Stable amplification of the S-adenosylmethionine decarboxylase gene in Chinese hamster ovary cells.
A Chinese hamster ovary cell subline (CHO/664) > 1000-fold resistant to the S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, CGP-48664 (4-(aminoiminomethyl)-2,3-dihydro-1H-inden-1-one diaminomethylenehydrazone), has been developed and characterized. The cells were also cross-resistant to the highly specific nucleoside analog inhibitor of AdoMetDC, MDL-73811. These unique cells stably overexpress AdoMetDC due to a 10-16-fold amplification of the AdoMetDC gene, which resulted in a similar increase in AdoMetDC transcript levels. In the presence of 100 microM CGP-48664, the CHO/664 cells displayed AdoMetDC activities similar to the parental line. Following removal of the inhibitor, AdoMetDC activity increased steadily over 20 days to 10-12 times that found in parental CHO cells. Decarboxylated (dc) AdoMet pools accumulated rapidly from < 5 pmol/10(6) cells to approximately 1000-1500 pmol/10(6) cells at 3 days due to diffusion away of intracellular inhibitor and to the depletion of putrescine and spermidine as aminopropyl acceptors in dcAdoMet-mediated synthase reactions. Polyamine pools shifted as putrescine, and spermidine pools were processed forward to spermine. During the period from 3 days to 20 days, dcAdoMet pools fell steadily and eventually stabilized at 100-200 pmol/10(6) cells. Providing excess putrescine at this time as an aminopropyl acceptor rapidly lowered dcAdoMet pools and led to a near normalization of polyamine pools, indicating that both dcAdoMet and putrescine are essential in maintaining steady-state polyamine pool profiles. As with cell line variants that overproduce ornithine decarboxylase, polyamine transport was found to be increased in CHO/664 cells due to an apparent inability of the system to down-regulate polyamine transport in response to polyamine excess. Given the unique metabolic disturbances seen in these cells, we anticipate that in addition to providing a useful system for evaluating the specificity of newly developed AdoMetDC inhibitors, they will undoubtedly prove valuable for investigating the various regulatory interrelationships involved in polyamine homeostasis and possibly other aspects of purine metabolism.
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
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D
Kramer
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Mett
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Evans
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Regenass
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Diegelman
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Porter
D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors / Q000235:genetics*; D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells / Q000201:enzymology*; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004351:Drug Resistance; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic; D007189:Indans / Q000494:pharmacology; D011073:Polyamines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism
D000578:Amidines; D007189:Indans; D011073:Polyamines; D012333:RNA, Messenger; C082408:4-amidinoindan-1-one 2'-amidinohydrazone; D012436:S-Adenosylmethionine; D012437:Adenosylmethionine Decarboxylase