2. Academic Validation
  3. A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124

A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124

  • Biomedicines. 2023 Apr 28;11(5):1310. doi: 10.3390/biomedicines11051310.
Chia-Chi Chen 1 2 3 4,  Ruo-Yu Liao 5,  Fang-Yu Yeh 1,  Yu-Rou Lin 1,  Tze-You Wu 6,  Alexa Escobar Pastor 7,  Danny Danilo Zul 7,  Yun-Chien Hsu 1,  Kuan-Yo Wu 8,  Ke-Fang Liu 5,  Reiji Kannagi 9,  Jang-Yi Chen 10,  Bi-He Cai 1
Affiliations

Affiliations

  • 1 School of Medicine, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 2 Department of Physical Therapy, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 3 School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 4 Department of Pathology, E-Da Hospital, Kaohsiung City 82445, Taiwan.
  • 5 Department of Medical Laboratory Science, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 6 Department of Biomedical Engineering, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 7 School of Medicine for International Students, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 8 Department of Biological Science and Technology, I-Shou University, Kaohsiung City 82445, Taiwan.
  • 9 Institute of Biomedical Sciences, Academia Sinica, Taipei City 11529, Taiwan.
  • 10 Institute of Biology and Anatomy, National Defense Medical Center, Taipei City 11529, Taiwan.
PMID: 37238980 DOI: 10.3390/biomedicines11051310
Abstract

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human Cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 μM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

Keywords

PTC readthrough; PTC124; nonsense mutation; p53; site-directed mutagenesis.

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