Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase

A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N1-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 approximately 20- and approximately 40-fold higher than in non-transgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N1-acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts approximately 380-fold, whereas mRNA increased only approximately 3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased approximately 200-fold. By Western blot, enzyme protein was found to increase approximately 46 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.     

Polyamine analogue induction of programmed cell death in human lung tumor cells

The naturally occurring polyamines putrescine, spermidine, and spermine are required for cell growth. Based on this requirement, several polyamine analogues that interfere with polyamine function and metabolism have been synthesized as antineoplastic agents. The symmetrically substituted N1,N12-bis(ethyl)spermine (BESpm), and unsymmetrically substituted N1-ethyl-N11-[(cyclopropyl)methyl]-4, 8-diazaundecane (CPENSpm) have previously been shown to cause rapid cytotoxicity of NCI H157 cells, with concurrent high induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. However, the precise mechanism(s) of the cytotoxic action of the compounds is not known. We now demonstrate that treatment with either BESpm or CPENSpm results in morphological and biochemical changes consistent with the activation of programmed cell death pathways, and that the unsymmetrically substituted CPENSpm more rapidly activates the death program. These studies suggest that the cell type-specific cytotoxicity of these polyamine analogues may be a result of their ability to selectively activate the cell death pathway in sensitive phenotypes and indicate that the relationship between the structure of the polyamine analogues and the ability to induce programmed cell death should be investigated.     

Paracrine inducers of uterine endometrial spermidine/spermine N1-acetyltransferase gene expression during early pregnancy in the pig

The endogenous factors that underlie the transient induction of the gene encoding spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in cellular polyamine catabolism, in pig uterine endometrium during periimplantation are not known. The present study examined a number of peptide growth factors and regulatory molecules that are present within the uterine environment at early pregnancy, coincident with maximal SSAT gene expression, for their ability to manifest endogenous SSAT gene-inducing activity. Basal SSAT expression in luminal epithelial cells was higher (p < 0. 01) than that for glandular epithelial (GE) or stromal (ST) cells. Recombinant human insulin-like growth factor-I (IGF-I; 50 ng/ml) had no effect on steady-state SSAT mRNA levels, but it increased mitogenesis in all three cell types. In contrast, IGF-I caused a marked induction (p < 0.01) of SSAT mRNA levels in the human endometrial carcinoma cell line Hec-1-A. Uterine explants incubated with interleukin-6, transforming growth factor alpha, epidermal growth factor (each at 1, 10, and 100 ng/ml), retinoic acid and retinol (each at 0.01, 0.1, and 1 microM), and estradiol-17beta (10 nM) had SSAT mRNA levels similar to controls. By contrast, leukemia inhibitory factor (LIF; at 10 and 100 ng/ml) caused a modest, but significant (p < 0.05), increase in SSAT mRNA levels over those of untreated explants. This effect of LIF, however, did not approach the level of induction observed in GE or ST cells after addition of medium conditioned by Day 12 or 17 porcine conceptuses and in endometrial explants supplemented with medium conditioned by Day 21 porcine conceptuses or a continuous cell line (Jag-1) derived from Day 14 porcine trophoblast. We suggest that transient induction of endometrial SSAT gene expression at implantation is mediated by the functional interactions of specific conceptus-derived regulatory factors, distinct from estrogen, with endometrial-derived factor(s) such as LIF. These complex interactions are probably requisite for the transient, yet dramatic, induction of SSAT gene expression and may be critical for successful implantation.     

Preclinical antitumor efficacy of the polyamine analogue N1, N11-diethylnorspermine administered by multiple injection or continuous infusion

