RESUMO
Globally, preterm birth (PTB) is a primary cause of mortality and morbidity in infants, with PTB rates increasing worldwide over the last two decades. Biomarkers for accurate early prediction of PTB before the clinical event do not currently exist. Given their roles in the development and progression of many disease states, there has been increasing interest in the utility of microRNAs (miRNAs) as early biomarkers for pregnancy-related disorders, including PTB. The present study was designed to examine potential differences in miRNA abundances in maternal plasma from mothers with infants born following a moderate to late (28-36 weeks' gestation, n = 54) spontaneous PTB (SPTB) compared to mothers with matched term infants (n = 54). Maternal plasma collected at 15 weeks' gestation were utilised from the Auckland and Adelaide cohorts from the Screening for Pregnancy Endpoints (SCOPE) study. miRNAs in plasma were quantified using the NanoString nCounter expression panel (800 miRNAs). The top four most abundant miRNAs were significantly decreased in the plasma of mothers in the SPTB group with results consistent across both cohorts and pathway analysis was undertaken to examine the biological processes linked to the dysregulated miRNAs. The top candidate miRNAs (miRs-451a, -223-3p, let-7a-5p, and -126-3p) were linked to gene pathways associated with inflammation, apoptosis, and mitochondrial biogenesis. Moreover, miRNAs were consistently less abundant in the plasma of mothers of preterm infants across both sites, suggesting potential global dysregulation in miRNA biogenesis. This was supported by a significant downregulation in expression of key genes that are involved in miRNA biogenesis (DROSHA, DICER, and AGO2) across both sites in the SPTB group. In summary, the present study has identified miRNAs in maternal plasma that may provide predictive utility as early biomarkers for the risk of later SPTB. Importantly, these observations were conserved across two independent cohorts. Further, our data provide evidence for a persistent decrease in miRNA abundance in mothers who later experienced an SPTB, which is likely to have widespread consequences for gene regulation and epigenetic processes.
Assuntos
Biomarcadores , MicroRNAs , Nascimento Prematuro , Humanos , Nascimento Prematuro/sangue , Nascimento Prematuro/genética , Feminino , MicroRNAs/sangue , MicroRNAs/genética , Gravidez , Biomarcadores/sangue , Adulto , Recém-Nascido , Idade GestacionalRESUMO
Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.
Assuntos
Cisteína , Hormônio do Crescimento , Animais , Dimerização , Escherichia coli , Humanos , Camundongos , Proteínas RecombinantesRESUMO
Cancer cell lines often have large structural variants (SVs) that evolve over time. There are many reported differences in large scale SVs between HL-60 and HL-60/S4, two cell lines derived from the same acute myeloid leukemia sample. However, the stability and variability of inter- and intra-chromosomal structural variants between different sources of the same cell line is unknown. Here, we used Hi-C and RNA-seq to identify and compare large SVs in HL-60 and HL-60/S4 cell lines. Comparisons with previously published karyotypes identified novel SVs in both cell lines. Hi-C was used to characterize the known expansion centered on the MYC locus. The MYC expansion was integrated into known locations in HL-60/S4, and a novel location (chr4) in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. Collectively we demonstrate the usefulness of Hi-C and with RNA-seq data for the identification and characterization of SVs.
Assuntos
Cromossomos Humanos , Variação Genética , Cromatina , Fusão Gênica , Genoma Humano , Células HL-60 , Humanos , Cariótipo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/genética , RNA-SeqRESUMO
Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the nine amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper-catalyzed click reaction yielded high purity, site-specific conjugates with >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.
