Restoring wtp53 activity in HIPK2 depleted MCF7 cells by modulating metallothionein and zinc

Exp Cell Res. 2009 Jan 1;315(1):67-75. doi: 10.1016/j.yexcr.2008.10.018. Epub 2008 Oct 28.

Abstract

The maintenance of p53 transactivation activity is important for p53 apoptotic function. We have shown that stable knockdown of HIPK2 induces p53 misfolding with inhibition of p53 target gene transcription. In this study we established a lentiviral-based system for doxycyclin (Dox)-induced conditional interference of HIPK2 expression to evaluate the molecular mechanisms involved in p53 deregulation. We found that HIPK2 knockdown induced metallothionein 2A (MT2A) upregulation as assessed by RT-PCR analysis, increased promoter acetylation, and increased promoter luciferase activity. The MT2A upregulation correlated with resistance to Adriamycin (ADR)-driven apoptosis and with p53 inhibition. Thus, acute knockdown of HIPK2 (HIPK2i) induced misfolded p53 protein in MCF7 breast cancer cells and inhibited p53 DNA-binding and transcription activities in response to ADR treatment. Previous works show that MT may modulate p53 activity through zinc exchange. Here, we found that inhibition of MT2A expression by siRNA in the HIPK2i cells restored p53 transcription activity. Similarly zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis. These data support the notion that MT2A is involved in p53 deregulation and strengthen the possibility that combination of chemotherapy and zinc might be useful to treat tumors with inactive wtp53.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • DNA, Neoplasm / metabolism
  • Doxorubicin / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Lentivirus
  • Metallothionein / metabolism*
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Protein Binding / drug effects
  • Protein Conformation / drug effects
  • Protein Folding / drug effects
  • Protein Serine-Threonine Kinases / deficiency*
  • Protein Serine-Threonine Kinases / metabolism
  • Transcription, Genetic / drug effects
  • Transcriptional Activation / drug effects
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Up-Regulation / drug effects
  • Zinc / metabolism*
  • Zinc / pharmacology

Substances

  • Carrier Proteins
  • DNA, Neoplasm
  • MT2A protein, human
  • Tumor Suppressor Protein p53
  • Phosphoserine
  • Doxorubicin
  • Metallothionein
  • HIPK2 protein, human
  • Protein Serine-Threonine Kinases
  • Zinc