RESUMEN
The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious. In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters. The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells. We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications. From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes. Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Portadoras/metabolismo , Genes MDR , Errores Innatos del Metabolismo Lipídico/genética , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Anciano , Transporte Biológico , Membrana Celular/metabolismo , Cromosomas Humanos Par 7 , Resistencia a Múltiples Medicamentos , Expresión Génica , Marcadores Genéticos , Humanos , Errores Innatos del Metabolismo Lipídico/metabolismo , Repeticiones de MicrosatéliteRESUMEN
A series of omega-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3beta-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C).oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.
Asunto(s)
Antivirales/síntesis química , VIH-1/efectos de los fármacos , Triterpenos/síntesis química , Triterpenos/farmacología , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , VIH-2/efectos de los fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Triterpenos Pentacíclicos , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Triterpenos/química , Células Tumorales Cultivadas , Ácido BetulínicoRESUMEN
A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.
Asunto(s)
Antivirales/síntesis química , VIH-1/efectos de los fármacos , Triterpenos/síntesis química , Triterpenos/farmacología , Antivirales/farmacología , ADN Nucleotidiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos , Proteína gp120 de Envoltorio del VIH/metabolismo , Inhibidores de la Proteasa del VIH , VIH-1/enzimología , Humanos , Integrasas , Estructura Molecular , Triterpenos Pentacíclicos , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Triterpenos/química , Células Tumorales Cultivadas , Ácido BetulínicoRESUMEN
The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatin um(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t 1/2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleo-protein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisplatino/metabolismo , Aductos de ADN/metabolismo , ADN Helicasas , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Compuestos Organoplatinos/metabolismo , Quelantes , Reactivos de Enlaces Cruzados , Ácido Edético , Cinética , Unión Proteica , Dodecil Sulfato de SodioRESUMEN
A series of platinum dichloroethylenediamine complexes [PtCl2(R-en)] bearing a side chain on one carbon atom of the ethylenediamine ligand, with or without a functional group on the side chain, have been prepared and investigated for antitumor activity against L1210 leukemia. They were tested both in vitro, with cisplatin-sensitive and resistant cell lines, and in vivo, with cisplatin-sensitive and resistant tumors grafted i.p. in B6D2F1 mice. The rationale for this study was to test how charge, polarity and shape of the R side chain influence antitumor activity. Complexes carrying one or more ammonium groups on the side chain were all inactive. Derivatives with a carbamate function attached by the nitrogen atom, via a methylene group, to the ethylenediamine moiety ('N-bound' carbamate) were highly active in vitro and in vivo. The best results were obtained with these carbamates bearing hydrophobic substituents of intermediate size. Replacement of N-bound by O-bound carbamate or by urea groups led to decreased in vivo activity. Sulfonamide derivatives were all inactive. Good to excellent activities were also recorded for complexes bearing bulky bicycloalkyl substituents, without any functional group, attached to one ethylenediamine carbon atom. Thus, it is the steric features of the side chain rather than its polarity that appear to favor the antitumor activity of the complex. Compared to cisplatin and oxaliplatin, the present complexes do not exhibit advantages in terms of experimental antitumor activities in solid tumor models.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos Organoplatinos/síntesis química , Animales , Antineoplásicos/farmacología , Carbamatos/química , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Técnicas In Vitro , Leucemia L1210 , Masculino , Ratones , Ratones Endogámicos , Compuestos Organoplatinos/farmacología , Oxaliplatino , Compuestos de Amonio Cuaternario/química , Relación Estructura-Actividad , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
Potent inhibitors of EGF-dependent protein tyrosine kinase (PTK) activity were synthesized in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Several of these compounds inhibited EGF-dependent DNA synthesis in ER 22 cells with IC50 < 1 microM. In this series of PTK inhibitors, the role of the salicylate moiety as a potential divalent ion chelator was tested and found to be nonessential in all cases. The length and ramification of the substituting carboxyl group were investigated to improve cellular bioavailability, and this analysis provided compounds with increased inhibitory effect on EGF-induced DNA synthesis. Salicylates esterified with long hydrophobic chains were shown to be noncompetitive inhibitors of ATP, in contrast to the free acid and methyl salicylate. Moreover, all the tested inhibitors were shown to be noncompetitive inhibitors of the peptide substrate. Structure-activity relationships allowed us to suspect a hydrophobic pocket in the tyrosine kinase domain, preferentially interacting with aromatic rings. Finally, the selectivity of the best inhibitors was tested against other kinases, and they were found to be selective for tyrosine kinase. They were also shown to be good inhibitors of EGF-receptor autophosphorylation.
