MDM2-C has p53-independent transformation activity.

<div><p><b>A</b>. Analysis of exogenously expressed MDM2-FL or MDM2-C in H1299 cells with simultaneous expression of p53. Increasing amount of pcDNA3-<i>mdm2-FL</i> and pcDNA-P2mdm2<i>-C</i> plasmid DNAs were transiently transfected with a constant amount of SN3 plasmid (p53) into H1299 <i>p53-</i>null cells. PGL2 Basic plasmid was used as a DNA normalizer. Cells extracts were prepared 48 hours after transfection and analyzed for protein expression by western blot analysis using the Mdm2 monoclonal antibody mix (4B11) and p53 monoclonal antibody mix (240, 1801, 421). Actin was used as a loading control. HRP-conjugated anti-mouse and anti-rabbit were used as secondary antibodies. This is a representative of three independent experiments.</p> <p><b>B</b>. 48 hours after transfection, cells were lysed and protein extracts were utilized in a luciferase assay reaction. Samples were compared to PGL2 Basic plasmid and normalized for amount of protein. An average of three independent experiments is shown. Error bars represent standard error. </p> <p><b>C</b>. Colony formation assay in H1299. Cells were transiently transfected with the plasmid pBaBe-puro-<i>mdm2-FL</i> or pBaBe-puro-<i>mdm2-C</i> and after 24 hours, 2000 cells were plated into media (RPMI with 2ug/ml puromycin). Cells were allowed to grow for 3 weeks. Picture represents one experiment. Two experiments were carried out in duplicates. </p> <p><b>D</b>. Colony counts after colony formation assay. An average of two experiments carried in duplicates is shown above. Error bars represent standard error. * Asterisks represent p-value compared to vector control.</p></div

Endogenous Human MDM2-C Is Highly Expressed in Human Cancers and Functions as a p53-Independent Growth Activator

<div><p>Human cancers over-expressing <i>mdm2</i>, through a T to G variation at a single nucleotide polymorphism at position 309 (<i>mdm2</i> SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, <i>mdm2-C</i>. Alternatively spliced <i>mdm2</i> transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with <i>mdm2-A</i> and <i>mdm2-B</i>, but the <i>mdm2-C</i> isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with <i>in vitro</i> translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) <i>mdm2</i> G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in <i>p53</i>-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.</p> </div

An MDM2-C specific antibody named C410, detects the MDM2-C protein.

<div><p><b>A</b>. Schematic of full length MDM2 (MDM2-FL) and MDM2-C. The retained proteins’ biochemical functional domains are shown as color codes. The peptide sequence used as an immunogen containing the splice junction of human MDM2-C is shown. Glycine (G) and Cysteine (C) residues were added to the N-terminus of the peptide to facilitate conjugation to an immunoreactive protein, keyhole limpet hemocyanin (KLH).</p> <p><b>B</b>. ³⁵S methionine was used as a radioactivity source to label <i>in </i><i>vitro</i> translated proteins. <i>In </i><i>vitro</i> translations of pcDNA3-mdm2-FL and pcDNA3-P2mdm2-C using the TNT coupled wheat germ extract system. Resulting MDM2-FL and MDM2-C protein were electrophoresed on a10% SDS-PAGE in a 5:1:1 ratio. The gel was transferred to a nitrocellulose membrane and exposed to film for significant MDM2-C protein product detection. Wheat germ lysate without DNA was used as a negative control.</p> <p><b>C</b>. Immunoprecipitation of ³⁵S methionine radioactive-labeled <i>in </i><i>vitro</i> translated MDM2-FL and MDM2-C proteins using MDM2 C410 and pre-immune polyclonal serum antibodies. Protein ratios as shown in <b>B</b> were used in the pull down assay. Samples were electrophoresed on a 10% SDS-PAGE gel transferred to a nitrocellulose membrane and exposed to film for protein detection. This is representative of three independent experiments.</p> <p>D. <i>In </i><i>vitro</i> translated protein made in rabbit reticulocyte lysate (RRL lanes 1 and 2) were compared to protein translated in wheat germ extract (WGE lanes 3 and 4). These proteins were detected with either antibody 4B11 (top panel) or 2A9 (bottom panel). HRP-conjugated anti-mouse and anti-rabbit were used as secondary antibodies.</p></div

Human MDM2-C interacts with a variety of proteins <i>in</i><i>vivo</i>.

