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1.
Neuropathol Appl Neurobiol ; 46(2): 111-124, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31179566

RESUMO

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.


Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/imunologia , Linhagem da Célula/imunologia , Germinoma/diagnóstico , Germinoma/imunologia , Neoplasias Encefálicas/metabolismo , Perfilação da Expressão Gênica , Germinoma/metabolismo , Humanos , Prognóstico , Transcriptoma , Microambiente Tumoral/imunologia
2.
Adv Exp Med Biol ; 923: 351-357, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27526163

RESUMO

We have incorporated LiNc-BuO, an oxygen-sensing paramagnetic material, in polydimethylsiloxane (PDMS), which is an oxygen-permeable, biocompatible, and stable polymer. We fabricated implantable and retrievable oxygen-sensing chips (40 % LiNc-BuO in PDMS) using a 20-G Teflon tubing to mold the chips into variable shapes and sizes for in vivo studies in rats. In vitro EPR measurements were used to test the chip's oxygen response. Oxygen induced linear and reproducible line broadening with increasing partial pressure (pO2). The oxygen response was similar to that of bare (unencapsulated) crystals and did not change significantly on sterilization by autoclaving. The chips were implanted in rat femoris muscle and EPR oximetry was performed repeatedly (weekly) for 12 weeks post-implantation. The measurements showed good reliability and reproducibility over the period of testing. These results demonstrated that the new formulation of OxyChip with 40 % LiNc-BuO will enable the applicability of EPR oximetry for long-term measurement of oxygen concentration in tissues and has the potential for clinical applications.


Assuntos
Técnicas Biossensoriais , Dimetilpolisiloxanos/química , Espectroscopia de Ressonância de Spin Eletrônica , Metaloporfirinas/química , Músculo Esquelético/metabolismo , Oximetria/métodos , Consumo de Oxigênio , Oxigênio/metabolismo , Animais , Cristalização , Masculino , Miniaturização , Pressão Parcial , Ratos Wistar , Reprodutibilidade dos Testes , Fatores de Tempo
3.
Oncogene ; 31(36): 4054-66, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22139077

RESUMO

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPß. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.


Assuntos
Glioblastoma/irrigação sanguínea , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Neovascularização Patológica/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Interleucina-8/genética , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , NF-kappa B/antagonistas & inibidores , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Elementos de Resposta , Sulfonas/farmacologia , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Carga Tumoral , Proteínas ras/metabolismo
4.
Cancer Res ; 61(23): 8569-77, 2001 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11731444

RESUMO

The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Fatores de Crescimento Endotelial/fisiologia , Glioma/irrigação sanguínea , Linfocinas/fisiologia , Neovascularização Patológica/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Glioma/metabolismo , Humanos , Linfocinas/biossíntese , Linfocinas/farmacologia , Neovascularização Patológica/patologia , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Vitronectina/farmacologia
5.
J Neurosurg ; 95(3): 472-9, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11565870

