Differential changes in purine nucleotides after Doxorubicin treatment of human cancer cells in vitro.
The present investigation was performed to elucidate the role of purine nucleotides as potential indicators of chemosensitivity of malignant tumors. Drug-sensitive (s) and -resistant (r) tumor cell lines grown as monolayers (s: T47D, MCF-7 wild-type; r: NCI/ADR-RES, MCF-7/MDR) or as multicellular spheroids (T47D; NCI/ADR-RES) were exposed to 0.1, 1.0, and 10.0 microM Doxorubicin for up to 24 h. Purine nucleotides were assayed using HPLC and with some selected spheroids using imaging bioluminescence. The data show that in the time frame of the experiments reproducible and statistically significant changes in the nucleotides only occur at the highest drug concentration investigated. Under these conditions and using monolayer cultures, Doxorubicin caused a significant increase in ATP and GTP in sensitive but not in resistant cancer cells. Consequently, this differential change may be exploited for drug sensitivity testing in vitro. Doxorubicin exposure to spheroids was associated with significant increases in ATP and GTP in both sensitive and resistant variants. However, the kinetic of the changes in GTP was largely different between T47D and NCI/ADR-RES spheroids with a long-lasting, almost 3-fold elevation and a smaller, relatively short transient increase in GTP, respectively. Supplementing experiments with Doxorubicin treatment under inhibition of oxidative phosphorylation with Oligomycin abolished the drug-induced ATP and GTP peaks at persistent increases in ADP and AMP. Assuming that the spheroids may represent the in vivo situation to a better degree than monolayer cultures, experimental in vivo studies should clarify whether kinetic changes in GTP could be used as differential markers for the chemosensitivity of solid tumors. The experiments using Oligomycin support the hypothesis that purine nucleotides may be recycled from DNA fragments that result from the interaction of the drug with the DNA strands.
Institute of Physiology and Pathophysiology, Johannes Gutenberg-University of Mainz, D-55099 Mainz, Germany. walenta@uni-mainz.de
Stefan
S
Walenta
Barbara
B
Feigk
Ines
I
Wachsmuth
Torsten
T
Dunkern
Hadassa
H
Degani
Wolfgang
W
Mueller-Klieser
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004317:Doxorubicin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D004317:Doxorubicin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate