RESUMO
Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.
Assuntos
Adenoviridae/genética , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , DNA Polimerase Dirigida por DNA/genética , Vírus Oncolíticos/fisiologia , Regiões Promotoras Genéticas/genética , Replicação Viral , Proteínas E1A de Adenovirus/efeitos dos fármacos , Proteínas E1A de Adenovirus/genética , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Doxiciclina/farmacologia , Engenharia Genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Células HeLa , Humanos , Vírus Oncolíticos/genéticaRESUMO
p73 has been shown to transcriptionally activate genes positively responsive to wild-type p53. In order to undertake a comparative study of functions of p53 and p73 we have cloned the cDNA of p73 from MCF-7 cells. Adenovirus onco-protein E1A inhibits the transactivation by p73; a deletion mutant of E1A incapable of interacting with p300 and CREB-binding protein (CBP) fails to disrupt the transactivation. Furthermore, CBP increases the transactivation mediated by p73 suggesting that CBP may function as a co-activator and E1A inhibits p73-mediated transactivation by sequestering p300 or CBP. We show that p73 can transcriptionally inhibit a number of cellular and viral promoters. However, wild-type p53, p73 alpha and p73 beta differ in their ability to inhibit transcriptional activity of different promoters. While wild-type p53 inhibits the promoters of the human cytomegalovirus (CMV) immediate-early gene, the long terminal repeat of human immunodeficiency virus type 1 (HIV LTR), human cyclin A (cyc A) gene, and insulin-like growth factor receptor I (IGF-I-R), p73 alpha only inhibits the HIV LTR and cyc A promoters significantly; and p73 beta inhibits the CMV, HIV LTR and cyc A promoters. A mutant of p73 alpha having amino acid substitutions at positions 268 and 300 on the presumptive DNA-binding domain fails to transactivate the p21 promoter but represses the CMV and the HIV LTR promoter quite efficiently showing that the mechanisms of transactivation and repression by p73 are different. Interestingly, p73 alpha transactivates the IGF-I-R promoter, which is inhibited by wild-type p53; p73 beta has no significant effect on this promoter. This is a unique situation where p73 alpha differs from p73 beta as well as p53.
Assuntos
Proteínas E1A de Adenovirus/efeitos dos fármacos , Antígenos Virais/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Proteínas Imediatamente Precoces/efeitos dos fármacos , Proteínas Nucleares/farmacologia , Receptor IGF Tipo 1/efeitos dos fármacos , Proteína Supressora de Tumor p53/farmacologia , Proteínas rho de Ligação ao GTP/efeitos dos fármacos , Proteínas E1A de Adenovirus/metabolismo , Animais , Antígenos Virais/metabolismo , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Vetores Genéticos/farmacologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Ratos , Receptor IGF Tipo 1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucocorticoides/metabolismo , Proteínas do Leite , Prolactina/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Proteínas E1A de Adenovirus/efeitos dos fármacos , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Sítios de Ligação , Células COS/efeitos dos fármacos , Cricetinae , Proteínas de Ligação a DNA/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Histona Acetiltransferases , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Coativador 3 de Receptor Nuclear , Fosforilação , Prolactina/farmacologia , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição STAT5 , Sequências Repetidas Terminais , Transativadores/genética , Tirosina/metabolismoRESUMO
Cellular transformation by the adenovirus E1A oncoprotein requires its p300/CBP- and Rb-binding domains. We mapped inhibition of p53-mediated transactivation to the p300/CBP-binding region of E1A. An E1A mutant incapable of physically interacting with Rb retained the capacity to inhibit transactivation by p53, whereas E1A mutants of the p300/CBP-interacting domain failed to inhibit p53. The inhibitory effect of the p300/CBP-binding region of E1A on p53 was demonstrated with p53-activated reporters and endogenous p53 targets such as p21(WAF1/CIP1) or MDM2. E1A lacking the capacity to interact with Rb, but capable of p300/CBP interaction, was competent in suppression of a DNA-damage activated p53-dependent cell cycle checkpoint. Exogenous CBP and p300 were able to individually relieve E1A's inhibitory effect on p53-mediated transcription. Mutants of E1A that are not capable of interacting with p300 or CBP were found to efficiently stabilize endogenous p53 but were not competent in repression of p21 expression thus dissociating these two effects of E1A. Our results suggest that the p300/CBP-binding domain of E1A inhibits a p53-dependent cellular response which normally inhibits DNA replication following Adenovirus infection.
Assuntos
Acetiltransferases , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ciclo Celular/fisiologia , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1A de Adenovirus/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/efeitos dos fármacos , Ciclinas/metabolismo , Dano ao DNA , DNA de Neoplasias/metabolismo , Doxorrubicina/farmacologia , Inibidores Enzimáticos/metabolismo , Etoposídeo/farmacologia , Histona Acetiltransferases , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Retinoblastoma/metabolismo , Fatores de Transcrição , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Fatores de Transcrição de p300-CBPRESUMO
Squamous cell carcinomas can show different oncogenic alterations, histological patterns, and an unpredictable clinical behavior. We previously reported that the adenovirus E1a gene may induce sensitivity to DNA-damaging agents in mouse keratinocytes. In order to study whether E1a expression could be used as a therapeutic agent in different malignant cell lines carrying mutations on the p53 gene and other oncogenic alterations, we transfected and infected several murine and human carcinoma cell lines (HaCa4; MSC11A5; HeLa) with vectors containing the 13S or 12S E1a region. We evaluated the sensitivity to cisplatin (CDDP), doxorubicin (DOX) and gamma irradiation (RX) by the crystal violet method. The induction of apoptosis was assessed by flow cytometry and presence of DNA ladders in agarose gels. The expression of E1a and the tumor suppressor p53 protein was analysed by Western blotting. The carcinoma cell lines expressing E1a were about four- to tenfold more sensitive to CDDP and RX, respectively, than the control cells. Moreover, the reduction in cell viability and cell growth after exposure to CDDP or RX was very significant in the carcinoma cells expressing E1a. With these results, we conclude that expression of E1a may confer great sensitivity to DNA-damaging agents on squamous cell carcinoma cells independently of the p53 protein status and other oncogenic alterations.
