MicroRNA-9 inhibits growth and invasion of head and neck cancer cells and is a predictive biomarker of response to plerixafor, an inhibitor of its target CXCR4.

Head and neck squamous cell carcinomas (HNSCC) are associated with poor morbidity and mortality. Current treatment strategies are highly toxic and do not benefit over 50% of patients. There is therefore a crucial need for predictive and/or prognostic biomarkers to allow treatment stratification for individual patients. One class of biomarkers that has recently gained importance are microRNA (miRNA). MiRNA are small, noncoding molecules which regulate gene expression post-transcriptionally. We performed miRNA expression profiling of a cohort of head and neck tumours with known clinical outcomes. The results showed miR-9 to be significantly downregulated in patients with poor treatment outcome, indicating its role as a potential biomarker in HNSCC. Overexpression of miR-9 in HNSCC cell lines significantly decreased cellular proliferation and inhibited colony formation in soft agar. Conversely, miR-9 knockdown significantly increased both these features. Importantly, endogenous CXCR4 expression levels, a known target of miR-9, inversely correlated with miR-9 expression in a panel of HNSCC cell lines tested. Induced overexpression of CXCR4 in low expressing cells increased proliferation, colony formation and cell cycle progression. Moreover, CXCR4-specific ligand, CXCL12, enhanced cellular proliferation, migration, colony formation and invasion in CXCR4-overexpressing and similarly in miR-9 knockdown cells. CXCR4-specific inhibitor plerixafor abrogated the oncogenic phenotype of CXCR4 overexpression as well as miR-9 knockdown. Our data demonstrate a clear role for miR-9 as a tumour suppressor microRNA in HNSCC, and its role seems to be mediated through CXCR4 suppression. MiR-9 knockdown, similar to CXCR4 overexpression, significantly promoted aggressive HNSCC tumour cell characteristics. Our results suggest CXCR4-specific inhibitor plerixafor as a potential therapeutic agent, and miR-9 as a possible predictive biomarker of treatment response in HNSCC.

In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia.

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. Herein, we investigated the relationship between these two processes in normal and leukemic B cells. We found that leukemic B cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B cells. Furthermore, ligation of either surface CD79B, a B-cell receptor component required for downstream signaling, or surface Immunoglobulin M (IgM) by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to the dissociation of these two molecules in leukemic cells. A comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node- and peripheral blood-derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton's tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization.

Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group.

Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.

Interaction with vascular endothelium enhances survival in primary chronic lymphocytic leukemia cells via NF-kappaB activation and de novo gene transcription.

Chronic lymphocytic leukemia (CLL) cells rapidly undergo apoptosis in vitro, suggesting that the in vivo microenvironment provides crucial antiapoptotic signals. Overexpression of the antiapoptotic proteins Bcl-2 and Mcl-1 is a hallmark of CLL, and their expression is further enhanced in the lymphoid tissues. However, the high levels of Mcl-1 found in peripheral blood samples, coupled with its short half-life, led us to hypothesize that it must be actively maintained in the peripheral circulation. Coculture of CLL cells with human vascular endothelial cells significantly enhanced tumor cell survival, an effect that was not observed with normal B cells. This was associated with elevated levels of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-X(L) and marked increased expression of CD38 and CD49d, both of which are associated with clinically aggressive disease. Because CD38, CD49d, and some Bcl-2 family genes are transcriptional targets for NF-κB, we assessed NF-κB activation following coculture with endothelial cells. DNA binding of the NF-κB subunit Rel A was significantly increased and strongly correlated with changes in transcription of CD38, CD49d, BCL2, MCL1, and BCLXL, effects that were reversed by a peptide inhibitor of Rel A. These effects were not observed following coculture with nonendothelial cell lines. Therefore, CLL cells receive specific survival signals following interaction with endothelial cells mediated through the activation of NF-κB and the induction of downstream target genes. This type of interaction in the peripheral vasculature may explain the constitutive NF-κB activation and the overexpression of Bcl-2 family proteins commonly seen in this disease.

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling.

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.

Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs.

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.

