RESUMO
Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
Assuntos
Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Western Blotting , Regulação para Baixo , Ativação Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Fosfatidiletanolaminas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Processamento Pós-Transcricional do RNA/genética , RNA Antissenso/química , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease H/metabolismo , Ribonuclease P , Especificidade por Substrato , Transfecção/métodos , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteína bcl-X , Pequeno RNA não TraduzidoRESUMO
BACKGROUND: The B-20 study of the National Surgical Adjuvant Breast and Bowel Project (NSABP) was conducted to determine whether chemotherapy plus tamoxifen would be of greater benefit than tamoxifen alone in the treatment of patients with axillary lymph node-negative, estrogen receptor-positive breast cancer. METHODS: Eligible patients (n = 2306) were randomly assigned to one of three treatment groups following surgery. A total of 771 patients with follow-up data received tamoxifen alone; 767 received methotrexate, fluorouracil, and tamoxifen (MFT); and 768 received cyclophosphamide, methotrexate, fluorouracil, and tamoxifen (CMFT). The Kaplan-Meier method was used to estimate disease-free survival, distant disease-free survival, and survival. Reported P values are two-sided. RESULTS: Through 5 years of follow-up, chemotherapy plus tamoxifen resulted in significantly better disease-free survival than tamoxifen alone (90% for MFT versus 85% for tamoxifen [P = .01]; 89% for CMFT versus 85% for tamoxifen [P = .001]). A similar benefit was observed in both distant disease-free survival (92% for MFT versus 87% for tamoxifen [P = .008]; 91% for CMFT versus 87% for tamoxifen [P = .006]) and survival (97% for MFT versus 94% for tamoxifen [P = .05]; 96% for CMFT versus 94% for tamoxifen [P = .03]). Compared with tamoxifen alone, MFT and CMFT reduced the risk of ipsilateral breast tumor recurrence after lumpectomy and the risk of recurrence at other local, regional, and distant sites. Risk of treatment failure was reduced after both types of chemotherapy, regardless of tumor size, tumor estrogen or progesterone receptor level, or patient age; however, the reduction was greatest in patients aged 49 years or less. No subgroup of patients evaluated in this study failed to benefit from chemotherapy. CONCLUSIONS: Findings from this and other NSABP studies indicate that patients with breast cancer who meet NSABP protocol criteria, regardless of age, lymph node status, tumor size, or estrogen receptor status, are candidates for chemotherapy.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/uso terapêutico , Receptores de Estrogênio , Tamoxifeno/uso terapêutico , Antimetabólitos Antineoplásicos/administração & dosagem , Antineoplásicos Alquilantes/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Humanos , Metástase Linfática , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Receptores de Estrogênio/efeitos dos fármacos , Risco , Análise de Sobrevida , Falha de Tratamento , Resultado do TratamentoRESUMO
PURPOSE: To compare sequential methotrexate (M) and fluorouracil (F) (M-->F) with surgery (National Surgical Adjuvant Breast and Bowel Project [NSABP] B-13) and cyclophosphamide (C), M, and F with M-->F (NSABP B-19), in patients with estrogen receptor (ER)-negative tumors and negative axillary nodes. PATIENTS AND METHODS: A total of 760 patients were randomized to B-13; 1,095 patients with the same eligibility requirements were randomized to B-19. Disease-free survival (DFS), distant disease-free survival (DDFS), and survival were determined using life-table estimates. RESULTS: A significant benefit in overall DFS (74% v 59%; P < .001) was demonstrated at 8 years in all B-13 patients who received M-->F (69% v 56% [P = .006] in those
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores de Estrogênio/análise , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/química , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Leucovorina/administração & dosagem , Metástase Linfática , Mastectomia , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia , Taxa de SobrevidaRESUMO
The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta 1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta 1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-beta 1. Simian beta 1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.
Assuntos
Fragmentos de Peptídeos , Precursores de Proteínas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células CHO/metabolismo , Carboidratos/análise , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Precipitação Química , Cromatografia por Troca Iônica , Dicroísmo Circular , Cricetinae , DNA Complementar/genética , Amplificação de Genes , Pulmão/citologia , Pulmão/efeitos dos fármacos , Manosefosfatos/análise , Vison , Estrutura Secundária de Proteína , Proteínas/fisiologia , Tetra-Hidrofolato Desidrogenase/genética , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
Threonyl-tRNA synthetase was purified approximately 500-fold from a high-speed supernatant fraction of rat liver. The purified enzyme was estimated to be > 95% pure from acrylamide gel electrophoresis under denaturing and nondenaturing conditions. Based on a native molecular weight from sedimentation equilibrium of 154000 and a subunit molecular weight of 85000 obtained bo sodium dodecyl sulfate gel electrophoresis, the protein appears to be an alpha 2 dimer. The alpha 2 structure was also supported by cross-linking studies of the native enzyme. The purified protein has an S20,w of 7.2 and an isoelectric point of 6.4. Amino acid analyses revealed no unusual features, but attempts at automated sequence analyses suggested that both amino termini are blocked. Preliminary carbohydrate analyses suggested that the enzyme is a glycoprotein. Antibodies were raised against the purified protein which could inactivate and precipitate threonyl-tRNA synthetase.