RESUMO
Myxoid/round cell (RC) liposarcomas (MLS) were originally classified into two distinct populations based on histological differences; a myxoid component and a RC component. It is notable that, depending on an increase of the RC component, the prognosis significantly differs. Hence, the RC component is associated with metastasis and poor prognosis. However, the molecular mechanisms that contribute to the malignancy of the RC component still remain largely unknown. Here, we report microRNA-135b (miR-135b), a key regulator of the malignancy, highly expressed in the RC component and promoting MLS cell invasion in vitro and metastasis in vivo through the direct suppression of thrombospondin 2 (THBS2). Decreased THBS2 expression by miR-135b increases the total amount of matrix metalloproteinase 2 (MMP2) and influences cellular density and an extracellular matrix structure, thereby resulting in morphological change in tumor. The expression levels of miR-135b and THBS2 significantly correlated with a poor prognosis in MLS patients. Overall, our study reveals that the miR-135b/THBS2/MMP2 axis is tightly related to MLS pathophysiology and has an important clinical implication. This work provides noteworthy evidence for overcoming metastasis and improving patient outcomes, and sheds light on miR-135b and THBS2 as novel molecular targets for diagnosis and therapy in MLS.
Assuntos
Regulação Neoplásica da Expressão Gênica , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/mortalidade , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Inativação Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipossarcoma Mixoide/patologia , Neoplasias Pulmonares/secundário , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Metástase Neoplásica , Prognóstico , Interferência de RNA , Trombospondinas/genética , Carga TumoralRESUMO
BACKGROUND: Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC). METHODS: Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT-PCR. RESULTS: Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001). CONCLUSIONS: Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.
Assuntos
Neoplasias Colorretais/diagnóstico , Exossomos , MicroRNAs/sangue , Recidiva Local de Neoplasia/diagnóstico , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prognóstico , RNA Longo não CodificanteRESUMO
BACKGROUND: Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence. METHODS: We employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines. RESULTS: We found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recurrence. Decreased expression of miR-718 was associated with HCC tumour aggressiveness in the validated cohort series. We identified HOXB8 as a potential target gene of miR-718, and its upregulation was associated with poor prognosis. CONCLUSION: Circulating miRs in serum exosomes have potential as novel biomarkers for predicting HCC recurrence.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Transplante de Fígado , MicroRNAs/sangue , Adulto , Idoso , Carcinoma Hepatocelular/cirurgia , Células Cultivadas , Exossomos , Feminino , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Falha de Tratamento , Adulto JovemRESUMO
We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets. It is still unknown whether milk-derived RNA molecules play biological roles in infants; however, if milk-derived RNA do show functions in infants, our data will help guide future studies.
Assuntos
MicroRNAs/análise , Leite/química , RNA Mensageiro/análise , Animais , Bovinos , Colostro/química , Feminino , Humanos , Lactente , Fórmulas Infantis/química , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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
It is reported that neural stem cells (NSC) can arrest denervated muscle atrophy and promote nerve regeneration when transplanted into injured peripheral nerves, and that regenerated host axons can form synapses with transplanted and differentiated NSC. In this study, F344 rat nerve segments and F344 rat NSC were transplanted into host green fluorescence protein (GFP) transgenic F344 rats. This allowed transplanted F344 rat tissue to be used as a nonluminous background for the clear visualization of regenerated host GFP axons. Regenerated host axons grew into the transplanted F344 nerve segment 2 weeks after nerve anastomosis. Immunohistochemical staining and confocal microscope analysis revealed that regenerated host axons formed synapses with NSC-derived neurons. The findings confirmed that regenerated peripheral axons form synapses with neurons in peripheral nerves, possibly forming the basis for clinical application in peripheral nerve injury.
Assuntos
Axônios/transplante , Regeneração Nervosa/fisiologia , Células-Tronco Neurais/transplante , Neurônios/transplante , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Feminino , Técnicas Imunoenzimáticas , Masculino , Neurônios/citologia , Ratos , Ratos Endogâmicos F344RESUMO
To study the role of mutant p53 in the induction and cure of tumors, we generated transgenic mice carrying mutant p53 (mp53) containing a 9 bp deletion in exon 6 in addition to wild-type p53, expressing both p53 and mp53. The mp53 cDNA was cloned from a radiation-induced mouse tumor and ligated to the chicken beta-actin promoter/CMV-IE enhancer in the expression vector. The presence of mp53 suppressed p21 expression in primary fibroblasts after ionizing irradiation, indicating the dominant-negative activity of mp53 in the mice. These mice developed fibrosarcomas after the subcutaneous injection of 3-methylcholanthrene with an incidence 1.7-fold higher than that of wild-type mice (42% excess). The tumors were then treated via a potent atelocollagen delivery system with small interfering RNA (siRNA), that targeted the promoter/enhancer of the expression vector, resulting in the suppression of tumor growth in 30% of 44 autochthonous tumors, including four cures, and their transplants, the total fraction corresponding to the tumor excess. This suppressive effect involved the induction of apoptosis. These results indicate that mp53 activity causes tumors that can be suppressed by subsequent silencing of mp53 in the presence of wild-type p53 alleles.
Assuntos
Fibrossarcoma/induzido quimicamente , Fibrossarcoma/genética , Genes p53 , Mutação , RNA Interferente Pequeno/genética , Animais , Apoptose/genética , Western Blotting , Feminino , Inativação Gênica , Predisposição Genética para Doença , Masculino , Metilcolantreno , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.
Assuntos
Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos da radiação , Glicosídeo Hidrolases/fisiologia , Animais , Antineoplásicos/farmacologia , Northern Blotting , Western Blotting , Células Cultivadas , Cisplatino/farmacologia , Ensaio de Unidades Formadoras de Colônias , Raios gama , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfetos/farmacologiaRESUMO
Cholangiocarcinoma is an intractable cancer, with no effective therapy other than surgical resection. Elevated vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expressions are associated with the progression of cholangiocarcinoma. We therefore examined whether inhibition of VEGFR and EGFR could be a potential therapeutic target for cholangiocarcinoma. Vandetanib (ZD6474, ZACTIMA), a VEGFR-2/EGFR inhibitor, was evaluated. Four human cholangiocarcinoma cell lines were molecularly characterised and investigated for their response to vandetanib. In vitro, two cell lines (OZ and HuCCT1), both of which harboured KRAS mutation, were refractory to vandetanib, one cell line (TGBC24TKB) was somewhat resistant, and another cell line (TKKK) was sensitive. The most sensitive cell line (TKKK) had EGFR amplification. Vandetanib significantly inhibited the growth of TKKK xenografts at doses > or = 12.5 mg kg(-1) day(-1) (P<0.05), but higher doses (50 mg kg(-1) day(-1), P<0.05) of vandetanib were required to inhibit the growth of OZ xenografts. Vandetanib (25 mg kg(-1) day(-1)) also significantly (P=0.006) prolonged the time to metastasis in an intravenous model of TKKK metastasis. Inhibiting both VEGFR and EGFR signalling appears a promising therapeutic approach for cholangiocarcinoma. The absence of KRAS mutation and the presence of EGFR amplification may be potential predictive molecular marker of sensitivity to EGFR-targeted therapy in cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Piperidinas/uso terapêutico , Quinazolinas/uso terapêutico , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Japão , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, noncoding small RNAs that act as post-transcriptional gene regulators. Experimental evidence has shown that miRNAs can play roles as oncogenes or tumor suppressor genes, suggesting their contribution to cancer development and progression. Expression profiles of human miRNAs demonstrated that many miRNAs are deregulated in cancers and are differentially expressed in normal tissues and cancers. Therefore, miRNA profiling is used to create signatures for a variety of cancers, indicating that the profile will help further establish molecular diagnosis, prognosis and therapy using miRNAs. This paper introduces the aberrant expression of miRNAs in human cancer, and discusses the potential of these miRNAs as biomarkers and targets/molecules for molecular therapy.
