Pollock R E, Lang A, El-Naggar A K, Radinsky R, Hung M C
Department of Surgical Oncology MD Anderson Cancer Center 1515 Holcombe Blvd, Box 106 University of Texas Houston TX 77030 USA.
Sarcoma. 1997;1(1):23-9. doi: 10.1080/13577149778443.
Purpose. MDM2 is an oncogene whose protein product may promote tumorigenesis by blocking wild-type p53 tumor suppressor mediated G (0)/G(1) 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 (0)/G(1) 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.
目的。MDM2是一种癌基因，其蛋白质产物可能通过阻断野生型p53肿瘤抑制因子介导的G(0)/G(1)期细胞周期停滞来促进肿瘤发生，从而在细胞分裂前抑制受损DNA的修复。虽然在人类肉瘤中经常观察到MDM2基因扩增，但在软组织肉瘤患者中，将这种扩增与MDM2癌蛋白过度产生联系起来的机制及其功能意义尚未得到很好的描述。
方法。收集切除的软组织肉瘤和自体正常组织的组织库；所有标本在切除后15分钟内速冻。分别使用异戊醇和苯酚氯仿提取法从组织中提取DNA和RNA；使用PBSTDS裂解缓冲液制备细胞裂解物。以肌动蛋白（DNA）和甘油醛-3-磷酸脱氢酶（mRNA）作为上样对照，通过Southern印迹法检测MDM2基因扩增，通过Northern印迹法检测MDM2 mRNA过表达，以确认DNA和mRNA未降解。使用MDM2特异性单克隆抗体IF2（Oncogene Science公司）通过Western印迹法检测MDM2蛋白。使用p53特异性cDNA探针重新杂交Southern和Northern杂交膜，检测p53 DNA的存在和p53 mRNA的表达。
结果。对软组织肉瘤和自体正常组织进行MDM2基因扩增筛查，在30个筛查的肿瘤中有10个检测到扩增。筛查后，有6个标本有足够的生物材料用于后续的Northern和Western分析，以观察MDM2基因扩增是否与MDM2 mRNA和MDM2蛋白的过表达相关。此外，我们研究了其他机制是否可能导致MDM2癌蛋白的过表达。确定了MDM2癌蛋白过表达的几种可能机制。这些最常见的包括MDM2基因扩增、MDM2 mRNA过表达和MDM2癌蛋白过表达。然而，一些软组织肉瘤患者标本没有MDM2 mRNA过表达的证据，但相对于自体正常组织，肿瘤中存在MDM2癌蛋白过度产生，这意味着可能存在转录后调控。具有功能相关性的是，与自体正常组织细胞相比，肿瘤中MDM2癌蛋白过度产生与(0)/G(1)期细胞周期界面的细胞百分比大幅下降有关。
讨论。人类软组织肉瘤MDM2癌蛋白过度产生可能有多种机制。因此，减少MDM2过度产生的策略，如转录抑制以抑制MDM2启动子活性或RNA反义方法，最终可能提供最佳的治疗效果。
