Debnath Mukhopadhyay

C→U Editing of Neurofibromatosis 1 mRNA Occurs in Tumors That Express Both the Type II Transcript and apobec-1, the Catalytic Subunit of the Apolipoprotein B mRNA–Editing Enzyme

C-->U RNA editing of neurofibromatosis 1 (NF1) mRNA changes an arginine (CGA) to a UGA translational stop codon, predicted to result in translational termination of the edited mRNA. Previous studies demonstrated varying degrees of C-->U RNA editing in peripheral nerve-sheath tumor samples (PNSTs) from patients with NF1, but the basis for this heterogeneity was unexplained. In addition, the role, if any, of apobec-1, the catalytic deaminase that mediates C-->U editing of mammalian apolipoprotein B (apoB) RNA, was unresolved. We have examined these questions in PNSTs from patients with NF1 and demonstrate that a subset (8/34) manifest C-->U editing of RNA. Two distinguishing characteristics were found in the PNSTs that demonstrated editing of NF1 RNA. First, these tumors express apobec-1 mRNA, the first demonstration, in humans, of its expression beyond the luminal gastrointestinal tract. Second, PNSTs with C-->U editing of RNA manifest increased proportions of an alternatively spliced exon, 23A, downstream of the edited base. C-->U editing of RNA in these PNSTs was observed preferentially in transcripts containing exon 23A. These findings were complemented by in vitro studies using synthetic RNA templates incubated in the presence of recombinant apobec-1, which again confirmed preferential editing of transcripts containing exon 23A. Finally, adenovirus-mediated transfection of HepG2 cells revealed induction of editing of apoB RNA, along with preferential editing of NF1 transcripts containing exon 23A. Taken together, the data support the hypothesis that C-->U RNA editing of the NF1 transcript occurs both in a subset of PNSTs and in an alternatively spliced form containing a downstream exon, presumably an optimal configuration for enzymatic deamination by apobec-1.

CUGBP2 plays a critical role in apoptosis of breast cancer cells in response to genotoxic injury.

Abstract: Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase‐2 (COX‐2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE2) and is significantly upregulated in many types of adenocarcinomas. COX‐2 overexpression leads to increased PGE2 production, resulting in increased cellular proliferation. PGE2 enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX‐2 expression may lead to important therapeutic advances. Besides transcriptional control, COX‐2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU‐rich sequences to regulate COX‐2 mRNA translation. In the current study, we have determined that expression of both COX‐2 mRNA and CUGBP2 mRNA are induced in MCF‐7 cells, a breast cancer cell line, following exposure to 12 Gy γ‐irradiation. However, only CUGBP2 protein is induced, but COX‐2 protein levels were not altered. Silencer RNA (siRNA)‐mediated inhibition of CUGBP2 reversed the block in COX‐2 protein expression. Furthermore, MCF‐7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Apobec-1 mediated RNA editing of NF1 in colon cancer: Regulation through cis-acting elements and alternative splicing

with the SpectraCube system and analyzed with SKYview imaging software. Results: The average number of chromosomes was 84 in Caco-2, 73 in Colo-205, 62 in SW403, 46 in HCT-15, 45 in HCT-116, and 49 in Lovo. We found 11 structural alterations in Caco-2, 9 in Colo-205, 6 in SW403, 1 in HCT-15, 2 in HCT-116, and 2 in Lovo. The aberrations in NonRER colon cancer cells include complex rearrangements, such as der(13)t(13;6;13) in Colo-205, that have net been identified by conventional banding techniques. Most of the alterations in NonRER colon cancer cells were imbalanced, resulting in chromosomal gains and losses. Discussion: SKY enabled a detailed analysis of structural alterations, generating a complete karyotype. In comparison to NonRER-cells, RER-cells show a strikingly lower incidence of chromosomal gains and losses. Additionally, RER colon cancer cells displayed a markedly lower incidence of structural and numerical chromosomal alterations. Further studies have to show if the simple translocation pattern in RER colon tumors may be used for tumor classification and for prediction of therapeutic responses.