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 non-functional P53 protein (Toillon et al, 2002) and have no functional progesterone and estrogen receptors. The cells were grown in RPMI1640 medium (Gibco BRL) supplemented with 5% fetal bovine serum, glutamine and gentamicin and maintained in a 5% CO₂ humidified atmosphere in a 37 °C incubator.

Cells were washed twice with PBS buffer then the preheated (95°C) 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 directed against the N-terminal fragment between amino acid residues 26-169 of human MDM2 (Fakharzadeh et al, 1991) then incubated with peroxidase-conjugated anti-mouse IgG (Jackson Laboratory, USA) at 1/2000 dilution. Immunoreactive bands were visualized by incubation with luminol (according to manufacturer's instructions; ECL Western blotting detection system from Amersham). The blots were stripped and hybridized with P53 (DO1) monoclonal antibodies (Oncogene Research, Cambridge, MA, USA) and processed as for the MDM2 antibodies. The TATA- Binding Protein (TBP) monoclonal antibody was used as a loading control (kindly provided by IGBMC Core Facility, Illkirch, France). Another MDM2 antibody was used, MDM2 (C-18) purchased from Santa Cruz Biotechnology (California, USA). It is an affinity purified rabbit polyclonal antibody raised against a peptide mapping within the carboxyl terminal domain of MDM2 of human origin. A prestained SDS-PAGE molecular weight protein standards (low range) is used to estimate the molecular weights of proteins ((Bio-Rad, UK).

MCF-7 cells were maintained in RPMI1640 medium + 5% FBS + antibiotics at 37°C with 5% (v/v) CO₂ in 36 mm plates (6-well cluster, Nunc, Roskilde, Denmark). They were transfected either with 3 μg CMV expression vector (pcDNA3, obtained from Invitrogen, Paisley, UK) or with 3 μg CMV vector containing human P53 cDNA (kindly provided by the IGBMC core facility, Illkirch, France) by using lipofectamine (Gibco-BRL, Paisley, UK). MCF-7 cells that overexpress P53 as well as the control cells (transfected with the empty vector) were treated with vehicle alone or with 100 nM of progesterone for 24 hr. The total protein extracts were prepared and tested with DO-1, IF2 and TBP monoclonal antibodies as indicated above.

The expression of MDM2 proteins in MCF7, T47D, and MDA-MB231 human breast cancer cell lines was analyzed by Western blotting with anti-MDM2 (Ab-1) monoclonal antibody. Figure 1 shows that, with this antibody, four MDM2 isoforms were detected, the p57, p76, p80, and p90 kDa in all three cell lines. A prominent expression of MDM2 p57 protein as compared to the p90, p80 or p76 was observed in the three cell lines used in this study. MDM2 p90 protein level was dependent on p53 status in the cells. In MCF-7 cells that contained functional P53 protein, the endogenous MDM2 p90 protein level was higher than these in T47D and MDA-MB 230 cell lines (both have mutated P53 proteins). In contrast, MDM2 p57 appeared to be lower in MCF-7 as compared to its levels in the cells which have mutated P53. The high level of MDM2 p57 was observed in other breast cancer cells, a normal breast cell line and in other types of cancer cell lines (data not shown).

The MCF-7 cell line was used in this experiment because it has high levels of the four MDM2 isoforms and because we had already shown that progesterone down-regulated P53 protein in these cells (Alkhalaf and El-Mowafy, 2003). We investigated whether the P53 inhibition in this cell line would affect the endogenous levels of MDM2 proteins. When MCF-7 cells are treated with 100 nM of progesterone, expression of p90 was specifically inhibited, unlike that of the highly expressed p57 isoform or the TBP protein (Figure 2).

The observation that progesterone simultaneously inhibited endogenous MDM2 (Figure 2) and P53 (Alkhalaf and El-Mowafy, 2003) in MCF-7 cells prompted us to check the consequences of re-establishing the P53 expression in the cells by exogenous expression using transient transfection experiments. MCF-7 cells were transfected with expression vector (3 μg/well) for CMV vector and CMV vector containing human P53 cDNA. The cells were treated with vehicle or with 100 nM of progesterone for 24 hr. Western blot analysis was used to estimate the P53 levels achieved by transient transfection. CMV- transfected MCF-7 without progesterone treatment showed normal levels of P53, MDM2 p90 and MDM2 p57. When CMV-transfected MCF-7 cells were treated with 100 nM of progesterone, both MDM2 p90 and P53 were inhibited, unlike the MDM2p57 isoform. MCF-7 cells transfected with CMV+P53 vector showed approximately three fold higher P53 levels than CMV transfected cells (Figure 3) as revealed by densitometry. In these P53 overexpressing MCF-7 cells, the MDM2 p90 level was elevated to reach approximately the same level observed in the CMV transfected MCF7 cells without progesterone treatment. The increase in MDM2 p90 level in MCF-7 cells that overexpress P53 is associated with progesterone treatment suggesting that p53 reestablishing p53 expression enhances the expression of MDM2 p90 and that this increase in MDM2 level is progesterone-dependent.