Characterization of new cell line stably expressing CHI3L1 oncogene

Abstract

Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1). Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development

Cancer Genes and Chromosome Instability

“…Addition of CHI3L1 to cell medium increased mitogenic and proliferative properties of 293 cells (human embryonic kidney 293 cells, also often referred to as HEK293) [25,26]. 293 cells stably transfected with CHI3L1 have an accelerated growth rate relatively to the parental cells and can undergo anchorageindependent growth in soft agar that is one of the consistent indicators of oncogenic transformation [25,27].…”

PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly

“…Unspecific binding was blocked by 45 min incubation with 5 % FBS diluted in PBS-T. Afterwards staining with primary and secondary antisera was performed as described previously [12,13].…”