RESUMEN
Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.
Asunto(s)
Poliaminas Biogénicas/biosíntesis , Ácido Fólico/farmacología , Nucleótidos/metabolismo , S-Adenosilmetionina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Colon/citología , Colon/metabolismo , Humanos , Masculino , Próstata/citología , Próstata/metabolismoRESUMEN
While polyamine homoeostasis is clearly important in maintenance of normal cell function, the roles of these cations, as well as the enzymes that regulate their metabolism, in the neoplastic process are not clear. In particular, the polyamine catabolic enzyme SSAT (spermidine/spermine N(1)-acetyltransferase) seems to have different roles in tumorigenesis, depending upon the particular system being analysed. In attempts to clarify the function of SSAT in tumour development, we have utilized the Apc(Min/+) mouse, which carries a mutant allele of the Apc (adenomatous polyposis coli) gene, rendering it susceptible to the formation of multiple adenomas in the small intestine and colon. Using genetically engineered animals (i.e. transgenic and knockout mice), we have shown that SSAT acts as a tumour promoter in the Apc(Min/+) model. Modulation of tumorigenesis is not associated with changes in tissue levels of either spermidine or spermine. These findings, along with those made in other animal models of cancer, have prompted us to propose that metabolic flux through the polyamine biosynthetic and catabolic pathways, and the consequent changes in levels of various metabolites within the cell (i.e. the metabolome), is critical to tumour development. The metabolic flux model represents a novel way of thinking about the role of polyamines in cell physiology and the neoplastic process.
Asunto(s)
Genes APC , Neoplasias Intestinales/genética , Poliaminas/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Modelos Animales de Enfermedad , Ingeniería Genética , Neoplasias Intestinales/enzimología , Ratones , Ratones Mutantes , Ornitina Descarboxilasa/metabolismoRESUMEN
The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.
Asunto(s)
Poliaminas Biogénicas/fisiología , Ciclinas/fisiología , Fase G1/fisiología , Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Melanoma/patología , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Poliaminas Biogénicas/biosíntesis , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Desoxiadenosinas/farmacología , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Humanos , Melanoma/genética , Inhibidores de la Ornitina Descarboxilasa , Fosforilación , Proteína de Retinoblastoma/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Melanoma/patología , Transducción de Señal/efectos de los fármacos , Espermina/farmacología , Acetiltransferasas/biosíntesis , Poliaminas Biogénicas/metabolismo , Caspasa 3 , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Transducción de Señal/fisiología , Espermina/análogos & derivados , Células Tumorales CultivadasRESUMEN
Amantadine acetylation was demonstrated to occur both in vivo and in vitro using transgenic male mice overexpressing spermidine/spermine N(1)-acetyltransferase (SSAT). We previously reported that neither NAT1 nor NAT2 was responsible for catalyzing acetylation of the primary amine group of amantadine. We hypothesized that the inducible polyamine-catabolizing enzyme, SSAT, was an alternate pathway for acetylating amantadine. Transgenic mice injected s.c. with 3 mg/kg amantadine excreted 4.5 +/- 1% (mean +/- S.E.) of the administered dose as acetylamantadine in 24-h urine samples while, by contrast, nontransgenic control mice failed to excrete any detectable acetylamantadine in their urine. In vitro studies with the cytosolic liver fraction from transgenic mice as the source of SSAT demonstrated spermidine acetylation catalytic activity with an apparent K(m) = 267 +/- 46 microM and V(max) = 0.009 +/- 0.002 nmol/min/mg of protein. Amantadine competitively inhibited spermidine acetylation with an apparent K(i) = 738 +/- 157 microM. Incubation of amantadine, SSAT, and an acetyl CoA-regenerating system produced modest amounts of acetylamantadine. The NAT2 substrate, sulfamethazine, inhibited spermidine acetylation with a calculated K(i) = 3.5 mM, suggesting that SSAT may be an alternate pathway for acetylation of NAT2 substrates. The NAT1 substrate, p-aminobenzoic acid, had no inhibitory effect. These results provide evidence that amantadine can be acetylated by SSAT and may be a specific drug substrate for this enzyme. Further investigation of the role of SSAT as a potential drug-metabolizing pathway is warranted.
Asunto(s)
Acetiltransferasas/metabolismo , Amantadina/metabolismo , Acetilación , Acetiltransferasas/antagonistas & inhibidores , Animales , Catálisis , Ratones , Ratones TransgénicosRESUMEN
Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.
Asunto(s)
Antineoplásicos/farmacología , Espermina Sintasa/genética , Espermina/análogos & derivados , Espermina/metabolismo , Células Madre/efectos de los fármacos , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Adenosilmetionina Descarboxilasa/metabolismo , Animales , Poliaminas Biogénicas , División Celular/fisiología , Células Cultivadas , Embrión de Mamíferos/citología , Etopósido/farmacología , Silenciador del Gen , Ratones , Datos de Secuencia Molecular , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Espermina/farmacologíaRESUMEN
Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.
