Ordered cell cycle phase perturbations in Chinese hamster ovary cells treated with an S-adenosylmethionine decarboxylase inhibitor.
The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine-depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd-containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd-free medium before sampling (post-labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti-BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd-nonlabelled G1 cells into S phase during the post-labelling time, a measure of the G1/S transition was obtained. The time of appearence in G1 of BrdUrd-labelled cells which had divided was used to monitor the length of the G2+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd-labelled cells through S phase. CGP 48664-induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects on the G1/S transition or on the length of the G2+M phase were observed until 2 days after seeding.
Fredlund
J O
JO
Department of Animal Physiology, Lund University, Sweden.
Oredsson
S M
SM
D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors*; D000578:Amidines / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007189:Indans / Q000494:pharmacology; D007700:Kinetics; D013096:Spermine / Q000378:metabolism
D000578:Amidines; D004791:Enzyme Inhibitors; D007189:Indans; C082408:4-amidinoindan-1-one 2'-amidinohydrazone; D013096:Spermine; D004247:DNA; D012437:Adenosylmethionine Decarboxylase