RESUMO
OBJECTIVES: The aim of the study is to determine whether a positive OncoE6 Anal Test result has statistically significant higher odds of being associated with high-grade squamous intraepithelial lesion (HSIL) and to calculate sensitivity and specificity of this test for predicting HSIL in adult men who have sex with men and are living with HIV (MSMLWH). MATERIALS AND METHODS: Men living with HIV 18 years or older having ≥atypical squamous cells of undetermined significance-grade anal cytology results were eligible to enroll in this cross-sectional study. Anal samples were collected just before the high-resolution anoscopy procedure. OncoE6 Anal Test results were compared with histology, the reference standard. Sensitivity, specificity, and odds ratio were calculated using HSIL as the threshold. RESULTS: Two hundred seventy-seven consented MSMLWH were enrolled between June 2017 and January 2022. Of these, 219 (79.1%) had biopsies obtained and histology performed; 81 of 219 participants (37%) had 1 or more biopsies with HSIL results while the remaining 138 of 219 (63%) had only low-grade squamous intraepithelial lesion or were negative for dysplasia. Anal samples from 7 participants (8.6%, 7/81) with HSIL and 3 (2.2%, 3/138) with low-grade squamous intraepithelial lesion had positive OncoE6 Anal Test results. Odds of having HSIL were 4.26 times higher among participants testing positive for HPV16/HPV18 E6 oncoprotein(s) (OR = 4.26, 95% CI = 1.07-16.95, p = .04). The OncoE6 Anal Test demonstrated excellent specificity, 97.83% (93.78-99.55), but poor sensitivity, 8.64% (3.55-17.0). CONCLUSIONS: In this highest-risk population for anal cancer, one could combine the OncoE6 Anal Test, having excellent specificity, with the anal Pap test, having higher sensitivity. Patients found having both an abnormal anal Pap and positive OncoE6 Anal Test result could be triaged for rapid scheduling of their high-resolution anoscopy.
Assuntos
Neoplasias do Ânus , Carcinoma in Situ , Carcinoma de Células Escamosas , Infecções por HIV , Infecções por Papillomavirus , Minorias Sexuais e de Gênero , Lesões Intraepiteliais Escamosas , Adulto , Masculino , Humanos , Homossexualidade Masculina , Estudos Transversais , Canal Anal/patologia , Carcinoma in Situ/patologia , Carcinoma de Células Escamosas/patologia , Lesões Intraepiteliais Escamosas/patologia , Neoplasias do Ânus/patologia , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/patologia , PapillomaviridaeRESUMO
Glutaredoxin (Grx), peroxiredoxin (Prx), and thioredoxin (Trx) are redoxin family proteins that catalyze different types of chemical reactions that impact cell growth and survival through functionally distinct intracellular pathways. Much research is focused on understanding the roles of these redoxin proteins in the development and/or progression of human diseases. Grx and Prx are overexpressed in human cancers, including human lung cancers. BNP7787 is a novel investigational agent that has been evaluated in previous clinical studies, including non-small cell lung cancer (NSCLC) studies. Herein, data from activity assays, mass spectrometry analyses, and X-ray crystallographic studies indicate that BNP7787 forms mixed disulfides with select cysteine residues on Grx and Prx and modulates their function. Studies of interactions between BNP7787 and Trx have been conducted and reported separately. Despite the fact that Trx, Grx, and Prx are functionally distinct proteins that impact oxidative stress, cell proliferation and disease processes through different intracellular pathways, BNP7787 can modify each protein and appears to modulate function through mechanisms that are unique to each target protein. Tumor cells are often genomically heterogeneous containing subpopulations of cancer cells that often express different tumor-promoting proteins or that have multiple dysregulated signaling pathways modulating cell proliferation and drug resistance. A multi-targeted agent that simultaneously modulates activity of proteins important in mediating cell proliferation by functionally distinct intracellular pathways could have many potentially useful therapeutic applications.
