Marisa Gorrese

Transcriptional Repression of miR-34 Family Contributes to p63-Mediated Cell Cycle Progression in Epidermal Cells

p63, a p53 family member, is highly expressed in the basal proliferative compartment of the epidermis and its expression has been correlated with the growth ability and regenerative capacity of keratinocytes. In this study we report a mechanism through which p63 maintains cell cycle progression by directly repressing miR-34a and miR-34c. In the absence of p63, increased levels of miR-34a and miR-34c were observed in primary keratinocytes and in embryonic skin, with concomitant G1-phase arrest and inhibition of the cell cycle regulators cyclin D1 and cyclin-dependent kinase 4 (Cdk4). p63 directly bound to p53-consensus sites in both miR-34a and miR-34c regulatory regions and inhibited their activity. Concomitant downregulation of miR-34a and miR-34c substantially restored cell cycle progression and expression of cyclin D1 and Cdk4. Our data indicate that specific miR-34 family members have a significant role downstream of p63 in controlling epidermal cell proliferation.

CD200/OX2, a cell surface molecule with immuno-regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells

CD200 (formerly called OX2) is a transmembrane glycoprotein with immunosuppressive functions. It is expressed on normal B-lymphocytes, T-lymphocytes, dendritic cells and several solid tissues (Kawasaki & Farrar, 2008). CD200 receptor expression is limited to myeloid leucocytes and a subset of Tlymphocytes (Kawasaki & Farrar, 2008). In mouse systems, the binding of CD200 to its receptor (i) decreases the production of T-helper cell type 1 (Th1)-like cytokines, such as interleukin (IL)-2 and interferon c, (ii) increases the release of Th2-like cytokines, such as IL-10 and IL-4 (Gorczynski, 2001) and (iii) promotes the in vitro differentiation of T cells toward CD4CD25Foxp3 Treg lymphocytes (Gorczynski et al, 2008). CD200 is constantly overexpressed on chronic lymphocytic leukaemia (CLL) cells (McWhirter et al, 2006). The addition of CLL cells to mixed lymphocyte reactions causes an immunological shift from a Th1-like response to a Th2-like response, confirming that CD200 plays an important role in controlling T-cytotoxic immune response (McWhirter et al, 2006). Starting from these data, we extended the investigation of CD200 expression to another B-chronic lymphoproliferative disorder i.e. hairy cell leukaemia (HCL). Hairy cell leukaemia is a distinct disease entity in the World Health Organization (WHO) classification, displaying unique clinico-pathological and biological features (Tiacci et al, 2006). As hairy cells display a specific immunophenotype, multicolour flow cytometry is currently the best tool for HCL diagnosis. A total of 10 specimens (six peripheral blood samples and four bone marrow aspirates), collected from 10 patients with newly diagnosed HCL, were studied. As normal controls we analysed 10 peripheral blood specimens and two bone marrow aspirates from 12 healthy donors. An aliquot (50 ll) of each sample was incubated at 4 C for 30 min in the presence of appropriate amounts of monoclonal antibodies. The mixtures were then diluted 1:20 in ammonium chloride lysing solution, incubated at room temperature for 10 min and finally washed prior to flow cytometric analysis with the FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antigens were analysed: CD200, SmIg-kappa, SmIg-lambda, CD45, CD19, CD5, CD23, CD20, CD22, CD103, CD11c, CD25, CD43, CD10, CD3, CD56 and CD81. Hairy cells were gated as CD45CD19 ‘monocytoid cells’ (i.e. cells with light scatter features typical of monocytes). In addition, in the majority of cases, we also were able to perform a full immunological gate on CD45 CD19CD103CD11c cells. With regard to the normal controls included in our study, B-lymphocytes were simply gated as CD45CD19 cells. In all specimens cell doublets and debris were excluded from our analysis by forward-scatter versus side-scatter dotplot examination. To set the cut-off point to distinguish between CD200 negative and positive cells, we used the ‘Fluorescence Minus One’ technique as described by Perfetto et al (2004). A single case was arbitrarily judged CD200 positive when the percentage of positive cells (PPC) was higher than 30%. All HCL samples were CD200 positive with PPC and median fluorescence intensity (MFI) median values of 99 (25th–75th percentile 92–99) and 3016 (25th–75th percentile 1382–5430), respectively. Although CD200 was positive in 12 out of 12 normal controls, the PPC and MFI median values were of 71 (25th–75th percentile 64–83) and 582 (25th–75th percentile 406–725), respectively. Differences in PPCs and MFIs between HCLs and normal controls were statistically significant (Mann–Whitney U, two-tailed testing, P < 0Æ0001). Data regarding MFI analysis are shown in Fig 1. Whereas normal controls showed weak CD200 fluorescence intensity with a bimodal distribution, HCL samples showed bright CD200 expression in a homogeneous pattern (Fig 2). This is the first documented direct evidence of CD200 overexpression in HCL. As described above, CD200 promotes Th2-like cytokines synthesis. IL-4 and IL-10 are reported to reduce anti-tumour cytotoxic T cell response (McWhirter

Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas.

