Identification by suppression subtractive hybridization of p21 as a radio-inducible gene in human glioma cells.

Background: Radio-gene therapy involves the delivery, to tumor cells, of a therapeutic transgene whose expression is controlled by irradiation.

Materials And Methods: Here, we sought to identify novel radio-inducible transcripts in U87MG human malignant glioma cells using suppression subtractive hybridization (SSH).

Results: Of 998 clones from a subtracted library of irradiated U87MG cells, 24 candidate clones were identified by dot blot and 3 clones were confirmed as having been induced by irradiation by Northern blot analysis. All three clones showed 99-100% homology to the cyclin-dependent kinase (cdk) inhibitor, p21(Waf/Cip1). A screening of 12 human malignant glioma cell lines revealed that irradiation increased p21 mRNA expression and p21 protein levels levels in all of the five cell lines retaining p53 wild-type activity in a p53 reporter assay, but in none of seven p53 reporter-negative cell lines.

Conclusion: Irradiation induces p21 mRNA expression in a strictly p53-dependent manner and may only enhance the expression of a limited number of genes in glioma cells. We conclude that the identification of radio-inducible genomic sequences suitable for radio-gene therapy may turn out to be difficult.