RESUMO
Purpose: The strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD. Methods: Antibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF. Results: CTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium. Conclusions: Expanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.
Assuntos
Distrofia Endotelial de Fuchs/genética , Biossíntese de Proteínas , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Western Blotting , Proliferação de Células , Células Cultivadas , Endotélio Corneano/metabolismo , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/fisiologia , Humanos , Íntrons , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
With the advent of human induced pluripotent stem cell (hiPSC) technology, it is now possible to derive patient-specific cell lines that are of great potential in both basic research and the development of new therapeutics for human diseases. Not only do hiPSCs offer unprecedented opportunities to study cellular differentiation and model human diseases, but the differentiated cell types obtained from iPSCs may become therapeutics themselves. These cells can also be used in the screening of therapeutics and in toxicology assays for potential liabilities of therapeutic agents. The remarkable achievement of transcription factor reprogramming to generate iPSCs was recognized by the award of the Nobel Prize in Medicine to Shinya Yamanaka in 2012, just 6 years after the first publication of reprogramming methods to generate hiPSCs (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Cell 131, 861-872). This minireview series highlights both the promises and challenges of using iPSC technology for disease modeling, drug screening, and the development of stem cell therapeutics.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Transplante de Células-Tronco , Avaliação Pré-Clínica de Medicamentos , HumanosRESUMO
Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG·CAG triplet repeats in the 3' untranslated region of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG·CAG triplet-repeat instability is not fully understood. Herein, induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington's disease patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG·CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the expansion seen in somatic cells from DM1 patients. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. However, the overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared with fibroblasts, and only occupied the DMPK1 gene harboring longer CTG·CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG·CAG triplet-repeat expansion. The information gained from these studies provides new insight into a general mechanism of triplet-repeat expansion in iPSCs.
Assuntos
Distrofia Miotônica/genética , Células-Tronco Pluripotentes/metabolismo , Proteínas Serina-Treonina Quinases/genética , Expansão das Repetições de Trinucleotídeos/genética , Regiões 3' não Traduzidas/genética , Técnicas de Cultura de Células , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Proteína 2 Homóloga a MutS/biossíntese , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Distrofia Miotônica/patologia , Miotonina Proteína Quinase , Células-Tronco Pluripotentes/patologiaRESUMO
The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA·TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA·TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA·TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA·TTC triplet repeats expanded at a rate of about two GAA·TTC triplet repeats/replication. However, GAA·TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA·TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA·TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA·TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system.
Assuntos
Reparo de Erro de Pareamento de DNA , Ataxia de Friedreich/metabolismo , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Modelos Biológicos , Proteína 2 Homóloga a MutS/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/patologia , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Íntrons/genética , Proteínas de Ligação ao Ferro/genética , Fator 4 Semelhante a Kruppel , Camundongos , Proteína 2 Homóloga a MutS/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , FrataxinaRESUMO
We have previously demonstrated amelioration of Huntington's disease (HD)-related phenotypes in R6/2 transgenic mice in response to treatment with the novel histone deacetylase (HDAC) inhibitor 4b. Here we have measured the selectivity profiles of 4b and related compounds against class I and class II HDACs and have tested their ability to restore altered expression of genes related to HD pathology in mice and to rescue disease effects in cell culture and Drosophila models of HD. R6/2 transgenic and wild-type (wt) mice received daily injections of HDAC inhibitors for 3 days followed by real-time PCR analysis to detect expression differences for 13 HD-related genes. We find that HDACi 4b and 136, two compounds showing high potency for inhibiting HDAC3 were most effective in reversing the expression of genes relevant to HD, including Ppp1r1b, which encodes DARPP-32, a marker for medium spiny striatal neurons. In contrast, compounds targeting HDAC1 were less effective at correcting gene expression abnormalities in R6/2 transgenic mice, but did cause significant increases in the expression of selected genes. An additional panel of 4b-related compounds was tested in a Drosophila model of HD and in STHdhQ111 striatal cells to further distinguish HDAC selectivity. Significant improvement in huntingtin-elicited Drosophila eye neurodegeneration in the fly was observed in response to treatment with compounds targeting human HDAC1 and/or HDAC3. In STHdhQ111 striatal cells, the ability of HDAC inhibitors to improve huntingtin-elicited metabolic deficits correlated with the potency at inhibiting HDAC1 and HDAC3, although the IC50 values for HDAC1 inhibition were typically 10-fold higher than for inhibition of HDAC3. Assessment of HDAC protein localization in brain tissue by Western blot analysis revealed accumulation of HDAC1 and HDAC3 in the nucleus of HD transgenic mice compared to wt mice, with a concurrent decrease in cytoplasmic localization, suggesting that these HDACs contribute to a repressive chromatin environment in HD. No differences were detected in the localization of HDAC2, HDAC4 or HDAC7. These results suggest that inhibition of HDACs 1 and 3 can relieve HD-like phenotypes in model systems and that HDAC inhibitors targeting these isotypes might show therapeutic benefit in human HD.
