Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts.
SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT-EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N(1),N(11)-diethylnorspermine), CPENSpm (N(1)-ethyl-N(11)-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N(1)-ethyl-N(11)-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT-EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT-PCR (reverse transcription-PCR) results suggested that the analogue-affected regulation of SSAT-EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.
Uimari
Anne
A
Department of Biotechnology and Molecular Medicine, AI Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio, Kuopio, Finland. anne.uimari@uku.fi
Keinänen
Tuomo A
TA
Karppinen
Anne
A
Woster
Patrick
P
Uimari
Pekka
P
Jänne
Juhani
J
Alhonen
Leena
L
D000123:Acetyltransferases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006207:Half-Life; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009955:Ornithine Decarboxylase / Q000378:metabolism; D011073:Polyamines / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D049452:Green Fluorescent Proteins; D009955:Ornithine Decarboxylase; D011073:Polyamines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013096:Spermine; D000099228:Diamine N-Acetyltransferase; C118771:N(1)-ethyl-N-(11)-((cycloheptyl)methyl)-4,8-diazaundecane; C485184:enhanced green fluorescent protein; C059685:N(1),N(11)-diethylnorspermine; C083629:N(1)-ethyl-N-(11)-((cyclopropyl)methyl)-4,8-diazaundecane-1,11-diamine