A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N 1 -acetyltransferase (SSAT) was used to isolate primary fetal fibroblasts as a means to further elucidate the cellular consequences of activated polyamine catabolism. Basal levels of SSAT activity and steady-state mRNA in the transgenic fibroblasts were about ϳ20-and ϳ40-fold higher than in nontransgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N 1 -acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N 1 ,N 11 -diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts ϳ380-fold, whereas mRNA increased only ϳ3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased ϳ200-fold. By Western blot, enzyme protein was found to increase ϳ46 times higher in the treated transgenic fibroblasts than non-transgenic fibroblasts: a value comparable to 36-fold differential in enzyme activity. With DENSPM treatment, spermidine pools were more rapidly depleted in the transgenic fibroblasts than in nontransgenic fibroblasts. Similarly, transgenic fibroblasts were much more sensitive to DENSPM-induced growth inhibition. This was not diminished by co-treatment with an inhibitor of polyamine oxidase, suggesting that growth inhibition was due to polyamine depletion per se as opposed to oxidative stress. Since the two fibroblasts were genetically identical except for the transgene, the various metabolic and growth response differences are directly attributable to overexpression of SSAT.Deregulated polyamine biosynthesis as indicated by overexpressed ornithine decarboxylase (ODC) 1 activity is a well recognized characteristic of animal and human cancers. In at least one tumor type, the genetic basis for this has been attributed to a point mutation leading to stabilization of the enzyme protein (1). Although several groups have reported that ODC may play a causative role in the process of cell transformation in cultured systems (2)(3)(4), that function appears to be attenuated in vivo. Transgenic mice that systemically overexpress native ODC by at least 20-fold fail to show an increased incidence of tumors in any of their tissues (5). More likely, the enzyme interacts with other genes to fulfill a facilitating role in tumorigenesis not unlike that seen with oncogene cooperativity, a phenomenon that has been previously demonstrated between ODC and ras (6). This potential is clearly indicated in mouse skin carcinogenesis models where induction of ODC activity represents an early response to tumor promoters (7) and is known to be critical to tumor formation (8, 9). In this regard, O'Brien's group (10) recently reported that transgenic mice that overexpress a stabilized form of ODC in the skin are much more susceptible to 7,12-dimethylbenz(a)anthracene-induced carcinogenesis. In addition, their findings strongly suggest that am...

Cancer gene therapy with the aid of herpes simplex virus type 1 thymidine kinase gene (HSV-TK) and anti-herpes drug ganciclovir (GCV) has been widely used and its efficacy has been demonstrated in a variety of different malignant cells and animal tumor models. It is also commonly accepted, however, that this gene therapy regimen needs to be enhanced for a true clinical success. We studied whether polyamine biosynthesis inhibition by 2-difluoromethylornithine (DFMO), a clinically tested and well-tolerated chemotherapeutic drug, can increase the cytotoxicity of HSV-TK/GCV in 9L rat glioma cells. Our initial experiments showed that polyamine depletion actually protected the cells from cytotoxicity if GCV treatment was started too early after removal of DFMO. Analyses of cell growth, intracellular polyamine pools and cell cycle phase distribution suggested that later initiation of GCV treatment would be more beneficial due to increased proportion of cells in the middle of the cell cycle S phase. When the cells were exposed to GCV 3 or 4 days after removal of DFMO from growth medium, the cytotoxicity was increased up to 2.5-fold. We also verified whether cell cycle blockage per se could yield similar effect as DFMO. Our results from serum deprivation experiments showed that, despite of apparent cell growth and cell cycle phase distribution effects, serum starvation was weaker enhancer of HSV-TK/GCV cytotoxicity than DFMO. Finally, the general utility of HSV-TK/GCV ؉ DFMO combination was tested in another tumor cell type, human prostate carcinoma cell line DU-145. DFMO sensitized these cells to HSV-TK/ GCV cytotoxicity, but the effect was less prominent than in 9L cells. In conclusion, we have demonstrated that a correctly timed induction of DFMO-mediated polyamine biosynthesis inhibition can enhance the efficiency of HSV-TK/GCV gene therapy in vitro. The observed synergistic effect is potentially useful in clinical trials because, as opposed to use of other cell cycle-altering drugs, DFMO has already been tested in the treatment of human tumors and used as chemo preventive regimen with excellent tolerability.

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.

Abstract. We have shown earlier that polyamine biosynthesis inhibition is accompanied by cell cycle alterations that can be utilized to enhance the efficacy of herpes simplex virus thymidine kinase -ganciclovir (HSV-TK/GCV) cancer gene therapy. In the present study, we asked 1) can the activated polyamine catabolism instead of biosynthesis inhibition be utilized to enhance the efficacy of HSV-TK/GCV gene therapy, and 2) can other known cell cycle inhibitors be used to make tumor cells more sensitive to this form of gene therapy? We show, using rat (9L) and human (U251-MG) glioma cell populations with 15% of HSV-TK-positive cells that DENSPM-induced activation of polyamine catabolism caused a profound polyamine deprivation in U251-MG cells, but there were no associated cell cycle effects in these cells. Consequently, we did not see any enhancement of the HSV-TK/GCV system. Aphidicolin, hydroxyurea, mimosine and resveratrol, but not lovastatin induced an apparent cell cycle arrest, followed by an intense but transient increase of the S phase cells after removal of the drug. This effect was shown to potentiate the HSV-TK/GCV cytotoxicity to some extent, especially in 9L cells and when the GCV treatment was started 0-24 h before the drug treatment. However, the enhancement was weaker than observed earlier with DFMO-induced cell cycle arrest and a considerable degree of the effect appeared to result from the growth-inhibitory actions of the drugs. In summary, we demonstrate that polyamine deprivation via DENSPM action is not associated with cell cycle effects and is not sufficient to cause enhancement of the HSV-TK/GCV system. Also, drugs with a rapid effect to the cell cycle are weak boosters of the HSVTK/GCV gene therapy, thus being less useful than DFMO for enhancement of this gene therapy form in animal studies and clinical trials.

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