RESUMO

OBJECT: Activation of signaling by the epidermal growth factor receptor (EGFR) through gene amplification or rearrangement is common in human malignancy, especially in a large fraction of de novo glioblastomas multiforme (GBMs). The most common mutant EGFR, (AEGFR, also known as de2-7 EGFR and EGFRvIII) lacks a portion of the extracellular domain, enhances tumorigenicity in vivo, and causes resistance to the chemotherapeutic drug cisplatin (CDDP). This resistance is due to the suppression of CDDP-induced apoptosis by the constitutively active tyrosine kinase activity of the receptor. The authors have investigated whether inhibition of AEGFR signaling by the tyrosine kinase inhibitor, tyrphostin AG1478, could sensitize tumor xenografts to CDDP and, thereby, enhance its therapeutic efficacy in animals. METHODS: Nude mice were inoculated either subcutaneously or intracerebrally with human GBM cells expressing AEGFR and were then systemically treated with CDDP and/or AG1478. Tumor volumes were monitored and tumor sections were analyzed by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays or MIB-1 staining. Expression of AEGFR, but not wild-type EGFR, conferred CDDP resistance to the cells in vivo. Inhibition of receptor signaling by the EGFR-specific tyrosine kinase inhibitor, AG1478. sensitized the xenografts to the cytotoxic effects of CDDP. This combined CDDP/AG1478 treatment significantly suppressed growth of subcutaneous xenografts in nude mice in a synergistic manner (p < 0.01 compared with vehicle control) without causing generalized toxicity, whereas treatments with CDDP or AG1478 alone were ineffective. The synergistic growth suppression by the CDDP/AG1478 combination was not observed in xenografts overexpressing wild-type EGFR or kinase-deficient AEGFR. The combined CDDP/ AG1478 treatment induced tumor growth suppression, which correlated with increased apoptosis and reduced proliferation. This treatment also extended the life span of mice bearing intracerebral xenografts (p < 0.01 compared with controls). CONCLUSIONS: The results of this study may provide the basis for the development of a novel and safe therapeutic strategy for the very aggressive AEGFR-expressing GBM.


Assuntos
Neoplasias Encefálicas/genética , Sobrevivência Celular/genética , Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Glioblastoma/genética , Mutação/genética , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Tirfostinas/farmacologia , Animais , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Quinazolinas , Transplante Heterólogo , Células Tumorais Cultivadas
6.
Neurosurgery ; 49(2): 430-6; discussion 436-7, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11504120

RESUMO

OBJECTIVE: Sodium butyrate (SB), a differentiation-inducing agent, has been demonstrated to inhibit cellular proliferation in a number of human cell lines. Its precise mechanisms remain to be clarified, however. We investigated molecular mechanisms of SB-induced growth suppression as well as the effects of SB on the invasiveness of human glioma cells. METHODS: Human glioma U87MG and U251MG cells were treated with 1 or 2 mmol/L SB for 48 hours, and the inhibition of cell growth was assessed by spectrophotometric analysis. Cell cycle analysis was carried out by the 5-bromo-2'-deoxyuridine incorporation method, and expression of cell cycle-regulatory proteins was determined by immunoblotting. In addition, invasiveness was assessed using a Transwell chamber (Iwaki, Tokyo, Japan) with extracellular matrix substrate fibronectin or laminin (Iwaki). RESULTS: SB treatment resulted in significantly suppressed proliferation of both U87MG and U251MG cells in a dose-dependent manner. It inhibited the G1-S transition, which was associated with increased expression of p21 and cyclin D1 and reduced pRb phosphorylation. Treatment with antisense oligonucleotide for Rb abrogated SB-induced G1 arrest. p21 up-regulation was independent of the p53 status of the glioma cells. SB treatment also inhibited invasiveness on fibronectin and laminin. CONCLUSION: Our results indicate that SB may suppress the growth of human glioma cells through modulation of cell cycle progression and also may affect their invasiveness on extracellular matrix substrates, which suggests that SB may be a useful therapeutic agent in treating multiple aspects of malignant gliomas.


Assuntos
Neoplasias Encefálicas/patologia , Butiratos/farmacologia , Glioma/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Fase G1 , Humanos , Invasividade Neoplásica , Fase S , Células Tumorais Cultivadas
7.
Apoptosis ; 6(3): 191-7, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11388668

RESUMO

Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for clinical oncology. One potential adjunct to such conventional treatments is direct induction of cell death by activation of death receptor-mediated apoptosis. TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a recently identified member of the growing TNF superfamily, binds to its cognate "death" receptors DR4 and DR5 as well as "decoy" receptors DcR1 and DcR2. Upon binding, rapid apoptosis is enacted in a variety of human cancer cell lines independent of p53 status, but not in normal cell lines. TRAIL treatment results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. Furthermore, combination treatment of TRAIL with genotoxic chemotherapeutic agents synergistically suppresses growth of tumor xenografts which are otherwise resistant to treatment with TRAIL or chemotherapy alone. Unlike the other death ligands TNF-alpha or FasL, systemic administration of soluble human TRAIL does not cause toxicity in mice and non-human primates. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer.


