Search

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

The enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were approximately 4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were approximately 12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N(1)-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated approximately 70% reduction in the SSAT cofactor acetyl-CoA and a approximately 40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer.

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.

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N4 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 degrees C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at approximately 118 and approximately 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (approximately 100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

The effects of six cationic porphyrins on several enzymes involved in polyamine biosynthesis and catabolism have been examined. Both spermidine and spermine synthase were unaffected by the porphyrins at up to 2 mM. By contrast, omithine and S -adenosylmethionine decarboxylase were inhibited by the nickel and cobalt derivatives of meso -tetrakis( N -methyl-4-pyridiniumyl)porphyrin (T4MPyP) with ic 50 values in the 10–100 μM region. Spermidine/spermine N ′-acetyltransferase (SSAT) and polyamine oxidase (PAO) were highly sensitive to the six meso -substituted cationic porphyrins tested, with K i values as low as 6 nM for SSAT and 85 nM for PAO. These inhibitors may prove useful in defining the structural features of the active site of these enzymes.

In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 microM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA. By Northern blot analysis three RNA transcripts were found to hybridize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximately 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 microM N1,N11-bis(ethyl)norspermine (BENSPM) for 48-72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures--binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.

Pentamidine and berenil, clinical antiparasitic amidines, have been found to be potent competitive inhibitors of human spermidine/spermine acetyltransferase (SSAT). Ki values were found to be 2.4 and 2 microM, respectively, with spermidine as substrate. A second enzyme of polyamine back-conversion, murine polyamine oxidase (PAO), was found to be competitively inhibited by pentamidine, with a Ki of 7.6 microM when N-acetylspermine was the substrate. Berenil, on the other hand, was an extremely weak inhibitor (Ki = 120 microM). The implication of the effect of inhibition of polyamine back-conversion on the growth of mammalian parasites is discussed.

The progress reviewed here would seem to validate the regulatory approach to interference with polyamine biosynthesis as an antiproliferative strategy. To our knowledge, this is the first example, among anticancer drugs, of pharmacological intervention of a biochemical pathway based strictly on regulatory control. Several features of polyamine biology naturally favor this approach and may account for its relative success. These include (a) the nature of the regulatory mechanisms themselves, (b) the exquisite sensitivity of the pathway to regulatory control, (c) the rapid turnover of ODC and AdoMetDC, (d) the different structural specificity of ODC and AdoMetDC regulation versus growth-dependent functions, and (e) the direct dependence of growth on sustained polyamine biosynthesis. As such, the regulatory approach to interference with polyamine biosynthesis offers several advantages over the use of specific enzyme inhibitors (Table 10). Of these, perhaps, the more significant are the facts that more than one enzyme can be simultaneously and specifically suppressed and that compensatory mechanisms, which otherwise counter the effects of enzyme inhibitors (11), are not invoked. We are encouraged by the concurrence of in vitro mechanistic findings with the predictions of the hypothesis for the regulatory approach and by the in vitro and in vivo growth inhibitory effects of the analogs against murine leukemia. One disadvantage of the regulatory analogs, such as BESm, has been that, as with specific polyamine inhibitors such as DFMO, analog-induced polyamine depletion results in cytostatic growth inhibition. While this response may help to minimize host toxicities, it clearly compromises antitumor activity. An intriguing exception to this generality has recently been found among human lung carcinoma cell lines. Previously, Luk et al. (93, 94) and others (95) reported that, among a spectrum of human lung carcinoma lines, small cell carcinoma was exquisitely sensitive to the ODC inhibitor, DFMO. Not only did these cells display a cessation of growth but also an inability to survive during DFMO-induced polyamine depletion. Studies extending these findings to long term maintenance therapy in human small cell lung carcinoma implants in athymic mice revealed sustained growth inhibition of the tumor for longer than one year (96). Casero et al. (97) now find that human large cell carcinoma, which is otherwise refractory to chemotherapeutic intervention, displays a cytotoxic response in vitro to polyamine depletion induced by BES or BESm but not by DFMO. These findings suggest cytotoxic susceptibility to polyamine depletion appears to be agent and cell type specific. Based on these leads, current and future efforts will attempt to identify and exploit these specificities.

We have tested the notion that polyamines, particularly putrescine, mediate the response of mouse kidney to androgens. Hormonal effects were measured in female mice maintained on the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO), which results in a 85-90% reduction of ODC enzyme levels and a depletion of putrescine concentrations in kidney. These animals exhibited normal kidney cell hypertrophy in response to testosterone. In addition, androgen-inducibility of the RP2 gene was indistinguishable from that in normal mice. These results indicate that an increase in putrescine levels is not a prerequisite for androgen effects in mouse kidney and that putrescine does not mediate the hormonal response.

1. 1. The interactions of naturally occurring polyamines: putrescine, spermidine and spermine, with anticancer bis-guanylhydrazones: methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (DDUG) were investigated at the level of mitochondrial membrane. 2. 2. The effects of bis-guanylhydrazones on intact rat liver mitochondria were readily prevented or reversed by polyamines and these interactions were also affected by the mictochondrial transmembrane potential. 3. 3. Magnesium cations enhanced the protective action of polyamines. The data indicate that competition exists between the essential anticancer bis(guanylhydrazones) and polyamines for low affinity negatively charged binding sites at the outer surface of inner mitochondrial membrane. 4. 4. The study of drug interactions was extended to the level of isolated tumor mitochondria from rat HTC hepatoma and muraine L1210 leukemia cells. 5. 5. A complicated pattern of interactions between the anticancer bis-guanylhydrazones and phenethylbiguanide was obtained.

Polyamine oxidase from L1210 cells has been separated into two isozymes, A and B on the basis of chromatography on hydroxyapatite. The two isozymes also show different elution characteristics from MGBG Sepharose and Sephacryl-300. The molecular weights of A and B isozymes are estimated to be 260,000 and 200,000 daltons, respectively. The two isozymes show different kinetic characteristics with spermidine, spermine, N1-acetylspermine, and N1,N12-diacetylspermine.

Histochemical techniques were used successfully to investigate the oxidative and hydrolytic enzymes of a Cotylocercous cercaria tentatively identified as the larva of Plagioporus lepomis Dobrovolny. In tests for 11 hydrolytic enzymes, nonspecific esterase was detected in the nervous system, leucine aminopeptidase and acid phosphatase activities were found in the excretory system, and three phosphatases and phorylase were located in the subcuticular area, in the caudal region, or in the suckers. The possible functions of these enzymes are discussed with respect to their location. Results of tests for 13 oxidative enzymes suggest the presence of a glycolytic pathway, pentose phosphate pathway, Kreb's cycle, respiratory chain, and lipid synthesis. Sites of intensive oxidative enzyme activity include the subcuticular area, oral and ventral suckers, nervous system, and parenchyma.