Cancer Cell. with 100 nM of progesterone, MDM2 p90 was inhibited but the highly expressed MDM2 p57 F2rl1 isoform was not. The inhibition of MDM2 p90 protein by progesterone was abrogated in MCF-7 cells transfected with a P53 expressing vector. To our knowledge, this is the first report linking progesterone-induced growth inhibition with down-regulation of the MDM2 protein. We present evidence that reestablishing of P53 expression by transient transfection of P53 cDNA in these cells enhances the expression level of MDM2 p90 isoform. The data indicate that expression of MDM2 p90 is regulated through a P53-dependent pathway in response to progesterone. gene was originally cloned as an amplified gene on a murine double-minute chromosome in the tumorigenic 3T3DM murine cell line (Fakharzadeh et al, 1991). The corresponding human gene was also BMS-790052 (Daclatasvir) subsequently identified (Oliner et al, 1992). MDM2 expression is controlled at the transcriptional level from P53-independent (P1) and P53-responsive (P2) promoters (Zauberman et al, 1995), both encoding a 90 kDa full length MDM2 (p90) protein (Brown et al, 1999). In addition, MDM2 proteins of smaller sizes have been identified (Olson et al, 1993; Perry et al, 2000; Bartel et al, 2002). These differently sized proteins arise through either proteolytic cleavage (Pochampally et al, 1998), internal translational initiation (Saucedo et al, 1999) or alternative BMS-790052 (Daclatasvir) splicing (Sigalas et al, 1996; Matsumoto, 1998). Although the biochemical functions of these small proteins have not yet been determined, the MDM2-p90 isoform binds to and inactivates P53 tumor suppressor protein suggesting that MDM2 can function as a negative feedback regulator of P53 (Momand et al, 1992; Barak et al, 1993). Lukas et al (2001) suggested that MDM2 expression is altered in invasive breast cancer and is associated with more aggressive disease. We have recently demonstrated that the progesterone-induced growth inhibition of the MCF-7 human breast cancer cell line was associated with down-regulation of P53 BMS-790052 (Daclatasvir) endogenous levels (Alkhalaf and El-Mowafy, 2003). Because the regulation of MDM2 expression by P53 has been proposed by several authors to be the mechanism by which P53 balances its own activity (Juven et al, 1993; Midgley and Lane, 1997; Prives, 1998), we hypothesized that the decrease in P53 levels seen in MCF-7 cells treated with progesterone would affect MDM2 expression. We report here that in MCF-7 human breast cancer cells treated with progesterone, MDM2 p90 but not MDM2 p57 is down-regulated. To confirm the involvement of P53 in this down-regulation of MDM2, MCF-7 cells were transiently transfected with a P53 expression vector (Alkhalaf and El-Mowafy, 2003). Overexpression of P53 in MCF-7 cells stimulated the MDM2 expression and abrogated the effect of progesterone. The data suggest that expression of MDM2 p90 is regulated via a P53-dependent pathway in MCF-7 human breast cancer cells treated with progesterone. MATERIALS AND METHODS Cell lines and culture conditions The breast cancer cell lines MCF7, T47D, and MDA-MB231 were kindly provided by Bohdan Wasylyk (IGBMC Core Facility, Strasbourg, France). The MCF-7 cells contain functional P53 protein localized at the nucleus (Wasylyk et al, 1999) and classified as progesterone and estrogen receptor positive. The T47D cells have a mutated type of P53 which is localized in the cytoplasm (Schafer et al, 2000) and contain both estrogen and progesterone receptors. The MDA-MB231 cells have nonfunctional P53 protein (Toillon et al, 2002) and have no functional progesterone and estrogen receptors. The cells were grown in RPMI1640 medium (Gibco BMS-790052 (Daclatasvir) BRL) supplemented with 5% fetal bovine serum, glutamine and gentamicin and maintained in a 5% CO2 BMS-790052 (Daclatasvir) humidified atmosphere in a 37 C incubator. Western Blot Analysis Cells were washed twice with PBS buffer then the preheated (95C) lysis buffer [20 mM Tris-HCl pH 7.4, 20 mM dithiothreitol (DTT), 2 mM EDTA (sodium salt), 1% (v/v) Triton X-100, 1% (v/v) NP40, 1% (w/v) sodium deoxycholate, 1 mM sodium pyrophosphate, 1 mM sodium orthovandate (prepared in Tris buffer) and 1 mM phenylmethylsulphonyl-fluoride] was added directly to the cell monolayer. The cells were scraped and mixed with a rubber policeman, transferred to Eppendorf tubes and centrifuged at 13000 x g for 5 min. The resulting supernatant was saved and the protein was determined by the Bradford method. Extracts were boiled for 3 min in 2 x SDS buffer. Equal amounts of protein were loaded on 10% (w/v) polyacrylamide gels according to the method of Laemmli and then electrotransferred onto nitro-cellulose membranes. The blots were incubated first with anti-MDM2 (Ab-1, clone IF2) monoclonal antibody (Oncogene Research Product, Calbiochem). The antibody is.
