Novel Evidence That Extracellular Nucleotides and Purinergic Signaling Induce Innate Immunity-Mediated Mobilization of Hematopoietic Stem/Progenitor Cells

Pharmacological mobilization of hematopoietic stem progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) is a result of mobilizing agent-induced “sterile inflammation” in the BM microenvironment due to complement cascade (ComC) activation. Here we provide evidence that ATP, as an extracellular nucleotide secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates the mobilization process. This process is augmented in a P2X7 receptor-dependent manner, and P2X7-KO mice are poor mobilizers. Furthermore, after its release into the extracellular space, ATP is processed by ectonucleotidases: CD39 converts ATP to AMP, and CD73 converts AMP to adenosine. We observed that CD73-deficient mice mobilize more HSPCs than do wild-type mice due to a decrease in adenosine concentration in the extracellular space, indicating a negative role for adenosine in the mobilization process. This finding has been confirmed by injecting mice with adenosine along with pro-mobilizing agents. In sum, we demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs. Although ATP triggers and promotes this process, adenosine has an inhibitory effect. Thus, administration of ATP together with G-CSF or AMD3100 or inhibition of CD73 by small molecule antagonists may provide the basis for more efficient mobilization strategies

Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

Fast and efficient homing and engraftment of hematopoietic stem progenitor cells (HSPCs) is crucial for positive clinical outcomes from transplantation. We found that this process depends on activation of the Nlrp3 inflammasome, both in the HSPCs to be transplanted and in the cells in the recipient bone marrow (BM) microenvironment. For the first time we provide evidence that functional deficiency in the Nlrp3 inflammasome in transplanted cells or in the host microenvironment leads to defective homing and engraftment. At the molecular level, functional deficiency of the Nlrp3 inflammasome in HSPCs leads to their defective migration in response to the major BM homing chemoattractant stromal-derived factor 1 (SDF-1) and to other supportive chemoattractants, including sphingosine-1-phosphate (S1P) and extracellular adenosine triphosphate (eATP). We report that activation of the Nlrp3 inflammasome increases autocrine release of eATP, which promotes incorporation of the CXCR4 receptor into membrane lipid rafts at the leading surface of migrating cells. On the other hand, a lack of Nlrp3 inflammasome expression in BM conditioned for transplantation leads to a decrease in expression of SDF-1 and danger-associated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs

Circulating Hsa-miR-431-5p as Potential Biomarker for Squamous Cell Vulvar Carcinoma and Its Premalignant Lesions

Vulvar squamous cell carcinoma (VSCC) develops from high-grade squamous intraepithelial lesions (HSIL) and differentiated vulvar intraepithelial neoplasia (dVIN). This study aimed to assess the diagnostic value of circulating hsa-miR-431-5p in vulvar precancers and VSCC. Expression levels of hsa-miR-431-5p were analyzed by quantitative RT-PCR in plasma samples of 29 patients with vulvar precancers (HSIL or dVIN), 107 with VSCC as well as 15 healthy blood donors. We used hsa-miR-93-5p and hsa-miR-425-5p as normalizers. The levels of miR-431-5p were increased in the blood of patients with VSCC compared to those with vulvar precancers. Statistically significant differences in the survival rates (time to progression) were revealed for VSCC patients categorized by miR-431-5p levels. Low levels of circulating miR-431-5p were found to be indicative of unfavorable survival rates. In summary, our data reveal the diagnostic potential of circulating miR-431-5p in patients with vulvar precancers and VSCC

TET2 promoter DNA methylation and expression analysis in pediatric B-cell acute lymphoblastic leukemia

<em>TET2</em> is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the <em>TET2</em> promoter was found in human cancer. Previous analysis revealed no <em>TET2</em> mutations in acute lymphoblastic leukemia (ALL). Since the <em>TET2</em> promoter methylation status in pediatric ALL has not been reported, the aim of the present study was to determine if promoter hypermethylation may be a mechanism of <em>TET2</em> inactivation in a group of pediatric ALL cases. Methylation of <em>TET2</em> promoter region in one (1/45) ALL B-common patient was detected by methylation specific polymerase chain reaction (PCR) and subsequently analyzed by bisulfite sequencing. We found no correlation between promoter methylation and gene expression, measured by quantitative reverse transcriptase-PCR, however the level of <em>TET2</em> expression in ALL group was significantly decreased compared to children’s normal peripheral blood mononuclear cells and isolated B-cells. <em>TET2</em> promoter hypermethylation seems to have limited clinical relevance in childhood B-cell ALL due to its low frequency

Epigenetic-Mediated Downregulation of μ-Protocadherin in Colorectal Tumours

Carcinogenesis involves altered cellular interaction and tissue morphology that partly arise from aberrant expression of cadherins. Mucin-like protocadherin is implicated in intercellular adhesion and its expression was found decreased in colorectal cancer (CRC). This study has compared MUPCDH (CDHR5) expression in three key types of colorectal tissue samples, for normal mucosa, adenoma, and carcinoma. A gradual decrease of mRNA levels and protein expression was observed in progressive stages of colorectal carcinogenesis which are consistent with reports of increasing MUPCDH 5′ promoter region DNA methylation. High MUPCDH methylation was also observed in HCT116 and SW480 CRC cell lines that revealed low gene expression levels compared to COLO205 and HT29 cell lines which lack DNA methylation at the MUPCDH locus. Furthermore, HCT116 and SW480 showed lower levels of RNA polymerase II and histone H3 lysine 4 trimethylation (H3K4me3) as well as higher levels of H3K27 trimethylation at the MUPCDH promoter. MUPCDH expression was however restored in HCT116 and SW480 cells in the presence of 5-Aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Results indicate that μ-protocadherin downregulation occurs during early stages of tumourigenesis and progression into the adenoma-carcinoma sequence. Epigenetic mechanisms are involved in this silencing

Estimation of groin recurrence risk in patients with squamous cell vulvar carcinoma by the assessment of marker gene expression in the lymph nodes

Abstract Background Regional lymph node (LN) status is a well-known prognostic factor for vulvar carcinoma (VC) patients. Although the reliable LN assessment in VC is crucial, it presents significant diagnostic problems. We aimed to identify specific mRNA markers of VC dissemination in the LN and to address the feasibility of predicting the risk of nodal recurrence by the patterns of gene expression. Methods Sentinel and inguinal LN samples from 20 patients who had undergone surgery for stage T1-3, N0-2, M0 primary vulvar squamous cell carcinoma were analyzed. Gene expression profiles were assessed in four metastatic [LN(+)] and four histologically negative [LN(−)] lymph node samples obtained from four VC patients, by the Affymetrix U133 Plus 2.0 gene expression microarrays. Of the set of genes of the highest expression in the metastatic LNs compared to LN(−), seven candidate marker genes were selected: PERP, S100A8, FABP5, SFN, CA12, JUP and CSTA, and the expression levels of these genes were further analyzed by the real-time reverse transcription polymerase chain reaction (qRT-PCR) in 71 LN samples. Results All of the seven genes in question were significantly increased in LN(+) compared to LN(−) samples. In the initial validation of the seven putative markers of metastatic LN, the Cox proportional hazard model pointed to SFN, CA12 and JUP expression to significantly relate to the time to groin recurrence in VC patients. Conclusions Our findings first provided evidence that SFN, CA12 and JUP have a potential of marker genes for the prediction of the groin recurrence LN in VC patients.</p