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/imunologiaRESUMO
Type I interferon (IFN) is a pleiotropic cytokine regulating the cancer cell death and immune response. IFN-α can, as we have also reported, effectively induce an antitumor immunity by the activation of tumor-specific T cells and maturation of dendritic cells in various animal models. Unknown, however, is how the type I IFN alters the immunotolerant microenvironment in the tumors. Here, we found that intratumoral IFN-α gene transfer significantly decreased the frequency of regulatory T cells (Tregs) per CD4(+) T cells in tumors. The concentration of a Treg-inhibitory cytokine, interleukin (IL)-6, was correlated with the IFN-α expression level in tumors, and intratumoral CD11c(+) cells produced IL-6 in response to IFN-α stimulation. To confirm the role of IL-6 in the suppression of Tregs in tumors, an anti-IL-6 receptor antibody was administered in IFN-α-treated mice. The antibody increased the frequency of Tregs in the tumors, and attenuated systemic tumor-specific immunity induced by IFN-α. Furthermore, the IFN-α-mediated IL-6 production increased the frequency of Th17 cells in the tumors, which may be one of the mechanisms for the reduction of Tregs. The study demonstrated that IFN-α gene delivery creates an environment strongly supporting the enhancement of antitumor immunity through the suppression of Tregs.
Assuntos
Interferon Tipo I/genética , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Progressão da Doença , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Imunomodulação , Interferon Tipo I/metabolismo , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interleucina-6/metabolismo , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Camundongos , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Interleucina-6/antagonistas & inibidores , Células Th17/imunologia , Células Th17/metabolismo , Carga Tumoral/genética , Carga Tumoral/imunologiaRESUMO
A novel galacto-oligosaccharide (GOS) manufactured by a two-step enzyme reaction of lactose was examined in a comet assay for its potential to induce DNA damage in vivo by estimating the DNA fragmentation level in the cellular nuclei of the glandular stomach, colon, and peripheral blood. GOS was orally administered at doses of 0 (vehicle alone), 500, 1000, and 2000 mg/kg/day to five male Crl: CD(Sprague Dawley) rats per group three times (48, 24, and 3 h before the animals were terminated). The specimens were prepared in accordance with the standard protocol (version 14.2) of the "International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens" organized by the Japanese Center for the Validation of Alternative Methods. No significant differences in the percentage of DNA in the tail were observed between the GOS-treated groups and vehicle controls in any of the organs evaluated. Additionally, no GOS-related clinical signs or effects on body weight were seen. Based on these results, the comet assay of GOS in the glandular stomach, colon, and peripheral blood using rats was judged negative. Therefore, it is concluded that GOS did not induce DNA damage in vivo under the conditions employed in this study.
Assuntos
Ensaio Cometa , Galactose/química , Oligossacarídeos/análise , Administração Oral , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Oligossacarídeos/química , Oligossacarídeos/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Tolerância Imunológica , Interferon-alfa/administração & dosagem , Linfopenia/terapia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Apresentação de Antígeno , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Transplante de Células-Tronco Hematopoéticas , Imunoterapia/métodos , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon-alfa/uso terapêutico , Interleucina-6/metabolismo , Linfopenia/genética , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais , Plasmídeos/genética , Plasmídeos/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 degrees C. In the presence of 2 mmol CaCl(2)/l at 40 degrees C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca(2+), motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 degrees C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca(2+). These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca(2+), was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca(2+) and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca(2+) plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.
Assuntos
Reação Acrossômica/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Espermatozoides/enzimologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Reação Acrossômica/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting/métodos , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Cálcio/farmacologia , Células Cultivadas , Galinhas , Masculino , Toxinas Marinhas , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/antagonistas & inibidores , Motilidade dos Espermatozoides/efeitos dos fármacos , Estimulação Química , Sulfonamidas/farmacologia , Membrana Vitelina/metabolismoRESUMO
In 97 patients (60, chemotherapy; 22, chemoradiotherapy; 15, radiotherapy), histopathologic effects were evaluated microscopically, and histologic response rates were compared among three neoadjuvant treatment modalities. Predictive factors for neoadjuvant therapies were analyzed by logistic regression, including the results of p53 immunohistochemical staining. In the chemoradiotherapy group, the pathologic response rate was 86.4%, and was significantly higher than that for chemotherapy (P < 0.0001) or for radiotherapy (P = 0.0031). In patients with normal p53 protein expression, the histopathologic response rate to chemotherapy was 20.0%, a higher rate than that for patients with abnormal p53 overexpression. In the chemoradiotherapy or radiotherapy group, however, the response rates were almost the same, irrespective of p53 oncoprotein status. From multivariate analysis, the neoadjuvant treatment modality itself was identified as the most powerful predictive factor for the effect. Chemoradiotherapy had the most powerful effect on advanced esophageal cancer, and p53 status did not influence the clinical outcome in this group.
