Clinical impact of miR-223 expression in pediatric T-Cell lymphoblastic lymphoma.

Although probability of event-free survival in pediatric lymphoblastic T-cell lymphoma (T-LBL) is about 75%, survival in relapsed patients is very poor, so the identification of new molecular markers is crucial for treatment optimization. Here, we demonstrated that the over-expression of miR-223 promotes tumor T-LBL cell growth, migration and invasion in vitro. We found out that SIK1, an anti-metastatic protein, is a direct target of miR-223 and consequently is significantly reduced in miR-223-overexpressing tumor cells. We measured miR-223 expression levels at diagnosis in tumor biopsies from 67 T-LBL pediatric patients for whom complete clinical and follow up data were available, and we found that high miR-223 expression (above the median value) is associated with worse prognosis (PFS 66% vs 94%, P=0.0036). In addition, the multivariate analysis, conducted taking into account miR-223 expression level and other molecular and clinical characteristics, showed that only high level of miR-223 is an independent factor for worse prognosis. MiR-223 represents a promising marker for treatment stratification in pediatric patients with T-LBL and we provide the first evidence of miR-223 potential role as oncomir by SIK1 repression.

Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma.

Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management.

Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.

Plasma membrane calcium ATPases and related disorders.

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia.

Hair cells, plasma membrane Ca²âº ATPase and deafness.

Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K(+) and Ca(2+) into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tip-links, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype.

Mutations in PMCA2 and hereditary deafness: a molecular analysis of the pump defect.

The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K(+) and Ca(2+). Ca(2+) that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca(2+) pump (isoform PMCA2w/a): disturbances of the balance between Ca(2+) penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca(2+) binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca(2+) to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca(2+) homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca(2+), the human mutant instead failed to impair the Ca(2+) ejection by the pump.