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Related Experiment Videos

Polyomavirus large T antigen-dependent DNA amplification

P Stiegler1, S Schüchner, V Lestou

  • 1Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Austria.

Oncogene
|February 27, 1997
PubMed
Summary
This summary is machine-generated.

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Polyomavirus large T antigen promotes gene amplification in non-permissive cells by interacting with the retinoblastoma protein. This interaction is crucial for generating DNA amplification, a marker of genome instability.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • DNA amplification is a key indicator of genome destabilization and is common in immortalized or transformed cell lines.
  • Tumor virus gene products can induce permissivity for DNA amplification, often involving interaction with the tumor suppressor protein p53.
  • Polyomavirus large T antigen contributes to cell immortalization and transformation but does not interact with p53.

Purpose of the Study:

  • To investigate whether polyomavirus large T antigen can induce gene amplification in a non-permissive cell line (REF52).
  • To determine the role of polyomavirus large T antigen's interaction with the retinoblastoma protein in facilitating gene amplification.

Main Methods:

  • Conditional expression of wild-type or mutant polyomavirus large T antigen in REF52 cells.

Related Experiment Videos

  • Selection of phosphonoacetyl-L-aspartate (PALA)-resistant cells to quantify gene amplification frequency.
  • Analysis of chromosomal abnormalities and DNA structures in PALA-resistant cells using fluorescence in situ hybridization.

    • Main Results:

    • PALA-resistant cells, indicative of DNA amplification, arose at a frequency of approximately 5 x 10(-5).
    • The interaction of polyomavirus large T protein with the retinoblastoma protein or related pocket proteins was essential for this amplification.
    • PALA-resistant cells exhibited increased chromosome numbers and dicentric chromosomes, characteristic of amplified DNA structures.
    • Cells remained normal with respect to p53, and polyomavirus large T protein's interaction with E2F-pocket protein complexes partially overcame a PALA-induced G1 block.

    Conclusions:

    • Polyomavirus large T antigen's interaction with retinoblastoma protein family members is critical for inducing gene amplification in REF52 cells.
    • These findings suggest that E2F-pocket protein complexes are downstream targets in the pathway affected by polyomavirus large T antigen.
    • The study provides evidence that polyomavirus large T antigen can promote genome instability through mechanisms independent of p53 interaction.