Asunto(s)
Compuestos de Bencilo/síntesis química , Compuestos de Bencilo/farmacología , Receptores ErbB/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Salicilatos/síntesis química , Salicilatos/farmacología , Animales , Compuestos de Bencilo/química , Ratas , Salicilatos/química , Relación Estructura-ActividadRESUMEN
Ditercalinium (NSC 366241) is a 7H-pyrido[4,3-c]carbazole dimer with a diethylbipiperidine rigid chain linking the two heterocyclic rings. Ditercalinium is characterized by a high DNA affinity and bisintercalating ability, associated with potent antitumor properties, involving an original mechanism of action. Unfortunately as ditercalinium is hepatotoxic, its clinical evaluation has been interrupted. In order to eliminate or at least minimize the serious drawbacks related to its toxic effects, several chemical modifications have been made to the structure of ditercalinium, and their influence has been evaluated by measuring the DNA affinities, intercalation properties, and toxicity toward leukemia cells of the newly synthesized dimers. Reduction of the pyridinic moieties of ditercalinium, in order to suppress the permanent charges provided by the quaternizing chain, led to an almost complete loss of activity, although the DNA bisintercalating property of the dimer was preserved. Dimerization of the 7H-pyrido[4,3-c]carbazole rings by introduction of the rigid spacer on the N7- or C6-positions corresponding to the convex face of the pyridocarbazole, instead of the N2-position in ditercalinium, led to DNA bisintercalating dimers practically devoid of antitumor properties. However after quaternarization of the N2 atoms, the dimer linked by the N7 atoms exhibited a very high DNA affinity (greater than 10(9) M-1) and recovered antitumor activity, supporting the requirement of positive charges for the emergence of antitumor activity in these dimers. Introduction on the C6 of the 7H-pyridocarbazole ring of an aminomethyl or carboxyl group, a sugar residue, or C or N free amino acids such as Lys or Glu has also been carried out, in order to increase the hydrophilic properties of the molecules or to enable them to use amino acid transport systems. Although some of these compounds were active, none of them exhibited the pharmacological potency of ditercalinium.
Asunto(s)
Antineoplásicos/síntesis química , Carbazoles/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Carbazoles/química , Carbazoles/uso terapéutico , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Leucemia L1210/tratamiento farmacológico , Ratones , Ratones Endogámicos DBA , Relación Estructura-ActividadRESUMEN
The removal of inherent curving in Crithidia fasciculata kinetoplast DNA by various small DNA ligands, groove binders and mono- and bisintercalators, has been studied by gel retardation and electron microscopy. The migration of the kinetoplast DNA fragment is highly retarded during gel electrophoresis. We demonstrate that this retardation is suppressed by DNA ligands such as distamycin and ditercalinium, which have different modes of binding and sequence specificities. Observation by electron microscopy confirms that the effect of ditercalinium on gel migration of curved DNA is linked to DNA uncurving. As the drug is progressively added to DNA, a large broadening of the retarded band is observed during gel electrophoresis for distamycin and ditercalinium. In the case of distamycin, the retarded DNA band splits into two broad bands, whereas the noncurved DNA bands remain homogeneous. This indicates that the drug-DNA exchange is extremely slow in the gel and that a limited number of specific sites on DNA are critical for the removal of bending. GC-specific quinomycin, monointercalators, and bisintercalators act in a manner similar to that of AT-specific distamycin. This indicates that direct drug binding at the dAn tracts is not required for DNA uncurving. We propose that the uncurving of kinetoplast DNA by drugs is caused by a global alteration of DNA structure; subsequent increased flexibility leads to the suppression of rigid bending at the AT tract junctions.