Assuntos
Modelos Animais de Doenças , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases/metabolismo , Doença de Huntington/enzimologia , Doença de Huntington/genética , Peptídeos/fisiologia , Fenótipo , Animais , Células Cultivadas , Drosophila melanogaster , Sistemas de Liberação de Medicamentos/métodos , Células HCT116 , Histona Desacetilase 1/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos TransgênicosRESUMO
We have shown that a specific pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugate, 1R-Chl, alters the growth characteristics of various cancer cell lines in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without apparent cytotoxicity. This molecule has also shown efficacy in several mouse xenograft models, preventing tumor growth. Previous microarray studies have suggested that members of the histone H4 gene family, H4c and H4j/k, are the primary targets of this molecule, leading to reduced histone mRNA synthesis and growth arrest in cancer cells. In the present study, we examine the effects of 1R-Chl on transcription of other members of the H4 gene family, with the result that mRNA transcription of most genomic copies of H4 are down-regulated by 1R-Chl in a human pancreatic cancer cell line (MIA PaCa-2), but not in a cell line of non-cancerous origin (HEK293 cells). The basis for this differential effect is likely an open chromatin conformation within the H4 genes in cancer cells. Chromatin immunoprecipitation experiments show increased histone acetylation on the histone H4 genes in cancer cells, compared to HEK293 cells, explaining the differential activity of this molecule in cancer versus non-cancer cells.
Assuntos
Antineoplásicos Alquilantes/química , Clorambucila/química , Cromatina/metabolismo , Histonas/genética , Nylons/química , Acetilação , Antineoplásicos Alquilantes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Clorambucila/farmacologia , Cromatina/ultraestrutura , Imunoprecipitação da Cromatina , Regulação para Baixo , Células HEK293 , Humanos , Imidazóis/química , Nylons/farmacologia , Especificidade de Órgãos , Neoplasias Pancreáticas , Pirróis/química , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos dos fármacosRESUMO
The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAAâ TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAAâ TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAAâ TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells and occupies FXN intron 1, and shRNA silencing of MSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.
Assuntos
Ataxia de Friedreich/genética , Células-Tronco Pluripotentes Induzidas , Expansão das Repetições de Trinucleotídeos/genética , Células Cultivadas , Expansão das Repetições de DNA , Humanos , Proteínas de Ligação ao Ferro/genética , Instabilidade de Microssatélites , FrataxinaRESUMO
We designed and synthesized conjugates between pyrrole-imidazole polyamides and seco-CBI that alkylate within the coding regions of the histone H4 genes. DNA alkylating activity on the histone H4 fragment and cellular effects against K562 chronic myelogenous leukemia cells were investigated. One of the conjugates, 5-CBI, showed strong DNA alkylation activity and good sequence specificity on a histone H4 gene fragment. K562 cells treated with 5-CBI down-regulated the histone H4 gene and induced apoptosis efficiently. Global gene expression data revealed that a number of histone H4 genes were down-regulated by 5-CBI treatment. These results suggest that sequence-specific DNA alkylating agents may have the potential of targeting specific genes for cancer chemotherapy.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Genes/efeitos dos fármacos , Histonas/genética , Imidazóis/farmacologia , Nylons/farmacologia , Pirróis/farmacologia , Antineoplásicos Alquilantes/síntese química , Antineoplásicos Alquilantes/química , Apoptose/efeitos dos fármacos , DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Células K562 , Leucemia Eritroblástica Aguda/tratamento farmacológico , Nylons/síntese química , Nylons/química , Pirróis/síntese química , Pirróis/químicaRESUMO
Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Histona Desacetilases/metabolismo , Mutação , Animais , Brônquios/metabolismo , Membrana Celular/metabolismo , Cricetinae , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/química , Desnaturação Proteica , Dobramento de Proteína , RNA Interferente Pequeno/metabolismo , VorinostatRESUMO
We designed and synthesized a series of novel hybrid histone deacetylase inhibitors based on conjugation of benzamide-type inhibitors with either linear or cyclic peptides. Linear tetrapeptides (compounds 13 and 14), cyclic tetrapeptides (compounds 1 and 11), and heptanediamide-peptide conjugates (compounds 10, 12, 15 and 16) were synthesized through on-resin solid-phase peptide synthesis (SPPS). All compounds were found to be moderate HDAC1 and HDAC3 inhibitors, with IC(50) values ranging from 1.3 microM to 532 microM. Interestingly, compound 15 showed 19-fold selectivity for HDAC3 versus HDAC1.
