Aldonia Valasinas

Induction of the PAOh1/SMO polyamine oxidase by polyamine analogues in human lung carcinoma cells

PurposeThe induction of polyamine catabolism has been directly associated with the cytotoxic response of various tumor types to the antitumor polyamine analogues. Initially, human polyamine catabolism was assumed to be under the control of a rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (PAO). We have recently cloned a new polyamine analogue-inducible human polyamine oxidase (PAOh1/SMO) that efficiently uses spermine as a substrate. The induction of PAOh1/SMO in response to multiple polyamine analogues was examined in representative lung tumor cell lines.MethodsRepresentatives of three different classes of antitumor polyamine analogues were examined for their ability to induce PAOh1/SMO.ResultsThe human adenocarcinoma line, NCI A549 was found to be the most responsive line with respect to induction of PAOh1/SMO in response to analogue exposure. Similar to previous observations with SSAT expression, PAOh1/SMO induction was found to occur primarily in non-small-cell lung cancers cell lines. Using a series of polyamine analogues, it was found that the most potent inducers of PAOh1/SMO possessed multiple three-carbon linkers between nitrogens, as typified by N1,N11-bis(ethyl)norspermine.ConclusionsSince PAOh1/SMO is an analogue-inducible enzyme that produces H2O2 as a metabolic product, it may play a significant role in determining the sensitivity of various human tumors to specific polyamine analogues.

Antizyme induction by polyamine analogues as a factor of cell growth inhibition

The polyamines spermidine and spermine and their diamine precursor putrescine are essential for mammalian cell growth and viability, and strategies are sought for reducing polyamine levels in order to inhibit cancer growth. Several structural analogues of the polyamines have been found to decrease natural polyamine levels and inhibit cell growth, probably by stimulating normal feedback mechanisms. In the present study, a large selection of spermine analogues has been tested for their effectiveness in inducing the production of antizyme, a key protein in feedback inhibition of putrescine synthesis and cellular polyamine uptake. Bisethylnorspermine, bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane, longer oligoamine constructs and many conformationally constrained analogues of these compounds were found to stimulate antizyme synthesis to different levels in rat liver HTC cells, with some producing far more antizyme than the natural polyamine spermine. Uptake of the tested compounds was found to be dependent on, and limited by, the polyamine transport system, for which all these have approximately equal affinity. These analogues differed in their ability to inhibit HTC cell growth during 3 days of exposure, and this ability correlated with their antizyme-inducing potential. This is the first direct evidence that antizyme is induced by several polyamine analogues. Selection of analogues with this potential may be an effective strategy for maximizing polyamine deprivation and growth inhibition.

Antizyme induction mediates feedback limitation of the incorporation of specific polyamine analogues in tissue culture.

Spermidine, spermine and putrescine are essential for mammalian cell growth, and there has been a pervasive effort to synthesize analogues of these polyamines that will disrupt their function and serve as tools to inhibit cell proliferation. Recently, we demonstrated that a number of such polyamine analogues are also capable of inducing the regulatory protein AZ (antizyme). In the present study the incorporation of a few sample analogues [mimics of bis(ethyl)spermine] was shown to be significantly limited by a decrease in the V(max) for the polyamine transport system in response to analogue-induced AZ. This creates an unusual circumstance in which compounds that are being designed for therapeutic use actually inhibit their own incorporation into targeted cells. To explore the impact of this feedback system, cultures of rat hepatoma HTC cells were pre-treated to exhibit either low or high polyamine uptake activity and then exposed to polyamine analogues. As predicted, regardless of initial uptake activity, all cultures eventually achieved the same steady-state levels of the cellular analogue and AZ. Importantly, analogue-induced AZ levels remained elevated with respect to controls even after the native polyamines were reduced by more than 70%. To model the insufficient AZ expression found in certain tumours, GS-CHO (GS Chinese-hamster ovary) cells were transfected to express high levels of exogenic AZI (AZ inhibitor). As anticipated, this clone incorporated significantly higher levels of the polyamine analogues examined. This study reveals a potential limitation in the use of polyamine-based compounds as therapeutics, and strategies are presented to either circumvent or exploit this elegant transport feedback system.