Asunto(s)
Arginasa/genética , Hiperargininemia , Aminoácidos/sangre , Animales , Arginasa/fisiología , Arginina/sangre , Secuencia de Bases , Cartilla de ADN/genética , Marcación de Gen , Humanos , Ratones , Ratones Noqueados , Modelos Animales , Fenotipo , Poliaminas/metabolismoRESUMEN
A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.
Asunto(s)
Antineoplásicos/farmacología , Metionina/análogos & derivados , Metionina/farmacología , Ácidos Fosfínicos/farmacología , Animales , Antineoplásicos/metabolismo , División Celular/efectos de los fármacos , Leucemia L1210/metabolismo , Leucemia L1210/patología , Metionina/metabolismo , Ácidos Fosfínicos/metabolismo , Células Tumorales CultivadasRESUMEN
Acetylation of polyamines by spermidine/spermine N(1)-acetyltransferase (SSAT) has been implicated in their degradation and/or export out of the cell. The relationship of SSAT to polyamine pool dynamics and cell growth is not yet clearly understood. MCF-7 human breast carcinoma cells were transfected with tetracycline-regulated (Tet-off) SSAT human cDNA or murine gene. Doxycycline removal for >2 days caused a approximately 20-fold increase in SSAT RNA and a approximately 10-fold increase in enzyme activity. After 4 days, intracellular putrescine and spermidine pools were markedly lowered, and cell growth was inhibited. Growth inhibition could not be prevented with exogenous polyamines due to a previously unrecognized ability of SSAT to rapidly acetylate influxing polyamines and thereby prevent restoration of the endogenous pools. Instead, cells accumulated high levels of N(1)-acetylspermidine, N(1)-acetylspermine, and N(1), N(12)-diacetylspermine, a metabolite not previously reported in mammalian cells. Doxycycline deprivation before treatment with N(1), N(11)-diethylnorspermine markedly increased analog induction of SSAT mRNA and activity and enhanced growth sensitivity to the analog by approximately 100-fold. Overall, the findings demonstrate that conditional overexpression of SSAT lowers polyamine pools, inhibits cell growth, and markedly enhances growth sensitivity to certain analogs. The enzyme also plays a remarkably efficient role in maintaining polyamine pool homeostasis during challenges with exogenous polyamines.
Asunto(s)
Acetiltransferasas/genética , Acetiltransferasas/metabolismo , División Celular/fisiología , Poliaminas/metabolismo , Poliaminas/farmacología , Transcripción Genética , Acetilación , Neoplasias de la Mama , División Celular/efectos de los fármacos , Células Clonales , Doxiciclina/farmacología , Femenino , Homeostasis , Humanos , Ornitina Descarboxilasa/metabolismo , Putrescina/metabolismo , Proteínas Recombinantes/metabolismo , Espermidina/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11-diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM-treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.
Asunto(s)
Antineoplásicos/farmacología , Fase S/efectos de los fármacos , Espermina/análogos & derivados , Amidinas/farmacología , Animales , Poliaminas Biogénicas/análisis , Bromodesoxiuridina/metabolismo , Células CHO , Cricetinae , ADN/análisis , Fase G1/efectos de los fármacos , Indanos/farmacología , Espermina/farmacologíaRESUMEN
Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Lisosomas/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Poliaminas/metabolismo , Putrescina/análogos & derivados , Animales , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica , Eflornitina/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Interleucina-3/farmacología , Cinética , Lisosomas/fisiología , Lisosomas/ultraestructura , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , Putrescina/metabolismo , Putrescina/farmacología , Espermidina/análogos & derivados , Espermidina/metabolismo , Espermina/metabolismo , Proteína bcl-X , Poliamino OxidasaRESUMEN
We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.
Asunto(s)
Acetiltransferasas/biosíntesis , Poliaminas/metabolismo , Espermina/análogos & derivados , Acetiltransferasas/metabolismo , Animales , Activación Enzimática , Ratones , Ratones Transgénicos , Espermidina/metabolismo , Espermina/administración & dosificación , Espermina/metabolismo , Espermina/farmacologíaRESUMEN
Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.
Asunto(s)
Ciclinas/biosíntesis , Fase G1/efectos de los fármacos , Melanoma/metabolismo , Proteína de Retinoblastoma/biosíntesis , Espermina/análogos & derivados , Proteína p53 Supresora de Tumor/biosíntesis , Antineoplásicos/farmacología , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Humanos , Cinética , Melanoma/patología , Fosforilación , ARN Mensajero/biosíntesis , Proteína de Retinoblastoma/metabolismo , Espermina/farmacología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.
Asunto(s)
Acetiltransferasas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Metalotioneína/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Espermina/análogos & derivados , Transgenes , Acetiltransferasas/metabolismo , Animales , Poliaminas Biogénicas/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Hígado/ultraestructura , Ratones , Ratones Transgénicos , Microscopía Electrónica , Espermina/farmacología , Sulfato de Zinc/farmacologíaRESUMEN
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.
