Proximal 5′-Flanking Sequence of the Human γ-Glutamylcysteine Synthetase Heavy Subunit Gene Is Involved in Cisplatin-Induced Transcriptional Up-regulation in a Lung Cancer Cell Line SBC-3

The contribution of the 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh) gene to cisplatin-induced transcriptional up-regulation was studied using various human growth hormone reporter constructs which were transfected to a human lung cancer cell line SBC-3. Cisplatin at the concentration of 3 microM increased the transcriptional activity of the longest sequence from -1,413 to +91 bp of the gamma-GCSh gene to 246% of that in non-exposed cells. The distal sequence from -1,413 to -193 bp was shown to negatively regulate transcriptional activity in both cisplatin-exposed and non-exposed cells using deletion and thymidine kinase (TK) promoter-linked constructs. Cisplatin increased the transcriptional activity of the proximal GC-rich sequence from -192 to +91 bp to 340%, of which magnitude was the maximum among deletion constructs. A deletion from -108 to -28 bp, or +34 to +91 bp significantly decreased cisplatin-induced increases in transcriptional activity from 258 to 105%, or 340 to 160%, respectively. When the sequence from -108 to -22 bp, or +26 to +91 bp was linked to the heterologous TK promoter, cisplatin increased the transcriptional activity to 171 or 181%, respectively, from that of 128 or 137%, respectively, in non-exposed cells. These findings indicate that the proximal sequence from -192 to +91 bp of the gamma-GCSh gene, especially from -108 to -28 bp, and +34 to +91 bp, is involved in cisplatin-induced transcriptional up-regulation in SBC-3 cells.