CHK1 expression in Gastric Cancer is modulated by p53 and RB1/E2F1: implications in chemo/radiotherapy response.

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient's samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1's expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgery.

Evaluation of cardiac phenotype in horses with type 1 polysaccharide storage myopathy.

BACKGROUND: Type 1 polysaccharide storage myopathy (PSSM1), an equine glycogen storage disorder caused by a gain of function mutation (R309H) in the gene encoding glycogen synthase (GYS1), is associated with the accumulation of amylase-resistant alpha-crystalline polysaccharide inclusions within skeletal muscle. Several glycogenoses in humans have a cardiac phenotype, and reports exist of horses with PSSM and polysaccharide inclusions in cardiac muscle. HYPOTHESIS/OBJECTIVES: To investigate the hypothesis that horses with PSSM1 display a cardiac phenotype. Our objectives were to compare plasma cardiac troponin I (cTnI) concentration and the incidence of cardiac arrhythmias in PSSM1 homozygotes, heterozygotes, and control horses. METHODS: One hundred and twenty-five Belgian and Percheron horses under the same management were genotyped for the R309H GYS1 mutation. From these, 8 age-, breed-, and sex-matched cohorts of each genotype were identified. Plasma cTnI concentration and incidence of cardiac arrhythmias (determined by 24-hour Holter ECG) were compared between the groups. RESULTS: Although some PSSM1-affected horses had mildly increased plasma cTnI concentrations, there was no significant difference in cTnI concentrations between groups. There were no significant differences in the incidence of ectopic beats, cardiac conduction intervals or mean heart rate between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: We found no evidence of clinically relevant cardiac myocyte injury or arrhythmias in horses with PSSM1. Additional study is required to determine whether myocardial function may be compromised in this disorder.

Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy.

Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options.

Flow cytometry analysis: a quantitative method for collagen VI deficiency screening.

Mutations in COL6A1, COL6A2 and COL6A3 genes result in collagen VI myopathies: Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and intermediate phenotypes. At present, none of the existing diagnostic techniques for evaluating collagen VI expression is quantitative, and the detection of subtle changes in collagen VI expression remains challenging. We investigated flow cytometry analysis as a means of quantitatively measuring collagen VI in primary fibroblasts and compared this method with the standard method of fibroblast collagen VI immunohistochemical analysis. Eight UCMD and five BM molecularly confirmed patients were studied and compared to five controls. Flow cytometry analysis consistently detected a reduction of collagen VI of at least 60% in all UCMD cases. In BM cases the levels of collagen VI were variable but on average 20% less than controls. Flow cytometry analysis provides an alternative method for screening for collagen VI deficiency at the protein level in a quantitative, time and cost-effective manner.

Continuous on-line monitoring of secretion from rodent pituitary endocrine cells using fluorescent protein surrogate markers.

We have developed a system to use secreted fluorescent proteins (FPs) as surrogate markers for the continuous on-line monitoring of hormone release from perfused tissue slices. We have tested this system using GH-GFP transgenic rats with green fluorescent protein (GFP) targeted to the secretory vesicles (SVs) of pituitary growth hormone (GH) cells. Brief exposures of vibratome slices to GH secretagogues [GH-releasing hormone (GHRH), GH-releasing peptide-6 (GHRP-6)] or somatostatin caused changes in FP output that correlate with hormone secretion, subsequently measured in fractions of perfusate by radioimmunoassay. The temporal resolution of this method was capable of revealing differences in the kinetics of response to GHRH and GHRP-6 between wild-type and dwarf (dw/dw) rats harbouring the GH-GFP transgene. We further tested the utility of the system by generating transgenic mice with red FPs targeted to secretory vesicles (PRL-mRFP(sv)) and to the cytoplasm (PRL-DsRed(cyto)) of lactotrophs. Dopamine had no effect on the FP output from pituitary slices of PRL-DsRed(cyto) mice but inhibited output from those of PRL-mRFP(sv) animals, with a rebound increase of release after removal, which again correlated with hormone output measured in the perfusate by radioimmunoassay. The inhibition of monomeric RFP secretion by dopamine was dose-dependent, as was stimulation by low concentrations of oxytocin. The temporal resolution afforded by this method provides useful insight into the release kinetics from large populations of pituitary cells, and fills a temporo-spatial gap between single vesicle and single cell monitoring of exocytosis in milliseconds, and in vivo sampling studies of release into the bloodstream on a time scale of minutes.

[The growth plate in chronic renal insufficiency]. / La placa de crecimiento en la insuficiencia renal crónica.

Several alterations have been reported in the growth plate of young rats rendered uremic by subtotal nephrectomy, a widely used experimental model of growth failure secondary to renal insufficiency. In our lab's experience, uremia is associated with a markedly increased growth plate height which results from an elongation of the hypertrophic zone. These findings are not consistently observed in all studies, likely because of the different experimental conditions. Regardless of growth plate size, growth retardation induced by chronic renal failure is accompanied by an alteration of the dynamics of the growth plate with a decreased bone apposition rate at the metaphyseal end of growth cartilage and slower production and progression of chondrocytes from the resting zone up to the most distal hypertrophic zone adjacent to bone. These abnormal dynamics are associated with an irregular bone-cartilage interface and a disturbed process of chondrocyte maturation which becomes evident by a morphological criteria and by depressed expression of markers of chondrocyte maturation such as collagen X. The microscopic findings also suggest a disturbed process of capillary invasion, which precedes formation of new osseous tissue in the primary spongiosa, although the levels of vascular endothelial growth factor, as measured by immunohistochemistry, have been reported to be similar in the growth plate of uremic and control rats. The meaning of these findings in the pathogenesis of growth impairment secondary to chronic renal failure remains to be determined.

[Effect of growth hormone and calcitriol on the growth plate in uremic rats]. / Efecto de la hormona de crecimiento y del calcitriol sobre la placa de crecimiento de ratas urémicas.

This study analyzed the modifications induced by growth hormone (GH) and/or calcitriol treatments in the growth plate of growth retarded uremic rats. Four groups of 5/6 nephrectomized rats were studied: untreated (U), treated with GH (U + GH), treated with calcitriol (U + D), treated with GH and calcitriol (U + GH + D). Treatments were given intraperitoneally during the second week of renal failure. Uremic groups were compared with sham-operated rats fed ad libitum (C) or pair-fed with U (CP). In comparison with C and CP, histomorphometric analysis of tibial proximal ends of U group showed decreased bone formation, as estimated by osseous front advance (OFA), elongation of growth cartilage and its hypertrophic zone, and decreased size of most distal chondrocytes. The U + D group tended to normalize growth cartilage height, and that of its hypertrophic zone, as well as the size of chondrocytes. In U + GH group OFA improved and chondrocyte size became normal, but growth cartilage remained elongated. Similar results were found in the U + GH + D group. These findings indicate that, in chronic renal insufficiency, the beneficial effect of GH on growth is not associated with normalization of growth cartilage morphology and that calcitriol facilitates chondrocyte maturation. When given together the effect of GH prevails.

La placa de crecimiento en la insuficiencia renal crónica / The growth plate in chronic renal failure

Se han descrito diversas alteraciones en la placa de crecimiento de la tibia de ratas jóvenes urémicas por nefrectomía subtotal, un modelo experimental de hipocrecimiento secundario a fallo renal crónico ampliamente utilizado. En nuestra experiencia, la uremia severa se acompaña de un marcado aumento en la altura del cartílago de crecimiento consecutivo a un acusado incremento de la zona de condrocitos hipertróficos. Esta elongación del cartílago no se observa consistentemente en todos los estudios lo que puede depender de diferentes condiciones experimentales. Con independencia del tamaño del cartílago epifisario, el hipocrecimiento de la insuficiencia renal crónica se asocia a una alteración en la dinámica de la placa con disminución tanto del ritmo de aposición de hueso primario en el extremo metafisario del cartílago de crecimiento como de la producción y progresión de los condrocitos desde la zona germinal hasta la hipertrófica adyacente al hueso. Esta dinámica anormal se acompaña de una irregular interfase hueso-cartílago y de alteración del proceso de maduración de los condrocitos evidente tanto por criterios morfológicos como por disminución de marcadores de maduración condrocitaria, por ejemplo, colágeno X. Los hallazgos microscópicos indican también una perturbación del fenómeno fisiológico de invasión capilar y formación de nuevo tejido óseo en la espongiosa primaria, aunque los niveles del factor de crecimiento endotelial vascular, valorados por inmunohistoquímica, no han demostrado que estén modificados en la placa de crecimiento de ratas urémicas con respecto a los controles. El significado de estos hallazgos en relación con la patogenia del hipocrecimiento del fallo renal crónico está aún por aclarar (AU)

Several alterations have been reported in the growth plate of young rats rendered uremic by subtotal nephrectomy, a widely used experimental model of growth failure secondary to renal insufficiency. In our lab’s experience, uremia is associated with a markedly increased growth plate height which results from an elongation of the hypertrophic zone. These findings are not consistently observed in all studies, likely because of the different experimental conditions. Regardless of growth plate size, growth retardation induced by chronic renal failure is accompanied by an alteration of the dynamics of the growth plate with a decreased bone apposition rate at the metaphyseal end of growth cartilage and slower production and progression of chondrocytes from the resting zone up to the most distal hypertrophyc zone adjacent to bone. These abnormal dynamics are associated with an irregular bone-cartilage interface and a disturbed process of chondrocyte maturation which becomes evident by a morphological criteria and by depressed expression of markers of chondrocyte maturation such as collagen X. The microscopic findings also suggest a disturbed process of capillary invasion, which precedes formation of new osseous tissue in the primary spongiosa, although the levels of vascular endothelial growth factor, as measured by immunohistochemistry, have been reported to be similar in the growth plate of uremic and control rats. The meaning of these findings in the pathogenesis of growth impairment secondary to chronic renal failure remains to be determined (AU)

Efecto de la hormona de crecimiento y del calcitriol sobre la placa de crecimiento de ratas urémicas / Effect of growth hormone and calcitriol in the growrh plate of uremic rats

Este estudio analizó la placa de crecimiento de ratas urémicas hipocrecidas tras administración de hormona de crecimiento (GH) y/o calcitriol. Se estudiaron cuatro grupos: sin tratamiento (U), tratadas con GH (U + GH), con calcitriol (U + D), con GH y calcitriol (U + GH + D), diariamente durante la segunda semana de IRC. Estos grupos se compararon con ratas control con dieta ad libitum (C) o pareada con U (CP). La histomorfometría de la tibia proximal reveló en U una menor formación de hueso estimada por el avance del frente óseo (AFO), alargamiento del cartílago de crecimiento y de su zona hipertrófica, y condrocitos terminales más pequeños, en comparación con los grupos C y CP. El grupo U + D tendió a normalizar la altura del cartílago de crecimiento, de la zona hipertrófica, y el tamaño de los condrocitos terminales. En el grupo U + GH mejoró el AFO y se normalizó el tamaño condrocitario, pero el cartílago de crecimiento y la zona hipertrófica permanecieron alargados. El grupo U + GH + D mostró hallazgos similares al U + GH. Estos hallazgos indican que en la IRC el tratamiento con GH no normaliza la morfología de la placa y que el calcitriol facilita la maduración del condrocito. Cuando se administran conjuntamente, el efecto de la GH prevalece (AU)

This study analyzed the modifications induced by growth hormone (GH) and/or calcitriol treatments in the growth plate of growth retarded uremic rats. Four groups of 5/6 nephrectomized rats were studied: untreated (U), treated with GH (U + GH), treated with calcitriol (U + D), treated with GH and calcitriol (U + GH + D). Treatments were given intraperitoneally during the second week of renal failure. Uremic groups were compared with sham-operated rats fed ad libitum (C) or pair-fed with U (CP). In comparison with C and CP, histomorphometric analysis of tibial proximal ends of U group showed decreased bone formation, as estimated by osseous front advance (OFA), elongation of growth cartilage and its hypertrophic zone, and decreased size of most distal chondrocytes. The U + D group tended to normalize growth cartilage height, and that of its hypertrophic zone, as well as the size of chondrocytes. In U + GH group OFA improved and chondrocyte size became normal, but growth cartilage remained elongated. Similar results were found in the U + GH + D group. These findings indicate that, in chronic renal insufficiency, the benefitial effect of GH on growth is not associated with normalization of growth cartilage morphology and that calcitriol facilitates chondrocyte maturation. When given together the effect of GH prevails (AU)