Asunto(s)
Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Amidinas/farmacología , Antineoplásicos/farmacología , Indanos/farmacología , Lisosomas/metabolismo , Espermina/análogos & derivados , Adenosilmetionina Descarboxilasa/genética , Animales , Células CHO , Cricetinae , Resistencia a Antineoplásicos , Lisosomas/ultraestructura , ARN Mensajero/análisis , Espermina/metabolismo , Espermina/farmacología , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
BACKGROUND: The possibility was investigated that complex homeostatic mechanisms which maintain polyamine pools in prostate-derived tumors may differ from those which are typically seen in other tissues and tumors. METHODS: Growth sensitivity and various regulatory responses were investigated in three human prostate carcinoma cell lines (LNCaP, DU145, and PC-3) treated with the inhibitor of S-adenosylmethionine decarboxylase CGP-48664 or the polyamine analog N1,N11-diethylnorspermine (DENSPM), both of which are currently undergoing phase I clinical trial. RESULTS: Prostate tumor cell lines were all similarly growth-inhibited by the inhibitor CGP-48664 (IC50 values, 1-5 microM at 72 hr), but varied considerably in their sensitivity to DENSPM. The rank-order for cell-line growth inhibition by the analog was DU145 > PC-3 > LNCaP, with IC50 values of 1, 30, and 1,000 microM, respectively. Both compounds depleted intracellular polyamine pools to levels which seemed sufficient to account for inhibition of cell growth. While polyamine enzyme regulatory responses to both CGP-48664 and DENSPM were typical of those seen in other cell types, regulation of polyamine transport differed distinctly. Based on Vmax determinations, LNCaP cells failed to upregulate transport in response to CGP-48664, while PC-3 and LNCaP cells failed to downregulate transport in response to DENSPM. CONCLUSIONS: Relative to other cell lines, polyamine transport in prostate carcinoma cell lines was found to be uniquely insensitive to regulation by polyamines or analogs. Although this did not seem to correlate with growth sensitivity to polyamine analogs in vitro, it should be therapeutically exploitable in in vivo systems.
Asunto(s)
Adenosilmetionina Descarboxilasa/farmacología , Amidinas/farmacología , Inhibidores de Crecimiento/farmacología , Indanos/farmacología , Poliaminas/metabolismo , Neoplasias de la Próstata/patología , Espermina/análogos & derivados , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Amidinas/química , Transporte Biológico , Humanos , Indanos/química , Masculino , Estructura Molecular , Neoplasias de la Próstata/metabolismo , Espermidina/metabolismo , Espermina/química , Espermina/farmacologíaRESUMEN
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.
Asunto(s)
Acetiltransferasas/metabolismo , Espermina/análogos & derivados , Acetiltransferasas/genética , Animales , Western Blotting , Fibroblastos/enzimología , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Espermina/metabolismo , Factores de TiempoAsunto(s)
Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Melanoma/metabolismo , Proteína de Retinoblastoma/metabolismo , Espermina/análogos & derivados , Proteína p53 Supresora de Tumor/metabolismo , Acetiltransferasas/biosíntesis , Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inducción Enzimática , Humanos , Melanoma/enzimología , Melanoma/patología , Espermina/farmacología , Células Tumorales CultivadasRESUMEN
The spermine analogue N1,N11-diethylnorspermine (DENSPM) has been shown to induce the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase, disrupt polyamine pool homeostasis, and inhibit tumor growth. DENSPM is currently being developed as an anti-neoplastic agent and is about to enter Phase II clinical trials. In this report, the antitumor efficacy of DENSPM was evaluated against a human transitional cell bladder BL13 carcinoma xenograft implanted orthotopically and s.c. in nude athymic mice. DENSPM was administered via continuous s.c. infusion at 93 mg/kg/day for 5 days. Treatment with DENSPM was well tolerated and produced tumor regressions in all mice with a significant proportion (up to 50%) of apparent cures. On the basis of low toxicity and good therapeutic efficacy, there is a strong rationale for evaluation of the therapeutic efficacy of DENSPM against bladder carcinomas in Phase II clinical trials.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Espermina/análogos & derivados , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Carcinoma de Células Transicionales/metabolismo , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Putrescina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Espermina/farmacocinética , Espermina/uso terapéutico , Espermina/toxicidad , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
We have generated a transgenic mouse line that overexpresses the rate-controlling enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N1-acetylspermidine, not normally found in mouse tissues, a massive accumulation of putrescine, and decreases in spermidine and/or spermine pools. The most striking phenotypic change was permanent hair loss at the age of 3 to 4 weeks which was typified histologically by the appearance of extensive follicular cysts in the dermis. The effect seemed attributable to putrescine interference with hair development, possibly with differentiation/proliferation of epidermal cells located in hair follicles. Female members of the transgenic line were found to be infertile apparently due to ovarian hypofunction and hypoplastic uteri. The findings demonstrate the utility of spermidine/spermine N1-acetyltransferase overexpression as an effective means for genetically modulating total tissue polyamine pools in transgenic animals and examining the developmental and oncogenic consequences.