MDM2 RNA binding is blocked by novel monoclonal antibody h-MDM2-F4-14.

Amplification of MDM2 has been described in a variety of human cancers. Prognostic studies have revealed that abnormal MDM2 expression correlates with poor prognosis. Many of the consequences of mdm2/p53 interactions have been investigated, and mdm2-p53 dependent events characterized. In contrast, understanding of mdm2-p53 independent activities is comparatively in it's infancy amongst these the ability of mdm2 to bind RNA. However, although the significance of this activity has been the subject of some speculation, the precise role and impact of this function upon cell replication or apoptosis has yet to be fully defined. These studies have been obstructed by a lack of specific reagents able to interfere with this reaction. As a prelude to further exploring the significance of mdm2 RNA binding we report the inhibition of mdm2 RNA binding activity by newly produced MDM2 monoclonal antibodies anti-h-mdm2 F4-14 and F2-2. A variety of MDM2 specific antibodies have been produced and applied in research without complete knowledge of their reactivity profiles, but in the face of the growing number of mdm2 RNA isoforms, the results of such studies can be difficult to interpret. Each of the RNA binding inhibitory antibodies produced in this study was found to be reactive with full length MDM2 protein expressed in tumor cell lysates, transfected NIH3T3 cell lysates and via eukaryotic cell free rabbit reticulocyte in vitro translation. Antibody F4-14, the most potently inhibitory antibody, reacts strongly with the full length MDM2 together with protein isoforms A, B, C and D. In contrast, antibody F2-2 reacts only with full-length MDM2 protein. The ability of h-mdm2-F4-14 and to a lesser extent F2-2 to inhibit RNA binding presents the possibility of modulating human mdm2s ability to bind RNA, compromise this function and present opportunities to investigate in more detail the biological significance of this activity.

Recombinant human MDM2 oncoprotein shows sequence composition selectivity for binding to both RNA and DNA.

MDM2 is a 90 kDa nucleo-phosphoprotein that binds p53 and other proteins contributing to its oncogenic properties. Its structure includes an amino proximal p53 binding site, a central acidic domain and a carboxy region which incorporates Zinc and Ring Finger domains suggestive of nucleic acid binding or transcription factor function. It has previously been reported that a bacculovirus expressed MDM2 protein binds RNA in a sequence-specific manner through the Ring Finger domain, however, its ability to bind DNA has yet to be examined. We report here that a bacterially expressed human MDM2 protein binds both DNA as well as the previously defined RNA consensus sequence. DNA binding appears selective and involves the carboxy-terminal domain of the molecule. RNA binding is inhibited by an MDM2 specific antibody, which recognises an epitope within the carboxy region of the protein. Selection cloning and sequence analysis of MDM2 DNA binding sequences, unlike RNA binding sequences, revealed no obvious DNA binding consensus sequence, but preferential binding to oligopurine:pyrimidine-rich stretches. Our results suggest that the observed preferential DNA binding may occur through the Zinc Finger or in a charge-charge interaction through the Ring Finger, thereby implying potentially different mechanisms for DNA and RNA MDM2 binding.

High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma.

PURPOSE: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14(ARF) pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse. HYPOTHESIS: Inactivation of the p53/MDM2/p14(ARF) pathway develops during treatment and contributes to neuroblastoma relapse. METHODS: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14(ARF) methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization. RESULTS: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14(ARF) inactivation in 12 of 41 (29%) cases: 3 had p14(ARF) methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. CONCLUSIONS: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated.