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Functional repair of p53 mutation in colorectal cancer cells using trans-splicing.

He X, Liao J, Liu F, Yan J, Yan J, Shang H, Dou Q, Chang Y, Lin J, Song Y - Oncotarget (2015)

Bottom Line: The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously.Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo.In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT
Mutation in the p53 gene is arguably the most frequent type of gene-specific alterations in human cancers. Current p53-based gene therapy contains the administration of wt-p53 or the suppression of mutant p53 expression in p53-defective cancer cells. . We hypothesized that trans-splicing could be exploited as a tool for the correction of mutant p53 transcripts in p53-mutated human colorectal cancer (CRC) cells. In this study, the plasmids encoding p53 pre-trans-splicing molecules (PTM) were transfected into human CRC cells carrying p53 mutation. The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously. Intratumoral administration of adenovirus vectors carrying p53 trans-splicing cassettes suppressed the growth of tumor xenografts. Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo. In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

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Related in: MedlinePlus

Schematic illustration of trans-splicing used for the correction of mutant p53 transcripts and the detection of trans-spliced p53 RNA in transfected cellsA. the structure of p53 pre-trans-splicing molecules (PTM). The hybridization domain is antisense to intron 7 of p53 pre-mRNA. BD-A and BD-B differ only in their binding regions, domain B was more efficient than domain A in promoting trans-splicing. BD, binding domain; BP, branch point; PPT, polypyrimidine tract; SS, splicing site. B. Schematic representation of the trans-splicing mechanism. Cis-splicing of the mutant p53 pre-mRNA yields a mutant p53 transcript in codon 273. Mutant p53 transcripts were repaired through the approach of trans-splicing. Arrowheads indicate the PCR primers used for detection of trans-splicing-generated products. C. Detection of trans-spliced p53 RNA in transfected HT-29 cells. D. DNA sequence analysis of RT-PCR product of trans-splicing isolated from HT-29 cells transfected with p53-PTM plasmids.
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Figure 1: Schematic illustration of trans-splicing used for the correction of mutant p53 transcripts and the detection of trans-spliced p53 RNA in transfected cellsA. the structure of p53 pre-trans-splicing molecules (PTM). The hybridization domain is antisense to intron 7 of p53 pre-mRNA. BD-A and BD-B differ only in their binding regions, domain B was more efficient than domain A in promoting trans-splicing. BD, binding domain; BP, branch point; PPT, polypyrimidine tract; SS, splicing site. B. Schematic representation of the trans-splicing mechanism. Cis-splicing of the mutant p53 pre-mRNA yields a mutant p53 transcript in codon 273. Mutant p53 transcripts were repaired through the approach of trans-splicing. Arrowheads indicate the PCR primers used for detection of trans-splicing-generated products. C. Detection of trans-spliced p53 RNA in transfected HT-29 cells. D. DNA sequence analysis of RT-PCR product of trans-splicing isolated from HT-29 cells transfected with p53-PTM plasmids.

Mentions: To determine whether trans splicing repaired mutant p53 transcripts in cancer cells, we transfected p53-PTM (Figure 1A) and the controls (Figure S1) into two colorectal cancer cell lines (HT-29 and SW620) carrying p53 mutation in codon 273. Then RT-PCR was performed to detect trans-spliced p53 RNAs using specific primers that bridged the splice junction. To distinguish endogenous p53 transcripts, reverse primer was located in FLAG-tag (Figure 1B). The RT-PCR results demonstrated trans-spliced p53 RNAs were only detected in HT-29 cells transfected with p53-PTM, no detectable products were shown in HT-29 cells transfected with pcDNA3.1 or pGFPPTM (Figure 1C). Not only the amplified product size matched a RT-PCR product generated from intact p53 cDNA (Figure 1C), but also DNA sequence results confirmed trans-splicing-mediated repair of mutant p53 transcripts with high fidelity (Figure 1D). In addition, we demonstrated that mutant p53 transcripts in SW620 cells were also repaired by trans-splicing (Supplementary Figure S2). All these indicated trans-splicing-mediated repair of p53 mutation with high specificity and fidelity. To optimize the efficiency of trans-splicing-mediated correction of mutated p53 transcripts, the trans-splicer constructs with a hybridization domain complementary to different regions of p53 intron 7 were transfected into HT-29 cells, the results of semi-quantitative RT-PCR demonstrated the trans-splicer construct with hybridization domain B (p53-PTM-B) possessed better efficiency (data not shown). Therefore, this construct was used for all subsequent study.


