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Yeast As a Chassis for Developing Functional Assays to Study Human P53
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Yeast As a Chassis for Developing Functional Assays to Study Human P53

Published on: August 4, 2019

Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model

A Carnero1, J D Hudson, G J Hannon

  • 1Institute of Child Health, London, UK.

Nucleic Acids Research
|June 28, 2000
PubMed

Insights

Researchers identified antisense fragments that inactivate the p53 tumor suppressor gene by inhibiting its mRNA translation. This inactivation promotes cell immortalization and bypasses p53-induced growth arrest, offering a tool for gene function analysis.

Area of Science:

  • Molecular Biology
  • Cancer Genetics
  • Gene Regulation

Background:

  • The p53 tumor suppressor gene plays a critical role in preventing cancer by inducing cell cycle arrest or apoptosis in response to DNA damage.
  • Dysregulation or mutation of p53 is common in many human cancers, making it a key target for therapeutic strategies.
  • Understanding the precise mechanisms of p53 function and inactivation is crucial for developing effective cancer treatments.

Purpose of the Study:

  • To identify and characterize novel antisense fragments capable of functionally inactivating the p53 tumor suppressor gene.
  • To investigate the mechanism by which these antisense fragments inhibit p53 activity, specifically focusing on mRNA translation.
  • To explore the potential of using these antisense fragments for genetic analysis and understanding the role of p53 in cellular processes like growth arrest and immortalization.

Main Methods:

  • Development and application of an improved system for functional identification of active antisense fragments.
  • Isolation and mapping of antisense fragments targeting specific regions of the p53 coding sequence.
  • In vitro and in vivo assays to assess the impact of antisense fragments on p53 mRNA translation and protein function.
  • Cell culture experiments using cell lines with mutant p53 and primary mouse embryonic fibroblasts to evaluate effects on growth arrest, lifespan, and immortalization.
  • Analysis of downstream effectors and pathways influenced by p53 inactivation and restoration.

Main Results:

  • Successfully isolated antisense fragments that specifically inactivate the p53 tumor suppressor gene by inhibiting p53 mRNA translation.
  • Demonstrated that these antisense fragments can overcome p53-induced growth arrest in cells expressing a mutant p53.
  • Showed that continuous expression of p53 antisense fragments can lead to the immortalization of primary mouse fibroblasts, which requires ongoing p53 inactivation.
  • Identified MDM2 or SV40 large T antigen, but not E7 or oncogenic ras, as capable of overcoming p53-induced arrest upon restoration of p53 expression.
  • Found that functional inactivation of both p21 and BAX, but not individually, allows some bypass of p53-induced growth arrest, suggesting multiple targets mediate p53's antiproliferative action.

Conclusions:

  • The identified antisense fragments provide a novel tool for the conditional functional inactivation of the p53 gene.
  • These findings highlight the critical role of continuous p53 inactivation in the immortalization of certain cell types.
  • The study demonstrates the utility of antisense technology for dissecting gene function, particularly for genes involved in loss-of-function phenotypes relevant to cancer and aging.
  • Understanding the complex network of p53 transcriptional targets is essential for fully comprehending its tumor-suppressive functions.

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