Assuntos
Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Genes p53/genética , Terapia Neoadjuvante/métodos , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/análise , Biópsia por Agulha , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Terapia Combinada , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Esofagectomia/métodos , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
We report an elderly patient with squamous cell carcinoma who was successfully treated with chemotherapy using vinorelbine. A 76-year-old man was referred to our hospital for evaluation of a nodular shadow in the left lung. Chest CT scam showed a 3-cm tumor shadow in left S9 and a 1-cm small nodule in right S2. Transbronchial lung biopsy yielded a diagnosis of squamous cell carcinoma. The clinical stage was IV (cT2N2M1). The patient first underwent chemotherapy consisting of cisplatin (CDDP) 80 mg/m2 on day 1 and vinorelbine (VNB) 20 mg/m2 on days 1, 8, and 15, which generated tumor shrinkage of 48% as well as transient elevation of grade 1 in serum creatinine. The 2 cycles of chemotherapy using vinorelbine only (VNB 20 mg/m2 on days 1, 8, 15) produced a tumor reduction of 70% with grade-1 decrease of granulocytes. The low grade of toxicity enabled us to treat the patient in our outpatient office for the second cycle of the regimen. This case suggests that chemotherapy using low-dose vinorelbine might be suitable to treat elderly patients with NSCLC in outpatient settings.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Vimblastina/análogos & derivados , Vimblastina/administração & dosagem , Idoso , Cisplatino/administração & dosagem , Esquema de Medicação , Humanos , Masculino , VinorelbinaRESUMO
Recombinant adenovirus mediated p53 gene transfer combined with anti-cancer drugs has clinical potential for gene therapy of lung cancer. We constructed a recombinant adenoviral vector expressing wild-type p53 cDNA (Ad-p53), and assessed the efficacy of a combined treatment with Ad-p53 and six anti-cancer drugs (cisplatin, 5-fluorouracil, doxorubicin, docetaxel, irinotecan, and etoposide) for human lung cancer cell lines, H1299 (with deleted p53), RERF-LC-OK (with mutant p53), and A549 (with wild-type p53). The infection of the Ad-p53 vector into H1299 cells, RERF-LC-OK cells, or A549 cells increased the sensitivity to all six drugs regardless of the cellular p53 status, and a synergism was observed by the isobolic method in combination studies (D<1). We conclude that our strategy using adenoviral mediated p53 gene transfer to cancer cells can enhance the cytotoxic effect of anti-cancer drugs, which leading to an improvement of lung cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Genes p53/genética , Vetores Genéticos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Adenovírus Humanos/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Terapia Combinada , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Fase G1/efeitos dos fármacos , Fase G1/genética , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Neoplasias Pulmonares/metabolismo , Células Tumorais CultivadasRESUMO
Expression of the 37-kDa laminin binding protein (37LBP), a precursor protein of the 67-kDa laminin receptor, correlates well with the biological aggressiveness of cancer cells. Previously, we have established murine lung cancer cell lines T11 and T15, in which 37LBP expression was remarkably diminished, and reported that the mean survival time of the T11 and the T15-recipients was significantly prolonged compared with that of the control cell lines (P29 and T42). In the present study, immunohistochemical findings of the tumors demonstrated that the microvessel density in the T11 (28. 1+/-7.2/mm(2)) and in the T15 tumor (29.7+/-6.5/mm(2)) were significantly lower than that observed in P29 (46.3+/-8.7/mm(2)) or in T42 (50.5+/-4.4/mm(2)). Expression of vascular endothelial growth factor (VEGF) was repressed in T11 and T15 compared with its expression in P29 and T42. It was also shown that conditioned media of T11 and T15 cells exhibited significantly reduced proliferation and migration of the capillary endothelial cells. These results suggest that decreased expression of 37LBP in antisense-RNA transfectant may relate to its low tumorigenicity, and that this effect may be partly caused by the diminished tumor angiogenesis of murine lung cancer.
Assuntos
Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Neovascularização Patológica/metabolismo , Precursores de Proteínas/fisiologia , Receptores de Laminina , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Camundongos , Peso Molecular , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA Antissenso/genética , RNA Antissenso/farmacologia , Transfecção , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The laminin binding protein of 37 kDa (37LBP) is regarded as a precursor protein of the high-affinity 67-kDa laminin receptor (67LR). Expression of 67LR/37LBP is well correlated with biological aggressiveness of cancer, particularly with invasive and metastatic potential. To investigate in detail the role of 37LBP in cancer cells, we synthesized recombinant 37LBP (r37LBP) as a fusion protein and generated an IgG-type polyclonal antibody P4G against r37LBP. Western blot analysis with P4G showed a single band of 67LR under both nonreducing and reducing conditions using cell extract of human fibrosarcoma cells HT1080. It was shown that P4G inhibited cell attachment to immobilized laminin in a dose-dependent manner. Further, the intravenous injection of HT1080 cells pretreated with P4G, compared with that of cells pretreated with normal rabbit serum, resulted in a reduced number of experimental metastases (3.3+/-5.1 vs. 58.0+/-38.0 nodules per mouse, respectively) (P<0.005). These results suggest that P4G inhibits the colonization and growth of HT1080 cells in the lungs of mice, and that the blocking of r37LBP with the specific antibody P4G may offer a potential strategy for preventing cancer metastasis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fibrossarcoma/terapia , Precursores de Proteínas/imunologia , Receptores de Laminina/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Fibrossarcoma/secundário , Humanos , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Peso Molecular , Coelhos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
OBJECTIVE: Because of the relative inaccessibility of the heart for repeated gene therapy, it would be useful to regulate the expression of transgenes delivered in a single dose of a gene therapy vector. Incorporation into the vector of a regulatable promoter that is responsive to pharmacologic agents that are widely used and well tolerated in clinical practice represents such a control strategy. METHODS: A replication-deficient adenovirus or an adeno-associated virus containing a chimeric promoter composed of 5 glucocorticoid response elements and the murine thrombopoietin complementary DNA (AdGRE.mTPO or AAVGRE.mTPO) was administered to the hearts of Sprague-Dawley rats. Platelet levels were evaluated as a reporter of transgene activity with or without dexamethasone. For comparison, rats received a control adenovirus vector, AdCMV.mTPO or AdCMV.Null, and the control adeno-associated virus vector AAVCMV.luc, which encodes for the firefly luciferase (luc) gene. RESULTS: Platelet elevation in the AdGRE.mTPO group peaked 4 days after dexamethasone administration, with a return to baseline 1 week after the initial corticosteroid dose. Subsequent dexamethasone administration at 2 and 4 weeks resulted in similar but progressively decreased responses. The AAVGRE.mTPO group had 5 peak platelet levels to a minimum of 2.2-fold with respect to baseline without diminution with subsequent dexamethasone administrations out to 169 days. In contrast, the AdCMV.Null and AAVCMV.luc groups demonstrated no increase in platelet counts and the AdCMV.mTPO group demonstrated a slow rise to a single peak platelet count independent of dexamethasone administration. CONCLUSION: It may be possible to control on demand the expression of a gene transferred to the heart. This strategy should be useful in cardiac gene therapy.
Assuntos
Anti-Inflamatórios/administração & dosagem , Dexametasona/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Cardiopatias/terapia , Regiões Promotoras Genéticas/genética , Trombopoetina/genética , Transgenes/genética , Animais , Anti-Inflamatórios/farmacocinética , Plaquetas/efeitos dos fármacos , Dependovirus/genética , Dexametasona/farmacocinética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Cardiopatias/genética , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Fatores de TempoRESUMO
Expression of the 37-kDa laminin binding protein (37LBP/p40), a precursor of the 67-kDa laminin receptor, is well-correlated with the biological aggressiveness of cancer cells. To elucidate the direct role played by 37LBP/p40 in cancer cells, a murine lung cancer cell line T11, the 37LBP/p40 expression of which was remarkably diminished, was established by the introduction of the antisense 37LBP/p40-RNA using a retroviral vector. As a result, the population doubling time of T11 was prolonged (60 h) compared with that of P29, the non-transfected parental cell line (42 h), and TN2, a transfectant with vehicle only (40 h). In-vitro studies also showed that T11 cells adhered to immobilized laminin less firmly than P29 cells did. When 5 x 10(5) cells were subcutaneously inoculated into syngenic mice, the mean survival time of T11-recipients (77.0+/-14.8 days) was also significantly prolonged compared with that for P29 (34.8+/-5.5 days) and TN2 (36.7+/-6.1 days) recipients (P < 0.001). The electron-microscopic view of the tumour tissue revealed that T11 cells were loosely apposed and their intercellular space was markedly widened. Some of the T11 cells sporadically degenerated with the infiltration of lymphocytes and neutrophils. These results suggest that the suppressed expression of 37LBP/p40 reduces the capability of lung cancer cell proliferation in vitro and tumour formation in vivo.
