Detection of p53 Allele Deletions in Human Cancer by Quantification of Genomic Copy Number

Abstract

In most cases cancer is a genetic disease, characterized by a multiple step process [1], which involves the inactivation of tumor suppressor genes (e.g., by deletion of alleles) and the activation of oncogenes (e.g., by amplification of alleles). In healthy cells these misregulated genes are important regulators of cellular growth and cellular division, DNA repair, and apoptosis. p53 is the most prominent and most important tumor suppressor gene which becomes inactivated in nearly all cancer types, roughly 50% of all human tumors being affected by p53 inactivation [2]. The deletion of an allele or the inactivation by point mutations are the most prominent mechanisms for loss of function of p53. Tumors containing inactivated p53 are highly aggressive and often resistant to chemo- as well as radiotherapy. As the functional status of p53 has many clinical and therapeutical implications [3, 4, 5], rapid and sensitive tests for the presence or absence of functional p53 genes are needed. Several techniques have been described for the detection of p53 allele deletions: (a) analysis of polymorphic DNA markers (RFLPs, minisatellites, microsatellites) for loss of heterozygosity [6, 7]; (b) Southern blotting techniques [2]; (c) fluorescence in situ hybridizations [5, 8]; (d) comparative genomic hybridization [8, 9]; and (e) quantitative competitive PCR [10] which all have several disadvantages, e.g., that they require large amounts of high molecular DNA (b); that they involve cell culture techniques and fluorescence microscopy (c, d); that they depend on the presence of heterozygous allelotypes of DNA markers, application of radioactivity, or fluorescent labels plus DNA sequencers (a); that they require internal control templates (e); and that they only detect allele imbalance but in most cases do not allow discrimination between deletions and amplifications (a).