Assuntos
MicroRNAs , Neoplasias , Antineoplásicos/uso terapêutico , Biomarcadores/análise , Biomarcadores Tumorais/análise , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , MicroRNAs/análise , MicroRNAs/genética , MicroRNAs/uso terapêutico , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológicoRESUMO
AIMS/HYPOTHESIS: It appears that the adult pancreas has limited regenerative ability following beta cell destruction by streptozotocin (STZ). However, it is not clear if this limitation is due to an inability to respond to, rather than an absence of, regenerative stimuli. In this study we aimed to uncouple the regenerative signal from the regenerative response by using an exogenous stem cell source to detect regenerative stimuli produced by the STZ-injured pancreas at physiological blood glucose levels. METHOD: Adult nude mice received 150 mg/kg STZ and 1x10(6) J1 mouse embryonic stem (ES) cells by i.p. injection. Permanent beta cell depletion of 50% was estimated from the ratio of beta:alpha cells in pancreata from STZ-treated mice compared with control animals after 24 days. RESULTS: Transplanted ES cells homed to the STZ-injured pancreas and formed tumours. Immunocytochemical analysis of pancreas-associated ES tumours revealed foci containing insulin/PDX-1 double-positive and glucagon-positive/PDX-1-negative cell clusters associated with PDX-1-positive columnar lumenal epithelium and extensive alpha-amylase-positive pancreatic acini comprising approximately 0.1% of ES tumour volume. CONCLUSIONS/INTERPRETATION: These data indicate that (1) the adult pancreas produces a milieu of regenerative stimuli following beta cell destruction, and (2) this is not dependent on hyperglycaemic conditions; (3) these regenerative stimuli appear to recapitulate the signalling pathways of embryonic development, since both exocrine and endocrine lineages are produced from PDX-1-positive precursor epithelium. This model will be useful for characterising the regenerative mechanisms in the adult pancreas.
Assuntos
Células-Tronco Embrionárias/transplante , Células Secretoras de Insulina/citologia , Pâncreas/crescimento & desenvolvimento , Estreptozocina/farmacologia , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Divisão Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Nus , MorfogêneseRESUMO
To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Endorribonucleases/metabolismo , Impressão Genômica , RNA Helicases/genética , Canais de Cátion TRPC/metabolismo , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/genética , RNA Helicases DEAD-box , Primers do DNA , Endorribonucleases/genética , Regulação Enzimológica da Expressão Gênica , Genótipo , Íntrons , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA Helicases/metabolismo , Ribonuclease III , Canais de Cátion TRPC/genéticaRESUMO
Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.
Assuntos
Neuropatias Amiloides/terapia , Marcação de Genes/métodos , Terapia Genética/métodos , Pré-Albumina/genética , Neuropatias Amiloides/genética , Animais , Sequência de Bases , Colágeno/genética , Reparo do DNA/genética , Conversão Gênica , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Oligonucleotídeos/genética , TransfecçãoRESUMO
We have previously demonstrated that Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery. To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by precoating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded. The complexes with a nanoparticle form were efficiently transduced into cells without use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.
Assuntos
Colágeno , Técnicas de Transferência de Genes , Adenoviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Portadores de Fármacos , Perfilação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Camundongos , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/genética , Análise de Sequência com Séries de Oligonucleotídeos , TransfecçãoRESUMO
Poly(ADP-ribose) polymerase (Parp) monitors DNA strand breaks and poly(ADP-ribosyl)ates nuclear proteins using NAD as a substrate. The participation of Parp in DNA damage responses has been demonstrated by recent studies using Parp knockout mice. On the other hand, accumulated evidence has shown that Parp is involved in the regulation of gene expression and cell differentiation. In this study, the role of Parp in tumorigenesis and differentiation was studied with Parp-/- embryonic stem (ES) cells. When Parp+/+, Parp+/-, and Parp-/- ES cells were injected subcutaneously into nude mice, teratocarcinoma-like tumors developed from ES cells. However, only tumors derived from Parp-/- ES cells showed trophoblast giant cells (TGCs) containing single or multiple megalo-nuclei. These TGCs are located in a large blood-lake like hemorrhage. This example suggests that Parp is not essential for tumor formation, however, it is involved in trophoblastic cell differentiation and could consequently affect tumor phenotype.
Assuntos
Diferenciação Celular , Transformação Celular Neoplásica , Poli(ADP-Ribose) Polimerases/fisiologia , Trofoblastos/patologia , Animais , Camundongos , Células-Tronco , Trofoblastos/citologiaRESUMO
A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.
Assuntos
Carcinógenos/toxicidade , Genes ras , Neoplasias Mamárias Experimentais/genética , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia/toxicidade , Polimorfismo Conformacional de Fita Simples , Proto-Oncogene Mas , RatosRESUMO
Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.
