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The MDM2 oncoprotein promotes apoptosis in p53-deficient human medullary thyroid carcinoma cells
T Dilla1, J A Velasco, D L Medina
1Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Spain.
Abstract:
The MDM2 oncoprotein has been shown to inhibit p53-mediated growth arrest and apoptosis. It also confers growth advantage to different cell lines in the absence of p53. Recently, the ability of MDM2 to arrest the cell cycle of normal human fibroblasts has also been described. We report a novel function for this protein, showing that overexpression of MDM2 promotes apoptosis in p53-deficient, human medullary thyroid carcinoma cells. These cells, devoid of endogenous MDM2 protein, exhibited a significant growth retardation after stable transfection with mdm2. Cell cycle distribution of MDM2 transfectants [medullary thyroid tumor (MTT)-mdm2] revealed a fraction of the cell population in a hypodiploid status, suggesting that MDM2 is sufficient to promote apoptosis. This circumstance is further demonstrated by annexin V labeling. MDM2-induced apoptosis is partially reverted by transient transfection with p53 and p19ARF. Both MTT and MTT-mdm2 cells were tumorigenic when injected into nude mice. However, the percentage ofapoptotic nuclei in tumor sections derived from MDM2-expressing cells was significantly higher relative to that in the parental cell line. MDM2-mediated programmed cell death is at least mediated by a down-regulation of the antiapoptotic protein Bcl-2. Protein levels of caspase-2, which are undetectable in the parental cell line, appear clearly elevated in MTT-mdm2 cells. Caspase-3 activation does not participate in MDM2-induced apoptosis, as determined by protein levels or poly(ADP-ribose) polymerase fragmentation. The results observed in this medullary carcinoma cell line show for the first time that the product of the mdm2 oncogene mediates cell death by apoptosis in p53-deficient tumor cells.
Insights
Overexpression of MDM2 oncogene induces apoptosis in p53-deficient thyroid cancer cells. This MDM2-mediated cell death involves Bcl-2 down-regulation and caspase-2 activation, offering new therapeutic insights.
Area of Science:
- Molecular Oncology
- Cancer Biology
- Cell Death Mechanisms
Background:
- The MDM2 oncoprotein typically inhibits p53-mediated apoptosis and promotes cell growth, even in p53-deficient cells.
- MDM2 has also been observed to induce cell cycle arrest in normal human fibroblasts.
- The role of MDM2 in apoptosis induction in p53-deficient tumor cells remained largely unexplored.
Purpose of the Study:
- To investigate a novel function of MDM2 in promoting apoptosis in p53-deficient human medullary thyroid carcinoma cells.
- To elucidate the molecular mechanisms underlying MDM2-induced apoptosis in this specific cancer context.
Main Methods:
- Stable transfection of p53-deficient medullary thyroid carcinoma cells (MTT) with the mdm2 oncogene.
- Cell cycle analysis (hypodiploidy), Annexin V labeling, and Western blotting for apoptosis-related proteins (Bcl-2, caspases).
- Tumorigenicity assays in nude mice and analysis of apoptosis in tumor sections.
Main Results:
- Stable MDM2 overexpression in MTT cells (MTT-mdm2) led to significant growth retardation and induced apoptosis, evidenced by hypodiploidy and Annexin V staining.
- MDM2-induced apoptosis was partially reversed by co-expression of p53 and p19ARF.
- MDM2-mediated apoptosis involved down-regulation of anti-apoptotic Bcl-2, up-regulation of caspase-2, but not caspase-3 activation.
Conclusions:
- MDM2 oncogene product is sufficient to promote apoptosis in p53-deficient medullary thyroid carcinoma cells.
- MDM2-induced programmed cell death is mediated, at least in part, by down-regulation of Bcl-2 and activation of caspase-2.
- These findings reveal a novel pro-apoptotic role for MDM2 in p53-deficient tumors, distinct from its known functions.