Alterations in gene expression and sensitivity to genotoxic stress following HdmX or Hdm2 knockdown in human tumor cells harboring wild-type p53

Aging (Albany NY). 2009 Jan;1(1):89-108. doi: 10.18632/aging.100008.

Abstract

While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth.Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown.Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.

Keywords: Hdm2; HdmX; RNAi; gene expression profiling; p53.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actins / genetics
  • Breast Neoplasms / metabolism
  • Cell Cycle Proteins
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cisplatin / pharmacology
  • Cyclin A2 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • DNA Damage / drug effects
  • DNA Damage / genetics*
  • Down-Regulation / genetics
  • Doxorubicin / pharmacology
  • E2F1 Transcription Factor / genetics
  • Female
  • G1 Phase / genetics
  • Gene Expression / genetics
  • Gene Expression Profiling*
  • Gene Expression Regulation, Neoplastic / physiology*
  • Humans
  • Immediate-Early Proteins / genetics
  • Nuclear Proteins / genetics*
  • Oligonucleotide Array Sequence Analysis
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-mdm2 / genetics*
  • RNA Interference*
  • RNA, Small Interfering / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transfection
  • Tumor Suppressor Protein p53 / genetics*
  • Tumor Suppressor Protein p53 / metabolism
  • Tumor Suppressor Proteins / genetics
  • Up-Regulation / genetics

Substances

  • ACTA2 protein, human
  • Actins
  • CCNA2 protein, human
  • CDKN1A protein, human
  • Cell Cycle Proteins
  • Cyclin A2
  • Cyclin-Dependent Kinase Inhibitor p21
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • Immediate-Early Proteins
  • MDM4 protein, human
  • Nuclear Proteins
  • PMAIP1 protein, human
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Small Interfering
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • BTG2 protein, human
  • Doxorubicin
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Cisplatin