Purpose. MDM2 is an oncogene whose protein product may promote tumorigenesis by blocking
wild-type p53 tumor suppressor mediated G0/G1 cell cycle arrest, thereby inhibiting repair of damaged DNA prior to cell division. While
MDM2 DNA amplification is frequently observed in human sarcoma, the mechanisms linking this amplification to MDM2
oncoprotein over-production as well as its functional significance have not been well characterized in patients with soft
tissue sarcoma.Methods. A tissue bank of resected soft tissue sarcomas and autologous normal tissues was assembled; all specimens were
snap frozen within 15 min of resection. DNA and RNA were extracted from tissues using isoamyl alcohol and phenol
chloroform extraction methods, respectively; cell lysates were prepared using PBSTDS lysis buffer. DNA and mRNA were
confirmed as being non-degraded and were then examined for MDM2 DNA amplification (Southern blots) and mRNA
over-expression (Northern blots) using actin (DNA) and glyceraldehyde-3-phosphate dehydrogenase (mRNA) as loading
controls. The MDM2 protein was examined on Western blots using the MDM2-specific monoclonal antibody IF2
(Oncogene Science, Inc). The presence of p53 DNA and expression of p53 mRNA was examined by rehybridizing the
Southern and Northern filters using a p53-specific cDNA probe.Results. Soft tissue sarcomas and autologous normal tissues were screened for MDM2 DNA amplification, which was
detected in 10 of 30 tumors screened. After screening, there was sufficient biomaterials from six specimens for subsequent
Northern and Western analysis to see whether MDM2 gene amplification correlated with over-expression of MDM2
mRNA and MDM2 protein. In addition, we examined whether other mechanisms may lead to over-expression of the
MDM2 oncoprotein. Several possible mechanisms of MDM2 oncoprotein over-expression were identified. These most
commonly included MDM2 DNA amplification, MDM2 mRNA over-expression and MDM2 oncoprotein over-expression.
However, some soft tissue sarcoma patient specimens had no evidence of MDM2 mRNA over-expression yet had
MDM2 oncoprotein over-production in the tumor relative to autologous normal tissue, implying possible post-transcriptional
regulation. Of functional relevance, MDM2 oncoprotein over-production by tumors was associated with large
decreases in the percentage of cells in the G0/G1 cell cycle interface compared with autologous normal tissue cells.Discussion. It is likely that there are multiple mechanisms underlying human soft tissue sarcoma MDM2 oncoprotein
over-production. Consequently, strategies that decrease MDM2 over-production, such as transcriptional repression to
inhibit MDM2 promoter activity or RNA antisense approaches, may ultimately offer the best therapeutic efficacy.
