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Changes in Sarcoma 180 Cells Associated with Drug-induced Resistance to Adenosine Analogs¹

Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York 14203

A subline of mouse Sarcoma 180 (S-180) cells, resistant to adenosine analogs, was selected in cell culture by serial passage in the presence of increasing concentrations of N⁶-furfuryladenosine. These cells (S-180/KR) were cross-resistant to a number of other adenosine analogs, including N⁶-(²-isopentenyl)adenosine (IPAR). The resistance was unaltered after 5 months maintenance in absence of the drug. S-180/KR cells showed increased sensitivity to certain purine and pyrimidine nucleoside analogs such as 6-thiopurine ribonucleoside and 5-fluorodeoxyuridine.

The activity of purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) an enzyme that in crude cell extracts but not in whole cells cleaved IPAR-8-¹⁴C to the noncytotoxic free base at 10% the rate for inosine, was equal in sensitive and resistant cells. In contrast, the activity of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), an enzyme that provides an alternate metabolic pathway for utilization of adenosine (K_m 29 µM) was increased 45% in extracts of resistant cells. No cleavage of IPAR by this enzyme to inosine in cells or cell extracts was observed, but IPAR was a competitive inhibitor of adenosine deamination with a K_i of 1.3 mM. The change that appears to be solely responsible for resistance to adenosine analogs was the 20,000-fold reduction in the activity of adenosine kinase (adenosine 5'-triphosphate:adenosine 5'-phosphotransferase, EC 2.7.1.20). This enzyme converts adenosine and its analogs to their 5'-monophosphates. The elution pattern of adenosine kinase of both cells on diethylaminoethyl cellulose and K_m for adenosine (0.5 µM) were identical.

In resistant cells, deficient in adenosine kinase but containing increased adenosine deaminase, the rate of uptake of adenosine was slightly reduced; V_max 18.2 and 5.1 nmoles/hr/mg cells in S-180 (K_t 47 µM) and S-180/KR (K_t 20 µM), respectively. These changes were reflected in altered growth response to adenosine when de novo synthesis of purine nucleotides was blocked by amethopterin.

In sensitive, but not in resistant cells, IPAR accumulated in trichloroacetic acid-soluble pool in the form of 5'-monophosphate, which comprised 75% of cellular IPAR metabolites. In resistant cells deficient in adenosine kinase and unable to otherwise metabolize IPAR, the rate of uptake of IPAR was greatly reduced; V_max 3.3 and 0.08 nmoles/hr/mg cells in S-180 (K_t 24 µM) and S-180/KR (K_t 14 µM), respectively.

¹ Supported in part by USPHS Research Grants CA-04175 and CA-13038 (M. H. F.) from the National Cancer Institute and by a Training Grant in Clinical Biochemistry GM-09160 (H. K. S.).

Received 7/10/72. Accepted 8/14/72.

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