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Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover

S Lin1, W Wang, G M Wilson

  • 1Laboratory of Biological Chemistry, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.

Molecular and Cellular Biology
|October 12, 2000
PubMed

Insights

Prostaglandin A(2) (PGA(2)) reduces cyclin D1 expression by destabilizing its mRNA in lung cancer cells. This involves AUF1 binding to a specific region in the 3' untranslated region of cyclin D1 mRNA.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Pharmacology

Background:

  • Prostaglandin A(2) (PGA(2)) is an experimental chemotherapeutic agent known to reduce cyclin D1 expression and cause growth arrest in various cancer cell lines.
  • The precise molecular mechanisms by which PGA(2) down-regulates cyclin D1 expression remain to be fully elucidated, particularly in non-small-cell lung carcinoma.

Purpose of the Study:

  • To investigate the mechanisms underlying PGA(2)-induced down-regulation of cyclin D1 expression in human non-small-cell lung carcinoma H1299 cells.
  • To identify the specific mRNA elements and protein factors involved in regulating cyclin D1 mRNA stability upon PGA(2) treatment.

Main Methods:

  • Utilized a cyclin D1 promoter-luciferase construct and nuclear run-on assays to assess gene transcription rates.
  • Employed electrophoretic mobility-shift assays (EMSA) with radiolabeled RNA probes to detect protein-RNA interactions.
  • Performed RNA-binding protein cross-linking, antibody supershifting, and immunoprecipitation assays to identify and confirm protein binding.
  • Assessed mRNA stability and expression of reporter genes containing specific cyclin D1 3' untranslated region (UTR) elements in transfected cells.

Main Results:

  • PGA(2) treatment did not affect cyclin D1 gene transcription rates but significantly decreased cyclin D1 mRNA stability.
  • Electrophoretic mobility-shift assays revealed enhanced protein binding to cyclin D1 mRNA in PGA(2)-treated cells, correlating with mRNA loss.
  • Binding activity was localized to a 390-base element (K12) within the cyclin D1 3' UTR.
  • The RNA-binding protein AUF1 was identified as a key factor binding to cyclin D1 mRNA, confirmed by antibody-based assays.
  • Insertion of the K12 element into reporter gene 3' UTRs markedly reduced expression and half-life in PGA(2)-treated cells.

Conclusions:

  • PGA(2) down-regulates cyclin D1 expression primarily by decreasing cyclin D1 mRNA stability, not by affecting transcription.
  • A specific 390-base element in the 3' UTR of cyclin D1 mRNA is crucial for mediating this PGA(2)-induced destabilization.
  • The RNA-binding protein AUF1 plays a significant role in the PGA(2)-mediated decay of cyclin D1 mRNA.

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