RESUMO
Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.
Assuntos
Adenocarcinoma/virologia , Carcinoma Ductal Pancreático/virologia , Parvovirus H-1/fisiologia , Neoplasias Pancreáticas/virologia , Infecções por Parvoviridae/virologia , Proteína Smad4/genética , Proteína Smad4/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/secundário , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Luciferases/metabolismo , Mutação/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/patologia , Prognóstico , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad4/antagonistas & inibidores , Transfecção , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.
Assuntos
Vetores Genéticos , Vírus Miúdo do Camundongo/patogenicidade , Recombinação Genética , Vírion/patogenicidade , Montagem de Vírus , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Humanos , Camundongos , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/fisiologia , Plasmídeos , Transfecção , Ensaio de Placa Viral , Vírion/genética , Vírion/metabolismo , Replicação ViralRESUMO
Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Neoplasias/terapia , Parvovirus/fisiologia , Replicação Viral , Humanos , Neoplasias/genéticaRESUMO
Vector and helper plasmids for the production of recombinant H1 (rH1) parvovirus, an oncolytic virus and candidate vector for cancer gene therapy, were constructed with the aim of reducing the contamination of these preparations with replication-competent viruses (RCV). Split-helper plasmids were constructed by manipulating the splicing signals for the capsid proteins such that VP1 and VP2 were expressed from separate plasmids. H1 vectors with similarly mutated splice sites were packaged, using the split-helper plasmids, and the resulting recombinant H1 viruses were completely free of RCV because the generation of recombinants expressing both capsid proteins was prevented. Vector yields of rH1 produced with split-helper plasmids in combination with splice site-modified vectors were similar (in the range of 10(7) replication units/ml) to yields of rH1 produced with the standard vector/helper pair, in which case significant levels of RCV were generated (10(4)-10(5) plaque-forming units/ml). To assess the functionality of this approach in vivo, rH1 was produced that contained the human interleukin 2 (IL-2) transgene and that was devoid of RCV. This IL-2-carrying rH1 vector expressed IL-2 efficiently in human tumor cells (HeLa) in vitro and generated antitumor responses in nude mice xenografted with HeLa cells that had been infected ex vivo with this virus. These results should allow the large-scale production of recombinant oncotropic parvoviruses and their assessment for the gene therapy of cancer in a clinical setting.
