title: Identification of regulatory regions within the KAI1 promoter: a role for binding of AP1, AP2 and p53.
abstract: The mechanism underlying loss of KAI1 gene expression in invasive and metastatic tumour cells is unknown. A possible scenario could involve altered expression or function of protein factors normally involved in regulating KAI1 transcription. To explore this possibility, we have initiated a study to characterise regulatory elements of the KAI1 promoter, using as a model, two bladder cancer cell lines (BL13 and HT1376) expressing high levels of endogenous KAI1 messenger RNA (mRNA). Transfection experiments using reporter plasmids with progressive KAI1 promoter deletions, identified a 76 bp region upstream of the transcription initiation site which contained putative binding motifs for AP2, p53 and AP1, as essential for reporter activity. DNA-binding studies using nuclear extracts from both cell lines, showed that AP1 and AP2 formed specific complexes with oligonucleotides containing KAI1 promoter motifs. Mutation of either motif abrogated reporter activity and abolished specific complex formation. In BL13 cells (endogenous wildtype p53), but not in HT1376 cells (endogenous mutant p53), mutation of the p53-binding motif also abrogated reporter activity and abolished specific complex formation in gel shift assays. These data suggested that a combination of AP2, p53 and AP1 binding to specific motifs within the KAI1 promoter might be required for high level promoter activity and that loss of expression or function of these factors might contribute to loss of KAI1 expression in invasive tumours and tumour cell lines. To explore this possibility, we examined levels of these proteins in nuclear extracts of BL13 and HT1376, as well as three bladder cancer cell lines which expressed little or no KAI1 mRNA. Our data suggested that a loss of KAI1 mRNA was not simply due to absence of AP2, AP1 or p53 expression.
authors.affiliation: Oncology Research Centre, Level 2 Clinical Sciences Building, Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Marreiros
authors.forename: Robert
authors.initials: R
authors.lastname: Czolij
authors.forename: Gina
authors.initials: G
authors.lastname: Yardley
authors.forename: Merlin
authors.initials: M
authors.lastname: Crossley
authors.forename: Paul
authors.initials: P
authors.lastname: Jackson
mesh_terms: D015703:Antigens, CD; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051861:Kangai-1 Protein; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C497688:CD82 protein, human; D004268:DNA-Binding Proteins; D051861:Kangai-1 Protein; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase