Repression of human reduced folate carrier gene expression by wild type p53

The relationship between loss of functional p53 and human reduced folate ca rrier (hRFC) levels and function was examined in REH lymphoblastic leukemia cells, which express wild type p53, and in p53-null K562 cells (K562(pTet- on/p53)) engineered to express wild type p53 under control of a tetracyclin e-inducible promoter. Activation of p53 in REH cells by treatment with daun orubicin was accompanied by decreased (similar to5-fold) levels of hRFC tra nscripts and methotrexate transport. Treatment of K562(pTet-on/p53) cells w ith doxycycline resulted in a dose-dependent expression of p53 protein and transcripts, increased p21 protein, decreased dihydrofolate reductase, and G(1) arrest with decreased numbers of cells in S-phase. p53 induction was a ccompanied by up to 3-fold decreases in hRFC transcripts transcribed from t he upstream hRFC-B promoter and similar losses of hRFC protein and methotre xate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and di hydrofolate reductase without effects on hRFC levels or activity. When the hRFC-B promoter was expressed as full-length and basal promoter-luciferase reporter constructs in K562(pTet-on/p53) cells, induction of p53 with doxyc ycline resulted in a 3-fold loss of promoter activity, which was reversed b y cotransfection with a trans-dominant-negative p53. These studies show tha t wild type p53 acts as a repressor of hRFC gene expression, via a mechanis m that is independent of its effects on cell cycle progression.