Asunto(s)
ADN Circular/química , Sustancias Intercalantes/farmacología , Conformación de Ácido Nucleico , Poli dA-dT/química , Animales , Crithidia , ADN Circular/efectos de los fármacos , ADN de Cinetoplasto , Electroforesis en Gel de Poliacrilamida/métodos , Cinética , Ligandos , Sustancias Macromoleculares , Matemática , Mapeo Restrictivo , Relación Estructura-ActividadRESUMEN
The aim of genomic pharmacology is to discover substances able to influence the expression of specific genes. These substances could be used in many pathologies in which the expression of specific genes is abnormal and in the control of viral infection. The potential targets of genomic pharmacology may be classified in two groups: first, the proteins regulating genetic expression, and, second, the nucleic acids which carry the genetic information. The preliminary results obtained with antidirectional nucleic acids are presented and discussed.
Asunto(s)
Expresión Génica/efectos de los fármacos , Farmacología Clínica , Proteínas de Unión al ADN/farmacología , Biblioteca Genómica , Humanos , Leucina Zippers/fisiología , Oligonucleótidos Antisentido/farmacología , ARN Catalítico/farmacología , Dedos de Zinc/fisiologíaRESUMEN
The MDR P-glycoprotein has been described as a major factor of multidrug resistance. This transmembrane glycoprotein acts like an energy dependent efflux pump which possesses a broad specificity. It seems to be acting as a pump requiring drug fixation prior to extrusion. With the aim of investigating which parameters influence the recognition of drugs by the MDR system, we have determined the toxicities of different drugs on human and murine sensitive and resistant cell lines. For this purpose we have isolated and characterized a human adriamycin-resistant cell line, CEM/Adr, which presents an MDR phenotype. The tested drugs were ellipticine and olivacine derivatives which differ through discrete lateral chain substitutions. The influence of lateral chain lipophilicity and nitrogen quaternarization on drug recognition was studied. Small modifications in the chemical structure of the drugs have induced large changes in their toxicities and in the cross-resistance levels of the MDR cells to the tested compounds. The cross-resistances of the murine and human cells to the various compounds were strikingly different. The validity of murine screening models in the selection of anti-tumor drugs for human therapy must therefore be questioned.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Elipticinas/farmacología , Leucemia P388/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Medicamentos/genética , Ensayos de Selección de Medicamentos Antitumorales/métodos , Amplificación de Genes , Humanos , Leucemia P388/genética , Leucemia P388/patología , Metabolismo de los Lípidos , Glicoproteínas de Membrana/metabolismo , Ratones , Fenotipo , Solubilidad , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.
Asunto(s)
ADN , Porfirinas , Secuencia de Aminoácidos , Animales , Cationes , Bovinos , Fenómenos Químicos , Química , ADN/efectos de los fármacos , Transferencia de Energía , Etidio/farmacología , Datos de Secuencia Molecular , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Timo/metabolismoRESUMEN
The bending of kinetoplast DNA from Crithidia fasciculata is thought to be related to the periodic distribution of AA or TT cluster sequences. The sensitivity to DNase I of the two strands of this DNA was analyzed at nucleotide resolution by sequencing gel electrophoresis. The effect on the DNase I cleavage pattern of two drugs, ditercalinium and distamycin, that are able to remove bending was analyzed. The same analysis was done on a pBR 322 DNA fragment of random sequence as a control. The periodic distribution of the AA or TT clusters in the bent DNA fragment was first analyzed by computing the autocorrelation function of the AA or TT clusters in the bent DNA fragment. It is shown that the AT tracts are on average 10.5 base pairs apart. This value is almost identical with that of the B-DNA helix pitch in solution [10.5 (Wang, 1979); 10.6 +/- 0.1 (Rhodes & Klug, 1980)]. To reveal the periodic pattern of DNase I cleavage on this bent DNA, alone or in presence of drugs, the cross correlation between the different bands obtained from DNAse I cleavage and the presence of AA or TT sequences was computed. This shows that GC and mixed sequences are the most sensitive regions. These data also suggest that there is a periodic fluctuation in the width of the minor groove in the bent fragment. Ditercalinium and distamycin alter the DNase I cutting pattern of the bent DNA fragment but in an inverse fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Carbazoles/farmacología , Crithidia/genética , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Distamicinas/farmacología , Sustancias Intercalantes/farmacología , Pirroles/farmacología , Animales , Composición de Base , Sitios de Unión , Desoxirribonucleasa I/antagonistas & inhibidores , Conformación de Ácido Nucleico , Mapeo Nucleótido , Secuencias Repetitivas de Ácidos Nucleicos , Sensibilidad y EspecificidadRESUMEN
