Two other such genes encode the matrix metalloproteinase 2 and Rad51, where AP2a DNA binding and regulation was shown INCB18424 experimentally to require p53. Consistently, these genes were not recognized by purified AP2a recombinant protein but interaction was only observed when using nuclear extract. Thus, the PBM results suggest that indirect interactions of AP2a are much more widespread than previously known and that oncogenic transformation is accompanied by a change in AP2a target gene specificity mediated in part by the modulation of these indirect interactions. In this respect, novel AP2-bound genes from cellular datasets feature prominent cell kinase inhibitors cycle-related regulatory targets of the p53 and Rb tumor suppressors such as the E2F and cyclin gene families. Comparison of AP2 binding specificity from normal and tumor tissues yielded generally correlated results, as many genes that were bound by AP2a in the healthy tissue extract were also bound using tumor extracts. The higher number of genes bound from the tumor extracts can be attributed to the combined effects of differences in the activity of AP2a and of the proteins it synergizes or antagonizes with. Consistently, cancer-associated genes that had not been previously associated to AP-2 were identified in the tumor extracts datasets, as for instance the breast cancer susceptibility gene 2 and the cyclin-dependent kinase 2 gene. Comparison of the binding strength of regulatory sequences detected using the two types of extract yielded 149 sequences that were differentially bound by AP2a. When a similar comparison was performed between 2 sets of 4 randomly selected breast cancer extracts, to assess the experimental noise, 52 differentially bound sequences were obtained. Differences between the two sets of tumors may reflect the known heterogeneity of breast tumors. Nevertheless, the nearly three-fold higher number of differentially bound genes from healthy versus breast tumors tissue comparisons indicated that AP2a specificity differs much more between normal and cancerous tissues than among individual tumor types. One of the genes bound solely from the cancer biopsy extract was found to encode Bcl2, which is known to be down-regulated by AP2a to provoke tumor cell apoptosis upon chemotherapy. Functional analysis of networks associated with genes differentially bound by AP2a in normal and tumor extracts showed that genes are involved in genetic disorders and cancer, but also in reproductive system disease. Taken together, these results imply that the PBM-based approach may be used to detect bona fide direct and indirect targets of AP2 as well as reveal novel ones, and that it may differentiate healthy from cancerous breast tissues. They also support previous proposals that AP2a DNA binding may be subjected to antagonistic or synergistic interactions with numerous other nuclear proteins in tumor cells.