Selective cellular depletion of mitochondrial DNA by the polyamine analog N1,N12-bis(ethyl)spermine and its relationship to polyamine structure and function.
N1,N8-Bis(ethyl)spermidine (BESPD) and N1,N12-bis(ethyl)spermine (BESPM) are minimally modified analogs of spermidine and spermine that deplete cellular polyamine pools by suppressing key polyamine biosynthetic enzymes. The consequences of polyamine depletion and the concomitant analog replacement of these pools were compared on two cellular DNA targets, mitochondrial DNA (mtDNA) and a defined nuclear DNA episome present in 935.1 mouse fibroblasts. The spermidine analog, BESPD, depleted cellular putrescine and spermidine pools, but not spermine pools, and had no effect on either DNA target. Treatment with the corresponding analog of spermine, BESPM, resulted in a near-total depletion of all three polyamine pools and a greater than 80% reduction in the cellular content of mtDNA, without affecting the levels of the nuclear episome. Topological forms analysis by Southern blotting of mtDNA and episomal DNA from BESPM-treated cells failed to reveal any forms interconversion, indicating the absence of analog-induced single- or double-strand break damage to either DNA target. The growth-dependent loss of mtDNA is consistent with a rapid cessation of mtDNA replication and subsequent dilution of existing mtDNA copies by cell division. Similar decreases in polyamine pools and mtDNA were also observed in L1210 cells treated with BESPM. When a comparable level of polyamine depletion was produced in L1210 cells by specific enzyme inhibitors, there was no effect on the cellular content of mtDNA, and BESPD was not rendered capable of decreasing mtDNA levels. Because the analogs are structurally similar to the naturally occurring polyamines and would be expected to have similar binding properties, the loss in mtDNA may reflect dysfunctional replacement by BESPM at spermine-specific binding sites in the mitochondrion.
Vertino
P M
PM
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
Beerman
T A
TA
Kelly
E J
EJ
Bergeron
R J
RJ
Porter
C W
CW
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015139:Blotting, Southern; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism; D004278:DNA, Superhelical / Q000187:drug effects / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D007939:Leukemia L1210 / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D011073:Polyamines / Q000494:pharmacology*; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013997:Time Factors
D000970:Antineoplastic Agents; D004272:DNA, Mitochondrial; D004273:DNA, Neoplasm; D004278:DNA, Superhelical; D011073:Polyamines; D013096:Spermine; C052614:N(1), N(12)-diethylspermine; D004247:DNA; C050659:N(1),N(8)-diethylspermidine; D013095:Spermidine