Assuntos
Química Click , Hormônio do Crescimento Humano/análogos & derivados , Polietilenoglicóis/química , Tirosina/análogos & derivados , Azidas/química , Catálise , Cobre/química , Código Genético , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Humanos , Tirosina/genéticaRESUMO
Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR). The GHR antagonist, B2036, combines an amino acid substitution at 120 that confers GHR antagonist activity, with eight additional amino acid substitutions. Conjugation to polyethylene glycol (PEG) increases the serum half-life of these proteins due to reduced renal clearance. Recombinant forms of GH and its antagonists are mainly produced in prokaryotic expression systems, such as E. coli. However, efficient production in E. coli is problematic, as these proteins form aggregates as inclusion bodies resulting in poor solubility. In the present study, we demonstrate that N-terminal fusion to a thioredoxin (Trx) fusion partner improves soluble expression of codon-optimized B2036 in E. coli when expressed at 18 °C. Expression, purification and PEGylation protocols were established for three GHR antagonists: B2036, B20, and G120Rv. Following purification, these antagonists inhibited the proliferation of Ba/F3-GHR cells in a concentration-dependent manner. PEGylation with amine-reactive 5 kDa methoxy PEG succinimidyl propionate yielded a heterogeneous mixture of conjugates containing four to seven PEG moieties. PEGylation significantly reduced in vitro bioactivity of the conjugates. However, substitution of lysine to arginine at amino acid residue 120 in B2036 improved the in vitro activity of the PEGylated protein when compared to unmodified PEGylated B2036. Pharmacokinetic analysis demonstrated that the circulating half-life of PEGylated B20 was 15.2 h in mice. Taken together, we describe an effective strategy to produce biologically active PEGylated human GHR antagonists.
Assuntos
Escherichia coli/metabolismo , Hormônio do Crescimento Humano/análogos & derivados , Hormônio do Crescimento Humano/antagonistas & inibidores , Substituição de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Meia-Vida , Hormônio do Crescimento Humano/biossíntese , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/farmacocinética , Hormônio do Crescimento Humano/farmacologia , Humanos , Polietilenoglicóis/química , Transdução de Sinais/efeitos dos fármacos , SolubilidadeRESUMO
BACKGROUND: GBA mutations are numerically the most significant genetic risk factor for Parkinson's disease (PD), yet these mutations have low penetrance, suggesting additional mechanisms. OBJECTIVES: The objective of this study was to determine if the penetrance of GBA in PD can be explained by regulatory effects on GBA and modifier genes. METHODS: Genetic variants associated with the regulation of GBA were identified by screening 128 common single nucleotide polymorphisms (SNPs) in the GBA locus for spatial cis-expression quantitative trail locus (supported by chromatin interactions). RESULTS: We identified common noncoding SNPs within GBA that (1) regulate GBA expression in peripheral tissues, some of which display α-synuclein pathology and (2) coregulate potential modifier genes in the central nervous system and/or peripheral tissues. Haplotypes based on 3 of these SNPs delay disease onset by 5 years. In addition, SNPs on 6 separate chromosomes coregulate GBA expression specifically in either the substantia nigra or cortex, and their combined effect potentially modulates motor and cognitive symptoms, respectively. CONCLUSIONS: This work provides a new perspective on the haplotype-specific effects of GBA and the genetic etiology of PD, expanding the role of GBA from the gene encoding the ß-glucocerebrosidase (GCase) to that of a central regulator and modifier of PD onset, with GBA expression itself subject to distant regulation. Some idiopathic patients might possess insufficient GBA-encoded GCase activity in the substantia nigra as the result of distant regulatory variants and therefore might benefit from GBA-targeting therapeutics. The SNPs' regulatory impacts provide a plausible explanation for the variable phenotypes also observed in GBA-centric Gaucher's disease and dementia with Lewy bodies. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Gaucher , Doença de Parkinson , Doença de Gaucher/genética , Genes Modificadores , Glucosilceramidase/genética , Humanos , Corpos de Lewy , Mutação , Doença de Parkinson/genéticaRESUMO
BACKGROUND: Adult lactase non-persistence (LNP) is due to low lactase expression, resulting in lactose malabsorption (LM). LNP is a genetic trait, but is typically determined by LM markers including breath H2, blood glucose, and urinary galactose after a lactose tolerance test. Known validity of these markers using milk is limited, despite being common practice. Compositional variation, such as ß-casein variants, in milk may impact diagnostic efficacy. This study aimed to evaluate the diagnostic accuracy to detect LNP using these commonly measured LM markers after both lactose and milk challenges. METHODS: Fourty healthy young women were challenged with 50 g lactose then randomized for separate cross-over visits to ingest 750 mL milk (37.5 g lactose) as conventional (both A1 and A2 ß-casein) and A1 ß-casein-free (a2 Milk™) milk. Blood, breath and urine were collected prior to and up to 3 h following each challenge. The presence of C/T13910 and G/A22018 polymorphisms, determined by restriction fragment length polymorphism, was used as the diagnostic reference for LNP. RESULTS: Genetic testing