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  • Analysis of genes involved in response to doxorubicin and a GD2 ganglioside-specific 14G2a monoclonal antibody in IMR-32 human neuroblastoma cells.

Analysis of genes involved in response to doxorubicin and a GD2 ganglioside-specific 14G2a monoclonal antibody in IMR-32 human neuroblastoma cells.

Acta biochimica Polonica (2015-08-19)
Irena Horwacik, Małgorzata Durbas, Elżbieta Boratyn, Anna Sawicka, Paulina Węgrzyn, Sylwia Krzanik, Anna Górka, Joanna Drożniak, Ewa Augustyniak, Aleksandra Kowalczyk, Hanna Rokita
ABSTRACT

Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-SESN1 antibody produced in rabbit, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Anti-RPS27L antibody produced in rabbit, affinity isolated antibody