Biochemical consequences of resistance to a recently discovered IMP dehydrogenase inhibitor, benzamide riboside, in human myelogenous leukemia K562 cells.
Benzamide riboside (BR) exhibits potent antitumor activity in a variety of cultured human tumor cells. The drug is metabolized to benzamide adenine dinucleotide (BAD), which in turn functions as a selective inhibitor of IMP dehydrogenase (IMPDH) activity with a Ki of 0.118 microM. In vitro, BR is a more potent antitumor inhibitor of IMPDH than tiazofurin, another IMPDH inhibitor which has shown significant oncolytic activity in adult patients with end-stage leukemia. To elucidate the mechanism of resistance, a variant of human myelogenous leukemia K562 cells was developed by subculturing sensitive cells in sublethal concentrations of BR over 60 generations. The BR resistant line that emerged exhibited an IC50 (a concentration producing 50% reduction in cell proliferation) of 148 microM, compared to the sensitive line which had an IC50 of 1.6 microM. The activity of the target enzyme, IMPDH, was increased 3-fold in the resistant variant. Studies on BR metabolism revealed that resistant cells formed only 18% of the active metabolite, BAD, compared to sensitive cells. This finding, in turn, correlated with the specific activity of NAD pyrophosphorylase (the enzyme responsible for the synthesis of BAD) which was reduced to undetectable levels in the resistant variant. The basal levels of NAD and guanylates were also significantly decreased to 41% and 48%, respectively, in the resistant line compared to the parent line. Additionally, after treatment with BR a decrease in guanylate level was observed only in the sensitive cells. Sensitive and resistant cells exhibit comparable cytotoxicity to agents outside the tiazofurin family, suggesting that a multidrug resistance was unlikely to explain the resistance to BR. Moreover, BR resistant cells exhibit collatoral sensitivity to 6-aminopurine, cytarabine and 5-fluorouracil, which have different mechanisms of action. In conclusion, these studies establish that the primary mechanism of resistance to BR involves an increase in IMPDH (target enzyme) activity with a concurrent decrease in NAD pyrophosphorylase (BAD synthetic enzyme) activity.
Jayaram
H N
HN
Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200, USA.
O'Connor
A
A
Grant
M R
MR
Yang
H
H
Grieco
P A
PA
Cooney
D A
DA
D000328:Adult; D002460:Cell Line; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D009705:Nucleosides / Q000627:therapeutic use*; D009713:Nucleotidyltransferases / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism
D004791:Enzyme Inhibitors; D009705:Nucleosides; D012265:Ribonucleotides; C076145:3-(1-deoxyribofuranosyl)benzamide; D007168:IMP Dehydrogenase; D009713:Nucleotidyltransferases