The enzymatic incorporation of a dipyrrylmethane into uroporphyrinogen III.

Uroporphyrinogen III 2 is the biosynthetic precursor of heme, chlorophylls and all the natural porphyrins. During the enzymatic conversion of porphobilinogen 1 into uroporphyrinogen III 2 an intramolecular rearrangement takes place (see [l] and references therein for a literature survey). The polymerization of four units of porphobilinogen by the combined action of the enzymes porphobilinogen deaminase and uroporphyrinogen III cosynthetase does not afford uroporphyrinogen I 3 as could be expected from a repetitive head-to-tail condensation of porphobllinogen but the isomeric uroporphyrinogen III 2 where an inversion in the order of the p substituents took place. Since no chemically defined pyrrylmethanes were isolated during the enzymatic reaction that could help explain that inversion, the synthetic 2-aminomethyldipyrrylmethane 4 resulting from the formal head-to-tail condensation of two units of porphobilinogen was examined as a first intermediate of the enzymatic system involved in porphobilinogen polymerization [l] . It was found that in the presence of porphoblllnogen it was incorporated exclusively into uroporphyrinogen I, and not into uroporphyrinogen III. We then proposed [l] that both isomers originate by different pathways from the start of the polymerization and that the dipyrrylmethane 5 resulting from the formal head-to-head condensation of two units of

The enantioselective participation of (S)- and (R)-diaminovaleric acids in the formation of δ-aminolevulinic acid in cyanobacteria

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.

The enzymatic incorporation of a tripyrrane into uroporphyrinogen I

Porphyrins, chlorins and vitamin B12 share a common metabolic pathway through a long range of initial steps. Of crucial importance to this pathway is the biochemical mechanism involved in the transfortion of porphobilinogen into uroporphyrinogens III and I and into cobyrinic acid. Indeed, this mechanism keeps the secret of why all natural porphyrins, chlofins, and vitamin B12 derivatives are type III porphyfins. The polymerization of four units of porphobilinogen 1 by the combined action of the enzymes porphobilinogen deaminase and uroporphyrinogen III cosynthetase does not afford uroporphyrinogen ( as could be expected from a repetivtive head-totail condensation of porphobilinogen but the isomeric uroporphyfinogen III where an inversion in the order of the t3-substituents took place [ 1 ] (fig. 1). Porphobilinogen deaminase is the porphobilinogen

Biliverdin reductase: substrate specificity and kinetics.

The substrate specificity of the different forms of rat liver biliverdin reductase was examined using synthetic biliverdins. Biliverdins carrying methyl, ethyl and one propionate residue in their structure were not substrates of biliverdin reductase. Biliverdins with one propionate and one acetate residue or with two acetate residues were not reduced by the enzyme either. The presence of two propionates in the biliverdin structure gave a biliverdin with substrate activity. Increasing the number of propionates to four, as in coprobiliverdins, did not affect substrate activity, while the octaacid urobiliverdins were also good substrates of the enzymes. The beta isomer of urobiliverdin III and coprobiliverdin III were reduced at much higher rates by molecular form 3 of the enzyme as compared to molecular form 1, a fact which had already been observed with the beta isomer of biliverdins IX, XIII and hematobiliverdin. All the biliverdins mentioned above were readily reduced to bilirubins by sodium borohydride. The purified molecular forms 1 and 3 displayed sigmoidal kinetics with most of the biliverdins tested. The data were analyzed by nonlinear regression in a microcomputer and it was found that they fitted a model of a moderate cooperative dimer where both ES and ES2 are catalytically active. The Vm, Ks and the Hill numbers, nH, for biliverdin IX alpha and beta, hematobiliverdin IX alpha and beta, and several synthetic biliverdin isomers are given. Molecular form 2 showed classical Michaelian kinetics.