identified 14 out of 40 subjects as having LNP (C/C13910 and G/G22018). All three LM markers (breath H2, plasma glucose and urinary galactose/creatinine) discriminated between lactase persistence (LP) and LNP following lactose challenge with an area under the receiver operating characteristic (ROC) curve (AUC) of 1.00, 0.75 and 0.73, respectively. Plasma glucose and urinary galactose/creatinine were unreliable (AUC < 0.70) after milk ingestion. The specificity of breath H2 remained high (100%) when milk was used, but sensitivity was reduced with conventional (92.9%) and a2 Milk™ (78.6%) compared to lactose (sensitivities adjusted for lactose content). The breath H2 optimal cut-off value was lower with a2 Milk™ (13 ppm) than conventional milk (21 ppm). Using existing literature cut-off values the sensitivity and specificity of breath H2 was greater than plasma glucose to detect LNP following lactose challenge whereas values obtained for urinary galactose/creatinine were lower than the existing literature cut-offs. CONCLUSION: This study showed accurate diagnosis of LNP by breath H2 irrespective of the substrate used, although the diagnostic threshold may vary depending on the lactose substrate or the composition of the milk. TRIAL REGISTRATION: ACTRN12616001694404 . Registered prospectively on December 9, 2016.
Assuntos
Intolerância à Lactose , Lactose , Adulto , Animais , Testes Respiratórios , Ingestão de Alimentos , Feminino , Humanos , Hidrogênio/análise , Lactase/genética , Intolerância à Lactose/diagnóstico , Intolerância à Lactose/genética , Leite/químicaRESUMO
BACKGROUND: SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). METHODS: SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα- early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. RESULTS: As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc- [HR (95% CI) = 0.52 (0.34-0.78), p = 0.002]. Meanwhile, in ERα- patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto- [HR (95% CI) = 0.50 (0.34-0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc- or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28-27.03), p = 0.012], or SHON-Cyto- [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18-25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc- [HR (95% CI) = 0.41 (0.19-0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto- patients [HR (95% CI) = 4.63 (1.05-20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13-44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. CONCLUSION: SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Oncogênicas/metabolismo , Tamoxifeno/uso terapêutico , Adolescente , Adulto , Idoso , Antraciclinas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Núcleo Celular/metabolismo , Quimioterapia Adjuvante , Intervalo Livre de Doença , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Recidiva Local de Neoplasia/epidemiologia , Prognóstico , Adulto JovemRESUMO
BACKGROUND: Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells. RESULTS: Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-µm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin. CONCLUSION: Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease.
Assuntos
Núcleo Celular/química , Cromatina/química , Neutrófilos/química , Células HL-60 , HumanosRESUMO
Evidence from human clinical, epidemiological, and experimental animal models has clearly highlighted a link between the early life environment and an increased risk for a range of cardiometabolic disorders in later life. In particular, altered maternal nutrition, including both undernutrition and overnutrition, spanning exposure windows that cover the period from preconception through to early infancy, clearly highlight an increased risk for a range of disorders in offspring in later life. This process, preferentially termed "developmental programming" as part of the developmental origins of health and disease (DOHaD) framework, leads to phenotypic outcomes in offspring that closely resemble those of individuals with untreated growth hormone (GH) deficiency, including increased adiposity and cardiovascular disorders. As such, the use of GH as a potential intervention strategy to mitigate the effects of developmental malprogramming has received some attention in the DOHaD field. In particular, experimental animal models have shown that early GH treatment in the setting of poor maternal nutrition can partially rescue the programmed phenotype, albeit in a sex-specific manner. Although the mechanisms remain poorly defined, they include changes to endothelial function, an altered inflammasome, changes in adipogenesis and cardiovascular function, neuroendocrine effects, and changes in the epigenetic regulation of gene expression. Similarly, GH treatment to adult offspring, where an adverse metabolic phenotype is already manifest, has shown efficacy in reversing some of the metabolic disorders arising from a poor early life environment. Components of the GH-insulin-like growth factor (IGF)-IGF binding protein (GH-IGF-IGFBP) system, including insulin-like growth factor 1 (IGF-1), have also shown promise in ameliorating programmed metabolic disorders, potentially acting via epigenetic processes including changes in miRNA profiles and altered DNA methylation. However, as with the use of GH in the clinical setting of short stature and GH-deficiency, the benefits of treatment are also, in some cases, associated with potential unwanted side effects that need to be taken into account before effective translation as an intervention modality in the DOHaD context can be undertaken.
