RESUMO
Disparities in longitudinal growth within a species can be partly explained by endocrinological differences. We hypothesized that regulatory networks acting locally in the growth plate may also be important. We tested this hypothesis by evaluating the IGF/IGFBP expression, the vitamin D pathway, and the PTHrP-Indian hedgehog (IHH) feedback loop in rib growth plates from 10- and 21-wk-old small- (Miniature Poodles, MP) and large-breed dogs (Great Danes, GD) using immunohistochemistry and quantitative (q)PCR. The rib growth plates of GD were 1.7 times thicker compared with those of MP, with larger proliferative (in absolute terms) and larger hypertrophic (in absolute and relative terms) zones. IGF/IGFBP gene expression profiling of the growth plates revealed decreased gene expression of igfbp2, -4, and -6 and an unaltered expression of igf-I and igf-II and their respective receptors in GD vs. MP. Immunohistochemistry and qPCR findings showed that the vitamin D pathway was more active in GD than in MP. Staining for 1α- and 24-hydroxylase was more abundant and intense in GD and the gene expressions of 1α-hydroxylase and the vitamin D receptor-driven 24-hydroxylase were six- and eightfold higher in GD vs. MP, respectively. Consistent with the immunohistochemistry findings, the expression of mRNA for components of the parathyroid hormone-related peptide (PTHrP)-IHH loop was different in GD compared with MP, with there being a relative threefold downregulation of Pthrp and a tenfold upregulation of Ihh in GD vs MP. These differences suggest that the effects of IHH in the regulation of chondrocyte proliferation and hypertrophy, both independently of PTHrP, can become more dominant during rapid growth rates. In conclusion, our data suggest that, in addition to modest endocrine differences, more pronounced changes in the expression of locally acting regulatory networks, such as the IGF system, vitamin D pathway, and PTHrP-IHH feedback loop are important contributors to within-species disparities in growth rates.
Assuntos
Cães/crescimento & desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Costelas/crescimento & desenvolvimento , Animais , Cães/genética , Cães/metabolismo , Feminino , Expressão Gênica , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Costelas/metabolismo , Especificidade da EspécieRESUMO
Resistance to glucocorticoids (GC) is an important adverse risk factor in the treatment of acute lymphoblastic leukemia (ALL). To induce apoptosis, GC bind to the GC receptor (GR), which is regulated by various (co)chaperone proteins such as heat-shock protein 70 (HSP-70), HSP-40, HIP (HSP-70-interacting protein), BAG-1 (BCL-2-associated gene product-1), HOP (HSP-70/HSP-90-Organizing protein), HSP-90, P-23, FKBP-51, FKBP-52 and CYP-40. In this study, we tested the hypothesis that mRNA expression levels of these molecules are determinants of GC resistance in childhood ALL. In all, 20 children with ALL cells in vitro sensitive to prednisolone (LC(50) < 0.1 microg/ml) were compared each with a resistant patient (LC(50) >150 mug/ml), matched for immunophenotype, age and white blood cell count. mRNA expression levels of the (co)chaperone molecules were measured by quantitative real-time RT-PCR and normalized to GAPDH and RNaseP levels. In vitro resistance to prednisolone was measured by MTT assay. HSP-90 mRNA expression levels were 2000-fold higher as compared to HSP-70. Using matched pair analysis, mRNA expression levels of the various (co)chaperone molecules were not significantly different between in vitro-sensitive and -resistant patients. GC resistance in childhood ALL cannot be attributed to different mRNA expression levels of the investigated (co)chaperone molecules involved in GC binding and transport to the nucleus.
Assuntos
Glucocorticoides/farmacologia , Chaperonas Moleculares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Glucocorticoides/metabolismo , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactente , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Receptores de Glucocorticoides/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
About 30% of T cell acute lymphoblastic leukemias (T-ALL) carry TAL1 gene aberrations. In the majority of cases (approximately 25%), this concerns a submicroscopic deletion of approximately 90 kb in chromosome region 1p32, which deletes the coding regions of the SIL gene and the untranslated region of the TAL1 gene, thereby placing the TAL1 gene under control of the SIL promoter region. Translocation (1;14)(p32;q11) involving the TAL1 gene occurs at a much lower frequency (3%), whereas some other rare variant translocations have been described as well. In this study we developed a set of TAL1 FISH probes based on the split-signal FISH principle that enables detection of both types of TAL1 gene aberrations in single test. For this purpose, one probe was designed downstream of the TAL1 gene (TAL1-D) and the second probe in the region upstream of the TAL1 gene, partly covering the SIL gene (SIL-U). We show that this split-signal FISH probe set allows reliable detection of the unaffected SIL-TAL1 gene region with a fusion signal, SIL-TAL1 fusion genes with loss of the SIL-U signal, and TAL1 gene translocations with a split-signal, independent of the involved partner gene.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas , Translocação Genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Quebra Cromossômica , Cromossomos Humanos Par 1/genética , Deleção de Genes , Biblioteca Gênica , Sequências Hélice-Alça-Hélice , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia-Linfoma de Células T do Adulto/imunologia , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genéticaRESUMO
A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.