Alterations in gene expression and sensitivity to genotoxic stress following HdmX or Hdm2 knockdown in human tumor cells harboring wild-type p53

Abstract

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.

Outcome of the p53-mediated DNA damage response in neuroblastoma is determined by morphological subtype and MYCN expression

“…Microarray analysis of these cells revealed a subset of p53 target genes with anti-proliferative properties and a further subset of genes which are transactivated by E2F as cells transverse the G 1 -S boundary. 38 In conclusion, in this study we have shown that both the cell type N or S and MYCN are involved in the downstream response to irradiation induced DNA damage and that these effects are at least partially p53-dependent. Also, there were no MYCN coamplified genes that were found to be associated with failure to G 1 arrest.…”

p53 regulation: Teamwork between RING domains of Mdm2 and MdmX

“…These recent advancements in the field assigned an essential role of the RING domain of MdmX in negative regulation of p53 in vivo, just like Mdm2 RING domain, through p53 degradation. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] because activation of p53 can lead to apoptosis, quiescence and senescence. [21][22][23][24][25][26][27][28][29][30][31] Furthermore, the ability of p53 to choose between quiescence and senescence is determined by its ability to inhibit mTOR.…”

Deep Insight Into YPEL3

“…In order to study novel p53 regulated genes, a microarray experiment was completed where the negative regulators of p53 (Hdm2 and HdmX) were silenced in MCF7 breast carcinoma cells using siRNA technology (Heminger et al, 2009). MCF7 cells contain wild-type p53; therefore, knocking down Hdm2 and HdmX allowed for the nongenotoxic stress induction of p53.…”

“…MCF7 cells contain wild-type p53; therefore, knocking down Hdm2 and HdmX allowed for the nongenotoxic stress induction of p53. This led to a G1 phase cell cycle arrest along with sensitization of the arrested MCF7 cells to DNA damage (Heminger et al, 2009). From analysis of the microarray data, a novel p53 target was discovered named YPEL3.…”