Assuntos
Benzamidas/química , Inibidores Enzimáticos/síntese química , Inibidores de Histona Desacetilases , Fármacos Neuroprotetores/síntese química , Peptídeos/química , Ciclização , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Humanos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Peptídeos/síntese químicaRESUMO
Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.
Assuntos
Proliferação de Células/efeitos dos fármacos , Clorambucila/farmacologia , Proteínas de Fusão bcr-abl/genética , Nylons/farmacologia , Animais , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/farmacologia , Sequência de Bases/efeitos dos fármacos , Benzamidas , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Células Cultivadas , Clorambucila/administração & dosagem , Clorambucila/química , Avaliação Pré-Clínica de Medicamentos , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Modelos Biológicos , Nylons/química , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Transdução GenéticaRESUMO
Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC(50) values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K(i) of approximately 14 nm, 15 times lower than the K(i) for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.
Assuntos
Inibidores Enzimáticos/química , Ataxia de Friedreich/tratamento farmacológico , Inibidores de Histona Desacetilases , Doença de Huntington/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ácidos Pimélicos/química , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ataxia de Friedreich/enzimologia , Histona Desacetilases/metabolismo , Humanos , Doença de Huntington/enzimologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Camundongos , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Ácidos Pimélicos/farmacologiaRESUMO
Targeting specific genes or gene products by small molecules is novel approach of cancer chemotherapy. We have developed sequence-specific DNA alkylating agents, conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. These compounds efficiently alkylate DNA at a targeted sequence and inhibit gene expression caused by alkylation at template strand of coding region. Recently, histone H4c gene could be targeted by polyamide-chlorambucil (Chl) conjugates. Thus, we designed and synthesized polyamide seco-CBI conjugates 1-5 targeting histone H4c coding sequence. High resolution denaturing polyacrylamide gel electrophoresis (PAGE) showed polyamide seco-CBI conjugates alkylated at the histone H4c coding sequence.
Assuntos
Antineoplásicos Alquilantes/química , Histonas/genética , Imidazóis/química , Nylons/química , Pirróis/química , Antineoplásicos Alquilantes/síntese química , Inativação Gênica , Nylons/síntese químicaRESUMO
We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, which alkylates and down-regulates transcription of the human histone H4c gene and inhibits the growth of several cancer cell lines in vitro and in a murine SW620 xenograft model, without apparent animal toxicity. In this study, we analyzed the effects of 1R-Chl in the chronic myelogenous leukemia cell line K562 and identified another polyamide conjugate, 6R-Chl, which targets H4 genes and elicits a similar cellular response. Other polyamide conjugates that do not target the H4 gene do not elicit this response. In a murine model, both 1R-Chl and 6R-Chl were found to be highly effective in blocking K562 xenograft growth with high-dose tolerance. Unlike conventional and distamycin-based alkylators, little or no cytotoxicities and animal toxicities were observed in mg/kg dosage ranges. These results suggest that these polyamide alkylators may be a viable treatment alternative for chronic myelogenous leukemia.
Assuntos
Proliferação de Células/efeitos dos fármacos , Clorambucila/uso terapêutico , Histonas/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Nylons/farmacologia , Alquilação , Animais , Sequência de Bases , Western Blotting , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorambucila/síntese química , Clorambucila/química , Feminino , Humanos , Imidazóis/química , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Nylons/síntese química , Nylons/química , Pirróis/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hairpin polyamide-chlorambucil conjugates containing an alpha-diaminobutyric acid (alpha-DABA) turn moiety are compared to their constitutional isomers containing the well-characterized gamma-DABA turn. Although the DNA-binding properties of unconjugated polyamides are similar, the alpha-DABA conjugates display increased alkylation specificity and decreased rate of reaction. Treatment of a human colon carcinoma cell line with alpha-DABA versus gamma-DABA hairpin conjugates shows only slight differences in toxicities while producing similar effects on cell morphology and G2/M stage cell cycle arrest. However, striking differences in animal toxicity between the two classes are observed. Although mice treated with an alpha-DABA hairpin polyamide do not differ significantly from control mice, the analogous gamma-DABA hairpin is lethal. This dramatic difference from a subtle structural change would not have been predicted.