Certain N-alkylated analogues of the natural polyamine spermine have been found to disrupt polyamine pool homeostasis and inhibit tumor cell growth. The most effective of these analogues, N1, N11-diethylnorspermine (DENSPM), apparently depletes intracellular polyamine pools primarily by inducing the polyamine acetylating enzyme spermidine/spermine N1-acetyltransferase, which contributes to polyamine depletion via increased polyamine excretion and catabolism. In this report, the experimental therapeutic efficacy of DENSPM was further examined with the use of other human solid tumor xenografts, including A121 ovarian carcinoma, A549 lung adenocarcinoma, HT29 colon carcinoma, and SH-1 melanoma, and compared with previously obtained findings with MALME-3M and PANUT-3 human melanomas. In vitro studies indicated that the growth sensitivity of most tumor cell lines to DENSPM was similar, with characteristically flat dose-response curves and IC50s ranging between 0.1 and 1 micrometer the only exception was the HT29 colon carcinoma cell line, which had an IC50 of >100 micrometer. For in vivo studies, DENSPM was administered by i.p. injection to female nude athymic mice at 40 and/or 80 mg/kg 3 times a day (every 8 h) for 6 days or by continuous s.c. infusion with the use of Alzet pumps at 120, 240, or 360 mg/kg/day for 4 days. Treatment began after s.c. tumor xenografts had reached 100-200 mm3. The SH-1 melanoma, A549 lung adenocarcinoma, and A121 ovarian carcinoma xenografts responded well to the i.p. administration of analogue with obvious tumor regressions, long-term tumor growth suppressions, and a significant proportion (up to 40%) of apparent cures (i.e., lack of tumor regrowth). However, in similarity to in vitro findings, HT29 colon carcinoma xenografts responded poorly to DENSPM treatment. Massive induction of N1-acetyltransferase activity and extensive depletion of polyamine pools were consistent findings in most tumor types after in vivo or in vitro treatment with DENSPM. The rapidly growing human LOX melanoma xenograft, however, demonstrated poor induction of N1-acetyltransferase activity and the poorest response to DENSPM treatment. In nude athymic mice with MALME-3M melanoma xenografts, constant infusion delivery of DENSPM resulted in prolonged inhibition of tumor growth and long-term tumor regressions comparable to those produced by multiple i.p. injections. On the basis of the unique structure of DENSPM, novel target and mode of intervention, mild host toxicity, and activity against different human solid tumor xenografts, DENSPM is currently being developed as an antitumor agent in humans.     

Growth inhibition of hormone-responsive and -resistant human breast cancer cells in culture by N1, N12-bis(ethyl)spermine

Previous studies have documented differential sensitivity of human lung cancer and melanoma cell lines to the cytotoxic effects of N1, N12-bis(ethyl)spermine (BESpm). We show here that BESpm can significantly inhibit the growth of six human breast cancer cell lines with 50% inhibitory concentration in the microM range. The degree of inhibition does not correlate with estrogen receptor status. Detailed studies with estrogen receptor-positive MCF-7 and estrogen receptor- negative Hs578t cells show a similar dose-response curve with concentrations of 1-10 microM resulting in maximal growth inhibition. Growth inhibition in both lines is associated with an 8-12-fold induction of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase, and a progressive decrease in intracellular polyamine levels over 6 days even though steady-state levels of BESpm are achieved within 24 h. Similar studies on WTMCF7 and AdrRMCF7 cells show that the acquisition of resistance to hormonal or doxorubicin therapy is not associated with resistance to the growth-inhibitory effects of BESpm. These results suggest that BESpm exerts similar growth-inhibitory effects against both hormone-responsive and -unresponsive human breast cancer cells, a finding which has significance for the potential use of polyamine analogues in treating human breast cancer.     

Conformationally restricted analogues of 1N,12N-bisethylspermine: synthesis and growth inhibitory effects on human tumor cell lines

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.     

Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli

A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl beta-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to N1-acetylspermidine, much of which was excreted. Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine. These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more sensitive to the growth inhibitory action of the antitumor agent N1,N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5 alpha cells containing pINSAT2. The purified enzyme had a similar specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.     

Localization of MOC-1 cell surface antigen in small-cell lung carcinoma cell lines: an immunohistochemical and immunoelectron microscopic study

Expression of cell surface antigens of the neural cell adhesion molecule (N-CAM) class was recently shown to be shared by both fetal and neoplastic neuroendocrine cells, including those of the lung. We investigated the expression and localization of MOC-1 antigen on small-cell (neuroendocrine) lung carcinoma cell lines with immunohistochemical methods at the light (LM) and electron microscopy (EM) level and by Western blot. At LM level, using monoclonal antibody (MAb) MOC-1 with the ABC method and immunofluorescence, positive staining was observed on surfaces of cells from all tumor lines examined. Strongest immunostaining was found on cell surfaces of pulmonary small-cell carcinoma-derived cell line NCI-H69 with the majority of cells showing positive staining. An adherent variant of NCI-H69 cell line, H69V, exhibited positive staining in about 60% of cells, whereas only occasional cells of NCI-H727 cell line derived from pulmonary carcinoid tumor were positive for MOC-1 antigen. Western blot analysis confirmed these findings, showing a strong MOC-1-specific band in cell extracts of NCI-H69, with weaker band densities for H69V and NCI-H727. Immunoelectron microscopy (IEM) revealed that MOC-1 was not uniformly distributed on the outer surface of plasma membrane; immunogold particles appeared concentrated in areas of thick cell surface "fuzz" coating, surface microvilli, and in areas of cell-cell contact. In some cells, areas of plasma membrane invaginations and a few intracytoplasmic vesicles were also labeled, suggesting endocytosis. Surface labeling for SEM confirmed the finding of more dense labeling over the microvilli, cell membrane folds, and in areas of cell-cell contact. The cell lines derived from pulmonary neuroendocrine cell tumors can provide a useful model to study the role and function of neural adhesion molecules in pulmonary neoplasia and during lung development.     

