Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
 6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
Sarcoma 1997
PMID:Enhanced MDM2 Oncoprotein Expression in Soft Tissue Sarcoma: Several Possible Regulatory Mechanisms. 1852 Nov 97

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.
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PMID:Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip. 3047