Assuntos
Hormônio do Crescimento/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Animais , Epigênese Genética , Regulação da Expressão Gênica , Desenvolvimento Humano , Humanos , Modelos AnimaisRESUMO
Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development, and sexual maturation. Accumulated studies have reported that autocrine/paracrine hGH is an orthotopically expressed oncoprotein that promotes normal mammary epithelial cell oncogenic transformation. Autocrine/paracrine hGH has also been reported to promote mammary epithelial cell epithelial-mesenchymal transition (EMT) and invasion. However, the underlying mechanism remains largely obscure. MicroRNAs (miRNAs) are reported to be involved in regulation of multiple cellular functions of cancer. To determine whether autocrine/paracrine hGH promotes EMT and invasion through modulation of miRNA expression, we performed microarray profiling using MCF-7 cells stably expressing wild type or a translation-deficient hGH gene and identified miR-96-182-183 as an autocrine/paracrine hGH-regulated miRNA cluster. Forced expression of miR-96-182-183 conferred on epithelioid MCF-7 cells a mesenchymal phenotype and promoted invasive behavior in vitro and dissemination in vivo. Moreover, we observed that miR-96-182-183 promoted EMT and invasion by directly and simultaneously suppressing BRMS1L (breast cancer metastasis suppressor 1-like) gene expression. miR-96 and miR-182 also targeted GHR, providing a potential negative feedback loop in the hGH-GHR signaling pathway. We further demonstrated that autocrine/paracrine hGH stimulated miR-96-182-183 expression and facilitated EMT and invasion via STAT3 and STAT5 signaling. Consistent with elevated expression of autocrine/paracrine hGH in metastatic breast cancer tissue, miR-96-182-183 expression was also remarkably enhanced. Hence, we delineate the roles of the miRNA-96-182-183 cluster and elucidate a novel hGH-GHR-STAT3/STAT5-miR-96-182-183-BRMS1L-ZEB1/E47-EMT/invasion axis, which provides further understanding of the mechanism of autocrine/paracrine hGH-stimulated EMT and invasion in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Hormônio do Crescimento Humano/metabolismo , MicroRNAs/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Microscopia de Fluorescência , Família Multigênica , Invasividade Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismoRESUMO
To investigate the relationship between maternal serum concentrations of placental growth hormone (GH-V), insulin-like growth factor (IGF)-1 and 2, IGF binding proteins (IGFBP)-1 and 3 and birth weight in appropriate-for-gestational-age (AGA), large-for-gestational-age (LGA) and small-for-gestational-age (SGA) cases in a nested case-control study. Maternal serum samples were selected from the Screening for Pregnancy Endpoints (SCOPE) biobank in Auckland, New Zealand. Serum hormone concentrations were determined by ELISA. We found that maternal serum GH-V concentrations at 20 weeks of gestation in LGA pregnancies were significantly higher than in AGA and SGA pregnancies. Maternal GH-V concentrations were positively correlated to birth weights and customized birth weight centiles, while IGFBP-1 concentrations were inversely related to birth weights and customized birth weight centiles. Our findings suggest that maternal serum GH-V and IGFBP-1 concentrations at 20 weeks' gestation are associated with fetal growth.
Assuntos
Peso ao Nascer , Hormônio do Crescimento/sangue , Hormônios Placentários/sangue , Adulto , Estudos de Casos e Controles , Feminino , Desenvolvimento Fetal , Humanos , Recém-Nascido de Baixo Peso/sangue , Recém-Nascido , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/metabolismo , GravidezRESUMO
Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.
