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1.
Cell Rep ; 33(13): 108559, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33378670

RESUMO

The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50E1035Δ) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain. This mutation represents a human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins indicate that RAD50E1035Δ impairs MRE11 nuclease activity. The corresponding mutation in Saccharomyces cerevisiae causes severe thermosensitive defects in both DNA repair and Tel1ATM-dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled coil suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize the importance of the RAD50 coiled coil to regulate MRE11-dependent DNA end resection in humans.


Assuntos
Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transtornos da Insuficiência da Medula Óssea/genética , Criança , Pré-Escolar , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Deficiências do Desenvolvimento/genética , Humanos , Ligação Proteica , Domínios Proteicos , Análise de Sequência de Proteína , Deleção de Sequência , Transdução de Sinais
2.
Future Oncol ; 8(9): 1167-81, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23030491

RESUMO

AIM: There is considerable interest in approaches that could improve the therapeutic window of radiotherapy. In this study, hafnium oxide nanoparticles were designed that concentrate in tumor cells to achieve intracellular high-energy dose deposit. MATERIALS & METHODS: Conventional methods were used, implemented in different ways, to explore interactions of these high-atomic-number nanoparticles and ionizing radiation with biological systems. RESULTS: Using the Monte Carlo simulation, these nanoparticles, when exposed to high-energy photons, were shown to demonstrate an approximately ninefold radiation dose enhancement compared with water. Importantly, the nanoparticles show satisfactory dispersion and persistence within the tumor and they form clusters in the cytoplasm of cancer cells. Marked antitumor activity is demonstrated in human cancer models. Safety is similar in treated and control animals as demonstrated by a broad program of toxicology evaluation. CONCLUSION: These findings, supported by good tolerance, provide the basis for developing this new type of nanoparticle as a promising anticancer approach in human patients.


Assuntos
Háfnio/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/radioterapia , Óxidos/administração & dosagem , Radiossensibilizantes/administração & dosagem , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Simulação por Computador , Endossomos/metabolismo , Endossomos/patologia , Feminino , Háfnio/efeitos adversos , Háfnio/farmacocinética , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Método de Monte Carlo , Nanopartículas/efeitos adversos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Óxidos/efeitos adversos , Óxidos/farmacocinética , Radiossensibilizantes/efeitos adversos , Radiossensibilizantes/farmacocinética , Radiografia , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Neoplasia ; 9(4): 341-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17460778

RESUMO

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.


Assuntos
Neoplasias Colorretais/patologia , Receptores ErbB/fisiologia , Receptores ErbB/efeitos da radiação , Genes ras/fisiologia , Genes ras/efeitos da radiação , Neoplasias Pancreáticas/patologia , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Neoplasias Colorretais/genética , Neoplasias Colorretais/radioterapia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia
4.
Anticancer Res ; 25(6B): 4357-62, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16309240

RESUMO

Ritonavir, a protease inhibitor, has been successfully applied in the treatment of HIV infection. Reports of dramatic improvement of AIDS-related cancers, such as primary central system lymphoma after radiation therapy as well as Kaposi's sarcoma, led to the recent discovery of the "non viral" antitumor activity of HIV protease inhibitors. This study was designed to detect the antitumor effect of Ritonavir when combined with ionizing radiation both in vitro and in vivo in the HEP-2 head and neck carcinoma model. Inhibition of tumor growth was observed when mice were treated with Ritonavir alone and this effect was enhanced when combined with ionizing radiation. No adverse effect or significant toxicity in the hosts' body weights was seen between the different treatment and control groups throughout the experiments. A marked antiproliferation effect of the combination was observed in vitro. A marked reduction of angiogenesis was detected within the tumor sections from the Ritonavir combined with irradiation group as compared with the Ritonavir or irradiation alone groups. Western blot analysis showed that apoptosis was induced by an increased expression of Bax and decreased expression of Bcl-2 after treatment with Ritonavir and ionizing radiation. Thus, the antitumor effect of the latter combination is associated with the enhancement of radiation-induced apoptosis and inhibition of angiogenesis. These data suggested that Ritonavir could clinically improve the tumor response to radiation therapy, especially in head and neck carcinoma.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Ritonavir/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/irrigação sanguínea , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Neovascularização Patológica/radioterapia , Radiossensibilizantes/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cancer Ther ; 4(11): 1717-28, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16275993

