RESUMEN

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.

Asunto(s)

Aminoglicósidos , Antibacterianos/farmacología , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN/biosíntesis , ADN Viral/antagonistas & inhibidores , ADN Viral/metabolismo , Proteína Quinasa Activada por ADN , Enediinos , Activación Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Proteínas Nucleares , Fosforilación/efectos de los fármacos , Proteína de Replicación A

RESUMEN

Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with "classical" groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.

Asunto(s)

Bencimidazoles/farmacología , ADN/efectos de los fármacos , Distamicinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , Pirroles/farmacología , Células 3T3 , Animales , Bencimidazoles/química , Sitios de Unión , Sistema Libre de Células , Proteínas de Unión al ADN/metabolismo , Distamicinas/química , Células HeLa , Humanos , Ligandos , Ratones , Estructura Molecular , Proteínas Nucleares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/química , Pirroles/química , Factor de Respuesta Sérica , Transcripción Genética/efectos de los fármacos

RESUMEN

The interactions of Hoechst 33377 (H1) with 20 different oligomeric duplexes have been investigated via spectrofluorometric titrations and/or thermal denaturation experiments. H1 is shown to form 2:1 complexes with dsDNA binding sites of at least four contiguous A/T base pairs. H1 is also shown to possess the rare ability to meaningfully distinguish between different A.T rich sequences. For example, the combined equilibrium constants for complexation of the oligomeric duplex 5'-GCAATTGC-3' (15) by H1 are found to be 110-fold greater than for the duplex 5'-GCTTAAGC-3' (16). It is believed that the 5'-TpA-3' dinucleotide step in 16 disrupts the rigid "A-tract" conformation of 15 and discourages minor groove binding by agents capable of recognizing longer dsDNA sequences. Molecular models are presented which elucidate the structure of the (H1)(2)-dsDNA minor groove complex. The two H1 molecules bind to an A/T rich sequence of 6 bp in a slightly staggered, side-by-side, and antiparallel arrangement. Evidence suggests that the piperazine rings of the H1 side-by-side complex are capable of resting in the minor groove of G/C base pairs. Fluorescence microscopy studies using NIH3T3 cells indicate that H1 is capable of traversing the cytoplasmic membrane and selectively localizing to nuclear DNA. H1 also demonstrated the ability to inhibit endogenous transcription of the c-fos gene in NIH3T3 cells at micromolar concentrations. Cytotoxicity studies employing the same cell type show H1 to possess an LD(50) of 3.5 microM.

Asunto(s)

Bencimidazoles/metabolismo , ADN/metabolismo , Sustancias Intercalantes/metabolismo , Células 3T3 , Animales , Bencimidazoles/síntesis química , Bencimidazoles/toxicidad , Sitios de Unión , Bisbenzimidazol/metabolismo , Bisbenzimidazol/toxicidad , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Genes fos/efectos de los fármacos , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/toxicidad , Ligandos , Ratones , Microscopía de Contraste de Fase , Modelos Moleculares , Ácidos Nucleicos Heterodúplex/metabolismo , ARN Mensajero/biosíntesis , Espectrometría de Fluorescencia , Fracciones Subcelulares/metabolismo , Termodinámica

RESUMEN

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.

Asunto(s)

Antineoplásicos Alquilantes/farmacología , Proteínas de Ciclo Celular , Ácidos Ciclohexanocarboxílicos/farmacología , Replicación del ADN/efectos de los fármacos , Indoles , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Benzofuranos , Quinasa de Punto de Control 2 , Cromosomas Fúngicos/efectos de los fármacos , Cromosomas Fúngicos/genética , Ciclohexenos , Daño del ADN , Replicación del ADN/fisiología , ADN de Hongos/biosíntesis , ADN de Hongos/efectos de los fármacos , ADN de Hongos/genética , Duocarmicinas , Proteínas Fúngicas/genética , Péptidos y Proteínas de Señalización Intracelular , Mutación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética

RESUMEN

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.

Asunto(s)

Aminoglicósidos , Antibacterianos/toxicidad , Núcleo Celular/metabolismo , Daño del ADN , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , ADN/antagonistas & inhibidores , Activación Transcripcional/efectos de los fármacos , Cinostatina/toxicidad , Antibióticos Antineoplásicos/toxicidad , Western Blotting , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Sistema Libre de Células/efectos de los fármacos , ADN/biosíntesis , ADN Viral/antagonistas & inhibidores , ADN Viral/biosíntesis , Electroforesis en Gel de Poliacrilamida , Enediinos , Humanos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Fosforilación/efectos de los fármacos , Proteína de Replicación A , Virus 40 de los Simios/genética , Solubilidad , Moldes Genéticos , Cinostatina/análogos & derivados

RESUMEN

Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).

Asunto(s)

Bleomicina/farmacología , Daño del ADN , Replicación del ADN/efectos de los fármacos , Sustancias Intercalantes/farmacología , Animales , Antígenos Virales de Tumores/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular/citología , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Línea Celular/virología , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/metabolismo , Sistema Libre de Células/virología , Chlorocebus aethiops , ADN Viral/antagonistas & inhibidores , ADN Viral/biosíntesis , Inhibidores de Crecimiento/farmacología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Líquido Intracelular/virología , Virus 40 de los Simios/genética

RESUMEN

Previous work has demonstrated that sequence-selective DNA-binding drugs can inhibit transcription factors from binding to their target sites on gene promoters. In this study, the potency and effectiveness of DNA-binding drugs to inhibit transcription were assessed using the c-fos promoter's serum response element (SRE) as a target. The drugs chosen for analysis included the minor groove binding agents chromomycin A(3) and Hoechst 33342, which bind to G/C-rich and A/T-rich regions, respectively, and the intercalating agent nogalamycin, which binds G/C-rich sequences in the major groove. The transcription factors targeted, Elk-1 and serum response factor (SRF), form a ternary complex (TC) on the SRE that is necessary and sufficient for induction of c-fos by serum. The drugs' abilities to prevent TC formation on the SRE in vitro were nogalamycin > Hoechst 33342 > chromomycin. Their potencies in inhibiting cell-free transcription and endogenous c-fos expression in NIH3T3 cells, however, were chromomycin > nogalamycin > Hoechst 33342. The latter order of potency was also obtained for the drugs' cytotoxicity and inhibition of general transcription as measured by [(3)H]uridine incorporation. These systematic analyses provide insight into how drug and transcription factor binding characteristics are related to drugs' effectiveness in inhibiting gene expression.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Preparaciones Farmacéuticas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Elementos de Respuesta/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Células 3T3/efectos de los fármacos , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Unión Competitiva/genética , Northern Blotting , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/metabolismo , Cromomicina A3/metabolismo , Cromomicina A3/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Nogalamicina/metabolismo , Nogalamicina/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/biosíntesis , Factor de Respuesta Sérica , Factores de Tiempo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo

RESUMEN

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Asunto(s)

Amidas/farmacología , Genes erbB-2 , Imidazoles/farmacología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Amidas/síntesis química , Amidas/química , Secuencia de Bases , Sitios de Unión , Línea Celular , Huella de ADN , Humanos , Imidazoles/síntesis química , Imidazoles/química , Cinética , Modelos Moleculares , Proteínas Proto-Oncogénicas c-ets , Pirroles/síntesis química , Pirroles/química , Pirroles/farmacología , Receptor ErbB-2/genética

RESUMEN

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.

Asunto(s)

Antineoplásicos Alquilantes/farmacología , Ácidos Ciclohexanocarboxílicos/farmacología , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Indoles , Virus 40 de los Simios/genética , Afidicolina/farmacología , Benzofuranos , Línea Celular Transformada , Ciclohexenos , Duocarmicinas , Humanos , Cinética , Fosforilación , Proteína de Replicación A , Fase S , Virus 40 de los Simios/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).

Asunto(s)

Alquilantes/farmacología , Antivirales/biosíntesis , Antivirales/farmacología , Indoles/farmacología , Virus 40 de los Simios/efectos de los fármacos , Urea/análogos & derivados , Replicación Viral/efectos de los fármacos , Animales , Antivirales/fisiología , Línea Celular , Sistema Libre de Células , Chlorocebus aethiops , Aductos de ADN/biosíntesis , Daño del ADN , Replicación del ADN/efectos de los fármacos , ADN Viral/antagonistas & inhibidores , ADN Viral/biosíntesis , ADN Viral/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Duocarmicinas , Electroforesis en Gel Bidimensional , Células HeLa , Calor , Humanos , Virus 40 de los Simios/química , Virus 40 de los Simios/genética , Transactivadores/biosíntesis , Urea/farmacología , Replicación Viral/genética

RESUMEN

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.

