RESUMEN
Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-x(L) and Mcl-1, known to regulate cell survival and death, in a panel of fourteen NSCLC cell lines. NSCLC cell lines express high levels of Mcl-1 and Bcl-x(L), but not Bcl-2. Silencing the expression of Mcl-1 with small interfering RNA (siRNA) oligonucleotides potently killed a subgroup of NSCLC cell lines. In contrast, Bcl-x(L) siRNA had no effect in these lines unless Mcl-1 siRNA was also introduced. Interestingly, high MCL1 to BCL-xl messenger RNA determines whether the cells depend on Mcl-1 for survival. We further investigated the role of Mcl-1 in NSCLC cells using a Mcl-1-dependent cell line, H23. The expression of a complementary DNA containing only the coding region of MCL1 rescued H23 cells from the toxicity of a 3' untranslated region (UTR) targeting Mcl-1 siRNA but not a siRNA targeting the coding region of MCL1. Furthermore, we show that Mcl-1 sequesters the BH3-only protein Noxa and Bim and the apoptotic effector Bak. Not surprisingly, Noxa, Bim, or Bak knockdown partially rescued H23 cells from toxicity mediated by Mcl-1 siRNA to different degrees. Collectively, our results indicate that targeting Mcl-1 may improve therapy for a subset of NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Línea Celular Tumoral , Humanos , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
AZD1152 is a highly selective Aurora B kinase inhibitor currently undergoing Phase I and II clinical evaluation in patients with acute myelogenous leukemia and advanced solid malignancies. We have established two AZD1152-resistant cell lines from SW620 colon and MiaPaCa pancreatic carcinoma lines, which are >100-fold resistant to the active metabolite of AZD1152, AZD1152 HQPA and interestingly, cross-resistant to the pan-Aurora kinase inhibitor, VX-680/MK0457. Using whole-genome microarray analysis and comparative genomic hybridization, we were able to identify MDR1 and BCRP as the causative genes that underlie AZD1152 HQPA-resistance in these models. Furthermore, the upregulation of either of these genes is sufficient to render in vivo tumor growth insensitive to AZD1152. Finally, the upregulation of MDR1 or BCRP is predictive of tumor cell sensitivity to this agent, both in vitro and in vivo. The data provide a genetic basis for resistance to Aurora kinase inhibitors, which could be utilized to predict clinical response to therapy.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Organofosfatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinazolinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aurora Quinasa B , Aurora Quinasas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Hibridación Genómica Comparativa , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica/métodos , Humanos , Concentración 50 Inhibidora , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Piperazinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Several modified nucleoside 5'-triphosphates were synthesized containing adenine-mimicking pyrimidine derivatives as an aglycone. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.
Asunto(s)
Adenosina Trifosfato/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Nucleótidos de Desoxiadenina/química , Tionucleótidos/química , Adenosina Trifosfato/metabolismo , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiadenina/metabolismo , Especificidad por Sustrato , Tionucleótidos/metabolismoRESUMEN
Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5'-triphosphates do not contain a substituent in position 3' DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3'-hydroxy group in L-dNTP prevents their incorporation into the DNA chain.
Asunto(s)
Replicación del ADN , Nucleótidos/metabolismo , Especificidad por SustratoAsunto(s)
Virus de la Mieloblastosis Aviar/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , VIH/enzimología , Nucleótidos/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo , Catálisis , Humanos , Cinética , Nucleótidos/química , Compuestos Organofosforados/química , Especificidad por SustratoRESUMEN
To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-beta (PKC-beta) and several stable PKC-beta transfectants, we found that PRKX gene expression is under control of PKC-beta; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Macrófagos/citología , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteínas Serina-Treonina Quinasas/genética , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Variación Genética , Granulocitos/citología , Granulocitos/enzimología , Células HL-60 , Humanos , Linfocitos/citología , Linfocitos/enzimología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/enzimología , Monocitos/citología , Monocitos/enzimología , Proteínas Nucleares , Oligodesoxirribonucleótidos/farmacología , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción , Transcripción Genética , TransfecciónRESUMEN
Phosphorothioate analogues of oligonucleotides (PS-oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [R(P)] configuration, while the presence of the linkage(s) of the [S(P)] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3'-end. The presence of [S(P)] linkage at this position strongly reduces the enzyme activity while the [R(P)] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.
