Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues.
We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.
Niiranen
Kirsi
K
A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P. O. Box 1627, Finland.
Pietilä
Marko
M
Pirttilä
Terhi J
TJ
Järvinen
Aki
A
Halmekytö
Maria
M
Korhonen
Veli-Pekka
VP
Keinänen
Tuomo A
TA
Alhonen
Leena
L
Jänne
Juhani
J
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015317:Biogenic Polyamines / Q000378:metabolism*; D017931:DNA Primers; D004622:Embryo, Mammalian / Q000166:cytology*; D006706:Homeostasis; D051379:Mice; D013234:Stem Cells / Q000201:enzymology*; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D015317:Biogenic Polyamines; D017931:DNA Primers; D000099228:Diamine N-Acetyltransferase