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Fig. 6. Intracellular polyamine profile of LNCaP ornithine decarboxylase/Tet-Ind cells after the addition of 30 mMexogenous putrescine for 3, 6, 9, 12, and 24 h. Putrescine was added directly to the media. After incubation in putrescine for the times indicated, the cells were harvested and their intracellular polyamine levels determined. Values are means ± SEM of data repeated in triplicate.

although not to the same degree as observed with Id-1. In contrast, c-fos (unlike with TET-mediated ODC overexpression treatments) yielded no consistent change of mRNA levels with exogenous putrescine treatments. These results showed that the Id-1,

Fig. 7. Northern analysis of LNCaP ornithine decarboxylase/Tet-Ind cells after the addition of 30 mMexogenous putrescine for 3, 6, 9, 12, and 24 h. Putrescine was added directly to the media. After incubation in putrescine for times indicated, total RNA was isolated and subjected to Northern blot analysis with probes for RING3, c-jun, Id-1, and c-fos. Filters were then stripped and reprobed for GAPDH.

Fig. 7. Northern analysis of LNCaP ornithine decarboxylase/Tet-Ind cells after the addition of 30 mMexogenous putrescine for 3, 6, 9, 12, and 24 h. Putrescine was added directly to the media. After incubation in putrescine for times indicated, total RNA was isolated and subjected to Northern blot analysis with probes for RING3, c-jun, Id-1, and c-fos. Filters were then stripped and reprobed for GAPDH.

RING3, and c-jun genes increased in a time- and dose-dependent manner with exogenous putrescine. Id-1 showed the most dramatic increases in steady-state mRNA levels, while RING3 and c-jun were less affected. The c-fos mRNA levels were not visible after treatment with exogenous putrescine, but again ODC overexpression did lead to increases in c-fos (Fig. 2).

1.5. Increased SSAT Activity is Not Responsible for Induction of These Genes

The observed reduction in intracellular spermidine and spermine after treatment with exogenous putrescine or TET-mediated ODC overexpression might have been a result of increased SSAT activity. To determine the extent to which putrescine induces SSAT activity, the LNCaP ODC/Tet-Ind cells were grown for 24 h in media containing either 15 mM exogenous putrescine or 10 |iM DENSPM (N1, N11-diethylnorspermine, a spermine analog that induces high levels of SSAT activity) (30) (Table 1) before being harvested and assayed for SSAT activity. The cells receiving exogenous putrescine displayed an eightfold higher SSAT activity than the control cells (Table 1). The DENSPM-treated cells showed an 18-fold higher SSAT activity than the control cells. Our previous studies have shown that TET-mediated ODC overexpressing cells also raised SSAT activity 115-fold higher than control treated cells (data not shown).

ODC overexpression and exogenous putrescine raised intracellular putrescine levels, increased SSAT activity, and further caused a reduction in intracellular spermidine and spermine. We wished to determine whether it was the increase in intracellular putrescine or the reduction in intracellular spermidine or spermine that led to the increase in RING3, Id-1, and c-jun mRNA levels. Because DENSPM caused an increase in SSAT coupled with a reduction of intracellular putrescine (as well as spermidine and spermine), yet did

Table 1

SSAT Activity, Intracellular Polyamine Profile, and Relative Expression Levels of the Genes in LNCaP ODC/Tet-Ind Cells After Treatment of 15 mM Exogenous Putrescine or 10 |M DENSPM for 24 h

Table 1

SSAT Activity, Intracellular Polyamine Profile, and Relative Expression Levels of the Genes in LNCaP ODC/Tet-Ind Cells After Treatment of 15 mM Exogenous Putrescine or 10 |M DENSPM for 24 h

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