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A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon

Chia-Chi Chen1,2,3,4, Ruo-Yu Liao5, Fang-Yu Yeh1

  • 1School of Medicine, I-Shou University, Kaohsiung City 82445, Taiwan.

Biomedicines
|May 27, 2023
PubMed

Insights

The drug PTC124 can help restore full-length p53 protein by promoting premature termination codon (PTC) readthrough. It is more effective for C-terminal p53 nonsense mutations than N-terminal ones.

Area of Science:

  • Molecular Biology
  • Cancer Genetics
  • Drug Discovery

Background:

  • Nonsense mutations create premature termination codons (PTCs), leading to truncated proteins.
  • Approximately 3.8% of human cancers harbor p53 nonsense mutations.
  • The drug PTC124 shows potential for promoting PTC readthrough and restoring protein function.

Purpose of the Study:

  • To develop a cost-effective method for creating p53 nonsense mutation clones.
  • To investigate the PTC readthrough activity of PTC124 on different p53 nonsense mutations.

Main Methods:

  • A modified inverse PCR-based site-directed mutagenesis method was employed.
  • Four p53 nonsense mutations (W91X, S94X, R306X, R342X) were cloned.
  • Clones were transfected into p53-null H1299 cells and treated with PTC124.

Main Results:

  • PTC124 induced p53 re-expression in clones with C-terminal mutations (R306X, R342X).
  • PTC124 did not induce p53 re-expression in clones with N-terminal mutations (W91X, S94X).

Conclusions:

  • PTC124 demonstrates differential efficacy based on the location of p53 nonsense mutations.
  • PTC124 is more effective at rescuing C-terminal p53 nonsense mutations.
  • A rapid and affordable site-directed mutagenesis method was established for p53 nonsense mutation cloning and drug screening.
Keywords:
PTC readthroughPTC124nonsense mutationp53site-directed mutagenesis

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