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1.
Mol Ther ; 10(1): 150-61, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15233950

RESUMO

We recently published the construction and evaluation of a beta-catenin-dependent, highly active promoter, CTP1, and its possible application for the treatment of colorectal cancer using gene-directed enzyme prodrug therapy with adenoviral (Ad) vectors. Alternative Ad-based approaches such as tumor-specific, replication-competent vectors and/or exploiting therapeutic gene products with intrinsic toxic activity, such as gibbon ape leukemia virus fusogenic membrane glycoprotein, diphtheria toxin A (DTA), and ricin, would demand a very tightly regulated promoter to avoid breakthrough replication and toxicity in nontumor tissue and Ad producer cell lines. In this study we optimized the activity/specificity profile of the synthetic beta-catenin-dependent promoter by varying its basal promoter, the number of Tcf binding sites, and the distance between these and the basal promoter. The optimal promoter, CTP4, showed virtually undetectable expression in cells with normal beta-catenin regulation but high level expression in cells deregulated for beta-catenin. Using CTP4 we were able to generate, for the first time to our knowledge, an Ad vector expressing fully active wild-type DTA without the need for time-consuming and cumbersome production systems. CTP4 should be the promoter of choice for Ad-based gene therapies of tumors deregulated for beta-catenin. We provide preliminary evidence that these may include prostate and ovarian as well as colorectal cancer.


Assuntos
Adenoviridae/genética , Proteínas do Citoesqueleto/metabolismo , Toxina Diftérica/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias/terapia , Fragmentos de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo , Sítios de Ligação , Carcinoma/química , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Toxina Diftérica/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Neoplasias Ovarianas/química , Neoplasias Ovarianas/metabolismo , Fragmentos de Peptídeos/metabolismo , Neoplasias da Próstata/química , Neoplasias da Próstata/metabolismo , TATA Box/genética , Fatores de Transcrição TCF , Transativadores/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina
2.
J Virol ; 76(9): 4441-55, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11932411

RESUMO

Human cytomegalovirus (HCMV) ie1 deletion mutant CR208 is profoundly growth deficient after low-multiplicity infection of primary fibroblasts. Previously, we showed that many fewer cells infected with CR208 at low multiplicity accumulated the delayed-early (DE) protein ppUL44 than accumulated the immediate-early 2 (IE2) p86 protein, indicating a high frequency of abortive infections. We now demonstrate that accumulation of all DE proteins tested was defective after low-multiplicity infection in the absence of IE1 p72. Accumulation of the DE proteins pUL57, pUL98, and pUL69 followed a pattern very similar to that of ppUL44 during low-multiplicity CR208 infection. Accumulation of the ppUL112-113 proteins occurred in a greater proportion of cells than other DE proteins during low-multiplicity CR208 infection, but was still deficient relative to wild-type virus. We also show for the first time that steady-state levels of many DE RNAs were reduced during low-multiplicity CR208 infection and that by in situ hybridization of the abundant cytoplasmic 2.7-kb TRL4 DE (beta2.7) RNA, a viral DE RNA followed a defective pattern of accumulation similar to that of ppUL44. Furthermore, transfected DE promoter-reporter constructs were found in transient assays to be considerably less responsive to CR208 infection than to infection by wild-type Towne virus. Our results indicate a general defect in DE gene expression following low-multiplicity HCMV infection in the absence of functional IE1 p72, most probably mediated by reduced transcription of DE genes and by the reduced accumulation of DE RNAs.


Assuntos
Citomegalovirus/patogenicidade , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Mutação , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/genética , Efeito Citopatogênico Viral , Humanos , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , RNA Viral/genética , RNA Viral/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Proteínas Virais/genética
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