Asunto(s)
ADN Nucleotidilexotransferasa/química , ADN Nucleotidilexotransferasa/metabolismo , Desoxirribonucleósidos/química , Desoxirribonucleósidos/metabolismo , Activación Enzimática , Oligonucleótidos , Relación Estructura-ActividadRESUMEN
All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Timina/metabolismo , Cartilla de ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Transcriptasa Inversa del VIH/metabolismo , Humanos , Estereoisomerismo , Relación Estructura-Actividad , Moldes GenéticosRESUMEN
Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.
Asunto(s)
Antivirales/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Fosfatos/química , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Animales , Antivirales/sangre , Antivirales/química , Didesoxinucleótidos , Estabilidad de Medicamentos , Humanos , Especificidad por Sustrato , Nucleótidos de Timina/sangre , Nucleótidos de Timina/química , Zidovudina/sangre , Zidovudina/química , Zidovudina/metabolismoRESUMEN
9-[2-(phosphonomethylcarbonylamino)ethyl]adenine, 9-[(2-phosphonoethyl)aminocarbonylmethyl]adenine, 9-[2-[(2-phosphonoethyl)carbonylamino]ethyl]adenine, and their diphosphates were synthesized. All three diphosphates were shown to incorporate into the 3'-terminus of the DNA chain during the synthesis of the avian myeloblastosis virus catalyzed by reverse transcriptase. However, they do not serve as substrates for DNA polymerase alpha from human placenta, polymerase beta from calf thymus, or terminal deoxynucleotidyl transferase from calf thymus.
Asunto(s)
Adenina/síntesis química , Compuestos Organofosforados/química , Adenina/análogos & derivados , Adenina/metabolismo , Animales , Secuencia de Bases , Bovinos , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/metabolismo , Especificidad por SustratoRESUMEN
2'-Deoxythymidine 5'-triphosphate and 2'-deoxyadenosine 5'-triphosphate analogs containing a methylene group between the alpha phosphorus and 5' oxygen were synthesized. The substrate properties of these compounds toward some mammalian DNA polymerases and retroviral reverse transcriptases were evaluated using a system containing phage M13mp10 DNA, a synthetic oligonucleotide, and the enzyme. The compounds containing a hydroxyl at the 3' position were incorporated into the DNA chain by DNA polymerase alpha and terminal deoxynucleotidyl transferase, but were not recognized by retroviral reverse transcriptases and mammalian DNA polymerases epsilon and beta. The selectivity of the compounds synthesized was capitalized on during simultaneous isolation of DNA polymerases alpha and epsilon from human placenta. A methylene group was also introduced into the acyclovir molecule. It was shown that this modification inactivates furanose-related nucleotide analogs, but has a minor effect on the substrate properties of acyclic nucleotide analogs.
Asunto(s)
ADN Polimerasa II/antagonistas & inhibidores , Nucleótidos de Desoxiadenina/farmacología , Nucleótidos de Timina/farmacología , Animales , Secuencia de Bases , Bovinos , ADN Nucleotidilexotransferasa/antagonistas & inhibidores , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa II/metabolismo , Cartilla de ADN , Nucleótidos de Desoxiadenina/química , Escherichia coli/enzimología , Humanos , Datos de Secuencia Molecular , Especificidad por Sustrato , Nucleótidos de Timina/químicaRESUMEN
A new series of nucleotide analogs, (Z)-pyrophosphoryl (phosphonyloxymethyl) but-2-enyl derivatives of pyrimidines and purines, was synthesized. Their substrate and inhibitory properties toward some DNA polymerases and reverse transcriptases were evaluated. They were shown to be selective inhibitors of HIV reverse transcriptase. The structure-substrate properties relationships for nucleotide analogs were discussed.
Asunto(s)
Nucleótidos/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , VIH/enzimología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Transcriptasa Inversa , Especificidad por SustratoRESUMEN
Two 2'-deoxynucleoside 5'-alpha-methylenephosphonyl-beta, gamma-diphosphates were synthesized. They were incorporated into the DNA chain by DNA polymerase alpha from human placenta. Meanwhile, they were not recognized by DNA polymerase epsilon and beta of the same origin as well as by reverse transcriptases from human immunodeficiency virus and avian myeloblastosis virus.
