RESUMO
There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.
Assuntos
Processamento Alternativo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/genética , Precursores de RNA/metabolismo , Neoplasias da Mama , Morte Celular , Feminino , Citometria de Fluxo , Genes Reguladores , Variação Genética , Humanos , Masculino , Neoplasias da Próstata , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Precursores de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tapsigargina/farmacologia , Transfecção , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
It has been estimated that greater than 35% of all human genes undergo alternative splicing. The process of alternative splicing is highly regulated and disruption of a splicing pattern can produce splice variants that have different functions. Certain splice variants that are associated with induction of cell death, regulation of cellular proliferation and differentiation, cell signaling, and angiogenesis are present in a variety of cancers. Several of these cancer-related alternatively spliced genes will be discussed in this review. In addition, alternative splicing is associated with several genetic disorders such as beta-thalassemia, cystic fibrosis, and muscular dystrophy. Control of pre-mRNA splicing patterns with antisense oligonucleotides presents an attractive way to potentially treat and manage a variety of diseases. This review will discuss potential gene targets for antisense oligonucleotide induced modification of alternative splicing patterns. Furthermore, the chemistries and delivery strategies of antisense oligonucleotides will be discussed.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos Antissenso/farmacologia , Processamento Alternativo/genética , Animais , Fibrose Cística/genética , Fibrose Cística/terapia , Desenho de Fármacos , Genes Supressores de Tumor , Humanos , Distrofias Musculares/genética , Distrofias Musculares/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Oncogenes/genética , RNA Mensageiro/genética , Talassemia beta/genética , Talassemia beta/terapiaRESUMO
The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.
