Caroline Bret

An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization

Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1(+) and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell-to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138− and CD138+ plasma cells

Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10+CD27−CD38+, 6±6 cells/μL), naïve (CD10−CD27−CD38−, 125±90 cells/μL), memory B lymphocytes (CD10−CD27+CD38−, 58±42 cells/μL), and plasma cells (CD10−CD27++CD38++, 2.1±2.1 cells/μL) within circulating CD19+ cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138− (57±12%) and CD138+ (43±12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67+ and show weak CXCR4 expression.

Specific Activity of Class II Histone Deacetylases in Human Breast Cancer Cells

Although numerous studies have underlined the role of histone deacetylases (HDAC) in breast physiology and tumorigenesis, little is known on the particular contribution of the various classes of HDACs in these processes. Using estrogen receptor-α (ERα)–positive MCF-7 breast cancer cells, the effects of MC1575 and MC1568, two novel class II–specific HDAC inhibitors, were analyzed on cell proliferation, apoptosis, and estrogen signaling. The specificity of these HDAC inhibitors was validated by measuring histone and α-tubulin acetylation and by the specific in vitro inhibition of recombinant HDAC4 using histone and nonhistone substrates, contrasting with the lack of inhibition of class I HDACs. In addition, MC1575 did not inhibit class I HDAC gene expression, thus confirming the specific targeting of class II enzymes. Similar to trichostatin A (TSA), MC1575 displayed a dose-dependent antiproliferative effect and induced cell cycle arrest although this blockade occurred at a different level than TSA. Moreover, and in contrast to TSA, MC1575 had no effect on MCF-7 cells apoptosis. Interestingly, MC1575 was able to increase p21waf1/CIP1 mRNA levels but did not regulate the expression of other genes such as cyclin D1, p27, p14ARF, Bcl2, Baxα, Trail-R1, and Trail-R2. Finally, MC1575 strongly induced ERβ gene expression but did not decrease ERα expression, nor did it switch hydroxytamoxifen to an agonist activity. Altogether, these data suggest that the class II HDAC subfamily may exert specific roles in breast cancer progression and estrogen dependence. (Mol Cancer Res 2008;6(12):1908–19)

SULFs in human neoplasia: implication as progression and prognosis factors.

BackgroundThe sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on cell surface. SULF1 and SULF2 are two endosulfatases able to cleave specific 6-O sulfate groups within the heparan chains. Their action can modulate signaling processes, many of which with key relevance for cancer development and expansion. SULF1 has been associated with tumor suppressor effects in various models of cancer, whereas SULF2 dysregulation was in relation with protumorigenic actions. However, other observations argue for contradictory effects of these sulfatases in cancer, suggesting the complexity of their action in the tumor microenvironment.MethodsWe compared the expression of the genes encoding SULF1, SULF2 and heparan sulfate proteoglycans in a large panel of cancer samples to their normal tissue counterparts using publicly available gene expression data, including the data obtained from two cohorts of newly-diagnosed multiple myeloma patients, the Oncomine Cancer Microarray database, the Amazonia data base and the ITTACA database. We also analysed prognosis data in relation with these databases.ResultsWe demonstrated that SULF2 expression in primary multiple myeloma cells was associated with a poor prognosis in two independent large cohorts of patients. It remained an independent predictor when considered together with conventional multiple myeloma prognosis factors. Besides, we observed an over-representation of SULF2 gene expression in skin cancer, colorectal carcinoma, testicular teratoma and liver cancer compared to their normal tissue counterpart. We found that SULF2 was significantly over-expressed in high grade uveal melanoma compared to low grade and in patients presenting colorectal carcinoma compared to benign colon adenoma.We observed that, in addition to previous observations, SULF1 gene expression was increased in T prolymphocytic leukemia, acute myeloid leukemia and in renal carcinoma compared to corresponding normal tissues. Furthermore, we found that high SULF1 expression was associated with a poor prognosis in lung adenocarcinoma.Finally, SULF1 and SULF2 were simultaneously overexpressed in 6 cancer types: brain, breast, head and neck, renal, skin and testicular cancers.ConclusionsSULF1 and SULF2 are overexpressed in various human cancer types and can be associated to progression and prognosis. Targeting SULF1 and/or SULF2 could be interesting strategies to develop novel cancer therapies.

Expression of genes encoding for proteins involved in heparan sulphate and chondroitin sulphate chain synthesis and modification in normal and malignant plasma cells

Syndecan‐1 is a proteoglycan that concentrates heparin‐binding factors on the surface of multiple myeloma cells, and probably plays a major role in multiple myeloma biology. As heparan sulphate and chondroitin sulphate are the bioactive components of syndecan‐1, we analysed the signature of genes encoding 100 proteins involved in synthesis of these chains, i.e. from precursor uptake to post‐translational modifications, using Affymetrix microarrays. The expression of enzymes required for heparan sulphate and chondroitin sulphate biosynthesis was shown to increase in parallel with syndecan‐1 expression, throughout the differentiation of memory B cells into plasmablasts and normal bone marrow plasma cells. Sixteen genes were significantly different between normal and malignant plasma cells, nine of these genes –EXT2, CHSY3, CSGALNACT1, HS3ST2, HS2ST1, CHST11, CSGALNACT2, HPSE, SULF2 – encode proteins involved in glycosaminoglycan chain synthesis or modifications. Kaplan–Meier analysis was performed in two independent series of patients: B4GALT7, CSGALNACT1, HS2ST1 were associated with a good prognosis whereas EXT1 was linked to a bad prognosis. This study provides an overall picture of the major genes encoding for proteins involved in heparan sulphate and chondroitin sulphate synthesis and modifications that can be implicated in normal and malignant plasma cells.

Assessment of RNA Quality Extracted from Laser-Captured Tissues Using Miniaturized Capillary Electrophoresis

Assessment of RNA Quality Extracted from Laser-Captured Tissues Using Miniaturized Capillary Electrophoresis

Expression and role of RIP140/NRIP1 in chronic lymphocytic leukemia

RIP140 is a transcriptional coregulator, (also known as NRIP1), which finely tunes the activity of various transcription factors and plays very important physiological roles. Noticeably, the RIP140 gene has been implicated in the control of energy expenditure, behavior, cognition, mammary gland development and intestinal homeostasis. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. During the past years, several papers have reported evidences linking RIP140 to hematologic malignancies. Among them, two recent studies with correlative data suggested that gene expression signatures including RIP140 can predict survival in chronic lymphocytic leukemia (CLL). This review aims to summarize the literature dealing with the expression of RIP140 in CLL and to explore the potential impact of this factor on transcription pathways which play key roles in this pathology.

Nucleotide excision DNA repair pathway as a therapeutic target in patients with high-risk diffuse large B cell lymphoma.

Gene expression profiling studies revealed that DLBCL comprises two main subgroups displaying distinct molecular gene signatures and clinical outcome: one subgroup, termed germinal-center B-cell-like (GCB), associated with a gene expression profile (GEP) of healthy germinal-center B cells and with a good outcome, and another subgroup, termed activated B-cell-like (ABC), with GEP of healthy peripheral blood activated B cells and with a poorer outcome. GCB and ABC subgroups represent 50% and 30%, respectively, of DLBCL cases. The residual 20% of patients are unclassifiable. 1 We used previously described methods to build powerful risk scores in hematological malignancies and designed a 12-gene expression-based risk score (GERS) predictive for overall survival (OS) in two independent cohorts of patients with DLBCL. 2 GERS allows identifying 12.3% of patients within GCB and high risk and 33.7% of patients within ABC and high risk. GERS is an independent prognostic factor when compared with previously published factors, including the International Prognostic Index (IPI). 2

Identifying high-risk adult AML patients: epigenetic and genetic risk factors and their implications for therapy

ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous disease at molecular level, in response to therapy and prognosis. The molecular landscape of AML is evolving with new technologies revealing complex panorama of genetic abnormalities where genomic instability and aberrations of epigenetic regulators play a key role in pathogenesis. The characterization of AML diversity has led to development of new personalized therapeutic strategies to improve outcome of the patients.

DNA repair in diffuse large B-cell lymphoma: a molecular portrait

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