Procedure for the quantitation of Gadd45 expression levels in clonal hematopoietic progenitor cells by competitive polymerase chain reaction

Clin Biochem. 1999 Feb;32(1):1-8. doi: 10.1016/s0009-9120(98)00092-7.

Abstract

Objective: The growth arrest and DNA damage-inducible (gadd) genes represent a family of stress-inducible genes that are coordinately regulated at transcriptional level. Gadd45, in particular, has been linked to a p53-dependent inducible network required for regulated transition from G1 to S phase of cell cycle following genotoxic insult and growth arrest treatments and has seemingly a pivotal role in DNA repair.

Design and methods: Here we show that competitive polymerase chain reaction (PCR) is an adequate method to quantitate gadd45 expression levels in hematopoietic progenitor cell line 32D, whose constitutive gene expression is very low.

Results: The sensitivity and reproducibility of our strategy support its usefulness for clinical purposes, to assess the DNA repair capacity of highly purified early myeloid progenitors, whose failure may be responsible for either short-term chemotherapy side effects (bone marrow hypoplasia and peripheral blood cytopenia) or long-term consequences of antiblastic drugs (leukemia and myelodysplasia).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Northern
  • Cell Division
  • Clone Cells
  • DNA Damage
  • DNA Primers
  • Dose-Response Relationship, Drug
  • GADD45 Proteins
  • Hematopoietic Stem Cells / metabolism*
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Polymerase Chain Reaction / methods*
  • Proteins / isolation & purification
  • Proteins / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • DNA Primers
  • Intracellular Signaling Peptides and Proteins
  • Proteins
  • Tumor Suppressor Protein p53