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 (N1,N11-diethylnorspermine), CPENSpm (N1-ethyl-N11-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-[(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.

Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes

The effects of spermine and spermine analogues on the B-Z transition of poly(dG-me5dC) and on the aggregation and ‘melting’ temperature of calf thymus DNA were studied by spectroscopic methods. The association constants of these polyamines with double- and single-stranded calf thymus DNA were calculated from their effects on the melting temperature. The effect of these compounds on the release of ethidium bromide (EB) from an EB-DNA complex were measured by a spectrofluorimetric method. This efficiency of the polyamine-induced B-Z transition strongly depended on the length of the central carbon chains of the compounds and on the functional groups attached to the carbon chains. Both the terminal primary amino groups and the length of the central carbon chain affected the aggregation of DNA. The affinity of the analogues for DNA increased as the number of n-butyl groups increased, but decreased with either an increase or a decrease in the length of the central carbon chain. The effect of spermine and spermine analogues on the release of EB from an EB-DNA complex did not always correlate with the affinities of analogues for calf thymus DNA. In particular, tetra-amines with more than one n-butyl group bound better to DNA than did spermine, but released bound EB and induced aggregation of DNA less well than did spermine. We postulate that either a bend and/or other localized conformational changes of DNA are responsible for the spermine-induced aggregation of DNA and the release of EB from the EB-DNA complex.