RESUMO
The cellular Brn-3a transcription factor is known to activate transcription of the genes encoding the human papilloma virus E6 and E7 proteins and is over-expressed in women with cervical neoplasia. We show that cervical cell lines with reduced Brn-3a expression show a greatly reduced ability to form tumours in nude mice compared to control cells and also show reduced expression of the HPV E6 and cellular Bcl-2 oncogenes. These effects are also observed in cervical cells over-expressing the related Brn-3b factor, which is known to antagonize activation of HPV gene expression by Brn-3a. These results demonstrate, for the first time, that inhibition of Brn-3a expression or enhanced Brn-3b expression can inhibit cervical cell-derived tumour growth in vivo as well as in vitro. Hence they establish Brn-3a as a key factor in cervical tumorigenesis and as a potential therapeutic target in human cervical neoplasia.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Neoplasias do Colo do Útero/patologia , Divisão Celular , Proteínas de Ligação a DNA/análise , Feminino , Humanos , Papillomaviridae/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/análise , Fator de Transcrição Brn-3 , Fator de Transcrição Brn-3A , Fator de Transcrição Brn-3B , Fatores de Transcrição/análise , Neoplasias do Colo do Útero/virologiaRESUMO
Human immunoglobulin G4 (IgG4) exists in two molecular forms due to the heterogeneity of the inter-heavy chain disulphide bridges in the hinge region in a proportion of secreted human IgG4. This heterogeneity is only revealed under denaturing, non-reducing conditions in which an HL "half antibody" is detected, a phenomenon not seen in other human IgG isotypes. In native conditions noncovalent interactions hold the antibody together as the H2L2 tetramer. Analysis of the hinge sequences of human IgG heavy chains suggested that the presence of serine at residue 241 might be the cause of this heterogeneity. We therefore changed the serine at 241 to proline (found at that position in IgG1 and IgG2) in a mouse/human chimeric heavy chain. This single residue substitution leads to the production of a homogeneous antibody. Further, the variant IgG4 has significantly extended serum half-life and shows an improved tissue distribution compared to the original chimeric IgG4.
Assuntos
Imunoglobulina G/genética , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Ligação Competitiva , Linhagem Celular , Cricetinae/genética , Relação Dose-Resposta Imunológica , Eletroforese em Gel de Poliacrilamida , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes de Fusão , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.