Functional repair of p53 mutation in colorectal cancer cells using trans-splicing.

He X, Liao J, Liu F, Yan J, Yan J, Shang H, Dou Q, Chang Y, Lin J, Song Y - Oncotarget (2015)

Schematic illustration of trans-splicing used for the correction of mutant p53 transcripts and the detection of trans-spliced p53 RNA in transfected cellsA. the structure of p53 pre-trans-splicing molecules (PTM). The hybridization domain is antisense to intron 7 of p53 pre-mRNA. BD-A and BD-B differ only in their binding regions, domain B was more efficient than domain A in promoting trans-splicing. BD, binding domain; BP, branch point; PPT, polypyrimidine tract; SS, splicing site. B. Schematic representation of the trans-splicing mechanism. Cis-splicing of the mutant p53 pre-mRNA yields a mutant p53 transcript in codon 273. Mutant p53 transcripts were repaired through the approach of trans-splicing. Arrowheads indicate the PCR primers used for detection of trans-splicing-generated products. C. Detection of trans-spliced p53 RNA in transfected HT-29 cells. D. DNA sequence analysis of RT-PCR product of trans-splicing isolated from HT-29 cells transfected with p53-PTM plasmids.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385834&req=5

Figure 1: Schematic illustration of trans-splicing used for the correction of mutant p53 transcripts and the detection of trans-spliced p53 RNA in transfected cellsA. the structure of p53 pre-trans-splicing molecules (PTM). The hybridization domain is antisense to intron 7 of p53 pre-mRNA. BD-A and BD-B differ only in their binding regions, domain B was more efficient than domain A in promoting trans-splicing. BD, binding domain; BP, branch point; PPT, polypyrimidine tract; SS, splicing site. B. Schematic representation of the trans-splicing mechanism. Cis-splicing of the mutant p53 pre-mRNA yields a mutant p53 transcript in codon 273. Mutant p53 transcripts were repaired through the approach of trans-splicing. Arrowheads indicate the PCR primers used for detection of trans-splicing-generated products. C. Detection of trans-spliced p53 RNA in transfected HT-29 cells. D. DNA sequence analysis of RT-PCR product of trans-splicing isolated from HT-29 cells transfected with p53-PTM plasmids.
Mentions: To determine whether trans splicing repaired mutant p53 transcripts in cancer cells, we transfected p53-PTM (Figure 1A) and the controls (Figure S1) into two colorectal cancer cell lines (HT-29 and SW620) carrying p53 mutation in codon 273. Then RT-PCR was performed to detect trans-spliced p53 RNAs using specific primers that bridged the splice junction. To distinguish endogenous p53 transcripts, reverse primer was located in FLAG-tag (Figure 1B). The RT-PCR results demonstrated trans-spliced p53 RNAs were only detected in HT-29 cells transfected with p53-PTM, no detectable products were shown in HT-29 cells transfected with pcDNA3.1 or pGFPPTM (Figure 1C). Not only the amplified product size matched a RT-PCR product generated from intact p53 cDNA (Figure 1C), but also DNA sequence results confirmed trans-splicing-mediated repair of mutant p53 transcripts with high fidelity (Figure 1D). In addition, we demonstrated that mutant p53 transcripts in SW620 cells were also repaired by trans-splicing (Supplementary Figure S2). All these indicated trans-splicing-mediated repair of p53 mutation with high specificity and fidelity. To optimize the efficiency of trans-splicing-mediated correction of mutated p53 transcripts, the trans-splicer constructs with a hybridization domain complementary to different regions of p53 intron 7 were transfected into HT-29 cells, the results of semi-quantitative RT-PCR demonstrated the trans-splicer construct with hybridization domain B (p53-PTM-B) possessed better efficiency (data not shown). Therefore, this construct was used for all subsequent study.

Bottom Line: The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously.Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo.In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT
Mutation in the p53 gene is arguably the most frequent type of gene-specific alterations in human cancers. Current p53-based gene therapy contains the administration of wt-p53 or the suppression of mutant p53 expression in p53-defective cancer cells. . We hypothesized that trans-splicing could be exploited as a tool for the correction of mutant p53 transcripts in p53-mutated human colorectal cancer (CRC) cells. In this study, the plasmids encoding p53 pre-trans-splicing molecules (PTM) were transfected into human CRC cells carrying p53 mutation. The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously. Intratumoral administration of adenovirus vectors carrying p53 trans-splicing cassettes suppressed the growth of tumor xenografts. Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo. In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

Show MeSH
Related in: MedlinePlus