Role of human Bone Marrow-Mesenchymal Stromal Cells-mediated Notch signaling on acute myeloid leukemia cells survival and proliferation in vitro

Il signaling di Notch \ue8 noto per contribuire alla patogenesi di uno spettro di neoplasie umane, comprese le leucemie a cellule T, le leucemie a cellule B, ecc. Tuttavia, studi recenti hanno implicato la via di Notch come soppressore del tumore nelle neoplasie mieloproliferative e diversi tumori solidi. Tuttavia, il suo ruolo nella umana leucemia mieloide acuta ( AML) rimane poco chiaro. Nel presente studio, abbiamo studiato il ruolo di Notch pathway nello sviluppo, nella progressione e la ricaduta della leucemia mieloide acuta umana. L'espressione dei recettori Notch (Notch1, Notch2, Notch3 e Notch4) e loro ligandi (DLL1, DLL3, Dll4, Jagged1 e Jagged2) sono stati analizzati in linee cellulari di AML umane HL-60, THP1, U937, K562 e in cellule primarie di AML provenienti da pazienti, attraverso citometria a flusso, western immunoblotting e RT- PCR. Inoltre, il fenotipo dei recettori Notch \ue8 stata valutata nelle stesse popolazioni cellulari co-coltivate sia con cellule staminali mesenchimali umane da donatori sani ( HBM- MSC) e da pazienti AML (HBM - AML - MSC) . Inoltre, abbiamo analizzato la sopravvivenza delle cellule AML e la proliferazione in seguito al trattamento con l'inibitore Notch GSI -XII e farmaci chemioterapici. Abbiamo osservato che i campioni AML umani hanno espresso i recettori Notch e ligandi a livello attivati e anche gli obiettivi Notch a valle, suggerendo che la via di Notch \ue8 funzionale a livelli basali di AML umano. Inoltre, le MSC proteggono le cellule AML dall'apoptosi anche in presenza di farmaci chemioterapici. Questo studio mostra nuove possibili interazioni tra stromale microambiente del midollo osseo e cellule leucemiche.Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. However, its role in human Acute Myelogenous Leukemia (AML) remains unclear. In the present study, we investigated the role of Notch pathway in the development, progression and relapse of human AML. The expression of Notch receptors (Notch1, Notch2, Notch3 and Notch4) and their ligands (DLL1, DLL3, DLL4, Jagged1 and Jagged2) have been analyzed in human AML cell lines HL-60, THP1, U937, K562 and primary AML samples by flow cytometry, western immunoblotting and RT-PCR approaches. In addition, the phenotype of Notch receptors has been evaluated in the same cell populations cocultured both with human MSCs from healthy donors (hBM-MSCs) and from AML patients (hBM-AML-MSCs). Furthermore, we analyzed AML cell survival and proliferation upon treatment with Notch inhibitor GSI-XII and chemotherapeutic drugs. We observed that human AML samples expressed Notch receptors and ligands at activated levels and also downstream Notch targets, suggesting that Notch pathway is functional at basal levels in human AML. In addition, MSCs protect AML cells from apoptosis even in the presence of chemotherapeutic drugs. This study shows new possible interactions between the bone marrow stromal microenvironment and leukemia cell

Haplotype affinities resolve a major component of goat (<i>Capra hircus</i>) MtDNA D-loop diversity and reveal specific features of the Sardinian stock

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using Bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity. We identified haplotype groups overrepresented in Sardinian goats as a result of founder effects. We found that breeders maintain diversity of matrilines most likely through equalization of the reproductive potential. Moreover, the relevant amount of inter-farm mtDNA diversity found does not increase proportionally with distance. Our results illustrate the effects of breeding practices on the composition of maternal gene pool and identify mtDNA types that may be considered in projects aimed at retrieving the maternal component of the oldest breeds of Sardinia.</br

Haplotype Affinities Resolve a Major Component of Goat (Capra hircus) MtDNA D-Loop Diversity and Reveal Specific Features of the Sardinian Stock

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using Bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity

High Resistance Rate of Chronic Myeloid Leukaemia (CML) to Imatinib Myselate (IM) Might be Related to Protein Tyrosine Phosphatase Receptor Type Gamma (PTPRG) Down-Regulation

CML is the most common myeloproliferative disease observed among adults, its 1st line of treatment is IM with a response rate ranging between 55 - 90%. In Qatar the resistance rate is higher than 45%. Our collaborators in Italy recently reported on the relation between CML and PTPRG.Our team reported previously on the high rate of resistance of CML to IM (45%). During this forum the team is further reporting on the possible underlying mechanisms behind this resistance (see Al-Dewik et al at this forum).Despite a few positive findings, no pattern could be identified to delineate a significant underlying mechanism. Our collaborators in Italy, identified that down-regulation of PTPRG increased colony formation in the PTPRG+ve megakaryocytic MEG-01 and LAMA-84, but had no effect in the PTPRG-ve K562 and KYO-1. Its over-expression had an oncosuppressive effect in all four cell lines and is associated with inhibition of BCR/ABL-dependent signalling. PTPRG was downregulated at the mRNA and protein levels in CML patients in both PB and BM, including CD34+ cells, and is re-expressed following molecular remission of disease. This re-expression was associated with loss of methylation of a CpG island of PTPRG promoter in 55% patients. In K562 cells, the hypomethylating agent 5-aza-2’-deoxycytidine induced PTPRG expression and caused inhibition of colony formation that was partially reverted by antisense-mediated downregulation of PTPRG expression. Conclusions: Although this study was done on 2 independent patient populations, it suggests that in CML populations with high resistance rate it might be worth examining the PTPRG expression level and correlate it with the pattern of resistance. Our group has secured 3 years funding from QNRF (NPRP 4-157-3-052) to investigate PTPRG signalling in CML, including the study of a possible link among the high CML resistance and the PTPRG expression levels

Karyotyping and FISH of secondary culture.

<p>(A) Trisomy of chromosome 12 and structural chromosome aberration involving chromosomes 7 and 9 on secondary culture detected by karyotype; (B) Chromosome 9 staining by FISH of secondary culture.</p

Immunophenotype.

<p>Immunophenotyping of VR09 cell line (grey and filled curves) and cell suspensions from <i>in vivo</i> tumor mass (black and filled curves), as compared with isotype control (white curves).</p

Representative histological markers detected on tumors.

<p>High magnification (20X) of histological sections of tumors developing after <i>in vivo</i> injection of VR09 cell line. Lymphoid infiltrates display large size, plasmablastic-plasmacytic features and high Ki-67 index. Cells express CD19, CD20, CD138, CD79a, IgM, IgG and EBV protein (EBER).</p

Primers used to amplify and sequence P53, Card 11, CD79B, MYD88 AND RPMS1 genes.

<p>Isoform 1: 858 pb; Isoform 2: 546 pb; Isoform 3: 861 pb.</p

EBV positivity in the original tumor and in the VR09 cell line.

<p>PCR products analysis by Agilent 2100 Bioanalyzer showed the presence of the same 151 bp specific amplicon for EBV RPMS1 gene, thus demonstrating that EBV infection was present in the original cells from patient. A normal DNA from pancreas was used as negative control.</p