Among histological aggressive non-Hodgkin lymphomas (NHL), the overall risk of central nervous system (CNS) relapse is approximately 5%, a figure which is too low to offer prophylaxis to all patients. The aim of this work is to demonstrate the utility of flow cytometry (FCM) in detecting occult leptomeningeal disease in this subtype of NHL. We studied cerebrospinal fluid (CSF) involvement in 42 newly diagnosed aggressive NHL patients at risk for CNS involvement. We used multicolour FCM to detect CSF infiltrating neoplastic cells. Among the 42 patients studied, 11 had CSF involvement as detected by FCM. Of these, only four were also positive for conventional morphology (p=0.046). These results designate that FCM as the first choice technique in NHL CSF clinical cell analysis.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BackgroundAldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients.ResultsIn normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA).ConclusionOur study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

UbcH10 expression in human lymphomas

Aims:  The UbcH10 ubiquitin‐conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas.

Culture Conditions Allow Selection of Different Mesenchymal Progenitors from Adult Mouse Bone Marrow

The use of adult stem cells in tissue engineering approaches will benefit from the establishment of culture conditions that allow the expansion and maintenance of cells with stem cell-like activity and high differentiation potential. In the field of adult stem cells, bone marrow stromal cells (BMSCs) are promising candidates. In the present study, we define, for the first time, conditions for optimizing the yields of cultures enriched for specific progenitors of bone marrow. Using four distinct culture conditions, supernatants from culture of bone fragments, marrow stroma cell line MS-5, embryonic fibroblast cell line NIH3T3, and a cocktail of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), we isolated four different sub-populations of murine BMSCs (mBMSCs). These cells express a well-known marker of undifferentiated embryonic stem cells (Nanog) and show interesting features in immunophenotype, self-renewal ability, and differentiation potency. In particular, using NIH3T3 conditioned medium, we obtained cells that showed impairment in osteogenic and chondrogenic differentiation while retaining high adipogenic potential during passages. Our results indicate that the choice of the medium used for isolation and expansion of mBMSCs is important for enriching the culture of desired specific progenitors.

Polychromatic flow cytometry analysis of CD34+ hematopoietic stem cells in cryopreserved early preterm human cord blood samples.

During the last decades, extended characterizations were performed of human full‐term cord blood (hTCB) cells, but little information is available on human early preterm cord blood (hEPCB) hematopoietic stem cells (HSCs). In our study, we analyzed by flow cytometry 19 hEPCB and 17 hTCB samples. First, we observed that the percentage of CD34PosCD45Dim cells was higher in hEPCB compared with hTCB and that it decreased during 16th–20th week of pregnancy. Within the CD34PosCD45Dim population, we examined the expression of CD29, CD31, CD38, CD90, CD117, CD133, CD135, CD200, CD243, and CD338. We found that CD135 intensity and CD243Pos cells percentage were lower in hEPCB compared with hTCB. As to CD38, we observed that hEPCB samples were richer in undifferentiated CD34PosCD45DimCD38Neg HSCs compared with hTCB counterparts. We also compared the expression of the above‐mentioned molecules in undifferentiated and committed HSCs residing in hEPCB and hTCB. In particular, although CD34PosCD45DimCD38Pos HSCs from both hEPCB and hTCB expressed relatively higher amounts of CD29, CD71, and CD135 compared with CD34PosCD45DimCD38Neg cells, a higher expression of CD31 was restricted to CD34PosCD45DimCD38Pos cells from hEPCB samples, and a higher expression of CD117 was demonstrated in CD34PosCD45DimCD38Pos cells from hTCB samples. Moreover, our data showed that CD34PosCD45Dim cell population from hEPCB displayed higher percent of undifferentiated CD38NegCD133Pos cells compared with hTCB samples. Finally, analyzing monocytes and lymphocytes within the two samples, we observed that T‐cell percentages were higher in hTCB, whereas B‐cell percentages were higher in hEPCB. We, therefore, studied the B‐cell lineage maturation and found a higher percent of pro‐B and pre‐B cells in hEPCB compared with hTCB samples. Taken together, these results evidence the hematopoietic peculiarity of hEPCB, potentially useful for highlighting early steps of human hematolymphopoiesis as well as for developing novel strategies of stem cell‐based therapy.

Molecular evidence for a thymus-independent partial T cell development in a FOXN1-/- athymic human fetus.

The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1−/− human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1−/− fetus, in which we previously described a total blockage of CD4+ and partial blockage of CD8+ cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3+ and CD8+, but not of CD4+ cells, a few of them exhibiting a CD45RA+ naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process.

Inhibition of PID1/NYGGF4/PCLI1 gene expression highlights its role in the early events of the cell cycle in NIH3T3 fibroblasts

Abstract The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.