Assuntos
Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/uso terapêutico , Neoplasias/tratamento farmacológico , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Proteínas Reguladoras de Apoptose , Humanos , Ligantes , Camundongos , Modelos Biológicos , Transplante de Neoplasias , Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF
8.
Cancer Lett ; 162 Suppl: S17-S21, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11164186

RESUMO

Alterations of the epidermal growth factor receptor (EGFR) occur frequently in malignant gliomas through gene amplification or rearrangement, especially in a large fraction of de novo type glioblastomas. The most common of these mutant EGFRs (variously named de2-7 EGFR, deltaEGFR or EGFRvIII) lacks a portion of the extracellular ligand-binding domain. Here, we review the evidence that shows that expression of deltaEGFR bestows in vivo growth advantages to human glioma cells through its constitutively active tyrosine kinase activity. Thus, deltaEGFR may provide a novel therapeutic target for the most aggressive type of glioblastoma.


Assuntos
Receptores ErbB/fisiologia , Glioma/genética , Transdução de Sinais , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Mutação , Quinazolinas , Tirfostinas/farmacologia
9.
Brain Tumor Pathol ; 18(2): 73-81, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11908877

RESUMO

The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.


Assuntos
Neoplasias Encefálicas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Glioma/patologia , Bradicinina/farmacologia , Adesão Celular , Meios de Cultura Livres de Soro/farmacologia , Ecdisona/farmacologia , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes p16 , Humanos , Insulina/farmacologia , Integrinas/biossíntese , Integrinas/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/ultraestrutura
10.
Biosci Biotechnol Biochem ; 65(12): 2773-5, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11826976

RESUMO

An electro-transformation method was established for facultatively alkaliphilic Bacillus pseudofirmus OF4. A high osmolarity electroporation medium with a high electric field strength was effective. Transformation efficiency improved 110-fold in glycine-treated cells compared to non-glycine-treated cells. Under optimum conditions, the transformation efficiency was 1.69 x 10(6) transformants per microg of pUB110.


Assuntos
Bacillus/metabolismo , Glicina/metabolismo , Meios de Cultura , Eletroporação , Concentração Osmolar
11.
Cancer Res ; 60(4): 847-53, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10706092

RESUMO

The intractability of malignant gliomas to multimodality treatments plays a large part in their extremely poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel member of the tumor necrosis factor (TNF) family that induces apoptosis preferentially in tumor cells through binding to its cognate death receptors, DR4 and DR5. Here we show that the DNA-damaging chemotherapeutic drugs, cis-diamminedichloroplatinum(II) (CDDP) and etoposide, elicited increased expression of DR5 in human glioma cells. Exposure of such cells in vitro to soluble human TRAIL in combination with CDDP or etoposide resulted in synergistic cell death that could be blocked by soluble TRAIL-neutralizing DR5-Fc or the caspase inhibitors, Z-Asp-CH2-DCB and CrmA. Moreover, systemic in vivo administration of TRAIL with CDDP synergistically suppressed both tumor formation and growth of established s.c. human glioblastoma xenografts in nude mice by inducing apoptosis without causing significant general toxicity. The combination treatment resulted in complete and durable remission in 29% of mice with the established s.c. xenografts and also significantly extended the survival of mice bearing intracerebral xenografts. These results provide preclinical proof-of-principle for a novel therapeutic strategy in which the death ligand, TRAIL, is safely combined with conventional DNA-damaging chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Glioma/tratamento farmacológico , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteínas Reguladoras de Apoptose , Cisplatino/farmacologia , DNA/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Feminino , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Ligante Indutor de Apoptose Relacionado a TNF , Transplante Heterólogo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia
12.
Cancer Res ; 59(15): 3596-601, 1999 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10446968

