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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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The effect of adenovirus vector expressing p53-PTM on the growth of xenograft tumors in nude mice developed by inoculating HT-29 cellsA. Fluorescent microscopy showed efficient transduction of HT-29, as indicated by eGFP expression, 4 days following adenovirus vector administration. B. RT-PCR analysis evaluated trans-splicing-mediated repair of mutant p53 transcripts in xenograft tumors. Adenovirus vectors carrying p53-PTM or the controls were injected into xenograft tumors. 48 hours after injection, RNA were isolated from tumor tissues and then subjected to RT-PCR analysis (left panel) for trans-spliced p53 transcripts. DNA sequence analysis of RT-PCR product (right panel) demonstrated mutant site of p53 in codon 273 was repaired. C. Effects of p53-PTM on the growth of pre-established HT-29 xenografts at a gross morphology level. D. Tumor growth curves (left panel, a) and the average volume fold increase of tumors at the sacrifice with respect to the first measurements (right panel, b) demonstrated antitumor effects of p53-PTM in vivo. *p<0.05.
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Figure 4: The effect of adenovirus vector expressing p53-PTM on the growth of xenograft tumors in nude mice developed by inoculating HT-29 cellsA. Fluorescent microscopy showed efficient transduction of HT-29, as indicated by eGFP expression, 4 days following adenovirus vector administration. B. RT-PCR analysis evaluated trans-splicing-mediated repair of mutant p53 transcripts in xenograft tumors. Adenovirus vectors carrying p53-PTM or the controls were injected into xenograft tumors. 48 hours after injection, RNA were isolated from tumor tissues and then subjected to RT-PCR analysis (left panel) for trans-spliced p53 transcripts. DNA sequence analysis of RT-PCR product (right panel) demonstrated mutant site of p53 in codon 273 was repaired. C. Effects of p53-PTM on the growth of pre-established HT-29 xenografts at a gross morphology level. D. Tumor growth curves (left panel, a) and the average volume fold increase of tumors at the sacrifice with respect to the first measurements (right panel, b) demonstrated antitumor effects of p53-PTM in vivo. *p<0.05.

Mentions: To determine the effect of p53-PTM on the growth of HT-29 cells in vivo, we constructed adenovirus vectors expressing p53 trans-splicing cassettes and delivered them into xenograft tumors developed by the inoculation of HT-29 cells. These adenovirus vectors efficiently transduced HT-29 cells with MOI above 10 (Figure 4A). As a prelude to the analysis of their in vivo effects, trans-splicing-mediated repair of mutant p53 transcripts was evaluated in xenograft tumors after intratumoral injection of Ad-p53-PTM or the controls into xenograft tumors. As show in Figure 4B, the results of RT-PCR (left panel) and DNA sequence (right panel) confirmed mutant p53 transcripts was repaired in vivo by trans-splicing. To assess the ability of adenovirus-mediated trans-splicing to suppress the growth of tumors in vivo, tumor size was monitored over time after the administration of adenovirus vectors. The mice which had received the injection of Ad-p53-PTM showed a marked suppression of xenograft tumors growth, which could be reflected by gross morphology (Figure 4C) and growth curves (Figure 4D). As shown in Figure 5A, the proliferation of CRC cells indicated as ki67 staining was inhibited in tumor tissues of nude mice which received the administration of Ad-p53-PTM. Then, we analyzed the expression of the proteins involved in cell cycle and apoptosis in xenograft tumors to probe the inhibitory mechanism of p53-PTM in vivo. Proapoptotic regulatory proteins such as caspase-3 and Bax were barely detectable in tumor tissues of AdNull-treated mice, and augmented remarkably in tumors of mice which received injection of Ad-p53-PTM (Figure 5B). A reduction in cyclin D1, a cell cycle regulatory protein, was observed in tumors of Ad-p53-PTM-treated mice (Figure 5B). In addition, immunohistochemical analysis of tumor tissues showed that the administration of Ad-p53-PTM reduced staining of Bcl-2 identified as antiapoptotic protein (Figure 5B). Enhanced expression of p53-dependent downstream target genes mdm2 was detected in Ad-p53-PTM-treated mice (Figure 5B). These indicated p53-PTM induced p53-depedent downstream target molecules involved in cell cycle arrest and apoptosis, and then reduced the growth of xenograft tumors.


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)

The effect of adenovirus vector expressing p53-PTM on the growth of xenograft tumors in nude mice developed by inoculating HT-29 cellsA. Fluorescent microscopy showed efficient transduction of HT-29, as indicated by eGFP expression, 4 days following adenovirus vector administration. B. RT-PCR analysis evaluated trans-splicing-mediated repair of mutant p53 transcripts in xenograft tumors. Adenovirus vectors carrying p53-PTM or the controls were injected into xenograft tumors. 48 hours after injection, RNA were isolated from tumor tissues and then subjected to RT-PCR analysis (left panel) for trans-spliced p53 transcripts. DNA sequence analysis of RT-PCR product (right panel) demonstrated mutant site of p53 in codon 273 was repaired. C. Effects of p53-PTM on the growth of pre-established HT-29 xenografts at a gross morphology level. D. Tumor growth curves (left panel, a) and the average volume fold increase of tumors at the sacrifice with respect to the first measurements (right panel, b) demonstrated antitumor effects of p53-PTM in vivo. *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The effect of adenovirus vector expressing p53-PTM on the growth of xenograft tumors in nude mice developed by inoculating HT-29 cellsA. Fluorescent microscopy showed efficient transduction of HT-29, as indicated by eGFP expression, 4 days following adenovirus vector administration. B. RT-PCR analysis evaluated trans-splicing-mediated repair of mutant p53 transcripts in xenograft tumors. Adenovirus vectors carrying p53-PTM or the controls were injected into xenograft tumors. 48 hours after injection, RNA were isolated from tumor tissues and then subjected to RT-PCR analysis (left panel) for trans-spliced p53 transcripts. DNA sequence analysis of RT-PCR product (right panel) demonstrated mutant site of p53 in codon 273 was repaired. C. Effects of p53-PTM on the growth of pre-established HT-29 xenografts at a gross morphology level. D. Tumor growth curves (left panel, a) and the average volume fold increase of tumors at the sacrifice with respect to the first measurements (right panel, b) demonstrated antitumor effects of p53-PTM in vivo. *p<0.05.
Mentions: To determine the effect of p53-PTM on the growth of HT-29 cells in vivo, we constructed adenovirus vectors expressing p53 trans-splicing cassettes and delivered them into xenograft tumors developed by the inoculation of HT-29 cells. These adenovirus vectors efficiently transduced HT-29 cells with MOI above 10 (Figure 4A). As a prelude to the analysis of their in vivo effects, trans-splicing-mediated repair of mutant p53 transcripts was evaluated in xenograft tumors after intratumoral injection of Ad-p53-PTM or the controls into xenograft tumors. As show in Figure 4B, the results of RT-PCR (left panel) and DNA sequence (right panel) confirmed mutant p53 transcripts was repaired in vivo by trans-splicing. To assess the ability of adenovirus-mediated trans-splicing to suppress the growth of tumors in vivo, tumor size was monitored over time after the administration of adenovirus vectors. The mice which had received the injection of Ad-p53-PTM showed a marked suppression of xenograft tumors growth, which could be reflected by gross morphology (Figure 4C) and growth curves (Figure 4D). As shown in Figure 5A, the proliferation of CRC cells indicated as ki67 staining was inhibited in tumor tissues of nude mice which received the administration of Ad-p53-PTM. Then, we analyzed the expression of the proteins involved in cell cycle and apoptosis in xenograft tumors to probe the inhibitory mechanism of p53-PTM in vivo. Proapoptotic regulatory proteins such as caspase-3 and Bax were barely detectable in tumor tissues of AdNull-treated mice, and augmented remarkably in tumors of mice which received injection of Ad-p53-PTM (Figure 5B). A reduction in cyclin D1, a cell cycle regulatory protein, was observed in tumors of Ad-p53-PTM-treated mice (Figure 5B). In addition, immunohistochemical analysis of tumor tissues showed that the administration of Ad-p53-PTM reduced staining of Bcl-2 identified as antiapoptotic protein (Figure 5B). Enhanced expression of p53-dependent downstream target genes mdm2 was detected in Ad-p53-PTM-treated mice (Figure 5B). These indicated p53-PTM induced p53-depedent downstream target molecules involved in cell cycle arrest and apoptosis, and then reduced the growth of xenograft tumors.

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