RESUMO

In this study, we have evaluated the interactions between ionizing radiation and a matrix metalloproteinase (MMP) inhibitor. Using Matrigel invasion assays, we show that ionizing radiation induced a dose-dependent increase in the invasive phenotype of cultured B16 melanoma cells and that conditioned medium from these irradiated B16 cells promoted endothelial cell [human microvascular endothelial cells (HMEC)] invasiveness. To determine whether the radiation-induced changes in invasive phenotype could be due to changes in MMP activation, we have tested the ability of the MMP inhibitor Metastat to modulate the ionizing radiation-induced invasive phenotype using both an in vitro melanoma model and a mouse s.c. tumor model. In these studies, Metastat inhibited the ionizing radiation-induced invasive phenotype in cultured B16 cells and similarly inhibited the increase in HMEC invasion induced by conditioned medium from irradiated B16 cells. Conversely, ionizing radiation increased B16 MMP-2 activity and the conditioned medium from irradiated B16 induced HMEC MMP-2 activity. To further investigate the interaction between ionizing radiation and MMP activation, we then studied the effects of ionizing radiation on downstream effectors of the MMP system. We found that ionizing radiation induced vascular endothelial growth factor (VEGF) secretion by B16 melanoma cells and that this secretion was inhibited by Metastat. Similarly, conditioned medium from irradiated B16 was also able to increase VEGF secretion in HMECs. Moreover, ionizing radiation-induced melanoma cell invasiveness was partially inhibited by an anti-VEGF monoclonal antibody. In vivo, ionizing radiation plus concomitant Metastat yielded the greatest growth inhibition of melanoma s.c. tumors and this effect correlated with inhibition of angiogenesis as measured by both Doppler ultrasonography and platelet/endothelial cell adhesion molecule-1 staining. Finally, ionizing radiation modulated MMP-2, VEGF, and VEGF receptor expression in these tumor samples using immunohistochemistry. Taken together, these results suggest that there is an ionizing radiation-induced tumor survival pathway and a possible paracrine ionizing radiation-induced stimulatory pathway emanating from tumor cells toward the endothelial bed that is impeded when Metastat is given simultaneously. This model could provide in vivo evidence of the antitumor efficacy of combining a MMP inhibitor with ionizing radiation to target radiation-induced invasion and angiogenesis.


Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Metaloproteinases de Matriz , Neoplasias Induzidas por Radiação/patologia , Neovascularização Patológica , Animais , Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Colágeno/química , Colágeno/farmacologia , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta à Radiação , Combinação de Medicamentos , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Laminina/química , Laminina/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Melanoma Experimental/tratamento farmacológico , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Proteoglicanas/química , Proteoglicanas/farmacologia , Radiação Ionizante , Fatores de Tempo , Ultrassonografia , Ultrassonografia Doppler , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Lancet Oncol ; 5(5): 303-13, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15120667

RESUMO

Prostate cancer is the sixth most common cancer in the world and the third leading cause of cancer in men. The increase in the understanding of prostate carcinogenesis over the past 15 years has helped to define crucial steps in the natural history of the disease, namely initiation and progression to androgen independence. This heterogeneous disease encompasses a range of environmental and familial factors, which provides strong support for the use of chemopreventive strategies. Most patients with advanced prostate cancer are treated with androgen-deprivation therapy, which leads to a striking regression of androgen-responsive cancer cells. A transition from an androgen-responsive to an androgen-unresponsive stage is seen during the clinical course in almost all patients with prostate cancer. This transition also signals a substantial worsening of prognosis. Here, we review the most important findings in prostate carcinogenesis and the molecular anomalies associated with the androgen-refractory stage.