Assuntos
Antineoplásicos/farmacocinética , Cisteína/metabolismo , Glutarredoxinas/química , Mesna/análogos & derivados , Peroxirredoxinas/química , Sítios de Ligação , Cristalografia por Raios X , Glutarredoxinas/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Espectrometria de Massas , Mesna/farmacocinética , Modelos Moleculares , Peroxirredoxinas/metabolismo , Estrutura Terciária de ProteínaRESUMO
The role of the primary mammalian DNA methyltransferase, DNMT1, in maintaining CpG island methylation in human colon cancer cells has recently been questioned. This controversy has arisen from discrepancies between genetic knockout and RNA interference-mediated knockdown studies. Here, we re-examined the RNA interference-based approach and found that hypermethylation of single-copy genes is maintained in cells transiently and stably depleted of DNMT1.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Sequência de Bases , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Inativação Gênica , Genes Supressores de Tumor , Células HCT116 , Humanos , Regiões Promotoras Genéticas , Interferência de RNA , RNA Neoplásico/genética , RNA Interferente Pequeno/genéticaRESUMO
Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inativação Gênica , Glicoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas WntRESUMO
Molecular epidemiology (ME) is one tool used to end the HIV epidemic in the United States. We combined clinical and behavioral data with HIV sequence data to identify any overlap in clusters generated from different sequence datasets; to characterize HIV transmission clusters; and to identify correlates of clustering among people living with HIV (PLWH) in Washington, District of Columbia (DC). First, Sanger sequences from DC Cohort participants, a longitudinal HIV study, were combined with next-generation sequences (NGS) from participants in a ME substudy to identify clusters. Next, demographic and self-reported behavioral data from ME substudy participants were used to identify risks of secondary transmission. Finally, we combined NGS from ME substudy participants with Sanger sequences in the DC Molecular HIV Surveillance database to identify clusters. Cluster analyses used HIV-Transmission Cluster Engine to identify linked pairs of sequences (defined as distance ≤1.5%). Twenty-eight clusters of ≥3 sequences (size range: 3-12) representing 108 (3%) participants were identified. None of the five largest clusters (size range: 5-12) included newly diagnosed PLWH. Thirty-four percent of ME substudy participants (n = 213) reported condomless sex during their last sexual encounter and 14% reported a Syphilis diagnosis in the past year. Seven transmission clusters (size range: 2-19) were identified in the final analysis, each containing at least one ME substudy participant. Substudy participants in clusters from the third analysis were present in clusters from the first analysis. Combining HIV sequence, clinical and behavioral data provided insights into HIV transmission that may not be identified using traditional epidemiological methods alone. Specifically, the sexual risk behaviors and STI diagnoses reported in the substudy survey may not have been disclosed during Partner Services activities and the survey data complemented clinical data to fully characterize transmission clusters. These findings can be used to enhance local efforts to interrupt transmission and avert new infections.
Assuntos
Epidemias , Infecções por HIV , District of Columbia/epidemiologia , Infecções por HIV/epidemiologia , Humanos , Epidemiologia Molecular , Assunção de Riscos , Estados UnidosRESUMO
Although anal cancers represent just 0.5 % of all new cancer cases in U.S., rates have increased markedly, with highest rates in HIV-infected MSM. American Cancer Society estimates there will be â¼9090 new cases and â¼1420 deaths in 2021. We compared Roche Linear Array HPV Genotyping (Roche) and AmpFire HPV Genotyping (AmpFire) assays for concordance and agreement to detect 15â¯hr-HPV types from 151 anal specimens. Within run precision of AmpFire was assessed on 50 anal specimens. Specimens with Roche Combo-positive and HPV33, HPV35 and/or HPV58-positive results were further tested using HPV52-specific TaqMan assay. AmpFire generated valid results on 149/151 (98.7 %) specimens; 135/149 (90.6 %) and 134/149 (89.9 %) had detectable HR-HPV DNA by AmpFire or Roche, respectively. Overall concordance was 89.8 % (2007/2235, κâ¯=â¯0.65). HPV16 showed highest overall concordance at 93.3 % (139/149, κâ¯=â¯0.84). HPV68 had lowest overall concordance at 77.2 % (115/149, κâ¯=â¯0.28). Kappa values were interpreted as being moderate or good for all other HR-HPV types. Within run precision generated 744/750 concordant results; R2 valueâ¯=â¯0.97 (pâ¯<â¯0.0001) (Mantel Test). In conclusion, AmpFire and Roche demonstrated good inter-assay agreement for detecting most HR-HPV types from anal samples, with AmpFire detecting a broader range of HPV68 subtypes and detecting HPV52 without the need for confirmatory testing.
Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Minorias Sexuais e de Gênero , Alphapapillomavirus/genética , DNA Viral/genética , Genótipo , Homossexualidade Masculina , Humanos , Masculino , Papillomaviridae/genética , Infecções por Papillomavirus/diagnósticoRESUMO
We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Mutação , Sequência de Bases , Ilhas de CpG , Primers do DNA , Humanos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Washington, DC continues to experience a generalized HIV-1 epidemic. We characterized the local phylodynamics of HIV-1 in DC using next-generation sequencing (NGS) data. Viral samples from 68 participants from 2016 through 2017 were sequenced and paired with epidemiological data. Phylogenetic and network inferences, drug resistant mutations (DRMs), subtypes and HIV-1 diversity estimations were completed. Haplotypes were reconstructed to infer transmission clusters. Phylodynamic inferences based on the HIV-1 polymerase (pol) and envelope genes (env) were compared. Higher HIV-1 diversity (n.s.) was seen in men who have sex with men, heterosexual, and male participants in DC. 54.0% of the participants contained at least one DRM. The 40-49 year-olds showed the highest prevalence of DRMs (22.9%). Phylogenetic analysis of pol and env sequences grouped 31.9-33.8% of the participants into clusters. HIV-TRACE grouped 2.9-12.8% of participants when using consensus sequences and 9.0-64.2% when using haplotypes. NGS allowed us to characterize the local phylodynamics of HIV-1 in DC more broadly and accurately, given a better representation of its diversity and dynamics. Reconstructed haplotypes provided novel and deeper phylodynamic insights, which led to networks linking a higher number of participants. Our understanding of the HIV-1 epidemic was expanded with the powerful coupling of HIV-1 NGS data with epidemiological data.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Epidemias/estatística & dados numéricos , Infecções por HIV/epidemiologia , HIV-1/genética , Adulto , Idoso , Fármacos Anti-HIV/uso terapêutico , Estudos Transversais , District of Columbia/epidemiologia , Feminino , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Haplótipos , Heterossexualidade/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Taxa de Mutação , Filogenia , Fatores Sexuais , Minorias Sexuais e de Gênero/estatística & dados numéricos , Pessoas Transgênero/estatística & dados numéricos , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Astrocytes are ubiquitous CNS cells that support tissue homeostasis through ion buffering, neurotransmitter recycling, and regulation of CNS vasculature. Yet, despite the essential functional roles they fill, very little is known about the physiology of astrocytes in the ventral midbrain, a region that houses dopamine-releasing neurons and is critical for reward learning and motivated behaviors. Here, using a combination of whole-transcriptome sequencing, histology, slice electrophysiology, and calcium imaging, we performed the first functional and molecular profiling of ventral midbrain astrocytes and observed numerous differences between these cells and their telencephalic counterparts, both in their gene expression profile and in their physiological properties. Ventral midbrain astrocytes have very low membrane resistance and inward-rectifying potassium channel-mediated current, and are extensively coupled to surrounding oligodendrocytes through gap junctions. They exhibit calcium responses to glutamate but are relatively insensitive to norepinephrine. In addition, their calcium activity can be dynamically modulated by dopamine D2 receptor signaling. Taken together, these data indicate that ventral midbrain astrocytes are physiologically distinct from astrocytes in cortex and hippocampus. This work provides new insights into the extent of functional astrocyte heterogeneity within the adult brain and establishes the foundation for examining the impact of regional astrocyte differences on dopamine neuron function and susceptibility to degeneration.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/metabolismo , Mesencéfalo/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Cálcio/metabolismo , Forma Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feminino , Junções Comunicantes/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Camundongos , Norepinefrina/farmacologiaRESUMO
The DC Cohort is an ongoing longitudinal observational study of persons living with HIV. To better understand HIV-1 drug resistance and potential transmission clusters among these participants, we performed targeted, paired-end next-generation sequencing (NGS) of protease, reverse transcriptase and integrase amplicons. We elected to use free, publicly-available software (HyDRA Web, Stanford HIVdb and HIV-TRACE) for data analyses so that laboratory personnel without extensive bioinformatics expertise could use it; making the approach accessible and affordable for labs worldwide. With more laboratories transitioning away from Sanger-based chemistries to NGS platforms, lower frequency drug resistance mutations (DRMs) can be detected, yet their clinical relevance is uncertain. We looked at the impact choice in cutoff percentage had on number of DRMs detected and found an inverse correlation between the two. Longitudinal studies will be needed to determine whether low frequency DRMs are an early indicator of emerging resistance. We successfully validated this pipeline against a commercial pipeline, and another free, publicly-available pipeline. RT DRM results from HyDRA Web were compared to both SmartGene and PASeq Web; using the Mantel test, R2 values were 0.9332 (p<0.0001) and 0.9097 (p<0.0001), respectively. PR and IN DRM results from HyDRA Web were then compared with PASeq Web only; using the Mantel test, R2 values were 0.9993 (p<0.0001) and 0.9765 (p<0.0001), respectively. Drug resistance was highest for the NRTI drug class and lowest for the PI drug class in this cohort. RT DRM interpretation reports from this pipeline were also highly correlative compared to SmartGene pipeline; using the Spearman's Correlation, rs value was 0.97757 (p<0.0001). HIV-TRACE was used to identify potential transmission clusters to better understand potential linkages among an urban cohort of persons living with HIV; more individuals were male, of black race, with an HIV risk factor of either MSM or High-risk Heterosexual. Common DRMs existed among individuals within a cluster. In summary, we validated a comprehensive, easy-to-use and affordable NGS approach for tracking HIV-1 drug resistance and identifying potential transmission clusters within the community.