Nucleotide metabolizing ectoenzymes are upregulated in A431 cells periodically treated with cytostatic ATP leading to partial resistance without preventing apoptosis

Abstract

Extracellular ATP, when added as a single dose at concentrations higher than 0.1 mM to the culture medium, was growth inhibitory or even cytotoxic for human epidermoid carcinoma cells (A431). Adenosine at the same concentrations was much less potent. The molecular mechanism underlying the inhibitory effect of extracellular ATP has been investigated. The cytostatic as well as the cytotoxic effects of ATP could be prevented by supplying uridine as a pyrimidine source and, alternatively, by simultaneous addition of dipyridamole, which inhibits the uptake of adenosine. The data suggest that the long-term production and continuous uptake of adenosine, which is enzymatically generated from the ATP in the medium, led to an intracellular nucleotide imbalance with pyrimidine starvation. This triggered suicidal processes ending up in apoptosis of the cells. The tumor cells have been adapted to extracellular ATP with the aim to obtain cells which are more resistant to ATP. Therefore, growing cells were periodically treated with extracellular ATP. These cells were characterized by an enlargement of cell size, a decreased proliferation rate, and a reduced but not abolished sensitivity to cytostatic and cytotoxic ATP doses. The calcium response of adapted cells was shortened. The nucleotide hydrolyzing ectoenzyme activities (ecto-ATPase, ecto-ADPase, ecto-AMPase, ecto-Ap4Aase) were simultaneously upregulated. All phenotypic alterations of the adapted cells disappeared after cultivation for several generations in the absence of extracellular ATP. Considering ATP as a potential chemotherapeutic agent the adaptive phenomena of treated cells might be important.