Asunto(s)
ADN Polimerasa II/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Virus de la Mieloblastosis Aviar/enzimología , Secuencia de Bases , ADN/metabolismo , VIH/enzimología , Humanos , Datos de Secuencia Molecular , Nucleótidos/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Especificidad por SustratoRESUMEN
5''-Derivatives of 3'-(tetrazole-2''-yl)-3'-deoxythymidines were synthesized by interaction of 5'-benzoyl-2',3'-anhydrothymidine with tetrazole or its 5-derivatives followed by debenzoylation. Structures of two of them, 3'-(tetrazole-2''-yl)-3'-deoxythymidine and 3'-(5''-methyltetrazole-2''-yl)-3'-deoxythymidine, elucidated by X-ray analysis, revealed anti conformation of thymine about the glycosidic bond and 2'-endo-3'-exo-conformation of the sugar residue with gauche+ orientation with respect to C4'-C5'-bond. 3'-(Tetrazole-2''-yl)-3'-deoxythymidine 5'-triphosphate demonstrated poor substrate properties for the avian myeloblastosis virus reverse transcriptase and none for several other DNA polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Timidina/análogos & derivados , Nucleótidos de Timina/química , Secuencia de Bases , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Molecular , Oligodesoxirribonucleótidos , Especificidad por Sustrato , Timidina/química , Timidina/metabolismo , Nucleótidos de Timina/metabolismoRESUMEN
The substrate properties of four carbocyclic D and L nucleoside 5'-triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the D-beta and L-beta series were found to be terminating substrates for these enzymes, while the derivatives of the D-alpha and L-alpha series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template-independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Virus de la Mieloblastosis Aviar/enzimología , ADN Nucleotidilexotransferasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , VIH/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sitios de Unión , ADN/biosíntesis , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Datos de Secuencia Molecular , ARN/metabolismo , Especificidad por Sustrato , Moldes GenéticosRESUMEN
A series of pyrophosphoryl (Z)-(phosphonomethoxy)but-2-enyl derivatives of pyrimidines and purines 9a-d and the corresponding phosphonates 10a-d were synthesized. The prepared compounds contain the phosphonate group as an alpha-phosphate mimic as well as an acyclic residue emulating the sugar moiety in 2',3'-dideoxy-2',3'-didehydronucleoside 5'-triphosphates known as highly potent chain terminators of DNA polymerases. Phosphonates 10a-d were obtained by alternative alkylations of the nucleic bases followed by condensation with ethyl [[(p-tolylsulfonyl)oxy]methyl]phosphonate. Pyrophosphorylation of 10a-d afforded phosphonate diphosphates 9a-d. Their substrate properties were evaluated in cell-free systems containing various DNA polymerases including viral reverse transcriptases. Compounds 9a-d manifested good terminating substrate properties toward HIV-1 and AMV reverse transcriptases. They exhibited high selectivity and were not recognized by human DNA polymerases alpha and epsilon, DNA polymerase beta from rat liver, Escherichia coli DNA polymerase I, and HSV-1 and CMV DNA polymerases. Phosphonates 10b-d displayed no activity in HIV-1-infected MT-4 cells cultures; 10a was moderately effective (ED50 = 9 microM).
Asunto(s)
Nucleótidos/síntesis química , Nucleótidos/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Transcriptasa Inversa del VIH , Humanos , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico , Ratas , Inhibidores de la Transcriptasa InversaRESUMEN
5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl) thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl) thymine and 2',3'-lyxoanhydrothymidine have been shown to be terminator substrates of human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases as well as DNA polymerase I from E. coli. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. The KM values of ltTTP, rtTTP and laTTP incorporation into DNA chain agree closely with each other, being 1.5-2.5 times higher than KM for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times less than Vmax for dTTP. The evidence favours the hypothesis of a great affinity of modified nucleosides with flattened ribose ring of glycone for DNA polymerases active sites.
Asunto(s)
ADN Polimerasa I/metabolismo , Nucleótidos/metabolismo , Virus de la Mieloblastosis Aviar/enzimología , Secuencia de Bases , Sitios de Unión , ADN/biosíntesis , ADN/química , ADN/metabolismo , ADN Polimerasa I/antagonistas & inhibidores , Escherichia coli/enzimología , VIH/enzimología , Datos de Secuencia Molecular , Nucleótidos/química , ADN Polimerasa Dirigida por ARN/metabolismo , Especificidad por SustratoRESUMEN
5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase beta from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.