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While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth. Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1 cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7 cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown. Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.