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 G1cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7cells, 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.

Keywords: Hdm2; HdmX; RNAi; gene expression profiling; p53.

Figure 1.

(A) RT-PCR analysis of hdmX and hdm2 gene expression in various human cell lines. The endogenous levels of hdmX and hdm2 were determined relative to H1299 cells. All samples were normalized to GAPDH. (B) RNAi knockdown of HdmX or Hdm2 triggers p53-dependent p21 induction. Western blot analysis of indicated proteins from the various siRNA or doxorubicin (Dox) treated MCF7 cells. Knockdowns of the indicated proteins were greater than 80%. Protein extracts were made 24 hours after the last siRNA transfection or treatment with 5 μg/ml doxorubicin.

Figure 2.. Loss of HdmX and/or Hdm2 inhibits MCF7 colony formation.

(A) Following siRNA transfections, MCF7 cells were seeded at 500 cells/well in 6-well plates. The cells were allowed to grow for ten days then the colonies were stained with crystal violet. Significantly fewer colonies were present following knockdown of HdmX and/or Hdm2. The cells transfected with sip53 or a non-targeting control (siCon) showed minimal effects on colony formation relative to non-transfected control (Con/Control). (B) The percent cell growth relative to untransfected control was determined by extracting the stain in 10% acetic acid and quantifying the stain by reading absorbance at 590 nm.

Figure 3.. Knockdown of HdmX enhances doxorubicin-induced cytotoxicity.

(A) Percent cell viability relative to untransfected untreated control cells. MCF7 cells were treated with doxorubicin (0.25-1.0 μg/mL) for 48 hours and cell viability was determined by absorbance at 590 nm. The loss of HdmX and/or Hdm2 showed an enhanced cytotoxicity relative to control cells. (B) RT-qPCR analysis of hdmX, hdm2, p21 and p53 gene expression in the indicated siRNA transfected MCF7 cells. The hdmX, hdm2, and p53 transcripts were effectively knocked down by siRNA prior to drug treatment.

Figure 4.. GeneChip expression of 13 known p53-regulated genes that were induced by knockdown of either siHdmX or siHdm2.

Y-axis represents the average fold change (log₂) for each of the genes in the indicated siRNA transfections relative to siCon (X-axis, conditions labeled at the top of the chart).

Figure 5.. RT-qPCR validation of siRNA knockdown in MCF7 cells. (A).

The hdmX, hdm2, and p21 mRNA expression relative to siCon (non-targeting siRNA) is shown. The p21 transcript is induced following loss of HdmX or Hdm2, and synergistically induced following loss of both HdmX and Hdm2. (B) BTG2, ACTA2, and NOXA mRNA expression relative to untransfected control (Con). The p53 target genes, BTG2 and ACTA2, are induced by loss of HdmX and/or Hdm2, while the expression of the proapoptotic gene, NOXA, is not altered.

Figure 6.. GeneChip expression of 13 reported E2F1-regulated genes that were repressed by knockdown of either siHdmX or siHdm2.

Y-axis represents the average fold change (log₂) for each of the genes in the indicated siRNA transfections relative to siCon (X-axis, conditions labeled at the top of the chart).

Figure 7.. Repression of E2F1-regulated genes by Hdm2 or HdmX knockdown is blocked by concurrent knockdown of p21.

MCF7 cells were transfected with the indicated siRNA combinations. Twenty-four hours later, RNA was isolated and subjected to RT-qPCR to quantify expression of CCNA2, p21 and E2F1 after normalization to GAPDH. Expression levels (Y-axis) were relative to siCon and reported as RQ values. Error bars represent the 95% confidence interval of the relative expression.