ATP-induced Calcium Mobilization and Proliferation in Cervical Cancer Cells

An, W S; Le, G H

Abstract

PURPOSE
Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors. To know the role of nucleotides in cervical cancer cells, we observed the effect of ATP on cytosolic free calcium [[Ca2 ]] mobilization and cell proliferation in four human cervical cancer cell lines, Caski, HeLaS3, ME180 and SiHa cells. MATERIAL AND METHOD: The materials used were purchased from the following sources: ATP from Boehringer [Mannheim, Germany]; U73122 from Research Biochemicals Inc, [Natic, USA]; adenosine, ionomycin, Fura-2 and Fura-2/AM from Sigma Chemical [St. Louis, USA]; Dulbecco`s modified Eagle medium [DMEM], penicillin- streptomycin and fetal bovine serum from GIBCO [Grand Island, NY]; suramin from Wako [Tokyo, Japan]. All the other chemicals of the highest grade are available for the experiment. Cells were harvested after trypsinization at five days before the experiments, and seeded onto 22X22mm cover glasses at the concentration of 105cells/dish. The cover glass was attached to a 1cm hole located at the bottom of 35mm plastic culture dishes. Cells were then washed with modified Hanks` solution consisting of the following elements [in mM]: NaCl, 127; 0.33 of each MgSO4 and Na2HPO4; KH2PO4, 0.44; MgCl2, 1; HEPES, 10; CaCl2, 1 [pH 7.4]; and loaded Fura-2/AM [10 microM] for 45 min at 37degrees C. Fluorescence-loaded cells were washed three times with the same solution for exclusion of unloaded Fura-2/AM. The fluorescence of C6 glioma cells were measured at room temperature using the InCaTM Imaging System manufactured by Intracellular Imaging Inc. [Cincinnati, OH, USA]. For the calcium-free experiment, 10microM [final concentration] of EGTA solution was added to the calcium-free bathing solution. The concentration of [Ca2 ]i was calculated from the standard curve generated in situ.
RESULTS
In these cells, extracellular ATP [10-7M-10-3M] elevated [Ca2 ]i in a dose-dependent manner. In all four cell lines, ATP-induced [Ca2 ]i increases were attenuated in the extracellular calcium-free conditions. The pretreatment of U73122, phospholipase C [PLC] inhibitor, suppressed ATP-induced [Ca2 ]i increment clearly. Among the four cell lines, SiHa showed the highest proliferation activity followed by Caski, ME180 and HelaS3. The significant increases in cell proliferation were observed at 4 and 5 days after ATP treatment in all cell lines.
CONCLUSION
These data suggest that ATP, possibly released at sites of tissue injury or inflammation, might induce the proliferation of cancer cell via purinoceptor-mediated intracellular free calcium mobilization.