Characterization of interaction between Bcl-2 oncogene promoter I-Motif DNA and flavonoids using electrospray ionization mass spectrometry and pressure-assisted capillary electrophoresis frontal analysis.

B-cell lymphoma 2 (Bcl-2) is an antiapoptotic protein which is believed to be a triggering factor in developing human tumors. The Bcl-2 C-rich promoter element has been shown to form the i-motif (IM) via cytosine-cytosine (C-C+) base pair building blocks, which can be targeted through the binding of ligands associated with Bcl-2 expression modulation. In this work, we monitored IM development and thermodynamic stability within the Bcl-2 promoter via circular dichroism (CD) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The results demonstrated that at an acidic pH, as well as in a crowded molecular environment, the Bcl-2 promoter element predominantly exists in a stable intramolecular IM folded state. We further explored the potential of targeting of the Bcl-2 IM to increase chemotherapeutic efficacy. We first used a rapid ESI-MS screening assay to identify possible ligands, finding that three natural flavonoids (P1, P5 and P6) exhibited a clear affinity for IM binding at 1:1 stoichiometry. Relative to P6, P1 and P5 were expected to form the more stable complexes with the Bcl-2 IM in gas phase according to MS/MS data. We further used ESI-MS and pressure-assisted capillary electrophoresis frontal analysis (PACE-FA) to assess the binding constants for these flavonoids in gas and liquid phases, respectively, with the latter considering both specific and non-specific binding. We found P5 and P6 to specifically bind the Bcl-2 IM with binding constants of ~104 M-1. P1 binding was confirmed to be due to both specific and nonspecific interactions, and the specific binding constant (8.67 × 103 M-1) was found much less significant than the binding constant in gas phase. Taken all these observations into consideration, the specific binding of selected flavonoids to the Bcl-2 IM may prove to be a potential ligand for modulating Bcl-2 gene expression.

EBV-associated myoid tumor with lipoblast-like cells in a patient with normal immunity.

EBV-associated myoid tumor (EBVMT) comprises a specific group of soft tissue tumors with divergent histologic appearances, which typically occur in immunocompromised patients. To the best of our knowledge, there have been no previous reported of EBVMT in patients with normal immunity. EBVMT with lipoblast-like cells (EBVMT-LIC) is an extremely rare variant of EBVMT. Here, a male patient with normal immunity and EBVMT-LIC is presented. Comprehensive EBV latency expression pattern and tumorigenesis molecular analyses are performed to detail the pathologic features of this disease. Our patient was a 14-year-old who suffered from Burkitt's lymphoma 6 years ago and got complete remission for 5 years. At his last visit, he presented with pain and weakness in arms and legs. Subsequent MRI revealed an extramedullary mass at the cervical areas. CT-guided resection was then performed and comprehensive histopathologic examination was conducted. We found a haemangiopericytoma-like pattern with EBER-positive lipoblasts exist in this neoplasm and these features were in accordance with the diagnosis of EBVMT-LIC. Also, the EBV type I latency pattern was observed and the activation of Akt/mTOR pathway and Bcl-2 overexpression were found to be involved in the tumorigenesis of EBVMT-LIC. In conclusion, the results of this study suggest that EBVMT can occur in patients with normal immunity. EBV could achieve a latency type I pattern and may promote the development of EBVMT by activation of Akt/mTOR pathway and Bcl-2 overexpression. The role of chronic latent EBV infection in the development of EBVMT may be more important than previously thought.

Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma.

Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer.