Overexpression of MDM2 in MCF‐7 Promotes Both Growth Advantage and p53 Accumulation in Response to Estradiol

Abstract

The overexpression of the oncogene product MDM2 is often observed in human breast cancer cells, especially in estrogen receptor (ER)-positive ones. To study the role of MDM2 protein in ERpositive breast cancer, we have established cell lines derived from MCF-7 which stably express increased and decreased levels of MDM2 by transfection of a mammalian expression vector containing human mdm2 cDNA in sense and antisense orientations, respectively. Interestingly, MDM2 overexpression in MCF-7 cells afforded a remarkable growth advantage under estradiol (E 2 )-supplemented condition. Then, we analyzed the expression of p53, which is an important regulator of growth and the cell cycle. Unexpectedly, the p53 accumulation induced by E 2 was remarkably higher in MCF-7 cells stably overexpressing MDM2 than in the parent MCF-7 cells. On the other hand, reduction of MDM2 suppressed the E 2 -induced increase in p53 protein. Moreover, mdm2 antisense oligonucleotides prevented E 2 -induced accumulation of p53. In the steady state, the cellular levels of p53 were also correlated with those of MDM2. These interactions are not consistent with the well-known role of MDM2, which acts as a negative regulator for p53 by inhibiting its function and promoting its rapid degradation. These results suggest that MDM2 may regulate the expression of p53 in the steady state and in response to E 2 in breast cancer cells, and imply a novel and important role of MDM2 during breast carcinogenesis. Key words: Breast cancer -MDM2 -p53 -Estrogen receptorThe mdm2 oncogene was originally identified as a highly amplified gene on a murine double-minute chromosome in the 3T3DM cell line, a spontaneously transformed derivative of BALB/c3T3 cells.1, 2) The corresponding human mdm2 gene was also identified.3) Overexpression of the mdm2 gene in NIH3T3 cells increases its tumorigenic potential, thus establishing mdm2 as an oncogene.2) The gene encodes a polypeptide consisting of 489 amino acids that contains a binding domain for the tumor suppressor p53, an acidic region, zinc finger motifs and a ring finger domain.2, 4-6) MDM2 is believed to bind to the N-terminal region of p53 and to inhibit its transcriptional activity by masking its transactivation domain. 3,4) Recently, it was also shown that MDM2 promoted the rapid degradation of p53. 7,8) In contrast, p53 induces the expression of MDM2 via binding to its promoter region, suggesting that MDM2 can function as a negative feedback regulator of p53. 4,9) In addition to regulating p53, MDM2 has also been shown to interact with many other molecules, such as the retinoblastoma protein pRB, 10) E2F transcriptional factor, 11) ribosomal protein L5, 12) RNA, 13) cell fate regulator Numb, 14) and cell cycle inhibitor p19Arf . 15,16) Interaction with pRB and with E2F1/DP1 promoted G1/S cell cycle progression through disrupting RB function and stimulating transcriptional activity of E2F1. 10,11) Binding to the ribosomal protein L5 suggests the possibility that MDM2 may enhance the translation process. The association ...

Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53

“…T47D human breast cancer cells, containing a mutant form of p53, restored elevated p53 levels following treatment with estradiol (17). In addition, wild-typep53 MCF7 breast cancer cell lines overexpressing Mdm2 demonstrated increased steady-state levels of p53 in the presence of estradiol (45). Studies using murine models assessing estrogen stimulation of the mammary gland not only reported increased levels of nuclear, functional p53 (21, 49) but also demonstrated that hormone stimulation is necessary for a maximal p53-mediated response to ionizing radiation (1).…”

MDM2 Enhances the Function of Estrogen Receptor α in Human Breast Cancer Cells

“…For experiments evaluating the effect of E2, cells were cultured in phenol red-free RPMI1640 (PRF-RPMI, Gibco BRL) containing 10% FBS stripped of steroids by absorption to dextran-coated charcoal (DCC-FBS). MCF-7/pCmdm2 clones S1 and S3 were established by transfection of mammalian expression vector containing human mdm2 cDNA (pCmdm2) into MCF-7 cells as described previously (29,30). Saos-2, a p53-deficient osteosarcoma cell line, was from American type culture collection (Manassas, VA) and grown in McCoy's 5A medium (Gibco BRL) containing 15% FBS and antibiotics.…”

Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

“…Lack of such protection in BALB/c p53-null mammary epithelium further strengthened the pivotal role of p53 in hormone-induced protection against carcinogenesis (25). In MDM2-overexpressing breast cancer cells, enhanced ERa function was observed and E2 stabilized wild-type p53 without any increase in p53 gene transcription (26,27). Further, p53 has been reported to be in a ternary complex with ERa and MDM2 (28).…”