Transcriptional activation of the human HMG1 gene in cisplatin-resistant human cancer cells.

The nonhistone chromosomal protein, high mobility group 1 (HMG1), which is ubiquitously expressed in higher eukaryotic cells, preferentially binds to cisplatin-modified DNA. The observation that HMG1 is overexpressed in cisplatin-resistant human cancer cells suggests that cisplatin resistance may be closely associated with HMG1. To decipher the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated two overlapping genomic DNA clones containing the entire human HMG1 gene. These clones, which span approximately 15 kb of contiguous DNA, include 5 kb of the 5' flanking region as well as the entire coding sequence. We sequenced 1500 bp upstream of the first exon. The segment proximal to the transcription initiation site did not contain a TATA box but did possess an activating transcription factor site, an activator protein-2 site, one CCAAT box, and two CCAAT-binding transcription factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3-10-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB cells. An in vivo footprint experiment showed several differences of dimethyl sulfate modifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showed that 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 from parental cells. Semiquantitative reverse transcription-PCR and Western blot analysis also showed that expression of CTF/NF-1 was 3-20-fold higher in the resistant cell line than in its parental counterpart. These findings suggest that, in cisplatin-resistant cells, the expression of HMG1 gene product is enhanced at the transcriptional level and that this probably occurs through the enhanced expression of the CCAAT binding factor, CTF/NF-1.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Interaction with p53 enhances binding of cisplatin-modified DNA by high mobility group 1 protein.

A nonhistone chromosomal protein, high mobility group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds preferentially to cisplatin-modified DNA. HMG1 also functions as a coactivator of p53, a tumor suppressor protein. We investigated physical interactions between HMG1 and p53 and the influence of p53 on the ability of HMG1 to recognize damaged DNA. Using immunochemical coprecipitation, we observed binding of HMG1 and p53. Interaction between HMG1 and p53 required the HMG A box of HMG1 and amino acids 363-376 of p53. Cisplatin-modified DNA binding by HMG1 was significantly enhanced by p53. An HMG1-specific antibody that recognized the A box of this protein also stimulated cisplatin-modified DNA binding. These data suggest that an interaction with either p53 or antibody may induce conformational change in the HMG1 A box that optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and p53-mediated DNA repair.

Structural characterization of the human interleukin-13 receptor alpha1 gene promoter.

Human cancer cells have been found to express a large number of IL-13 receptors. We have previously shown that mRNA encoding one of these receptors, IL-13Ralpha1, is increased in cisplatin-resistant cells and is upregulated in tumor cells cultured with cisplatin. To understand the molecular mechanism of IL-13Ralpha1 gene expression, we cloned approximately 52 kbp of the IL-13Ralpha1 gene and sequenced the first exon and about 1 kbp of the upstream DNA. The first exon is 211 bp and contains 88 bp of coding sequence, while the first intron is about 13 kbp in length. The promoter region, which is GC rich, was found to lack both TATA and CCAAT boxes. Transient expression assays revealed that transcription of the IL-13Ralpha1 gene was significantly higher in cisplatin-resistant cells than in parental, cisplatin-sensitive cells. Deletion analysis of the IL-13Ralpha1 promoter identified a 70-bp core promoter region upstream of the transcription initiation site. Electrophoretic gel mobility shift assays showed that a synthetic IL-13Ralpha1 oligonucleotide (nt -40 to nt -15) bound a nuclear factor from cisplatin-resistant cells to a significantly greater degree than the equivalent factor from parental cells. This oligonucleotide was found to contain a palindromic sequence with a BstEII recognition site at its center. This palindromic sequence functions to mediate upregulation of IL-13Ralpha1 promoter in cisplatin-resistant cells and deletion or disruption of this sequence also resulted in severe reduction of the promoter activity. These findings suggest that IL-13Ralpha1 expression is upregulated at the transcriptional level in cisplatin-resistant cells. The characterization of both the IL-13Ralpha1 promoter and the transcription factors binding to it may contribute to our understanding of IL-13Ralpha1 regulation in cancer cells.

Structural and functional analysis of the control region of the human DNA topoisomerase II alpha gene in drug-resistant cells.

We have previously shown that the DNA topoisomerase II alpha (topo II alpha) gene is down-regulated in VP16/VM26-resistant cells at the transcriptional level. To determine the DNA elements responsible for down-regulation, the transcriptional activities of luciferase reporter constructs containing various lengths of the promoter sequences were investigated by transient transfection of two resistant cell lines, KB/VP2 and KB/VM4. The transcriptional activities of the full-length promoter (-295 to +85) and of three deletion constructs (-197, -154 and -74 to +85) were significantly down-regulated in resistant cells. In contrast, the transcriptional activity of the minimal promoter (-20 to +85) in resistant cells was similar to that in parental KB cells. Furthermore, introduction of a mutation in ICE1 abolished the down-regulation of the topo II alpha promoter activity in drug-resistant cells. In vivo footprinting analysis of topo II alpha gene promoter revealed several specific protein-binding sites, a GC box, ICE1, ICE2 and ICE3. In vivo footprinting analysis also identified a cluster of hypersensitive sites. However, there was no marked difference in protein-binding sites between parental and resistant cells. To confirm our previous results, we have established the VP16-resistant cell lines T12-VP1 and T12-VP2 from T12 cells derived from human bladder cancer T24 cells stably transfected with the chloramphenicol acetyltransferase reporter gene driven by the topo II alpha gene promoter. The expression to topo II alpha was down-regulated in both cell lines. We also found that CAT gene expression was significantly decreased to one-fifth of that in T12 parental cells. These results suggest that the expression of the topo II alpha gene requires the binding of multiple factors to the core promoter and is down-regulated at the transcriptional level, probably through binding of a negative factor to ICE1 in drug-resistant cells.

Transcription factor Y-box binding protein 1 binds preferentially to cisplatin-modified DNA and interacts with proliferating cell nuclear antigen.

The Y-box binding protein (YB-1) binds to inverted CCAAT box sequences that are present in the promoter region of many genes. We previously showed that YB-1 is overexpressed in human cancer cell lines that are resistant to cisplatin and that the depletion of YB-1 by transfection of a vector expressing YB-1 antisense RNA increases the sensitivity of human cancer cells to cisplatin. To determine whether YB-1 can bind to cisplatin-modified DNA, we fused YB-1 cDNA to glutathione S-transferase (GST) cDNA and purified the resulting GST fusion protein. When we tested the fusion protein with unmodified or cisplatin-modified oligonucleotides, we found that GST-YB-1 bound more strongly to cisplatin-modified oligonucleotides, as did GST fusion proteins of high mobility group 1 (HMG1), HMG2, and xeroderma pigmentosum group A protein. When we assayed the ability of proliferating cell nuclear antigen (PCNA) to interact with the GST fusion proteins, we observed binding to YB-1 but not to HMG1, HMG2, or xeroderma pigmentosum group A. Subsequent experiments demonstrated that YB-1 and PCNA interact directly via the COOH-terminal region of YB-1. Using immunochemical coprecipitation methods, we observed binding of YB-1 and PCNA in vivo. These results suggest that YB-1 can function as a recognition protein for cisplatin-damaged DNA and that it may be important in DNA repair or in directing the cellular response to DNA damage.

Structural basis for the regulation of UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells.

The UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (Gal NAc-T3) gene, a member of the Gal NAc transferase gene family, is expressed in a tissue-specific manner. To elucidate the function of this gene, we have focused on the molecular mechanism underlying regulation of gene expression. We have cloned Gal NAc-T3 cDNA and used it to show that Gal NAc-T3 mRNA is expressed in tumor cell lines derived from secretory epithelial tissue adenocarcinomas but not in cell lines derived from bladder and epidermoid carcinomas. Using a polyclonal antibody to Gal NAc-T3, we observed protein expression in adenocarcinoma but not non-adenocarcinoma cell lines, and in breast carcinoma cells but not in normal breast tissue. We used Gal NAc-T3 cDNA to isolate three overlapping genomic clones containing the 5'-portion of the human Gal NAc-T3 gene, and we sequenced 1.6 kb around the first exon. A transient expression assay using the luciferase gene showed that promoter activity was much higher in MCF-7 cells than in KB cells. In vivo footprint experiments showed significant protection of a distal GC box, an NRF-1 site, and an AP-2 site in MCF-7 cells. A novel stem and loop structure extending from nucleotide -103 to nucleotide -165 and contiguous to these transcription factor binding sites seemed to be functional in regulating Gal NAc-T3 gene transcription, and a KMnO4 footprint experiment showed that this stem and loop structure could be formed in vivo. We also observed dimethyl sulfate hypersensitive sites in the untranslated region around nucleotide +50 in MCF-7 but not in KB cells. These findings indicate that Gal NAc-T3 gene expression is regulated by multiple systems, including transcription factor binding sites and a stem-and-loop structure, and that this regulation is restricted to cell lines derived from epithelial gland adenocarcinomas but not cells derived from nonsecretory epithelial tissue carcinomas. In addition, our immunohistochemical results suggest that our anti-Gal NAc-T3 antibody may be useful for diagnostic purposes in the early stages of breast cancer.