Assuntos
Neoplasias da Próstata/fisiopatologia , Neoplasias da Próstata/terapia , Exposição Ambiental , Predisposição Genética para Doença , Humanos , Masculino , Neoplasias Hormônio-Dependentes , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
7.
Cancer Res ; 63(10): 2513-7, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12750274

RESUMO

Frequent deregulation of cyclin-dependent kinase (CDK) activation associated with loss of cell cycle control was found in most of human cancers. A recent development of a new class of antineoplasic agents targeting the cell cycle emerged as a small molecule CDK inhibitor, roscovitine, which presents potential antiproliferative and antitumoral effects in human tumors. Additional studies reported that roscovitine combined with cytotoxic agents can cooperate with DNA damage to activate p53 protein. However, little is known about the biological effect of roscovitine combined with ionizing radiation (IR) in human carcinoma, and no studies were reported thus far in p53 mutated carcinoma. In the breast cancer cell line MDA-MB 231, which lacks a functional p53 protein, we found a strong radiosensitization effect of roscovitine in vitro by clonogenic survival assay and in vivo in MDA-MB 231 xenograft model. Using Pulse Field Gel Electrophoresis, a strong impairment in DNA-double-strand break rejoining was observed after roscovitine and IR treatment as compared with IR alone. Cell cycle analysis showed a G(2) delay and no increase in radiation-induced apoptosis in the cells treated with IR or roscovitine and IR. On the other hand, we found a significant induction in micronuclei frequency after roscovitine and IR treatment as compared with IR alone. This effect was also observed in BALB murine cells in contrast to SCID murine cells, which are deficient in DNA-PKcs, suggesting a possible DNA-double-strand break repair defect in the nonhomologous end joining pathways. In MDA-MB 231 cells, the radiosensitization effect of roscovitine was associated with an inhibition of the DNA-dependent protein kinase activity caused by a marked decrease in Ku-DNA binding by using the electrophoretic mobility shift assay. In conclusion, we found a novel effect on DNA repair of the CDK inhibitor roscovitine, which acts as a radiosensitizer in vitro and in vivo in breast cancer cells lacking a functional p53.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , DNA Helicases , Purinas/farmacologia , Radiossensibilizantes/farmacologia , Animais , Antígenos Nucleares/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Terapia Combinada , Reparo do DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Autoantígeno Ku , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Roscovitina , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncogene ; 21(15): 2334-46, 2002 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-11948417

RESUMO

High-risk human papillomaviruses (HPVs) have been associated to the development of cervical and some other human cancers. Most of them express E6 and E7 oncoproteins, able to bind to p53 and retinoblastoma (pRb) tumor suppressor proteins respectively and neutralize their function. Restoration of these pathways by blocking E6 and E7 expression would provide a selective therapeutic effect. Here, we show that a clinically approved antiviral agent Cidofovir reduced E6 and E7 expression in cervical carcinoma Me180 and head and neck squamous cell carcinoma HEP2 cells at the transcriptional level. Cidofovir induced the accumulation of active p53 and pRb associated to induction of cyclin dependent kinase inhibitor p21(WAF1/CIP1) in Me180 and HEP2 cells. p53 induction was also shown in Hela HPV-positive cervical carcinoma cell line. In addition, S phase cell cycle accumulation with concomitant decrease of cyclin A expression were associated to the antiproliferative activity of Cidofovir in HPV-treated cells. Combining Cidofovir to irradiation both in vivo and in nude mice xenografts resulted in a marked radiosensitization in HPV-positive cells, which was not observed in virus negative cells. This study provides the basis for a new anticancer strategy to enhance the antitumor effect of ionizing radiation in HPV-related cancers, without increase deleterious effects.


Assuntos
Antivirais/farmacologia , Carcinoma/terapia , Citosina/análogos & derivados , Citosina/farmacologia , Organofosfonatos , Compostos Organofosforados/farmacologia , Papillomaviridae , Infecções por Papillomavirus/terapia , Radiossensibilizantes/farmacologia , Infecções Tumorais por Vírus/terapia , Animais , Carcinoma/metabolismo , Carcinoma/virologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Cidofovir , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Camundongos , Camundongos Nus , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Radiação Ionizante , Proteína do Retinoblastoma/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de Xenoenxerto
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