The specificity of biliverdin reductase: a study with different biliverdin types

The specificity of rat liver biliverdin reductase was examined with the help of a series of synthetic biliverdins. The mixture of the four biliverdin isomers obtained by the chemical oxidation of protohemin I, protohemin XI, protohemin XIV and harderohemin were used as substrates of biliverdin reductase and were compared with the mixture of biliverdins IX alpha-delta. Biliverdin reductase (molecular form 1) from rat liver efficiently reduced the isomer mixtures of biliverdins I, XI, XIV and harderobiliverdins to the bilirubins in the presence of NADPH. The enzymatic reduction of the different biliverdin types was studied in the presence of different NADPH analogues. NADPH could be replaced by NADH, 3-acetyl NADPH and deamino-NADPH with retention of a good substrate activity only in the case of biliverdins of types I and IX and harderobiliverdins. Biliverdins XI and XIV were efficiently reduced only in the presence of NADPH and an excess of NADH. Bactobilin III-alpha was also very efficiently reduced by biliverdin reductase in the presence of both NADPH and NADH but not in the presence of the other analogues. These results indicate that biliverdin reductase reduced bilitriene acids substituted with non-polar and polar residues.

SL‐11158, a Synthetic Oligoamine, Inhibits Polyamine Metabolism of Encephalitozoon cuniculi

Polyamine metabolism as a target for drug intervention in rapidly proliferating cells has evolved within the past two decades. The initial impetus was to block de novo synthesis of putrescine and spermidine, targeting ornithine decarboxylase and S-adenosylmethionine decarboxylase (Fig. I) . This, however, did not take into account the ability of many cells, especially tumor cells, to transport spermidine or spermine, interconverting them as needed using the highly inducible enzyme spermidinelspermine N’-acetyltransferase (SSAT) and polyamine oxidase. Thus present attempts to interfere with polyamine metabolism are now directed to the use of poly-amine analogs which are transported, induce SSAT, and cause reductions in cellular polyamine levels but do not replace polyamines in function [7,9]. Agents such as bis-ethylspemine, bis-ethylhomospermine and BE-4x4 [ 1,20(ethylamine)-5,IO, 15-triazanonadecane] have become the forerunners of a rapidly evolving synthetic strategy. These and other polyamine analogs are now in clinical trials for various types of cancers [6, 71 and as anti-spasmodic agents in AIDS-related diarrhea [4].

Regioselective binding of spermine, N¹,N12-bismethylspermine, and N¹,N12-bisethylspermine to tRNA Phe as revealed by 750 MHz ¹H-NMR and its possible correlation with cell cycling and cytotoxicity

Neste trabalho foi feito o estudo de ligacao de espermina (SPM), N1, N12- bismetilespermina (BMS) e N1, N12-bisetilespermina (BES) ao tRNAfen usando RMN de 1H a 750 MHz. As poliaminas foram enriquecidas com 13C nos residuos 5-CH2 e 8-CH2, sendo obtidos os picos cruzados por efeito de Overhauser nuclear entre as ressonâncias dos hidrogenios dos metilenos marcados com 13C e varios hidrogenios de grupos imino de pares de bases do tRNAfen atraves de espectros 1D filtrados por 13C. Foi encontrado que, enquanto SPM e BMS se ligam ao N(3)-H dos pares de bases T54-m1A58, U50-A64 e U52-A62, BES liga-se so nos pares T54-m1A58 e U50-A64. Esta regiosseletividade de ligacao das tres poliaminas ao tRNA foi correlacionada com seus efeitos biologicos no crescimento celular. Usando celulas de câncer de melanoma humano (MALME-3M), foi encontrado que SPM e BMS nao tem efeito e e citostatico, respectivamente, enquanto que BES e claramente citotoxica. Esta ultima poliamina tambem afeta o ciclo celular e, contrario ao comportamento de SPM e BMS, acarreta a uma clara parada do ciclo celular G1 /S.