RESUMO
Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila's DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.
Assuntos
Drosophila/genética , Desenho de Fármacos , Heterocromatina/metabolismo , AnimaisRESUMO
Heterochromatin is essential for regulating global gene transcription and protecting genome stability, and may play a role in tumor suppression. Drugs promoting heterochromatin are potential cancer therapeutics but very few are known. In order to identify drugs that can promote heterochromatin, we used a cell-based method and screened NCI drug libraries consisting of oncology drugs and natural compounds. Since heterochromatin is originally defined as intensely stained chromatin in the nucleus, we estimated heterochromatin contents of cells treated with different drugs by quantifying the fluorescence intensity of nuclei stained with Hoechst DNA dye. We used HeLa cells and screened 231 FDA-approved oncology and natural substance drugs included in two NCI drug libraries representing a variety of chemical structures. Among these drugs, streptonigrin most prominently caused an increase in Hoechst-stained nuclear fluorescence intensity. We further show that streptonigrin treated cells exhibit compacted DNA foci in the nucleus that co-localize with Heterochromatin Protein 1 alpha (HP1α), and exhibit an increase in total levels of the heterochromatin mark, H3K9me3. Interestingly, we found that streptonigrin promotes heterochromatin at a concentration as low as one nanomolar, and at this concentration there were no detectable effects on cell proliferation or viability. Finally, in line with a previous report, we found that streptonigrin inhibits STAT3 phosphorylation, raising the possibility that non-canonical STAT function may contribute to the effects of streptonigrin on heterochromatin. These results suggest that, at low concentrations, streptonigrin may primarily enhance heterochromatin formation with little toxic effects on cells, and therefore might be a good candidate for epigenetic cancer therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Heterocromatina/fisiologia , Estreptonigrina/farmacologia , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Heterocromatina/efeitos dos fármacos , Histonas/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismoRESUMO
Heterochromatin is a tightly packed form of DNA involved in gene silencing, chromosome segregation, and protection of genome stability. Heterochromatin is becoming more recognized in tumor suppression and may thus serve as a potential target for cancer therapy. However, to date there are no drugs that are well established to specifically promote heterochromatin formation. Here, we describe a screening method using Drosophila to identify small molecule compounds that promote heterochromatin formation, with the purpose of developing epigenetic cancer therapeutics. We took advantage of a Drosophila strain with a variegated eye color phenotype that is sensitive to heterochromatin levels, and screened a library of 97 FDA approved oncology drugs. This screen identified methotrexate as the most potent small molecule drug, among the 97 oncology drugs screened, in promoting heterochromatin formation. Interestingly, methotrexate has been identified as a JAK/STAT inhibitor in a functional screen, causing reduced phosphorylation of STAT proteins. These findings are in line with our previous observation that unphosphorylated STAT (uSTAT) promotes heterochromatin formation in both Drosophila and human cells and suppresses tumor growth in mouse xenografts. Thus, Drosophila with variegated eye color phenotypes could be an effective tool for screening heterochromatin-promoting compounds that could be candidates as cancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Epigênese Genética , Heterocromatina/efeitos dos fármacos , Metotrexato/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Animais Geneticamente Modificados , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cor , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/anatomia & histologia , Olho/citologia , Olho/efeitos dos fármacos , Olho/metabolismo , Feminino , Variação Genética , Instabilidade Genômica , Heterocromatina/química , Ensaios de Triagem em Larga Escala , Histonas/genética , Histonas/metabolismo , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/metabolismo , Masculino , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Pigmentação/efeitos dos fármacos , Pigmentação/genética , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel's cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-ß) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-ß interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-ß signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a.
Assuntos
Padronização Corporal/fisiologia , Ossos Faciais/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Nucleares/biossíntese , Somitos/embriologia , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone demethylases has been implicated in development and tumorigenesis. Here we show that Drosophila Kdm4A and Kdm4B are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. We further show that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. Our studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters.