RESUMO
Synovial sarcoma is a soft-tissue sarcoma and a rare type of cancer. Unfortunately, effective chemotherapies for synovial sarcomas have not been established. In this report, we show that synovial sarcoma cell lines have reduced repair activity for DNA damage induced by ionizing radiation (IR) and a topoisomerase II inhibitor (etoposide). We also observed reduced recruitment of RAD51 homologue (S. cerevisiae; RAD51) at sites of double-strand breaks (DSBs) in synovial sarcoma cell lines that had been exposed to IR. These findings showed that synovial sarcoma cell lines are defective in homologous recombination (HR) repair. Furthermore, we found that a poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281; olaparib) effectively reduced the growth of synovial sarcoma cell lines in the presence of an alkylating agent (temozolomide). Our findings offer evidence that treatment combining a PARP inhibitor and an alkylating agent could have therapeutic benefits in the treatment of synovial sarcoma.
Assuntos
Reparo de DNA por Recombinação/genética , Sarcoma Sinovial/tratamento farmacológico , Sarcoma Sinovial/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Etoposídeo/administração & dosagem , Humanos , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Radiação Ionizante , Sarcoma Sinovial/patologia , Temozolomida , Inibidores da Topoisomerase II/administração & dosagemRESUMO
PSF1 is a subunit of the GINS complex that functions along with the MCM2-7 complex and Cdc45 in eukaryotic DNA replication. Although mammalian PSF1 is predominantly expressed in highly proliferating cells and organs, little is known about the roles of PSF1 in mature cells or cancer cells. We found that PSF1 was expressed at relatively high levels in breast tumor cells, but at low levels in normal breast cells. Knockdown of PSF1 expression using small interfering RNA (siRNA) slowed the growth of breast cancer cell lines by delaying DNA replication but did not affect proliferation of normal human mammary epithelial cells. Reduced PSF1 expression also inhibited anchorage-independent growth in breast cancer cell lines. These results suggest that PSF1 over-expression is specifically involved in breast cancer cell growth. Therefore, PSF1 inhibition might provide new therapeutic approaches for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Regulação para Cima , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Chromosomal translocations are frequently associated with soft-tissue sarcomas. Fusion proteins generated by such translocations often play critical roles in tumorigenesis. Therefore, it is important to understand the function of the fusion protein to develop therapeutic interventions. The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in a fusion between the SYT gene on chromosome 18 and an SSX gene on the X chromosome. Although SYT-SSX fusion proteins appear to trigger synovial sarcoma development, little is known about the downstream targets of SYT-SSX. We found that the SYT-SSX fusion protein produces a dominant-negative function for SYT, which is a transcriptional coactivator. We then analyzed the gene expression profiles of SYT-SSX1-expressing HeLa cells using oligonucleotide microarrays and found that the SYT-SSX1 fusion protein directly down-regulated the expression of COM1, a regulator of cell proliferation. COM1 was found to be expressed at relatively low levels in synovial sarcoma tissues and cell lines. We then investigated the impact of conditional COM1 expression in the synovial sarcoma cell line. Increased COM1 expression resulted in induced apoptosis and in reduced cell growth and colony formation activity. Our results suggested that restoration of COM1 expression may be of therapeutic benefit in synovial sarcoma.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos X/genética , Regulação para Baixo/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma Sinovial/genética , Translocação Genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Dominantes , Células HeLa , Humanos , Células-Tronco Neoplásicas , Proteínas de Fusão Oncogênica/química , Regiões Promotoras Genéticas/genética , Estrutura Quaternária de Proteína , Transporte Proteico , Sarcoma Sinovial/patologiaRESUMO
