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Molecular characterization of hybridoma subclones spontaneously switching at high frequencies in vitro

ABSTRACT: This is a comparative microarray analysis of 36-65 hybridomas switching at normal low frequencies in vitro vs. selected variants spontaneously switching at 100 fold higher frequencies in vitro. This analysis reveals differential gene expression of 2091 unique probe sets, 1637 of them corresponded to known genes (647 of which were up-regulated and 990 down-regulated in the variants spontaneously switching at higher frequencies), including genes known to be involved in regular class switching (such as Cd40, Lig4, H2afx,Ung, Xrcc4, Tcfe2a, Id2, Apex1, Bcl6 and Prdm1) as well as genes involved in processes known to be necessary for regular class switch recombination (CSR), such as transcription, response to DNA damage and DNA repair, DNA binding, chromatin modifications, etc.

ORGANISM(S): Mus musculus

PROVIDER: GSE15357 | GEO | 2009/03/23

SECONDARY ACCESSION(S): PRJNA115775

REPOSITORIES: GEO

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Project description:Alternative splicing (AS) is particularly relevant to cancer progression and apoptosis. Although previous studies have shown that the apurinic-apyrimidinic endonuclease-1 (APEX1) is involved in tumor progression, it is unclear whether APEX1 can regulate AS on cell proliferation and apoptosis of Non-small-cell lung cancer (NSCLC). We performed a comprehensive analysis of the APEX1 expression in 517 lung NSCLC samples from the TCGA (Cancer Genome Atlas) database, and selected two sets of cancer samples with differentially expression APEX1 to analyze potential APEX1-regulated alternative splicing events (ASEs). Functional analysis of the APEX1 in A549 cells were performed in vitro. The APEX1 was overexpression in A549 cell by gene transfection. We identified AS targets regulated by APEX1, analyzed the GO biological process and KEGG functional pathways, and validated APEX1-regulated ASEs detected by RNA-seq and RT-PCR in A549 cells, then in clinical NSCLC samples these results were verified. The expression of the APEX1 was up-regulated in NSCLC samples, and overexpression of the APEX1 resulted in cell proliferation reduction and apoptosis induction. AS of many genes regulated by the APEX1 were enrich in cancer-related functional pathways. Results from A549 cell model and clinical samples showed that the MAPK signaling pathway, the Wnt signaling pathway were shared among the top ten enriched GO processes and KEGG functional pathways. According to our research, the validated AS events regulated by APEX1 mostly located in genes encoding transcription regulation factor in various signaling pathways, including the AXIN1 (axis inhibition protein 1), GCNT2 (N-acetyl glucosaminyl transferase 2), SMAD3 (SMAD Family Member 3), CTBP2 (C-Terminal Binding Protein 2). In this study, we successfully applied RNA-seq technology to demonstrate APEX1 regulation of AS. Our results underline that APEX1 was efficiently up-regulated in NSCLC samples, while overexpression of the APEX1 in A549 cells resulted in proliferation reduction and apoptosis induction. We confirm that the APEX1 regulates the AS of many genes which involved in cancer proliferation and apoptosis functional pathways, such as the MAPK signaling pathway and the Wnt signaling pathway, leading to mediate lung cancer progression. We found that high expression of the APEX1 in NSCLC is an independent prognostic factor related to tumor progression. Therefore, the APEX1 can serve as a molecular marker or therapeutic target for NSCLC treatment.

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Molecular characterization of hybridoma subclones spontaneously switching at high frequencies in vitro

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