RESUMO

Alteration of chromosome 10 is common in human melanomas and usually entails the loss of an entire chromosome homologue. Although the reasons for monosomy in cancer has remained obscure, one possibility is that multiple tumor suppressor genes residing on this chromosome must be lost in unison during tumor progression, and this is easier to accomplish by chromosome segregation rather than by multiple mutational and/or deletion events. The localization and identification of these genes has been hampered by the monosomy itself, which has resulted in a paucity of small defining deletions in tumors. Here, we have addressed the issue of monosomy in tumor development by using functional complementation mapping to localize and demonstrate the existence of different melanoma suppressor genes on chromosome 10 and assigned each locus a distinct tumorigenic phenotype. We report that a locus on 10q distal to 10q23.1, likely involving the PTEN tumor suppressor, causes a severe reduction in the kinetics of melanoma tumor formation in animals. In contrast, a previously unrecognized region at 10p15.3 has a distinct, but lesser, effect on in vivo melanoma growth. Thus, the loss of both of these regions, which is accomplished by tumor-associated monosomy, provides a significant growth advantage over the individual loss of either region, thereby explaining the monosomy observed in sporadic melanomas.


Assuntos
Cromossomos Humanos Par 10/genética , Genes Supressores de Tumor , Perda de Heterozigosidade , Melanoma/genética , Monossomia , Proteínas Supressoras de Tumor , Animais , Cromossomos Humanos Par 10/ultraestrutura , Progressão da Doença , Humanos , Células Híbridas , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Transplante de Neoplasias , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Células Tumorais Cultivadas
13.
Cell Growth Differ ; 10(2): 73-86, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10074901

RESUMO

The early incidence of p53 mutation in astrocytomas suggests that it plays an important role in astrocyte transformation. Astrocytes isolated from homozygous p53 knockout mice grow rapidly, lack contact inhibition, and are immortal. Here we tested whether the loss of p53 is sufficient for progression to tumorigenicity of astrocytes. We grew primary astrocytes under three conditions for over 120 population doublings and assessed their antigenic phenotype, chromosome number, and expression of glioma-associated genes as well as their ability to form colonies in soft agarose and tumors s.c. and intracranially in nude mice. Under two conditions (10% FCS and 0.5% FCS plus 20 ng/ml EGF), cells acquired the ability to form colonies in soft agarose and tumors in nude mice, and this was accompanied by the expression of genes, including epidermal growth factor receptor, platelet-derived growth factor receptor alpha and beta, protein kinase Cdelta, and vascular endothelial growth factor, which are known to be aberrantly regulated in human astrocytomas. Under the third condition (0.5% FCS plus 10 ng/ml basic fibroblast growth factor), astrocytes gained the ability to form colonies in soft agarose and had abnormal chromosome numbers similar to cells in the first two conditions but did not form tumors in nude mice or overexpress glioma-associated genes. These data provide experimental evidence for the idea that the malignant progression initiated by the loss of p53 may be subject to modulation by extracellular environmental influences.


Assuntos
Astrócitos/metabolismo , Transformação Celular Neoplásica , Proteína Supressora de Tumor p53/deficiência , Aneuploidia , Animais , Astrócitos/citologia , Northern Blotting , Encéfalo/citologia , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Testes de Carcinogenicidade , Técnicas de Cultura de Células/métodos , Divisão Celular , Células Cultivadas , Meios de Cultura , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioma/genética , Humanos , Camundongos , Camundongos Nus , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Tempo , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genética
14.
Jpn J Clin Oncol ; 29(11): 527-34, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10678554