Previously we have identified the Drosophila orbit gene whose hypomorphic mutations cause abnormal chromosome segregation (Inoue et al., 2000). The orbit encodes Orbit/Mast, a 165-kDa microtubule-associated protein (MAP) with GTP-binding motifs. Two human homologues of the Orbit/Mast, CLASP1 (hOrbit1) and CLASP2 (hOrbit2) have been identified. Using an antibody to CLASP1/hOrbit1 polypeptide, we confirmed that the polypeptide of about 150 kDa associates with microtubule purified from the porcine brain. Thus, we conjectured that CLASP1 may be a human orthologue of the Drosophila Orbit/Mast, and therefore we named it h (human) Orbit1. We constructed the plasmid for expression of a fusion protein of the putative microtubule-binding domain (1-662 out of 1289 residues) of hOrbit1 and the green fluorescent protein (GFP), and then, transfected the plasmid into Tet off cells derived from HeLa cell. Confocal laser scanning microscopic observation revealed that the GFP-fluorescence associated with short and thin filaments in the perinuclear region during the short period after plasmid transfection, and colocalized with only part of the microtubules. GFP fluorescence was later detected on the abnormally longer and thick bundles of microtubule filaments. Finally the bundles formed networks in the perinuclear region. The results suggest that the GFP-hOrbit1 N-terminal fragment (GFP-hOrbit1 NF) binds to the newly formed microtubules rather than the pre-formed ones, and that displacement of the endogenous hOrbit by the fragment might cause abnormal bundling of microtubules. Interestingly, the expression of the GFP-hOrbit1 NF results in cell death associated with nuclear fragmentation.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Biopolímeros , Morte Celular , Fragmentação do DNA , DNA Complementar/genética , Células HeLa , Humanos , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Fuso Acromático/metabolismo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
Failure to expeditiously repair DNA at sites of double-strand breaks (DSB) ultimately is an important etiologic factor in cancer development. NBS1 plays an important role in the cellular response to DSB damage. A rare polymorphic variant of NBS1 that resulted in an isoleucine to valine substitution at amino acid position 171 (I171V) was first identified in childhood acute lymphoblastic leukemia. This polymorphic variant is located in the N-terminal region that interacts with other DNA repair factors. In earlier work, we had identified a remarkable number of structural chromosomal aberrations in a patient with pediatric aplastic anemia with a homozygous polymorphic variant of NBS1-I171V; however, it was unclear whether this variant affected DSB repair activity or chromosomal instability. In this report, we demonstrate that NBS1-I171V reduces DSB repair activity through a loss of association with the DNA repair factor MDC1. Furthermore, we found that heterozygosity in this polymorphic variant was associated with breast cancer risk. Finally, we showed that this variant exerted a dominant-negative effect on wild-type NBS1, attenuating DSB repair efficiency and elevating chromosomal instability. Our findings offer evidence that the failure of DNA repair leading to chromosomal instability has a causal impact on the risk of breast cancer development.
Assuntos
Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica , Reparo do DNA , Proteínas Nucleares/genética , Polimorfismo Genético , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Feminino , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ligação Proteica , Transporte Proteico , Alinhamento de Sequência , Transativadores/metabolismoRESUMO
Oxidative and electrophilic stresses are sensed by Keap1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-Keap1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low Keap1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer-derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in Keap1-null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Estresse OxidativoRESUMO
Murine intestinal intraepithelial lymphocytes (IEL) encompass a high proportion of TCRgammadelta cells. A vast majority of these TCRgammadelta IEL express CD8alpha, but not CD8beta (CD8alphaalpha homodimer), and are considered to develop in intestinal epithelial layers independently of a functional thymus. Here we show that TCRgammadelta cells expressing both CD8alpha and CD8beta (CD8alphabeta heterodimer) appear in athymic nu/nu mice, although their appearance is random. The IEL comprising CD8alphabeta(+) TCRgammadelta cells expressed pronounced cytolytic and IFN-gamma-producing activities after TCRgammadelta ligation, which were markedly stronger than activities of IEL lacking CD8alphabeta(+) TCRgammadelta cells. Purified CD8alphabeta(+) TCRgammadelta cells expressed strong cytolytic activities and produced large quantities of IFN-gamma after TCR engagement. CD8alphabeta(+) TCRgammadelta cells were also identified among IEL from euthymic C57BL/6 mice, although their abundance varied among individual animals. However, cytolytic and IFN-gamma-producing activities in euthymic C57BL/6 mice were markedly lower than those in athymic nu/nu mice. Our findings suggest that CD8alphabeta(+) TCRgammadelta cells can develop in the intestine independently of a functional thymus/thymic epithelial cells and that they perform biological functions in situ.
Assuntos
Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitélio/imunologia , Intestinos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Citotoxicidade Imunológica , Citometria de Fluxo , Imunoensaio , Interferon gama/metabolismo , Camundongos , Camundongos NusRESUMO
We tested the combination of phosphodiesterase (PDE) 3 and PDE4 inhibitors as an interventional approach to prevent the development of brain damage after Shiga toxin (Stx)-producing Escherichia coli (STEC) infection, using mice with protein calorie malnutrition. The combination consisted of pentoxifylline and rolipram; the dose of each inhibitor was 7.5 mg/kg. Treatment with this combination, which was administered intraperitoneally twice daily at 12-h intervals, increased serum concentrations of each inhibitor to >2 microg/mL and afforded significant levels of protection when it was continued for 3 days, starting on day 2 (95% survival rate; P<.001) or day 3 (63% survival rate; P<.01) of infection. The treatment reduced plasma levels of Stx2; consequently, immunoreactions of Stx2 were not found in the brain, and survivors did not show neurologic symptoms. Protection was associated with decreased levels of tumor necrosis factor (TNF)- alpha and increased production of interleukin-10 in serum, the brain, and the cecum. Although the combination at doses >2 microg/mL reduced Gb3 content of and Stx2 binding to Caco-2 cells, its ability to suppress production of TNF- alpha seemed to be more important for the decrease in cell-bound Stx2 in intestinal epithelial cells. Therefore, the combination of PDE3 and PDE4 inhibitors might be used as an interventional approach to prevent brain damage caused by STEC infection.
Assuntos
Encefalopatias/prevenção & controle , Infecções por Escherichia coli/tratamento farmacológico , Pentoxifilina/uso terapêutico , Inibidores de Fosfodiesterase/uso terapêutico , Rolipram/uso terapêutico , Toxina Shiga II/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/metabolismo , Encefalopatias/microbiologia , Encefalopatias/patologia , Ceco/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Fezes/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pentoxifilina/administração & dosagem , Inibidores de Fosfodiesterase/administração & dosagem , Desnutrição Proteico-Calórica/complicações , Rolipram/administração & dosagemRESUMO
Although macrophages play a central role in the pathogenesis of septic shock, NK1(+) cells have also been implicated. NK1(+) cells comprise two major populations, namely NK cells and V alpha 14(+)NKT cells. To assess the relative contributions of these NK1(+) cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of beta(2)-microglobulin (beta(2)m)(-/-) mice that are devoid of V alpha 14(+)NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that beta(2)m(-/-) mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-gamma following LPS challenge were significantly higher in beta(2)m(-/-) mice, and endogenous IFN-gamma neutralization or in vivo depletion of NK1(+) cells rescued beta(2)m(-/-) mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-gamma producers. The J alpha 281(-/-) mice that are exclusively devoid of V alpha 14(+)NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of V alpha 14(+)NKT cells and IFN-gamma from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.
Assuntos
Células Matadoras Naturais/fisiologia , Lipopolissacarídeos/toxicidade , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Choque Séptico/etiologia , Subpopulações de Linfócitos T/fisiologia , Animais , Interferon gama/biossíntese , Interferon gama/sangue , Interleucina-4/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fenômeno de Shwartzman , Microglobulina beta-2/fisiologiaRESUMO
LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers. Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.
Assuntos
Granulócitos/imunologia , Interleucina-12/biossíntese , Listeriose/genética , Listeriose/imunologia , Antígeno-1 Associado à Função Linfocitária/genética , Regulação para Cima/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Movimento Celular/genética , Movimento Celular/imunologia , Granulócitos/metabolismo , Granulócitos/patologia , Imunidade Ativa/genética , Imunidade Inata/genética , Injeções Intraperitoneais , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Interleucina-12/fisiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima/genéticaRESUMO
Challenge with low doses of LPS together with D-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1(-/-) mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-alpha and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1(+) cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1(+/-) mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1(-/-) mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1(-/-) mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1(+/-) mice were only marginally affected by macrophage depletion. However, in LFA-1(-/-) mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1(-/-) mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-alpha levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.
Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-10/fisiologia , Interleucina-12/fisiologia , Lipopolissacarídeos/administração & dosagem , Fígado/patologia , Antígeno-1 Associado à Função Linfocitária/genética , Choque Séptico/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Ácido Clodrônico/administração & dosagem , Citocinas/biossíntese , Relação Dose-Resposta Imunológica , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Granulócitos/imunologia , Granulócitos/metabolismo , Imunidade Inata/genética , Mediadores da Inflamação/antagonistas & inibidores , Injeções Intravenosas , Interleucina-10/sangue , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/sangue , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucocitose/genética , Leucocitose/imunologia , Leucocitose/patologia , Fígado/imunologia , Fígado/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contagem de Plaquetas , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Choque Séptico/genética , Choque Séptico/mortalidade , Choque Séptico/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.