Assuntos
Axônios/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Cones de Crescimento/fisiologia , Proteína Fosfatase 2/metabolismo , Proteínas/metabolismo , Animais , Fissura Palatina/fisiopatologia , Esôfago/anormalidades , Esôfago/fisiopatologia , Técnicas de Silenciamento de Genes , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Hipertelorismo/fisiopatologia , Hipospadia/fisiopatologia , Immunoblotting , Hibridização In Situ , Camundongos , Camundongos Knockout , Proteínas/genética , Proteólise , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Imagem com Lapso de Tempo , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Anthropometric indices associated with childhood growth and height attained in adulthood, have been associated with an increased incidence of certain malignancies. To evaluate the cancer-height relationship, we carried out a study using international data, comparing various cancer rates with average adult height of women and men in different countries. METHODS: An ecological analysis of the relationship between country-specific cancer incidence rates and average adult height was conducted for twenty-four anatomical cancer sites. Age-standardized rates were obtained from GLOBOCAN 2008. Average female (112 countries) and male (65 countries) heights were sourced and compiled primarily from national health surveys. Graphical and weighted regression analysis was conducted, taking into account BMI and controlling for the random effect of global regions. RESULTS: A significant positive association between a country's average adult height and the country's overall cancer rate was observed in both men and women. Site-specific cancer incidence for females was positively associated with height for most cancers: lung, kidney, colorectum, bladder, melanoma, brain and nervous system, breast, non-Hodgkin lymphoma, multiple myeloma, corpus uteri, ovary, and leukemia. A significant negative association was observed with cancer of the cervix uteri. In males, site-specific cancer incidence was positively associated with height for cancers of the brain and nervous system, kidney, colorectum, non-Hodgkin lymphoma, multiple myeloma, prostate, testicular, lip and oral cavity, and melanoma. CONCLUSION: Incidence of cancer was associated with tallness in the majority of anatomical/cancer sites investigated. The underlying biological mechanisms are unclear, but may include nutrition and early-life exposure to hormones, and may differ by anatomical site.
Assuntos
Estatura , Neoplasias/epidemiologia , Adolescente , Adulto , Índice de Massa Corporal , Ecologia , Feminino , Humanos , Incidência , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , Neoplasias/diagnóstico , Análise de Regressão , Projetos de Pesquisa , Fatores Sexuais , Adulto JovemRESUMO
Excess growth hormone (GH) has been implicated in multiple cancer types and there is increasing interest in the development of therapeutic inhibitors targeting GH-GH receptor (GHR) signalling. Here we describe a panel of anti-GH monoclonal antibodies (mAbs) generated using a hybridoma approach and identify two novel inhibitory mAbs (1-8-2 and 1-46-3) that neutralised GH signalling. mAbs 1-8-2 and 1-46-3 exhibited strong inhibitory activity against GH-dependent cell growth in a Ba/F3-GHR cell viability assay, with EC50 values of 1.00 ± 0.27 and 0.5 ± 0.1 µg/mL, respectively. Cross-reactivity with the human placental hormones, placental lactogen (PL) and placental GH, was observed by ELISA, but neither antibody cross-reacted with mouse GH or human prolactin (PRL). mAb 1-8-2 had a binding affinity for GH of KD 0.62 ± 0.5 nM, while mAb 1-46-3 had a KD of 2.68 ± 0.53 nM, as determined by bio-layer interferometry. mAb 1-46-3 inhibited GH-dependent signal transduction in T-47D and LNCaP cancer cell lines and reduced GH-dependent cell growth and migration in the breast cancer cell line T-47D. mAb 1-46-3 inhibited T-47D cell viability more effectively than the GHR antagonist B2036. In conclusion, we describe two novel inhibitory anti-GH mAbs and provide in vitro evidence supporting development of these entities as anti-cancer therapeutics.