RESUMO

BACKGROUND: In addition to traditional modalities such as surgical intervention and radiotherapy, chemotherapy is a common therapeutic method for human malignant brain tumors. However, the effectiveness of chemotherapy is frequently hampered by cancer cell chemoresistance, resulting in an unsatisfactory outcome. To overcome this disadvantage, the proper selection of efficacious anticancer agents is required. METHODS: The expression levels of chemoresistance-related genes, MGMT, mdr1, MRP, MTIIA and GST-pi, in 28 surgical specimens of human brain tumors and in 10 human glioma cell lines were examined by Northern blot analysis. In addition, the SD10 values of human glioma cell lines against ACNU, CDDP, ADM and VP16 were estimated by a cell survival assay. RESULTS: The expression levels of each of the chemoresistance-related genes, except MRP, were generally higher in brain tumors than those in non-neoplastic brain tissues. MGMT expression correlated exclusively with ACNU resistance in all glioma cell lines examined (p = 0.0002). The transcriptional level of mdr1 in the tumor cells correlated with the SD10 values of VCR (p = 0.04) and ADM (p = 0.034). In contrast, the expression levels of MTIIA and GST-pi did not correlate with resistance to any of the drugs tested. A correlation of MRP mRNA expression with multidrug resistance was not apparent in the 10 cell lines tested. CONCLUSIONS: The data indicate that knowledge of the expression levels of MGMT and mdr1 may be particularly useful for a more rational selection of drugs which are not influenced by these resistance genes and which have improved efficacy against human brain tumors.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Genes MDR , Glioma/genética , Glicoproteínas de Membrana , Antígenos CD/análise , Antígenos CD/genética , Neoplasias Encefálicas/patologia , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Glioma/patologia , Humanos , Nimustina/farmacologia , RNA Mensageiro/análise , Tetraspanina 29 , Células Tumorais Cultivadas/efeitos dos fármacos
15.
Am J Pathol ; 153(4): 1239-48, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9777955

RESUMO

We have previously demonstrated that vascular endothelial growth factor-165 (VEGF), a tumor-secreted angiogenic factor, can acutely and chronically induce fenestrations in microvascular endothelium (Cancer Res 1997, 57:765-772). Because the morphology and function of microvascular endothelium differs from tissue to tissue, we undertook studies to examine whether the neovasculature in tumors also differed depending upon tumor location. Four tumor types implanted in the brain or subcutis in nude mice were studied: a murine rhabdomyosarcoma (M1S), a murine mammary carcinoma (EMT), and two human glioblastomas (U87 and U251). In addition, we studied Chinese hamster ovary cells stably transfected with human VEGF165. As previously reported, tumors grown in the subcutaneous space had a microvasculature that was fenestrated and had open endothelial gaps. The identical tumors when grown in the brain also had fenestrated endothelium and vessels with open endothelial gaps, but they were drastically reduced in occurrence. Open endothelial gaps were not seen in all tumors implanted in the brain (EMT and M1S), although fenestrated endothelium was always seen. VEGF and VEGF receptors were measured in tumors from both locations by immunoblotting and competitive polymerase chain reaction, respectively. VEGF amount was not significantly different between the tumor locations. Interestingly, total tumor vascular mRNA expression of both Flk-1 and Flt-1 was greater in tumor vessels derived from the brain compared with tumor vessels derived from subcutaneous tissues. These results demonstrate that the host microvascular environment determines the morphology and function of the tumor vasculature and that endothelia from different tissues vary in their ability to express the VEGF receptors given identical stimuli.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Endotélio Vascular/patologia , Glioblastoma/irrigação sanguínea , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neovascularização Patológica/patologia , Rabdomiossarcoma/irrigação sanguínea , Neoplasias Cutâneas/irrigação sanguínea , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Células CHO/transplante , Células CHO/ultraestrutura , Cricetinae , Primers do DNA/química , DNA de Neoplasias/análise , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
16.
Proc Natl Acad Sci U S A ; 95(10): 5724-9, 1998 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-9576951

