Repression of Human Reduced Folate Carrier Gene Expression by Wild Type p53

Abstract

The relationship between loss of functional p53 and human reduced folate carrier (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 tetracycline-inducible promoter. Activation of p53 in REH cells by treatment with daunorubicin was accompanied by decreased (ϳ5-fold) levels of hRFC transcripts and methotrexate transport. Treatment of K562 pTet-on/p53 cells with doxycycline resulted in a dosedependent 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 accompanied by up to 3-fold decreases in hRFC transcripts transcribed from the upstream hRFC-B promoter and similar losses of hRFC protein and methotrexate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and dihydrofolate 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 doxycycline resulted in a 3-fold loss of promoter activity, which was reversed by cotransfection with a trans-dominant-negative p53. These studies show that wild type p53 acts as a repressor of hRFC gene expression, via a mechanism that is independent of its effects on cell cycle progression.

Identification of a Mouse Thiamine Transporter Gene as a Direct Transcriptional Target for p53

“…Thus, despite significant amino acid sequence homology between these two vitamin transporters, they are highly specific toward their substrates. Whereas our evidence supports that p53 activates mTHTR-1 expression at the transcriptional level, Ding et al (47) have recently shown that p53 suppressed the hRFC gene expression. The physiological significance of the differential regulation of these vitamin transporters by p53 remains to be established.…”

Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells

“…Co-incubation of these cells with MTX and 5-aza-CdR at a concentration of 1 M resulted in a net decrease in MTX accumulation, leading these authors to exclude DNA methylation at the RFC promoter as a cause of acute down-regulation of RFC in response to MTX. Recent studies have shown that MTX can up-regulate the p53 tumor suppressor (80), which in turn causes RFC down-regulation (78), which would provide a plausible basis for an acute response to MTX. Nevertheless, on the basis of the 5-aza-CdR data presented here, a role of DNA methylation in mediating RFC down-regulation after shortterm exposure to MTX cannot yet be entirely excluded.…”

The role of the proto-oncogene ETS2 in acute megakaryocytic leukemia biology and therapy

“…38 The K562pTet-on cell line was developed from wild-type K562 cells by transfection with the regulator pTet-on plasmid using Lipofectin (Invitrogen Life Technologies), as previously reported. 39 To construct the pTRE2hyg/ETS2 plasmid, wild-type ETS2 cDNA from CMK cells was PCR amplified with forward (5 0 -AACTCGGATCCGCAGCGGCAGGATGAATGATTTCGGA-3 0 ) and reverse (5 0 -AACTCCTCGAGGCTCAGGGTGGTCCCGGCGA CCTCAG-3 0 ) primers and cloned into the pTRE2hyg plasmid at the BamHI and SalI sites (underlined in the above primer sequences). The plasmid was then transfected into the K562pTet-on cell line by electroporation (200 V, 950 mF) and screened by selection with hygromycin (200 mg ml À1 ).…”