Asunto(s)

Aminoglicósidos , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Daño del ADN , Replicación del ADN/efectos de los fármacos , ADN Viral/efectos de los fármacos , Herpesvirus Humano 4/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/virología , ADN Mitocondrial/efectos de los fármacos , Electroforesis en Gel Bidimensional , Enediinos , Inhibidores de Crecimiento/farmacología , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/virología , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/virología , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos

Asunto(s)

ADN Viral/análisis , ADN Viral/aislamiento & purificación , Herpesvirus Humano 4/genética , Línea Celular Transformada , Clonación Molecular/métodos , ADN Viral/química , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Campo Pulsado , Endopeptidasa K , Genoma Viral , Humanos , Peso Molecular , Mapeo Restrictivo , Sefarosa

RESUMEN

In this study, we examined how DNA-binding drugs prevented formation of transcription factor-DNA complexes and influenced gene transcription from the hamster dihydrofolate reductase promoter, which is regulated by E2F1 and Sp1. Gel mobility shift assay data showed that GC-binding drugs (e.g., mitoxantrone) inhibited the DNA binding of both E2F1 and Sp1. In contrast, AT-binding drugs (e.g., distamycin) interfered only with E2F1-DNA complex formation. In an in vitro transcription assay using HeLa nuclear extracts, inhibition of transcription was observed when mitoxantrone or distamycin was added either before or after assembly of the transcription complex on the DNA, although for the latter, higher drug concentrations were needed. Mitoxantrone, which was a stronger inhibitor of transcription factor-DNA complex, was more effective than distamycin at preventing transcript formation. Time course transcription in a cell-free assay with addition of various drug concentrations indicated that high drug concentrations of either mitoxantrone or distamycin completely blocked transcription, while low drug concentrations could delay the synthesis of transcripts.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Expresión Génica , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Animales , Cricetinae , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Células HeLa , Humanos , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Tetrahidrofolato Deshidrogenasa/genética , Factor de Transcripción DP1

RESUMEN

Bizelesin, an AT-specific DNA-alkylating antitumor drug, is a potent inhibitor of genomic DNA replication in BSC-1 cells. Fifty percent inhibition of DNA synthesis was observed at 10 nM bizelesin compared to 160 nM needed for 50% inhibition of RNA synthesis while no inhibition of protein synthesis was observed up to 200 nM. Sedimentation analysis of nascent genomic DNA showed that bizelesin inhibited new replicon initiation and had significantly less effect on replicon maturation. Bizelesin also suppressed the intracellular synthesis of simian virus 40 (SV40) DNA in virus-infected BSC-1 cells. The analysis of nascent SV40 intermediates synthesized after bizelesin treatment confirmed an initiation-specific inhibition. The inhibitory effects on cellular DNA replication occurred at bizelesin levels resulting in infrequent adducts (one adduct per several replicons). Only one bizelesin adduct per several SV40 molecules was needed for a potent inhibition of intracellular SV40 replication. In contrast, only partial inhibition of SV40 replication in vitro was observed with bizelesin-treated naked SV40 DNA as a template. Overall, the results indicate that infrequent bizelesin lesions impede the cellular replication apparatus at the level of the initiation of new replicons.

Asunto(s)

Antineoplásicos Alquilantes/farmacología , Reactivos de Enlaces Cruzados/farmacología , Replicación del ADN/efectos de los fármacos , ADN Viral/biosíntesis , Indoles/farmacología , Urea/análogos & derivados , Animales , Línea Celular , Duocarmicinas , Haplorrinos , Replicón/efectos de los fármacos , Virus 40 de los Simios/genética , Urea/farmacología

RESUMEN

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

Asunto(s)

Proteínas de Unión al ADN/biosíntesis , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces , Regiones Promotoras Genéticas , Factores de Transcripción/biosíntesis , Transcripción Genética , Latencia del Virus , Animales , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/biosíntesis , Secuencia de Consenso , Citomegalovirus/genética , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Genes Reporteros , Herpesvirus Humano 1/genética , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , TATA Box , Factores de Transcripción/metabolismo , Activación Viral , Dedos de Zinc

RESUMEN

The central pyrrole of a site-selective DNA minor groove binding tripyrrole peptide 1 has been attached to a branched decaaza decabutylamine via a -(CH-2)3-NHCO-(CH2)-3 linker to provide the decaaza-microgonotropen (8). The decaaza decabutylamine moiety of 8 was designed to have a much greater affinity to the phosphodiester linkages of the backbone of DNA. Employing Hoechst 33258 (Ht) as a fluorescent titrant, the equilibrium constants for the binding for of 8 to the hexadecameric duplex d(GGCGCA3T3GGCGG)/d(CCGCCA3T3GCGCC) and to calf thymus DNA were determined. The log of the product of equilibrium constants (log Kl1Kl2) for 1:1 and 1:2 complexes formation at A3T3 is 17 (35 degrees C). Results of studies of the inhibition of the binding of several proteins to target DNA are discussed. Binding of the E2F1 transcription factor to its DNA target is 50% inhibited at approximately 2 nM concentration of 8.

