Ecto-nucleotidase expression and activity in endometrioid-type endometrial carcinoma cell lines

Abstract

Introduction: ATP and adenosine are known for their role in promoting an immunosuppressive environment at the tumor site. It is known that some ecto-nucleotidases, proteins that handle the hydrolysis of tri-, di- and monophosphate nucleotides, are overexpressed in endometrial tumor tissues although the mechanisms underlying these processes remain controversial. In the present study we characterized, with cytochemistry and i mmunofluorescence, the ecto-nucleotidase profiles in endometrioid-type endometrial carcinoma cell lines. Furthermore, we have developed a new approach capable of simultaneously detecting both alkaline phosphatase expression and activity.

Materials and methods: Cell lines: 3 endometrioid endometrial carcinoma cell lines (Ishikawa, HEC-1B and ECC-1) were used for cytochemistry and immunofluorescence experiments. In situ ecto-nucleotidase enzyme activities: ATPase, ADPase, and AMPase activity experiments were performed in all cell lines, based on the Wachstein/Meisel technique. Immunofluorescence experiments: cell immunolabeling was performed using primary antibodies against different members of the ecto-nucleotidases: anti-ectonucleoside triphosphate diphosphohydrolase 2 (E-NTPDase2), anti-E-NTPDase3, anti-CD73, and anti-placental-like alkaline phosphatase (PLAP). Alkaline phosphatase immunoactivity assay: for the detection of both activity and al kaline phosphatase expression we developed a combinatorial assay in which enzyme activity can be co-visualized with protein expression. Immunofluorescence followed by an activity assay was performed on AP-expressing cells.

Results: Cytochemistry enzyme assays showed moderate ATPase activity in both Ishikawa and ECC-1 cell lines. Moderate ADPase activity was found in ECC-1 cells and high AMPase activity was detected in HEC-1B cells. In immunolabeling experiments we found NTPDase2 protein expression in all three cell lines, with NTPDase3 expression restricted to ECC-1 cells. CD73 was detected in all three cell lines. Moderate alkaline phosphatase activity and protein expression were found in Ishikawa and ECC-1 cells. A wide differential range of activities and ecto-nucleotidase expression among the studied cell lines was observed. NTPDase2 and NTPDase3 expression correlated with ATPase activity. CD73 protein expression coincided with the high AMPase activity shown with HEC-1B.

Conclusions: Ishikawa, HEC-1B, and ECC-1 cell lines represent a useful cell model for the study of ecto-nucleotidases in the context of endometrioid-type endometrial carcinoma.