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Mutação/genética , Adulto , Estudos de Coortes , Análise de Dados , District of Columbia , Feminino , Soropositividade para HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homossexualidade/efeitos dos fármacos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Software , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
Previous work has shown that DNA hypermethylation of tumor suppressor genes in colorectal cancer cells may be maintained in the absence of the major mammalian methyltransferase, DNA methyltransferase 1 (DNMT1). In an effort to dissect the dependency on DNMT1 to maintain such hypermethylation in different cancer types, we performed a systematic analysis of depletion of DNMT1 in colorectal (SW48), bladder (T24), and breast (T47D) cancer cells by DNMT1-specific small hairpin RNA (shRNA) targeting. We show that although DNMT1-deficient SW48 and T24 cells exhibited no observable growth defects and were able to maintain promoter hypermethylation, DNMT1-deficient T47D breast cells failed to form comparable numbers of colonies when stably selected for the incorporation of the DNMT1-specific shRNA expression vector, suggesting a growth defect with reduced levels of DNMT1. Further treatment of T47D cells with transient transfection of small interfering RNA targeting DNMT1 revealed that severely DNMT1-deficient T47D cells could not fully maintain promoter hypermethylation, and gene silencing was partially reversed at two of the three assayed loci. These observations suggest that human cancer cells may differ in their reliance on DNMT1 for maintaining DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA (Citosina-5-)-Metiltransferase 1 , Feminino , Deleção de Genes , Humanos , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Transfecção , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.
Assuntos
Neoplasias do Colo/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA , Animais , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1 , Drosophila/genética , Engenharia Genética , Humanos , Células Tumorais CultivadasRESUMO
The GATA family of transcription factors participates in gastrointestinal (GI) development. Increases in GATA-4 and -5 expression occur in differentiation and GATA-6 expression in proliferation in embryonic and adult settings. We now show that in colorectal cancer (CRC) and gastric cancer promoter hypermethylation and transcriptional silencing are frequent for GATA-4 and -5 but are never seen for GATA-6. Potential antitumor target genes upregulated by GATA-4 and -5, the trefoil factors, inhibinalpha, and disabled-2 (Dab2) are also silenced, in GI cancers, with associated methylation of the promoters. Drug or genetically induced demethylation simultaneously leads to expression, in CRC cells, of all of the GATA-4, -5, and downstream genes. Expression of exogenous GATA-5 overrides methylation at the downstream promoters to activate the target genes. Selection for silencing of both upstream transcription factors and their target genes in GI cancers could indicate that epigenetic silencing of the involved genes provides a summated contribution to tumor progression.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Inativação Gênica , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/etiologia , Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/genética , Epigênese Genética , Fator de Transcrição GATA4 , Fator de Transcrição GATA5 , Fator de Transcrição GATA6 , Humanos , Oncogenes , Regiões Promotoras Genéticas , Neoplasias Gástricas/etiologiaRESUMO
Inhibitors of DNA methyltransferases (DNMT) and histone deacetylases can reactivate epigenetically silenced tumor suppressor genes and thereby decrease tumor cell growth. Little, however, is known on the effects of these compounds in endothelial cell biology and tumor angiogenesis. Here, we show that the DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine markedly decrease vessel formation in different tumor models. We show that DNMT inhibitors are antiproliferative for tumor-conditioned endothelial cells, without affecting endothelial cell apoptosis and migration. Furthermore, these compounds inhibit angiogenesis in vitro and in vivo as shown by inhibition of endothelial cells sprouting in a three-dimensional gel and inhibition of microvessel formation in the chorioallantoic membrane, respectively. 5-Aza-2'-deoxycytidine, as well as the histone deacetylase inhibitor trichostatin A, reactivates the growth-inhibiting genes TSP1, JUNB, and IGFBP3, which are suppressed in tumor-conditioned endothelial cells. Despite enhanced DNMT activity and increased overall genomic methylation levels in tumor-conditioned endothelial cells, silencing of these genes seemed not to be regulated by direct promoter hypermethylation. For IGFBP3, gene expression in endothelial cells correlated with histone H3 acetylation patterns. In conclusion, our data show that DNMT inhibitors have angiostatic activity in addition to their inhibitory effects on tumor cells. This dual action of these compounds makes them promising anticancer therapeutics.