Tumor-targeted apoptosis by a novel spermine analogue, 1,12-diaziridinyl-4,9-diazadodecane, results in therapeutic efficacy and enhanced radiosensitivity of human prostate cancer

Interference with polyamine transport and biosynthesis has emerged as an important anticancer strategy involving polyamine analogues and specific inhibitors of key biosynthetic enzymes. Because the prostate gland has a high polyamine content, by using the polyamine transporter for selective uptake into cancer cells, alkylating polyamines are likely to be highly effective against prostatic tumors. We have recently synthesized a novel class of spermine analogues, the lead compound of which has efficacy against human cancer cells (P. S. Callery et al., U. S. patent, 5,612,239, Issued March 17, 1997.). In this study, to investigate the potential therapeutic efficacy of the lead spermine analogue 1,12-diaziridinyl-4, 9-diazadodecane (BIS), against advanced prostate cancer, we examined the in vitro effect and in vivo efficacy of the compound in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. BIS exhibited a dose-dependent cytotoxic effect against prostate cancer cells via induction of apoptosis. Treatment of cells with BIS (1 microM) for 24 h resulted in a significant induction of apoptosis (24%). Exposure of BIS-treated PC-3 prostate cancer cells to gamma-irradiation resulted in a significant increase in the number of cells undergoing apoptosis and a subsequent decrease in the IC50. Furthermore, BIS treatment led to a significant enhancement of loss of clonogenic survival in irradiated prostate cancer cells (both PC-3 and DU-145). In vivo efficacy trials demonstrated a significant antitumor effect of BIS against both PC-3 and DU-145 tumor xenografts in severe combined immunodeficient mice in a dose-dependent pattern at maximally tolerated doses. Terminal transferase end-labeling analysis indicated that BIS-mediated tumor regression in vivo occurs via induction of apoptosis among prostatic tumor cells. These results suggest that the novel spermine analogue BIS: (a) has a potent antitumor effect against prostatic tumors via induction of apoptosis; and (b) increases the radiosensitivity of human prostate cancer cells by decreasing the apoptotic threshold to radiation. This study may have important clinical implications for the manipulation of this antitumor activity of the polyamine analogue for the optimization of the therapeutic efficacy of radiation in patients with advanced prostate cancer.     

Analysis of the acyl-CoAs that accumulate during the peroxisomal beta-oxidation of arachidonic acid and 6,9,12-octadecatrienoic acid

A critical step in the cytotoxic action mechanism of tumor necrosis factor-alpha (TNF-alpha) involves, among mitochondrial dysfunctions, an early change of the inner membrane permeability displaying the characteristics of permeability transition. Cytosolic polyamines, especially spermine, are known to inhibit it. Our results show that spermine is only detectable in the TNF-alpha resistant C6 cells while N1-acetylspermidine is present in the TNF-alpha sensitive WEHI-164 cells, and putrescine and spermidine are found in both. TNF-alpha treatment does not change this distribution but only induces a quantitative alteration in TNF-alpha sensitive cells. Omission of glutamine (energetic substrate) from the culture media alters neither the TNF-alpha responsiveness of both cell lines nor their polyamine distributions, only their quantitative polyamine contents.     

Lysosomal sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the S-adenosylmethionine decarboxylase inhibitor, CGP-48664

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al., J. Biol. Chem., 270: 2124-2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of N1,N11-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phase 1 clinical trials-despite accumulating approximately 5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3, 10, 30, and 100 microM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 microM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 microM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine N1-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664-resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM.     

A specific light chain of kinesin associates with mitochondria in cultured cells

High-resolution two-dimensional gel electrophoresis (2-DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2-DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P< 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor 5A, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine-5-monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http:// www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.     

Blast lung injury from an explosion on a civilian bus

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were tested in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by ODC inactivation and intracellular polyamine depletion.