There is multiple evidence linking the inhibition of DNA topoisomerases I and II with the cytotoxic effects of antitumor drugs such as camptothecin and the DNA intercalators, 4-(9-acridinylamino)methanesulfon-m-anisidine) (mAMSA) and Adriamycin. We have assessed the effect of the DNA intercalator 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the DNA topoisomerase I and II activities. NBQ has no effect on the activity of purified topoisomerase I, whereas it induced purified topoisomerase II binding to DNA without inducing DNA scission. Above 30 microM, NBQ stimulated formation of double- and single-strand breaks mediated by topoisomerase II in plasmid DNA. Using the alkaline elution technique we determined that NBQ induced single-strand and DNA-protein-associated breaks in the human promyelocytic leukemia cell line HL-60. At sublethal concentrations (less than or equal to 1 microM), NBQ induce HL-60 cells to differentiate. Topoisomerase II-mediated DNA cleavage induced by mAMSA was substantially reduced in NBQ-differentiated cells. Our data suggest that topoisomerase II could play a major role in the biological activity of NBQ in vivo.
Asunto(s)
Daño del ADN , ADN/efectos de los fármacos , Sustancias Intercalantes/farmacología , Compuestos de Quinolinio/farmacología , Inhibidores de Topoisomerasa II , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Reactivos de Enlaces Cruzados , Humanos , Técnicas In Vitro , Inhibidores de Topoisomerasa I , Células Tumorales CultivadasRESUMEN
Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator. It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain. It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors. 1H n.m.r. studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs. Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings. DNA must be bent toward the minor groove. In E. coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation. In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner. The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA. The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium. The cytotoxicity of ditercalinium in E. coli results from the induction of a futile and abortive DNA repair. The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent. The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin. The Uvr protein recognition process appears to be associated with specific DNA structural alterations. In eukaryotic cells, ditercalinium is concentrated in mitochondria. Mitochondrial DNA is rapidly and totally degraded. Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated. The stimulation of the glycolytic pathway allows the cells to continue growth for several generations. Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization. It becomes inactive after ditercalinium treatment. A drop of the pyrimidine pool is then observed. Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity. The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria. The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Reparación del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Proteínas de Escherichia coli , Animales , Carbazoles/farmacología , Carbazoles/toxicidad , ADN/metabolismo , Interacciones Farmacológicas , Ensayos de Selección de Medicamentos Antitumorales , Endodesoxirribonucleasas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Sustancias Intercalantes/farmacología , Sustancias Intercalantes/toxicidad , Leucemia L1210/tratamiento farmacológico , Mutación , Conformación de Ácido Nucleico/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Genistein has been shown to inhibit specifically in vitro the epidermal growth factor (EGF)-receptor tyrosine protein kinase activity (Akiyama et al., J Biol Chem 262: 5592-5597, 1987). When the effects of genistein on NIH-3T3 cells were studied, a cytostatic effect was observed at low concentration (less than 40 microM) and a cytotoxic effect at higher concentration (greater than 40 microM). Genistein was able to block the mitogenic effect mediated by EGF on NIH-3T3 cells (IC50 = 12 microM) or by insulin (IC50 = 19 microM). Genistein was also able to block the mitogenic effect mediated by thrombin (IC50 = 20 microM) although the thrombin receptor does not involve a protein tyrosine kinase activity. Genistein at cytostatic concentration (less than 40 microM) did not prevent the induction of c-myc mRNA synthesis caused by the activation of EGF receptor by EGF. Therefore the cytostatic effect of genistein on NIH-3T3 cells did not appear to be mediated by EGF receptor tyrosine kinase inhibition. This hypothesis was also supported by the absence of effect of genistein on the EGF-stimulated phosphorylation of several proteins and particularly of a cytosolic 80 kD protein. The stimulation of S6 kinase activity of cells treated by EGF was prevented by genistein. The stimulation by EGF of in situ S6 phosphorylation was also prevented by genistein. In addition, S6 kinase extracted from cells treated by EGF was inhibited by genistein. These effects occur at similar doses and maximal inhibition of S6 kinase was obtained at about 15 microM.
Asunto(s)
Fibroblastos/efectos de los fármacos , Flavonoides/farmacología , Isoflavonas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN/biosíntesis , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Genisteína , Ratones , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc , Proteínas Quinasas S6 RibosómicasRESUMEN
The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo, at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mitochondrial-staining fluorophore DiOC2(5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Sustancias Intercalantes/farmacología , Mitocondrias/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Carbazoles/metabolismo , Carbazoles/uso terapéutico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , ADN/metabolismo , Desoxiglucosa/farmacología , Membranas Intracelulares/fisiología , Leucemia L1210/tratamiento farmacológico , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Microscopía Fluorescente , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dilatación Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
To investigate a role for globotriaosylceramide (Gb3) as a tumor-associated antigen, variant cells resistant to treatment with complement and monoclonal antibody 38-13, which recognizes Gb3, were selected from a Burkitt's lymphoma cell line, Ramos. Variant cells displayed a clear decrease of antibody-binding capacity whereas the amount of Gb3 at their plasma membrane was not significantly different from that of Ramos parental cells. This demonstrated a reduced accessibility of Gb3 at the surface of variant cells. In parallel, no changes in other surface antigens were recorded as compared to those in Ramos cells. No changes of proliferative properties in suspension culture or of c-myc expression were observed although variant cells showed a decreased colony-forming capacity in agar. Variant cells showed a significant reduction in tumorigenic potential when injected s.c. into nude mice. The decreased tumorigenicity appeared related to the low antibody-binding capacity because both tumorigenicity and Gb3 antigenicity were recovered in parallel in revertant cells growing in suspension culture. In vivo, after two transplantations of variant cells into mice, cells isolated from the few induced tumors still retained the low antibody-binding capacity.
Asunto(s)
Linfoma de Burkitt/patología , Globósidos/inmunología , Glicoesfingolípidos/inmunología , Trihexosilceramidas , Animales , Antígenos de Neoplasias/análisis , Antígenos de Superficie/análisis , Linfoma de Burkitt/inmunología , División Celular , Citometría de Flujo , Galactosa Oxidasa , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc , Células Tumorales CultivadasRESUMEN
We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation of the drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercalinium-intercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA- and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA- or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA. UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine dimers, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA- or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.
Asunto(s)
Antineoplásicos/metabolismo , Carbazoles/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Plásmidos , Carbazoles/farmacología , Sustancias Intercalantes/metabolismo , Cinética , Unión Proteica , Especificidad por SustratoRESUMEN
Tyrosine phosphorylation plays a crucial role in cell proliferation and cell transformation which suggests that tyrosine kinase-specific inhibitors might be used as anticancer agents. When the cytotoxic effect of the potent tyrosine kinase inhibitor genistein on various cell lines was studied, we observed that 9-hydroxyellipticine-resistant Chinese hamster lung cells (DC-3F/9-OH-E) were markedly more resistant to genistein than the parental cell line (DC-3F). The DC-3F/9-OH-E cells have been shown to have an altered DNA topoisomerase II activity. We therefore examined the effects of genistein on DNA topoisomerase II-related activities of nuclear extracts from DC-3F cells as well as on purified DNA topoisomerase II from calf thymus. Our results show that genistein (a) inhibits the decatenation activity of DNA topoisomerase II and (b) stimulates DNA topoisomerase II-mediated double strand breaks in pBR322 DNA on sites different from those of 4'-(9-acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium. Structure-activity studies with six chemically related compounds show that only genistein has an effect on the cleavage activity of DNA topoisomerase II in the concentration range studied. Finally, genistein treatment of DC-3F cells results in the occurrence of protein-linked DNA strand breaks as shown by DNA filter elution. Viscometric (lengthening) studies demonstrate that genistein is not a DNA intercalator. Genistein is therefore an interesting compound because it induces cleavable complexes without intercalation. Taken together, our results show that genistein is an inhibitor of both protein tyrosine kinases and mammalian DNA topoisomerase II. This could be accounted for by the sharing of a common structure sequence between the two proteins at the ATP binding site.