Transcriptional Activation of the Human HMG1 Gene in Cisplatin-resistant Human Cancer Cells

ABSTRACT The nonhistone chromosomal protein, high mobility group 1 (HMG1),which is ubiquitously expressed in higher eukaryotic cells, preferentiallybinds to cisplatin-modified DNA. The observation that HMG1 is overex-pressed in cisplatin-resistant human cancer cells suggests that cisplatinresistance may be closely associated with HMG1. To decipher the mech-anism of HMG1 overexpression in cisplatin-resistant cells, we isolated twooverlapping genomic DNA clones containing the entire human HMG1gene. These clones, which span ;15 kb of contiguous DNA, include 5 kbof the 5* flanking region as well as the entire coding sequence. Wesequenced 1500 bp upstream of the first exon. The segment proximal tothe transcription initiation site did not contain a TATA box but didpossess an activating transcription factor site, an activator protein-2 site,one CCAAT box, and two CCAAT-binding transcription factor/nuclearfactor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3–10-fold higherin cisplatin-resistant KB-CP20 cells than in parental KB cells. Anin vivofootprint experiment showed several differences of dimethyl sulfate mod-ifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showedthat binding of a nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was significantly higher than the binding of the same factor fromparental cells. Semiquantitative reverse transcription-PCR and Westernblot analysis also showed that expression of CTF/NF-1 was 3–20-foldhigher in the resistant cell line than in its parental counterpart. Thesefindings suggest that, in cisplatin-resistant cells, the expression of HMG1gene product is enhanced at the transcriptional level and that this prob-ably occurs through the enhanced expression of the CCAAT bindingfactor, CTF/NF-1.