RESUMO

Alterations of the epidermal growth factor receptor (EGFR) gene occur frequently in human malignant gliomas. The most common of these is deletion of exons 2-7, resulting in truncation of the extracellular domain (DeltaEGFR or EGFRvIII), which occurs in a large fraction of de novo malignant gliomas (but not in progressive tumors or those lacking p53 function) and enhances tumorigenicity, in part by decreasing apoptosis through up-regulation of Bcl-XL. Here, we demonstrate that the DeltaEGFR concomitantly confers resistance to the chemotherapeutic drug cisplatin (CDDP) by suppression of CDDP-induced apoptosis. Expression of Bcl-XL was elevated in U87MG.DeltaEGFR cells prior to and during CDDP treatment, whereas it decreased considerably in CDDP-treated parental cells. CDDP-induced activation of caspase-3-like proteases was suppressed significantly in U87MG.DeltaEGFR cells. These responses were highly specific to constitutively kinase-active DeltaEGFR, because overexpression of kinase-deficient DeltaEGFR (DK) or wild-type EGFR had no such effects. Correspondingly, DeltaEGFR specific tyrosine kinase inhibitors reduced Bcl-XL expression and potentiated CDDP-induced apoptosis in U87MG.DeltaEGFR cells. Ectopic overexpression of Bcl-XL in parental U87MG cells also resulted in suppression of both caspase activation and apoptosis induced by CDDP. These results may have important clinical implications for the use of CDDP in the treatment of those malignant gliomas expressing DeltaEGFR.


Assuntos
Caspases , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Receptores ErbB/genética , Glioblastoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tirfostinas , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspase 3 , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Humanos , Nitrilas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Células Tumorais Cultivadas , Proteína bcl-X
17.
Proc Natl Acad Sci U S A ; 94(22): 12081-7, 1997 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-9342366

RESUMO

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF121, VEGF165, and VEGF189. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF121- and VEGF165-overexpressing cells developed intracerebral hemorrhages after 60-90 hr. In contrast, mice implanted with VEGF189-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF121 and VEGF165, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF189, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Hemorragia Cerebral/etiologia , Fatores de Crescimento Endotelial/biossíntese , Glioblastoma/irrigação sanguínea , Linfocinas/biossíntese , Neovascularização Patológica , Adulto , Processamento Alternativo , Neoplasias Encefálicas/patologia , Movimento Celular , Transplante de Células , Hemorragia Cerebral/patologia , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/efeitos dos fármacos , Glioblastoma/patologia , Humanos , Linfocinas/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
18.
Neurosurgery ; 41(2): 434-40; discussion 440-1, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9257312

RESUMO

OBJECTIVE: A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. METHODS: We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. RESULTS: C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. CONCLUSION: ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.


Assuntos
Elementos Antissenso (Genética)/uso terapêutico , Glioma/terapia , Metiltransferases/genética , RNA Complementar/uso terapêutico , RNA Mensageiro/genética , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glioma/genética , Glioma/patologia , Nimustina/efeitos adversos , Nimustina/uso terapêutico , O(6)-Metilguanina-DNA Metiltransferase , Biossíntese de Proteínas , Ratos , Transfecção , Células Tumorais Cultivadas
19.
Curr Opin Oncol ; 9(3): 215-22, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9229142

RESUMO

Malignant gliomas are the most common primary tumors of the central nervous system but remain clinically intractable. This has engendered substantial efforts to elucidate the molecular genetic and biologic basis of glioma formation. This review focuses on recent discoveries of the genetic aberrations that occur during the progression of gliomas. These include those that inactivate tumor suppressor genes and activate oncogenes. The biologic consequences of such genetic changes on various tumor cell-host environment interactions are also described.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Glioma/genética , Apoptose/genética , Ciclo Celular/fisiologia , Genes Supressores de Tumor , Humanos , Neovascularização Patológica/genética , Oncogenes
20.
J Biol Chem ; 272(5): 2927-35, 1997 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-9006938

RESUMO

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.


Assuntos
Neoplasias Encefálicas/patologia , Receptores ErbB/biossíntese , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/patologia , Fosfotirosina , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias Encefálicas/genética , Linhagem Celular , Primers do DNA , Regulação para Baixo , Endocitose , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/fisiologia , Glioblastoma/genética , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção , Transplante Heterólogo
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