Assuntos
Alquilantes/química , Alquilantes/toxicidade , Aminobutiratos/química , DNA/efeitos dos fármacos , Nylons/química , Nylons/toxicidade , Alquilantes/síntese química , Alquilação , Animais , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clorambucila/química , DNA/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nylons/síntese química , Plasmídeos/genética , Estereoisomerismo , Fatores de TempoRESUMO
A polyamide-chlorambucil conjugate (1R-Chl) arrests a wide range of human cancer cell lines at the G2/M phase of the cell cycle and downregulates histone H4c gene expression. However, an siRNA against H4c mRNA causes G1/S arrest. Here, we report that 1R-Chl downregulates H4c prior to G2/M arrest. G2/M arrest is the result of extensive DNA damage by 1R-Chl, which leads to phosphorylation of H2A.X at serine 139, recruitment of the Nbs1 repair protein, and a cascade of unknown events culminating with cdc2 phosphorylation at tyrosine 15 and abolishment of cdc2 kinase activity. A control polyamide-Chl conjugate, which neither binds to the H4c gene nor has an anti-proliferative effect by itself, causes G2/M arrest when cells are treated with siRNAs specific for H3 or H4c.
Assuntos
Clorambucila/farmacologia , Interfase , Modelos Biológicos , Neoplasias/patologia , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Clorambucila/uso terapêutico , Fase G2 , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/antagonistas & inibidores , Histonas/genética , Humanos , Polímeros/farmacologia , Polímeros/uso terapêutico , RNA Interferente Pequeno/farmacologiaRESUMO
(+)-Yatakemycin (1, Fig. 1) and (+)-duocarmycin SA (2) are exceptionally potent, naturally occurring antitumor agents that derive their biological properties through a characteristic sequence-selective DNA-alkylation reaction. Studies have shown that both the AT-rich binding selectivity (shape-selective recognition) and the alkylation catalysis (shape-dependent catalysis) that contribute to the alkylation selectivity are dependent on the DNA minor groove shape and size characteristics of an AT-rich sequence (ref. 6 and references therein; refs. 7,8). Here we report the alkylation properties of yatakemycin and duocarmycin SA on free DNA (alpha-satellite DNA) and the same sequence bound in a nucleosome core particle (NCP) modeling the state of DNA in eukaryotic cells. Both compounds showed a clear, relatively unaltered ability to alkylate DNA packaged in NCPs in terms of both alkylating efficiency and sequence selectivity, despite the steric and conformational perturbations imposed by NCP packaging. These findings highlight the dynamic nature of NCP-bound DNA and illustrate that cell- and protein-free DNA-alkylation studies of members of this class of antitumor drugs provide valuable insights into their properties.
Assuntos
Antineoplásicos Alquilantes/química , DNA/química , Duocarmicinas , Indóis/química , Nucleossomos , Pirróis/químicaRESUMO
Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Imidazóis/farmacologia , Conformação de Ácido Nucleico , Pirróis/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores de Estrogênio/antagonistas & inibidores , Ligação Competitiva , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Imidazóis/metabolismo , Ligantes , Modelos Químicos , Conformação de Ácido Nucleico/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Pirróis/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
A small library of pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugates was screened for effects on morphology and growth characteristics of a human colon carcinoma cell line, and a compound was identified that causes cells to arrest in the G2/M stage of the cell cycle. Microarray analysis indicates that the histone H4c gene is significantly downregulated by this polyamide. RT-PCR and Western blotting experiments confirm this result, and siRNA to H4c mRNA yields the same cellular response. Strikingly, reduction of H4 protein by >50% does not lead to widespread changes in global gene expression. Sequence-specific alkylation within the coding region of the H4c gene in cell culture was confirmed by LM-PCR. The compound is active in a wide range of cancer cell lines, and treated cells do not form tumors in nude mice. The compound is also active in vivo, blocking tumor growth in mice, without obvious animal toxicity.