Cloning and characterization of a p-53-inducible gene, WIG-1

Elucidating the mechanisms by which p53 inhibits tumor growth is a central issue in cancer research, as the TP53 gene is mutated in over half of all human cancers. Lack of functional p53 also contributes to the resistance of many tumor types to chemotherapy and radiotherapy. p53 is a transcription factor, which regulates the expression of a large number of genes. In fact, most of the knowledge about the molecular mechanisms by which p53 exerts its biological functions has been acquired by the identification and characterization of genes whose expression is regulated by p53. This thesis reports on the identification and characterization of a novel p53-inducble gene, designated WIG-1 (Wild type p53 Induced Gene 1). The mouse homologue was first identified by an improved differential display technique. A 7661 bp wig-1 transcript was cloned from the p53negative mouse T lymphoma cell line (J3D), transfected with a temperature-sensitive mouse mutant p53 (Val- 135), using the 5 Marathon cDNA amplification technique. Two mouse wig-1 transcripts of 7.6 kb and 2.2 kb were detected in Northern analysis. The 2.2 kb mouse wig-1 transcript was suggested to arise due to alternative polyadenylation and encodes the same zinc finger protein as the 7.6 kb transcript. Mouse wig-1 induction in response to DNA damage was shown to be p53 dependent. Induction of mouse wig-1 was demonstrated to occur in vivo, in a tissue-specific manner by Northern analysis. The coding region of human WIG-1 was cloned from a brain cDNA library. The human and mouse WIG-1 coding sequences are 84% identical at the nucleotide level and 86.9% identical at the amino acid level, with the three conserved zinc finger motifs. Two human WIG-1 transcripts of 8 kb and 6 kb were detected by Northern analysis. Their induction was demonstrated to occur in human cell lines containing wild type p53 but not in cell lines with mutant p53. A p53-specific binding site with perfect match to the p53 consensus-binding site was found in intron 1 of human WIG-1. In vitro binding of wild type p53 to this site was shown by electrophoretic mobility gel shift assay. Tissue- and species-specific basal expression of both human and mouse WIG-1 were demonstrated by Northern analysis. The abundance of both human and mouse WIG-1 mRNAs and the ratio between the transcripts were shown to vary among different tissues. Human and mouse WIG-1 were mapped to 3q26.3 and mouse chromosome 3B, respectively. Human WIG-1 was demonstrated to be amplified with increased expression in primary squamous cell carcinomas of the lung, using Southern blot and RT-PCR analysis. Polyclonal antibodies against the mouse wig-1 were raised and purified. Further investigation into the function of WIG-1 should contribute to a better understanding of the p53-regulated pathways. This in turn will provide further information for logical, interventional therapeutic strategies against p53-deficient tumors