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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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Induction of cell cycle arrest in human colorectal cancer cells (HT-29) through trans splicing-mediated repair of mutant p53 transcriptsTrans-splicing plasmids or the controls were transfected into HT-29 cell carrying p53 mutation (codon 273), and then the proliferative activity, cell cycle and cell cycle regulatory genes of HT-29 cells were evaluated. A. MTS assay demonstrated that the proliferative activity of HT-29 cell was inhibited after the introduction of p53-PTM-B. *p<0.05. B. Flow cytometry revealed p53-PTM induces cell-cycle arrest in transfected HT-29 cells. C. Real-time RT-PCR results showed that the regulatory genes for cell cycle in mRNA level decreased significantly in HT29 cells transfected with p53-PTM-B. *p<0.05. D. Expression patterns of the regulatory genes for cell cycle in transfected HT-29 cells were determined by western blot analysis. Densitometric quantification of the indicated proteins was shown. Partial repair of mutant p53 transcripts by trans-splicing resulted in the dramatic decrement in the expression of the regulatory proteins which were responsible for cell cycle.
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Figure 2: Induction of cell cycle arrest in human colorectal cancer cells (HT-29) through trans splicing-mediated repair of mutant p53 transcriptsTrans-splicing plasmids or the controls were transfected into HT-29 cell carrying p53 mutation (codon 273), and then the proliferative activity, cell cycle and cell cycle regulatory genes of HT-29 cells were evaluated. A. MTS assay demonstrated that the proliferative activity of HT-29 cell was inhibited after the introduction of p53-PTM-B. *p<0.05. B. Flow cytometry revealed p53-PTM induces cell-cycle arrest in transfected HT-29 cells. C. Real-time RT-PCR results showed that the regulatory genes for cell cycle in mRNA level decreased significantly in HT29 cells transfected with p53-PTM-B. *p<0.05. D. Expression patterns of the regulatory genes for cell cycle in transfected HT-29 cells were determined by western blot analysis. Densitometric quantification of the indicated proteins was shown. Partial repair of mutant p53 transcripts by trans-splicing resulted in the dramatic decrement in the expression of the regulatory proteins which were responsible for cell cycle.

Mentions: To determine whether the repaired p53 transcripts were translated to produce functional p53 protein in transfected cells, we analyzed proliferative activity, the distribution of cell cycle and the expression of regulatory genes responsible for cell cycle in HT-29 cells transfected with p53-PTM or the controls. As shown in Figure 2A, proliferative activity of HT-29 cells was inhibited after the transfection of p53-PTM compared with the controls (p<0.05). Then, we evaluated the effect of p53-PTM on the distribution of cell cycle in HT-29 cell. Cell cycle determined by flow cytometry demonstrated a significant accumulation of the cells in G1 phase following the transfection of p53-PTM into HT-29 cells (Figure 2B). In addition, to further investigate the effect of p53-PTM on cell cycle, we examined the expression patterns of cell-cycle-related genes through quantitative RT-PCR and western blot. As shown in Figure 2C, real-time RT-PCR revealed that partial correction of mutant p53 transcripts by trans-splicing resulted in down-regulation of the mRNA of the genes responsible for the G1/S cell cycle checkpoint such as cyclin A2, B1, D1 and E. In agreement with quantitative RT-PCR data, the similar change in cell-cycle-related proteins was observed in western blot analysis (Figure 2D). All these indicated that p53-PTM induced cell cycle arrest in HT-29 cells through partial correction of mutant p53 transcripts. Simultaneously, the results showed a weak induction of cell cycle arrest in GFP-PTM-transfected CRC cells compared with the negative control (pcDNA3.1) (Figure 2B, C and D).


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)

Induction of cell cycle arrest in human colorectal cancer cells (HT-29) through trans splicing-mediated repair of mutant p53 transcriptsTrans-splicing plasmids or the controls were transfected into HT-29 cell carrying p53 mutation (codon 273), and then the proliferative activity, cell cycle and cell cycle regulatory genes of HT-29 cells were evaluated. A. MTS assay demonstrated that the proliferative activity of HT-29 cell was inhibited after the introduction of p53-PTM-B. *p<0.05. B. Flow cytometry revealed p53-PTM induces cell-cycle arrest in transfected HT-29 cells. C. Real-time RT-PCR results showed that the regulatory genes for cell cycle in mRNA level decreased significantly in HT29 cells transfected with p53-PTM-B. *p<0.05. D. Expression patterns of the regulatory genes for cell cycle in transfected HT-29 cells were determined by western blot analysis. Densitometric quantification of the indicated proteins was shown. Partial repair of mutant p53 transcripts by trans-splicing resulted in the dramatic decrement in the expression of the regulatory proteins which were responsible for cell cycle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Induction of cell cycle arrest in human colorectal cancer cells (HT-29) through trans splicing-mediated repair of mutant p53 transcriptsTrans-splicing plasmids or the controls were transfected into HT-29 cell carrying p53 mutation (codon 273), and then the proliferative activity, cell cycle and cell cycle regulatory genes of HT-29 cells were evaluated. A. MTS assay demonstrated that the proliferative activity of HT-29 cell was inhibited after the introduction of p53-PTM-B. *p<0.05. B. Flow cytometry revealed p53-PTM induces cell-cycle arrest in transfected HT-29 cells. C. Real-time RT-PCR results showed that the regulatory genes for cell cycle in mRNA level decreased significantly in HT29 cells transfected with p53-PTM-B. *p<0.05. D. Expression patterns of the regulatory genes for cell cycle in transfected HT-29 cells were determined by western blot analysis. Densitometric quantification of the indicated proteins was shown. Partial repair of mutant p53 transcripts by trans-splicing resulted in the dramatic decrement in the expression of the regulatory proteins which were responsible for cell cycle.
Mentions: To determine whether the repaired p53 transcripts were translated to produce functional p53 protein in transfected cells, we analyzed proliferative activity, the distribution of cell cycle and the expression of regulatory genes responsible for cell cycle in HT-29 cells transfected with p53-PTM or the controls. As shown in Figure 2A, proliferative activity of HT-29 cells was inhibited after the transfection of p53-PTM compared with the controls (p<0.05). Then, we evaluated the effect of p53-PTM on the distribution of cell cycle in HT-29 cell. Cell cycle determined by flow cytometry demonstrated a significant accumulation of the cells in G1 phase following the transfection of p53-PTM into HT-29 cells (Figure 2B). In addition, to further investigate the effect of p53-PTM on cell cycle, we examined the expression patterns of cell-cycle-related genes through quantitative RT-PCR and western blot. As shown in Figure 2C, real-time RT-PCR revealed that partial correction of mutant p53 transcripts by trans-splicing resulted in down-regulation of the mRNA of the genes responsible for the G1/S cell cycle checkpoint such as cyclin A2, B1, D1 and E. In agreement with quantitative RT-PCR data, the similar change in cell-cycle-related proteins was observed in western blot analysis (Figure 2D). All these indicated that p53-PTM induced cell cycle arrest in HT-29 cells through partial correction of mutant p53 transcripts. Simultaneously, the results showed a weak induction of cell cycle arrest in GFP-PTM-transfected CRC cells compared with the negative control (pcDNA3.1) (Figure 2B, C and D).

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