Assuntos
Materiais Biocompatíveis , Colágeno , Terapia Genética/métodos , Adenoviridae/genética , Animais , Materiais Biocompatíveis/economia , Biotecnologia , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Preparações de Ação Retardada , Sistemas de Liberação de Medicamentos , Implantes de Medicamento , Técnicas de Transferência de Genes , Terapia Genética/economia , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Marketing de Serviços de Saúde , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , SegurançaRESUMO
The endothelial-specific receptor tyrosine kinase flt-1 (VEGFR-1) is expressed early on during endothelial lineage commitment both in vivo and in vitro. However, the exact function of flt-1 in vascular development still remains unclear. Here we report that a 2.2-kb fragment 5' of the mouse flt-1 gene becomes transcriptionally active during endothelial cell differentiation in developing embryoid bodies derived from mouse ES cells. Reporter gene expression correlated well with PECAM-1 expression and mirrored the expression pattern of the endogenous flt-1 gene. The temporal and spatial activity of the 2.2-kb flt-1 promoter provides a means to (1) identify a living population of early committed endothelial/bipotential progenitors and (2) ectopically express biologically active genes during lineage commitment.
Assuntos
Embrião de Mamíferos/metabolismo , Endotélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Processamento Alternativo/genética , Animais , Biomarcadores/análise , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Clonagem Molecular , Embrião de Mamíferos/citologia , Endotélio/citologia , Citometria de Fluxo , Inativação Gênica , Genes Dominantes/genética , Genes Reporter/genética , Genoma , Camundongos , Dados de Sequência Molecular , Mutação/genética , Especificidade de Órgãos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Receptores de LDL/metabolismo , Recombinação Genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Transfecção , Células Tumorais Cultivadas , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
HST-1, a member of the fibroblast growth factor (FGF) family (FGF-4), has been shown to be a signaling molecule whose expression is essential for embryonic development. However, HST-1/FGF-4 expression has not been detected or reported in adult tissues so far analysed. To investigate whether there is a possible role of HST-1/FGF-4 in adult stage, we have carried out a highly sensitive RT-PCR analysis of Hst-1/Fgf-4 gene expression in adult mice tissues. Results show Hst-1/Fgf-4 gene expression in the nervous system, intestines, and testis of normal adult mice. In situ hybridization technique was used to localize Hst-1/Fgf-4 gene expression in the cerebellum and testis from 10-week-old mice. Cell type-specific gene expression was detected: Purkinje cells in the cerebellum and Sertoli cells in testis. These findings suggest that the Hst-1/Fgf-4 gene also plays an important role in adult tissues, and may offer insights into the biological significance of HST-1/FGF-4 in cerebellar and testicular functions.
Assuntos
Encéfalo/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Testículo/metabolismo , Células 3T3 , Animais , Encéfalo/embriologia , Células Cultivadas , Feminino , Fator 4 de Crescimento de Fibroblastos , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , RNA Mensageiro , Testículo/embriologia , Distribuição TecidualRESUMO
We have established a transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene with its own original promoter region, Jcl/SD-TgN(HrasGen)128Ncc (Hras128) rat. c-Ha-ras protein from expression of transduced and endogenous c-Ha-ras genes could be detected in the bladder epithelium of untreated transgenic rats. To examine their susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis, male transgenic and wild-type littermates were treated with 0.05% BBN in their drinking water for 10 weeks and then killed at week 20. The numbers and volumes of total macroscopic bladder tumors including both transitional cell papillomas and carcinomas (TCC) per rat were much greater in Hras128 rats than in their wild-type counterparts. The numbers of carcinomas per rat were also significantly greater in Hras128 rats. Two cases of TCC exhibiting invasion of the bladder muscle layer, which is extremely rare in the wild-type animals under the experimental conditions used, were also observed in Hras128 rats. The GGC-->GAC mutations at codon 12 of the transgene were observed in only two TCC out of 21 bladder tumors (9.5%), assessed by RFLP analysis and direct sequencing. SSCP analysis did not show any endogenous c-Ha-ras gene mutations. One of 25 tumors (4.0%) in wild-type rats had an endogenous c-Ha-ras gene mutation at codon 12 that was detected (GGA-->GAA) by single-strand conformation polymorphism and direct sequencing. These results indicate that the Hras128 rat is highly susceptible to BBN carcinogenesis and may be utilized as a rat model for analysis of bladder tumor development. The mutation findings indicate that the enhanced tumor development is not primarily due to mutations occurring in the transgene.