Spermine analogs regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

Spermidine/spermine N1-acetyltransferase (SSAT), the key enzyme in the catabolism of polyamines, is turned over rapidly and its content in the cell is low. In this study, the regulation of SSAT by spermine analogs, the inducers of the enzyme, was studied in the wild-type mouse fetal fibroblasts expressing endogenous SSAT and in the SSAT-deficient mouse fetal fibroblasts transiently expressing the SSAT- EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N1, N11 –bis(ethyl)norspermine), CPENSpm (N1-ethyl-N11-((cyclopropyl)methyl)-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-((cycloheptyl)methyl)-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity, respectively. The detected levels of activities correlated with the presence of SSAT and SSAT-EGFP proteins, the last one localizing both in the cytoplasm and nucleus. Reverse transcriptase -PCR results suggested that the analog-affected regulation of SSAT-EGFP expression occurred mainly at the levels beyond transcription. In the 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 the detected SSAT activities. Interestingly, the analogs 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 analogs involves regulatory actions at multiple levels and the spermine analogs, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.