Assuntos
Inibidores da Angiogênese/farmacologia , Azacitidina/farmacologia , Citidina/análogos & derivados , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neovascularização Patológica/enzimologia , Neovascularização Fisiológica/efeitos dos fármacos , Acetilação , Inibidores da Angiogênese/uso terapêutico , Animais , Azacitidina/uso terapêutico , Movimento Celular/efeitos dos fármacos , Citidina/farmacologia , Citidina/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Camundongos , Camundongos Mutantes , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Trombospondina 1/genéticaRESUMO
Microglia play critical roles in tissue homeostasis and can also modulate neuronal function and synaptic connectivity. In contrast to astrocytes and oligodendrocytes, which arise from multiple progenitor pools, microglia arise from yolk sac progenitors and are widely considered to be equivalent throughout the CNS. However, little is known about basic properties of deep brain microglia, such as those within the basal ganglia (BG). Here, we show that microglial anatomical features, lysosome content, membrane properties, and transcriptomes differ significantly across BG nuclei. Region-specific phenotypes of BG microglia emerged during the second postnatal week and were re-established following genetic or pharmacological microglial ablation and repopulation in the adult, indicating that local cues play an ongoing role in shaping microglial diversity. These findings demonstrate that microglia in the healthy brain exhibit a spectrum of distinct functional states and provide a critical foundation for defining microglial contributions to BG circuit function.
Assuntos
Gânglios da Base/fisiologia , Microglia/metabolismo , Animais , Gânglios da Base/patologia , Sinais (Psicologia) , Camundongos Transgênicos , Neurônios/fisiologia , FenótipoRESUMO
We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with 14C-labeled S-adenosyl-L-methionine (SAM) and dam methyltransferase as an internal control to standardize DNA quantity. This is followed by an incubation with 3H-labeled SAM and SssI methyltransferase for methylation quantification. By use of standard mixtures of cell line DNA with a defined methylation status in every assay, the ratio (3H/14C signal) of each sample can be converted into percentage values to assess the methylation density of the amplified sequence. This methylation-sensitive technique, termed ERMA (Enzymatic Regional Methylation Assay) provides several advantages over existing methods used for methylation analysis as it determines an exact measurement of the methylation density of the region studied. We demonstrate a use of this technique in determining the methylation density of the promoter region of the tumor suppressor gene p15INK4B and changes that occur after treatment with demethylating agents.
Assuntos
Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/química , Proteínas Supressoras de Tumor , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Ilhas de CpG/genética , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/química , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/genética , DNA-Citosina Metilases/metabolismo , Genes Supressores de Tumor , Células HL-60/química , Células HL-60/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Células Tumorais CultivadasRESUMO
The role of DNA methyltransferase 1, DNMT1, in human cancer cells has recently been contested. In this setting, DNMT1's function as the sole maintenance methyltransferase was based on the assumption that its biological activity is identical to the mouse homologue. However, the application of recent technological advances, including gene targeting and siRNA mediated ablation studies, has cast doubt on this presumed role. Here, we attempt to shed light on these new data within the context of previous experiments.
Assuntos
DNA (Citosina-5-)-Metiltransferases/fisiologia , Animais , DNA (Citosina-5-)-Metiltransferase 1 , Humanos , Mamíferos , Neoplasias/enzimologiaRESUMO
Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/fisiologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Neoplasias/genética , Sequência de Aminoácidos , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , DNA de Neoplasias/metabolismo , Marcação de Genes , Humanos , Fator de Crescimento Insulin-Like II/genética , Dados de Sequência Molecular , Neoplasias/enzimologia , Regiões Promotoras Genéticas , Inibidor Tecidual de Metaloproteinase-3/genética , Células Tumorais Cultivadas , DNA Metiltransferase 3BRESUMO
Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.