Assuntos
Carcinoma Pulmonar de Lewis/fisiopatologia , Adesão Celular , Laminina/biossíntese , RNA Antissenso/genética , Receptores de Laminina/fisiologia , Animais , Carcinoma Pulmonar de Lewis/patologia , Divisão Celular , Laminina/farmacologia , Camundongos , Receptores de Laminina/genética , Transfecção , Células Tumorais CultivadasAssuntos
Antineoplásicos/efeitos adversos , Doenças do Íleo/induzido quimicamente , Leucemia Promielocítica Aguda/tratamento farmacológico , Tretinoína/efeitos adversos , Vasculite/induzido quimicamente , Adulto , Antineoplásicos/uso terapêutico , Toxidermias/etiologia , Feminino , Humanos , Íleo/irrigação sanguínea , Íleo/efeitos dos fármacos , Íleo/patologia , Isquemia/induzido quimicamente , Leucemia Promielocítica Aguda/complicações , Necrose , Pele/irrigação sanguínea , Tretinoína/uso terapêuticoRESUMO
To evaluate the concept that in vivo transfer of perforin complementary DNA (cDNA) will suppress tumor growth, we constructed an adenovirus vector (AdGRE.PFP) carrying perforin cDNA driven by the glucocorticoid response element (GRE) promoter. We infected A549 lung carcinoma cells with this vector in vitro and in vivo, and evaluated cell growth over time. In the presence of dexamethasone, in vitro infection of A549 cells with the AdGRE.PFP vector yielded perforin messenger RNA (mRNA) transcripts and effectively suppressed A549 cell growth. In accord with these in vitro observations, administration of dexamethasone following direct injection of AdGRE. PFP into established subcutaneous A549 tumors in nude mice resulted in a marked reduction in tumor growth as compared with AdGRE.PFP infection without dexamethasone or with dexamethasone alone. These observations suggest that regulable, adenovirus-mediated gene expression of perforin cDNA may have potential as a strategy for local control of tumor cell growth.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Glucocorticoides/farmacologia , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas , Animais , Morte Celular , Divisão Celular , Dexametasona/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos , Células Tumorais CultivadasRESUMO
For many in vivo gene therapy clinical applications, it is desirable to control the expression of the transferred transgene using pharmacologic agents. To evaluate the feasibility of accomplishing this using corticosteroids, pharmacologic agents widely used in clinical medicine, we constructed replication deficient adenoviral (Ad) vectors containing an expression cassette with a chimeric promoter comprised of five glucocorticoid response elements (GRE) and the chloramphenicol acetyltransferase reporter gene (AdGRE.CAT) or the murine thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors functioned as expected, with marked glucocorticoid-induced upregulation of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE promoter in vivo, the AdGRE. CAT vector was administered intravenously to C57B1/6 mice, and CAT activity was quantified in liver before and after intraperitoneal administration of dexamethasone. The GRE promoter activity was dependent on the dexamethasone dose, with a 100-fold increase in CAT expression with 50 microg dexamethasone, similar to the levels observed in vivo with the Rous sarcoma virus long terminal repeat constitutive promoter. After dexamethasone administration, maximum CAT activity was observed at day 2, with a slow decline to baseline levels by 2 weeks. Based on these observations, we hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA would enable repetitive administration of corticosteroids to repetitively upregulate platelet levels for 1 to 2 weeks. The data show that this occurs, with dexamethasone administration every 3 weeks associated with 1-week elevations (at each 3-week interval) of serum mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels fourfold to sixfold over baseline. Thus, with the appropriate promoter, it is possible to use a commonly used pharmacologic agent to upregulate the expression of a newly transferred gene on demand.
Assuntos
Adenovírus Humanos/genética , Plaquetas/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Hematopoese/fisiologia , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/genética , Trombopoetina/genética , Animais , Cloranfenicol O-Acetiltransferase/genética , DNA Complementar/genética , DNA Recombinante/genética , Dexametasona/farmacologia , Feminino , Genes Reporter , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Células HeLa/metabolismo , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Proteínas Recombinantes de Fusão/fisiologia , Trombopoetina/biossíntese , Trombopoetina/fisiologiaRESUMO
Secretory leukoprotease inhibitor (SLPI) is a serine protease inhibitor, produced locally in respiratory and genital glands, but not in the liver. In the present study the promoter region of this gene was analyzed to better understand the molecular mechanisms involved in transcriptional regulation. DNase-I hypersensitive sites were detected within 1 kbp upstream of exon I in chromatin structures of type II pneumocyte cell line A549 and utero-cervical cell line HeLa, both of which express SLPI mRNA transcripts. The function of the SLPI promoter encompassing these DNase-I hypersensitive sites has been studied by deletion analysis with the luciferase gene as a transient expression vector. In this analysis, we found three transcription control regions that function in A549 cells but not in nonlung cell lines, such as HeLa and hepatoma Hep G2. Among three cis-regulatory regions, a proximal 41-bp region (-132 to -92 bp relative to the transcription start site) is responsible for the most striking magnitude of transcriptional activity. This region corresponds to the transcriptional activating sequence detected in another lung cell line, HS-24, indicating that this 41-bp sequence is required for lung cell-specific expression. An electrophoretic mobility shift assay demonstrated that this 41-bp promoter region contains an 11-bp recognition sequence for two nuclear binding proteins, one of which is abundant in lung cell lines, and the other in nonlung cell lines. These results suggest that the ratio of these two nuclear binding proteins confers the cell type specificity on the expression pattern of the SLPI gene.