Assuntos
Anticorpos Monoclonais , Hormônio do Crescimento , Animais , Feminino , Humanos , Camundongos , Gravidez , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Hormônio do Crescimento/imunologia , Placenta/metabolismo , Receptores da Somatotropina/metabolismo , Transdução de SinaisRESUMO
Genome wide association studies (GWAS) have identified a number of genomic loci that are associated with Parkinson's disease (PD) risk. However, the majority of these variants lie in non-coding regions, and thus the mechanisms by which they influence disease development, and/or potential subtypes, remain largely elusive. To address this, we used a massively parallel reporter assay (MPRA) to screen the regulatory function of 5254 variants that have a known or putative connection to PD. We identified 138 loci with enhancer activity, of which 27 exhibited allele-specific regulatory activity in HEK293 cells. The identified regulatory variant(s) typically did not match the original tag variant within the PD associated locus, supporting the need for deeper exploration of these loci. The existence of allele specific transcriptional impacts within HEK293 cells, confirms that at least a subset of the PD associated regions mark functional gene regulatory elements. Future functional studies that confirm the putative targets of the empirically verified regulatory variants will be crucial for gaining a greater understanding of how gene regulatory network(s) modulate PD risk.
RESUMO
Decades of published research support a role for growth hormone (GH) in cancer. Accordingly, there is increasing interest in targeting GH in oncology, with GH antagonists exhibiting efficacy in xenograft studies as single agents and in combination with anticancer therapy or radiation. Here we discuss challenges associated with using growth hormone receptor (GHR) antagonists in preclinical models and considerations for translation, such as the identification of predictive biomarkers for selecting patients and for monitoring drug efficacy. Ongoing research will determine whether suppressing GH signalling pharmacologically will also reduce the risk of developing cancer. An increase in GH-targeted drugs in preclinical development will ultimately provide new tools to test anticancer efficacy of blocking the GH signalling pathway.
Assuntos
Hormônio do Crescimento Humano , Neoplasias , Humanos , Hormônio do Crescimento/uso terapêutico , Hormônio do Crescimento/metabolismo , Receptores da Somatotropina/metabolismo , Hormônio do Crescimento Humano/metabolismo , Neoplasias/tratamento farmacológico , Transdução de SinaisRESUMO
Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.
Assuntos
Hormônio do Crescimento Humano , Receptores da Somatotropina , Humanos , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Receptores da Somatotropina/agonistas , Receptores da Somatotropina/antagonistas & inibidoresRESUMO
Growth hormone (GH) actions are mediated through binding to its cell-surface receptor, the GH receptor (GHR), with consequent activation of downstream signalling. However, nuclear GHR localisation has also been observed and is associated with increased cancer cell proliferation. Here we investigated the functional implications of nuclear translocation of the GHR in the human endometrial cancer cell-line, RL95-2, and human mammary epithelial cell-line, MCF-10A. We found that following GH treatment, the GHR rapidly translocates to the nucleus, with maximal localisation at 5-10 min. Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates identified 40 novel GHR binding partners, including the transcriptional regulator, HMGN1. Moreover, microarray analysis demonstrated that the gene targets of HMGN1 were differentially expressed following GH treatment, and co-immunoprecipitation showed that HMGN1 associates with the GHR in the nucleus. Therefore, our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs.
RESUMO
BACKGROUND: There has been extensive scrutiny of cancer driving mutations within the exome (especially amino acid altering mutations) as these are more likely to have a clear impact on protein functions, and thus on cell biology. However, this has come at the neglect of systematic identification of regulatory (non-coding) variants, which have recently been identified as putative somatic drivers and key germline risk factors for cancer development. Comprehensive understanding of non-coding mutations requires understanding their role in the disruption of regulatory elements, which then disrupt key biological functions such as gene expression. MAIN BODY: We describe how advancements in sequencing technologies have led to the identification of a large number of non-coding mutations with uncharacterized biological significance. We summarize the strategies that have been developed to interpret and prioritize the biological mechanisms impacted by non-coding mutations, focusing on recent annotation of cancer non-coding variants utilizing chromatin states, eQTLs, and chromatin conformation data. CONCLUSION: We believe that a better understanding of how to apply different regulatory data types into the study of non-coding mutations will enhance the discovery of novel mechanisms driving cancer.