación Ionizante

RESUMEN

The epidermal growth factor receptor (EGFR) is frequently dysregulated in malignant glioma that leads to increased resistance to cancer therapy. Upregulation of wild type or expression of mutant EGFR is associated with tumor radioresistance and poor clinical outcome. EGFR variant III (EGFRvIII) is the most common EGFR mutation in malignant glioma. Radioresistance is thought to be, at least in part, the result of a strong cytoprotective response fueled by signaling via AKT and ERK that is heightened by radiation in the clinical dose range. Several groups including ours have shown that this response may modulate DNA repair. Herein, we show that expression of EGFRvIII promoted gamma-H2AX foci resolution, a surrogate for double-strand break (DSB) repair, and thus enhanced DNA repair. Conversely, small molecule inhibitors targeting EGFR, MEK, and the expression of dominant-negative EGFR (EGFR-CD533) significantly reduced the resolution of gamma-H2AX foci. When homologous recombination repair (HRR) and non-homologous end joining (NHEJ) were specifically examined, we found that EGFRvIII stimulated and CD533 compromised HRR and NHEJ, respectively. Furthermore, NHEJ was blocked by inhibitors of AKT and ERK signaling pathways. Moreover, expression of EGFRvIII and CD533 increased and reduced, respectively, the formation of phospho-DNA-PKcs and -ATM repair foci, and RAD51 foci and expression levels, indicating that DSB repair is regulated at multiple levels. Altogether, signaling from EGFR and EGFRvIII promotes both HRR and NHEJ that is likely a contributing factor towards the radioresistance of malignant gliomas.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Receptores ErbB/metabolismo , Glioma/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Receptores ErbB/genética , Glioma/genética , Glioma/patología , Histonas/metabolismo , Humanos , Mutación/genética , Transducción de Señal , Células Tumorales Cultivadas