Asunto(s)

Butilaminas/metabolismo , Proteínas de Ciclo Celular , ADN/metabolismo , Pirroles/metabolismo , Factores de Transcripción/metabolismo , Animales , Bisbenzimidazol/química , Butilaminas/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Bovinos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Distamicinas/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión a Hierro , Cinética , Sondas de Oligonucleótidos , Pirroles/química , Proteína 1 de Unión a Retinoblastoma , TATA Box , Proteína de Unión a TATA-Box , Timo/metabolismo , Factor de Transcripción DP1 , Factores de Transcripción/genética , Proteínas de Unión a Transferrina , Dedos de Zinc

RESUMEN

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5'-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 microM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA3T3GGC)/d(CCGCT3A3CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1-DNA complex formation compared with disruption of a preexisting complex.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Distamicinas/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Cricetinae , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Humanos , Ratones , Proteína 1 de Unión a Retinoblastoma , Tetrahidrofolato Deshidrogenasa/genética , Factor de Transcripción DP1

RESUMEN

This study used 2-D agarose gel techniques to examine the effects of the DNA-strand scission enediyne C-1027 on DNA replication in SV40-infected BSC-1 cells. Replication of SV40 DNA was inhibited by C-1027 to a greater extent than was BSC-1 genomic DNA replication in infected cells. Low nanomolar concentrations (0.2-10 nM) of C-1027 affected a rapid, progressive decrease in SV40 replication activity and replication intermediates (RIs) within 15 min after drug addition. A concurrent decrease in the signal of both the SV40 bubble arc and replication activity with increasing concentrations of C-1027 suggested that C-1027 inhibited initiation of new RIs. Additionally, the reduction in bubble arc signal observed with C-1027 was prevented when elongation of nascent chains was blocked by aphidicolin. Thus, the C-1027-induced disappearance of RIs probably is related to the maturation of preformed replication molecules in the absence of initiation of new RIs. Strand damage to SV40 DNA was barely detectable at concentrations where inhibition of replication activity was nearly complete, indicating that C-1027 replication inhibition occurs in trans.

Asunto(s)

Aminoglicósidos , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Daño del ADN , Replicación del ADN/efectos de los fármacos , ADN Viral/biosíntesis , Péptidos , Virus 40 de los Simios/fisiología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , ADN Viral/aislamiento & purificación , Electroforesis en Gel de Agar , Enediinos , Cinética , Virus 40 de los Simios/efectos de los fármacos

RESUMEN

A kinetic analysis of cleavage of simian virus DNA (SV40 DNA) inside and outside green monkey BSC-1 cells by the enediyne-protein antibiotic C-1027 and its free chromophore is described. Information on rate constants was obtained by fitting populations of forms I (closed circular DNA), II (nicked circular DNA) and III (linear DNA) SV40 DNA as a function of drug concentration to a kinetic model which includes: cutting of form I to give form II with rate constant k1, cutting of form I to give form III with rate constant K4, and cutting of form II to give form III with rate constant k2. The ratio of single-strand (ss) to double-strand (ds) cutting for the holoantibiotic and the free chromophore, k1/k4, is approximately 1.8 for extracellular SV40 DNA. For intracellular DNA and extracellular DNA which has been post-treated with putrescine, ds cutting is much more probable, with k4 about four times as large as k1. This observation suggests that amine groups present in the cell are able to convert abasic sites opposite an ss break into a ds break in SV40 chromatin. The overall rate of cleavage of form-I DNA inside the cell is much larger than the rate outside, the sum k1 + k4 being about three times as large for intracellular DNA as for extracellular DNA.

Asunto(s)

Aminoglicósidos , Antibacterianos , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , ADN Viral/efectos de los fármacos , Péptidos , Virus 40 de los Simios/metabolismo , Animales , Antibacterianos/química , Antibióticos Antineoplásicos/química , Línea Celular , Chlorocebus aethiops , Electroforesis en Gel de Agar , Enediinos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Cinética , Biosíntesis de Proteínas , Putrescina/farmacología , Virus 40 de los Simios/efectos de los fármacos

RESUMEN

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces , Sustancias Intercalantes/farmacología , Regiones Promotoras Genéticas , Simplexvirus/genética , Factores de Transcripción/metabolismo , Latencia del Virus , Alquilantes/farmacología , Antraquinonas/farmacología , Composición de Base , Cromomicina A3/farmacología , Desoxirribonucleoproteínas/química , Distamicinas/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Sustancias Macromoleculares , Nogalamicina/farmacología , Proteínas Recombinantes de Fusión , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIA