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Inducible and Reversible Dominant-negative (DN) Protein Inhibition
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Transcriptional repression of the RET proto-oncogene by a mitogen activated protein kinase-dependent signalling

Scott D Andrew1, Amanda Capes-Davis, Patric J D Delhanty

  • 1Kolling Institute of Medical Research, Royal North Shore Hospital, Department of Molecular Medicine, University of Sydney, Sydney, NSW 2065, Australia. andrew@clinlabs.path.queensu.ca

Gene
|October 31, 2002
PubMed

Insights

Phorbol 12-myristate 13-acetate (PMA) treatment reduces medullary thyroid carcinoma cell differentiation by inducing early growth response gene 1 (Egr-1), which alters RET expression. This Egr-1 and Sp protein complex influences RET gene regulation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Transcription factors regulate critical cellular processes like growth and differentiation.
  • Medullary thyroid carcinoma (MTC) cell lines offer models for studying cancer development and gene regulation.

Purpose of the Study:

  • To investigate the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA) affects MTC cell phenotype and RET gene expression.
  • To identify the role of early growth response gene 1 (Egr-1) in PMA-induced changes in RET expression.

Main Methods:

  • Treatment of MTC TT cells with PMA.
  • Northern blotting to analyze gene expression (Egr-1 and RET).
  • Transient transfection assays with RET promoter constructs.
  • Gel shift and supershift assays to study protein-DNA interactions.
  • Overexpression studies of Egr-1.

Main Results:

  • PMA treatment reduced neurite outgrowth and induced Egr-1 expression while decreasing RET expression in TT cells.
  • A specific region (-70 to -33 bp) of the RET promoter was crucial for PMA-inducible expression.
  • PMA-induced Egr-1 formed a complex that bound to the RET minimal promoter, displacing Sp1 and Sp3.
  • Egr-1 induction correlated with RET downregulation in a raf-1 inducible TT cell line.

Conclusions:

  • PMA-induced Egr-1 plays a significant role in regulating RET expression in MTC cells.
  • The interaction between Egr-1 and Sp proteins on the RET promoter influences RET gene expression.
  • These findings suggest a mechanism for RET regulation in development and MTC involving Egr-1 and Sp proteins.

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