Cytohesins and centaurins control subcellular trafficking of macromolecular signaling complexes: regulation by phosphoinositides and ADP-ribosylation factors.

The ADP-ribosylation factor family of small GTP-binding proteins are implicated in the regulation of vesicular transport and control of cytoskeletal and cell adhesion events. The phosphoinositide 3-kinase, phosphoinositide 4-P 5-kinase and phospholipase D signaling pathways are major regulators of ARF signaling cascades. Two families of ARF regulatory molecules, the cytohesin ARF-Guanine nucleotide Exchange Factors and the centaurin GTPase-Activating Proteins provide key targets for the action of these lipids signals. A critical feature of the regulation of ARF signaling is coordinated recruitment of exchange factors, ARFs and GAPs to appropriate subcellular locations. These complexes drive repetitive cycles of ARF activation and membrane association that underlie the processes of cell movement as well as endosomal uptake and intracellular redistribution of signaling molecules. Cytohesins and centaurins bind specifically to a variety of other signaling proteins and these interactions may provide routes for regulated recruitment to the sites of ARF activation. Through their ability to control endosomal trafficking/recycling of these supramolecular signaling complexes ARF and phospholipid signaling pathways may have consequences that reach as far as the regulation of gene transcription and control of cell fate.

Application of the nitric oxide donor SNAP to cardiomyocytes in culture provides protection against oxidative stress.

Multiple data indicates that nitric oxide (NO) donors retain immediate protective effects against different disturbances in cardiovascular system. The aim of the present study was to investigate delayed effects of nitric oxide donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) application in cardiac H9c2 cell line. Cardiomyocytes were treated with SNAP for 2h followed by 24h wash with fresh growth medium. The concentration curve was constructed in range from 0.5 to 2mM, toxicity was observed at 2mM concentration of SNAP. For the study of SNAP-induced protection against t-butyl hydroperoxide-induced oxidative injury 1mM concentration was used. Cell viability was assessed by MTT reductase activity assay; mitochondrial transmembrane potential (mdeltapsi) was measured by flow cytometry with fluorescent dye DiOC(6). Synthesis of heat-shock proteins (hsps) was analyzed by Western blot. Analysis of the cell viability and mdeltapsi reflected delayed protective effect of 1mM SNAP application against oxidative injury. SNAP in 1mM concentration caused 70% induction of hsp75 synthesis in cardiomyocytes. However, the other analyzed hsps (hsp70, hsp27, hsp60, hsp10, and CyP A) did not display any significant induction after incubation with SNAP. Present work demonstrates that the NO donor SNAP causes delayed protection against oxidative stress in H9c2 cardiomyocyte cell line, reflected in cell viability increase and preservation of the mdeltapsi. We suppose the major pathway for the development of SNAP-induced protection is through mitochondria. Induction of hsp75 expression following SNAP pretreatment is one possible way to explanation the mechanisms of this protection.

Cytohesins and centaurins control subcellular trafficking of macromolecular signaling complexes: regulation by phosphoinositides and ADP-ribosylation factors

The ADP-ribosylation factor family of small GTP-binding proteins are implicated in the regulation of vesicular transport and control of cytoskeletal and cell adhesion events. The phosphoinositide 3-kinase, phosphoinositide 4-P 5-kinase and phospholipase D signaling pathways are major regulators of ARF signaling cascades. Two families of ARF regulatory molecules, the cytohesin ARF-Guanine nucleotide Exchange Factors and the centaurin GTPase-Activating Proteins provide key targets for the action of these lipids signals. A critical feature of the regulation of ARF signaling is coordinated recruitment of exchange factors, ARFs and GAPs to appropriate subcellular locations. These complexes drive repetitive cycles of ARF activation and membrane association that underlie the processes of cell movement as well as endosomal uptake and intracellular redistribution of signaling molecules. Cytohesins and centaurins bind specifically to a variety of other signaling proteins and these interactions may provide routes for regulated recruitment to the sites of ARF activation. Through their ability to control endosomal trafficking/recycling of these supramolecular signaling complexes ARF and phospholipid signaling pathways may have consequences that reach as far as the regulation of gene transcription and control of cell fate