<p>Immunoprecipitation from MANCA whole cell extracts using MDM2 C410 and pre-immune polyclonal serum antibodies antibodies. Samples were electrophoresed onto a 10% SDS-PAGE gel in duplicate. <b>A</b>. One half was stained with coomassie blue for protein detection and protein bands from A were excised for analysis via LC/MS/MS. <b>B</b>. Samples from half of gel A were transferred to nitrocellulose membrane and MDM2 protein was detected using the MDM2 monoclonal antibody mix (4B2, 2A9, 4B11). <b>A</b>. Bracket represents region where bands were cut out for LC/MS/MS analysis. <b>B</b>. Arrows depict MDM2-C protein. </p

MDM2-C is highly expressed in liposarcoma and breast carcinoma tissues.

<p><b>A</b>. Immunohistochemistry of lipoma and liposarcoma tissues using MDM2 C410 and pre-immune polyclonal serum antibodies. Biotinylated secondary antibody, ABC Reagent and DAB from Vector Labs were used. Pictures were obtained at 20X and 40x magnification. H&E refers to Hematoxylin and Eosin counterstaining. <b>B</b>. Breast tissue arrays (TMA-1008) were purchased from protein biotechnologies and automated histology was performed at the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center. Antibodies MDM2 C410 and MDM2 4B11 were used for staining. Mouse IGG was used for negative staining. H&E refers to Hematoxylin and Eosin counterstaining.</p

High expression of endogenous MDM2-C protein in G/G <i>mdm2</i> SNP309 MDM2 over-expressing cells.

<div><p><b>A</b>. Western blot analysis of whole cell extracts from: MANCA, SJSA-1, ML-1 and K562 cells. MDM2-C protein levels were analyzed via MDM2 C410 polyclonal serum antibodies (C410, lanes 1-4) and total MDM2 was detected with the monoclonal 4B11 (lanes 9-12 and long exposure for lane 12). Actin was used as a loading control. Pre immune polyclonal serum was used as a negative control. HRP-conjugated anti-mouse and anti-rabbit were used as secondary antibodies. This is representative of three independent experiments.</p> <p><b>Bi</b>. MANCA cells were lysed either as whole cell extracts (WCE) or into cellular compartments- cytosolic (CYTO) and Chromatin (CHR). Proteins were detected as in A. MDM2-C protein levels were analyzed via MDM2 C410 polyclonal serum antibodies (C410, lanes 1-3) and total MDM2 was detected with the monoclonal 4B11 (lanes 1-9 and long exposure for lane 9). </p> <p><b>Bii</b>. Tubulin and Fibrillarin were used to show efficient cellular fractionation of extract. </p> <p><b>C</b>. Spinning disk confocal microscopy of MANCA cells. Cells were fixed, permeabilized and incubated with p53, MDM2 C410 and pre-immune polyclonal serum antibodies. Slides were incubated with secondary Alexa-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse. DAPI was used to stain the cell nuclei. Pictures were taken at 60X magnification. Arrows indicate regions of Mdm2-C protein nuclear localization.</p></div

An <i>mdm2</i> splice variant transcript, <i>mdm2-C</i> is highly expressed in MDM2 over-expressing cells.

<div><p><b>A</b>. Quantitation using Image J after northern blot analysis of RNA from untreated MANCA, SJSA-1, ML-1 and K562 cell samples. An <i>mdm2</i> DNA probe to exon 12 was PCR generated and radiolabelled with α ³²P dCTP. Transcript levels were compared to K562 for basal expression and normalized for RNA levels to <i>gapdh</i>. An average of four independent experiments is shown. Error bars indicate standard error. </p> <p><b>B</b>. Schematic of <i>mdm2</i> messages detected using a Taqman probe for exons 6 and 7 (6-7 probe) or forward primer for 4:10 and reverse primer for 12 (4:10-12 primer) for <i>mdm2-C</i>. </p> <p><b>C</b>. qRT-PCR with 4:10 forward and exon 12 reverse vs. Taqman probe to exon 6 and 7 of <i>mdm2</i> were performed to detect <i>mdm2-C</i> and <i>mdm2</i> (with exons 6 and 7) transcripts. The <i>mdm2-C</i> transcripts were detected via Syber Green and <i>mdm2</i> (with exons 6 and 7) transcripts were detected via Taqman technology. Transcript levels were compared to K562 for basal expression and normalized for RNA levels to <i>gapdh</i>. An average of three independent experiments is shown. Error bars indicate standard error.</p> <p><b>D</b>. qRT-PCR of RNA using Taqman technology from p53 target genes, <i>p21</i> and <i>puma</i> after DNA damage treatment with 8μM etoposide for 3 hours. Data from each cell line is presented as normalized to its own untreated control sample for fold activation and normalized for RNA levels to <i>gapdh</i>. An average of three independent experiments is shown. Error bars indicate standard error.</p></div