Assuntos
Pulmão/química , Regiões Promotoras Genéticas/fisiologia , Proteínas/genética , Inibidores de Serina Proteinase/genética , Adenocarcinoma , Sequência de Bases , Carcinoma Hepatocelular , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/farmacologia , Epitélio/química , Epitélio/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Reporter , Teste de Complementação Genética , Células HeLa , Humanos , Luciferases , Pulmão/citologia , Neoplasias Pulmonares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Secretadas Inibidoras de Proteinases , Inibidor Secretado de Peptidases LeucocitáriasRESUMO
We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.
Assuntos
Carcinoma Pulmonar de Lewis/metabolismo , Laminina/química , Laminina/metabolismo , Animais , Ligação Competitiva , Western Blotting , Carcinoma Pulmonar de Lewis/patologia , Membrana Celular/metabolismo , Imunofluorescência , Laminina/genética , Proteínas de Membrana/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Células Tumorais CultivadasRESUMO
We studied the effects of 5 kinds of catechins on the adhesion of mouse lung carcinoma 3LL and melanoma B16F10 cells to the fibronectin substratum. (-)-Epicatechin gallate and (-)-epigallocatechin gallate were active in inhibiting the 3LL cell adhesion, while (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin were inactive. Gallate-containing catechins also impaired adhesion and/or spreading of B16F10 cells.
Assuntos
Catequina/farmacologia , Adesão Celular/efeitos dos fármacos , Fibronectinas/metabolismo , Neoplasias Pulmonares/patologia , Melanoma Experimental/patologia , Animais , Sítios de Ligação , Catequina/análogos & derivados , Camundongos , Células Tumorais CultivadasRESUMO
Laminin receptor polypeptide was immunodetected in human lung cancer with a polyclonal antibody raised against a 20-mer peptide of the putative high-affinity laminin receptor of 67 kDa (Wewer et al: Cancer Res 47: 5691-5698, 1987). As a result, immunoreactivity was recognized specifically in cancer cells both in freshly-prepared cell samples and in paraffin-embedded tissue specimens. It was shown that immunodetection of the laminin receptor polypeptide with this antipeptide antibody could be applied to diagnostic use for lung cancer. The results also suggest that the laminin receptor polypeptide is not necessarily a membrane-associated protein and may function without further processing to the 67 kDa-laminin receptor.
RESUMO
Previously we reported that over 75% of human non-small cell lung cancers overexpress the beta 1 integrin VLA-2 on their surface and show an increase in the mRNA encoding the alpha-2 chain of this integrin. These results suggested the possibility that the overproduction and overexpression of one or more of the beta 1 integrin may be involved in the pathogenesis of human lung tumors by modulating the invasive and/or metastatic potential of the tumor. We report here the generation and characterization of multiple clones of tumor cells derived from the primary culture of cells obtained from biopsy tissue of an aggressive human squamous cell lung tumor. We show that these tumor clones (or clonotypes) exhibit seven different yet stable phenotypes with respect to the expression of five members of the beta 1 integrin family. These results illustrate that a primary human lung tumor consists of multiple subpopulations of cells that while indistinguishable by ultrastructure are heterogeneous with respect to their beta 1 integrins. The availability of these distinct tumor clonotypes derived from a single tumor biopsy have made it possible to test the assumption that the beta 1 integrins play a role in tumor progression. The feasibility of this approach is demonstrated here by the intravenous inoculation of different human tumor clonotypes into severe combined immunodeficient (scid) mice. Our preliminary results with a pair of tumor clonotypes differing in VLA-1 and VLA-2 expression level reveal that the clonotype with high level of VLA-1 and VLA-2 displays a substantial increase in the experimental engraftment and metastasis of the human tumor cells in scid mice.