Gene Encoding Chitinase 3-Like 1 Protein (CHI3L1) is a Putative Oncogene

Abstract

An important task in understanding oncogenesis is the identification of those genes whose copy number and expression increase during tumorigenesis. Previously, in an effort to identify genes which could be used as molecular markers for glial tumors, we compared gene expression in glioblastoma to the normal brain cells. Among the genes with the most pronounced increased expression in tumors there was CHI3L1, encoding the secreted chitinase 3-like 1 protein (also known as HC gp-39 or YKL-40). Expression of CHI3L1 was found increased significantly in various tumors in comparison with corresponding normal tissues. Here we show that CHI3L1 can decrease the doubling time of 293 cells. We have also demonstrated that CHI3L1 allows the anchorage-independent growth in soft agar and, in addition, stable CHI3L1 expression made 293 cells tumorigenic: these cells stimulate the initiation of tumors after their xenograft transplantation into the Wistar rat brains. Thus, the overexpression of CHI3L1 is likely to be critical in the development of some tumors and when we gain more information about mechanisms of CHI3L1 oncogenicity, it could be used as one of the potential targets for anticancer therapy.

Keywords: chitinase 3-like 1 protein (CHI3L1); glioblastoma; oncogene.

Figure 1

CHI3L1 gene expression in glioblastomas and normal brain. Data according to GEO repository (http://ncbi.nlm.nih.gov/GEO), log coordinates. Comparison between two groups was done by using the independent samples t test, P<0.001. ■, Individual glioblastoma sample, ○, individual normal brain sample.

Figure 2

Effect of CHI3L1 on proliferation of 293 cells. Starved 293 cells were incubated in unsupplemented medium (▬□▬) or in medium containing 100 ng/ml CHI3L1 (־◊־) for time periods indicated, followed by cell harvesting and counting as described. Each individual point represents the mean of the values obtained from triplicate experiments.

Figure 3

Immunofluorescent analysis of CHI3L1. (A) Paraformaldehyde fixed 293 cells and 293 cells stably expressing CHI3L1 with polyclonal antibodies to CHI3L1; (B) cell nuclei – Hoechst 33342 staining; (C) the merged image indicates CHI3L1 localization in cytoplasm of 293 cells stably expressing CHI3L1. Red fluorescence: rabbit anti-goat Alexa Fluor 594.

Figure 4

Proliferation of 293 cells stably expressing CHI3L1. –♦– 293 cells, stably expressing CHI3L1, –▲– 293 cells, transfected with empty vector. Proliferation was measured by fluorescence after 3.5 hrs of exposure of the cells to MTT reagent. The data shown are the means±S.D. from four experiments for each cell line.

Figure 5

Oncogenic properties of CHI3L1. (A) Western blotting: total lysates of 293 cells stably expressing CHI3L1 (1), total lysates of 293 cells (2), recombinant CHI3L1 (3), glioblastoma total lysate, prepared earlier [19] (4); (M) prestained protein MW marker SM0441; (B) 293 cells in a soft agar colony formation assay (1•10⁴ cells\per well) stably transfected by pcDNA3.1\CHI3L1 (1), pcDNA3.1/GFP (2) and pcDNA3.1 (3).

Figure 6

Histological analysis of intracerebral implanted 293-CHI3L1 cells. (A) Rat brain tumor formed by intracranial inoculation of 293 cells stably expressing CHI3L1. Displacement of median cerebral structures and hydrocephaly in contralateral hemisphere. Tumors were characterized by lobular structure with dense superficial cell layer (B, green arrows) and prominent lobules (100-150 μm) and central newly ingrowing blood vessels (C, D, yellow arrow). Haematoxylin staining of paraffin embedded slices. Magnification ×5 (A), ×20 (B), ×100 (C), ×400 (D).

Figure 7

Immunofluorescence analysis of rat intracerebral tumor initiated by 293 cells stably expressing CHI3L1. Green fluorescence: anti-CHI3L1 goat polyclonal antibodies and rabbit anti-goat Alexa Fluor 488. Red fluorescence: monoclonal antibodies to GFAP and rabbit anti-mouse Alexa Fluor 594. Yellow bar=50 microns.