scholarly journals Proton-irradiated breast cells: molecular points of view

2019 ◽  
Vol 60 (4) ◽  
pp. 451-465 ◽  
Author(s):  
Valentina Bravatà ◽  
Francesco P Cammarata ◽  
Luigi Minafra ◽  
Pietro Pisciotta ◽  
Concetta Scazzone ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Proton Irradiation ◽  
Expression Profiles ◽  
Treatment Plan ◽  
Mcf10a Cell ◽  
Specific Cell ◽  
Dose Dependent

Abstract Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

Identification Of Galectin-1 and Galectin-3 As Novel Binding Partners For Factor VIII

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 28-28
Author(s):  
Jamie O' Sullivan ◽  
Orla Rawley ◽  
Vince Jenkins ◽  
Alain Chion ◽  
Teresa M Brophy ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Full Length ◽  
Dependent Manner ◽  
Binding Partners ◽  
Pngase F ◽  
Galectin 3 ◽  
Dose Dependent

Abstract During biosynthesis, Factor VIII (FVIII) undergoes complex post-translational modification including significant glycosylation. Consequently each FVIII molecule can contains 25 N- and 6 O-linked glycans. These carbohydrate structures are of physiological significance. For example, FVIII glycan expression modulates intracellular trafficking and also regulates FVIII clearance by dendritic cells. Nevertheless, the molecular mechanisms through which glycan structures influence FVIII biology remains poorly defined. Interestingly, carbohydrate-binding galectins (Gal) -1 and -3 have recently been reported to bind human VWF. Moreover, these galectin interactions significantly influence VWF function. In this study, based upon similar glycans expression profiles, we hypothesised that galectins might also constitute novel binding partners for human FVIII. In brief, His-tagged Gal-1 and Gal-3 were expressed in E-coli and purified using nickel chromatography. Recombinant FVIII (rFVIII) was purified from different commercial concentrates. Subsequently, FVIII glycosylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. Galectin-FVIII interaction was characterised using modified immunosorbant assays and surface plasmon resonance (SPR). In plate–binding assays using purified proteins and SPR studies, both Gal-1 and Gal-3 bound to full length rFVIII in a time- and dose-dependent manner. Interestingly the apparent affinities of the galectin-FVIII interactions (Kd of 0.11 ± 0.02nM for Gal-1 and 0.21 ± 0.1nM for Gal-3 respectively) were unusually high for these lectins. Digestion with PNGase F to remove N-linked glycans ablated FVIII binding to Gal-1 (8.6 ± 1%; p<0.0001). In contrast, PNG-FVIII retained significant ability to bind Gal-3 (30.3 ± 3%; p<0.0001). However, combined FVIII digestion with both PNGase F and O glycosidase further attenuated Gal-3 binding (16.5 ± 2%; p<0.05). Cumulatively these findings suggest that whilst Gal-1 binding is mediated predominantly through the N-linked glycans of FVIII, both N- and O-linked glycans modulate its interaction with Gal-3. The majority of FVIII glycans are contained within the B domain. Unsurprisingly, Gal-1 and Gal-3 binding were both markedly attenuated for B domain deleted rFVIII compared to full length rFVIII (42 ± 1% and 26 ± 0.8%; p<0.0001). Previous studies have described different glycosylation profiles for specific full length commercial rFVIII products. To investigate the relevance of this differential glycosylation, we compared the galectin-binding properties of Advate® (CHO cell line) and Helixate® (BHK cell line). Interestingly, Gal-1 and Gal-3 both displayed significantly enhanced affinity for Helixate (107 ± 2% and 124 ± 1%; p<0.05). These findings are consistent with the fact that the N-linked glycans of BHK-derived FVIII express galactose α1-3 galactose epitopes which constitute preferential galectin-binding ligands. To determine whether FVIII interacts with galectins in vivo, immunoprecipitation studies were performed using plasma from VWF-/- mice. We observed that that both Gal-1 and Gal-3 were co-precipitated with FVIII even in the absence of VWF. Consequently, both the VWF-FVIII complex and free FVIII in plasma are likely to circulate in a complex with galectins. Importantly, recent studies have reported a prothrombotic phenotype in Gal-1/Gal-3 double deficient mice compared to wild type controls following ferric chloride injury. To investigate whether galectin-binding influences FVIII function, FVIII activity was assessed using a one-stage clotting assay in the presence of increasing galectin concentrations. Interestingly, preincubation of FVIII with Gal-1 (0.5-17µM) resulted in a significant dose-dependent prolongation of the APTT (58 ± 0.2 sec compared to 26 ± 3 secs, p<0.001) In contrast, no such effect was observed for galectin-3 up to 20µM, suggesting these galectins may have differential effects on FVIII biology. In conclusion, we identify Gal-1 and Gal-3 as novel direct ligands for human FVIII. Both the N- and O-linked carbohydrates of FVIII contribute to galectin binding. Importantly, different commercial FVIII concentrates do not interact with galectins in the same manner. Finally, we also demonstrate that plasma FVIII can circulate in complex with both Gal-1 and Gal-3, and that Gal-1 binding can inhibit the procoagulant function of FVIII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals USP22-dependent HSP90AB1 expression promotes resistance to HSP90 inhibition in mammary and colorectal cancer

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Robyn Laura Kosinsky ◽  
Marlena Helms ◽  
Maria Zerche ◽  
Luisa Wohn ◽  
Anna Dyas ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Gene Expression Signature ◽  
Breast Cancer Cell Lines ◽  
Mcf10a Cell ◽  
Hsp90 Inhibition

AbstractAs a member of the 11-gene “death-from-cancer” gene expression signature, overexpression of the Ubiquitin-Specific Protease 22 (USP22) was associated with poor prognosis in various human malignancies. To investigate the function of USP22 in cancer development and progression, we sought to detect common USP22-dependent molecular mechanisms in human colorectal and breast cancer cell lines. We performed mRNA-seq to compare gene expression profiles of various colorectal (SW837, SW480, HCT116) and mammary (HCC1954 and MCF10A) cell lines upon siRNA-mediated knockdown of USP22. Intriguingly, while USP22 depletion had highly heterogeneous effects across the cell lines, all cell lines displayed a common reduction in the expression of Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1). The downregulation of HSP90AB1 was confirmed at the protein level in these cell lines as well as in colorectal and mammary tumors in mice with tissue-specific Usp22 deletions. Mechanistically, we detected a significant reduction of H3K9ac on the HSP90AB1 gene in USP22-deficient cells. Interestingly, USP22-deficient cells displayed a high dependence on HSP90AB1 expression and diminishing HSP90 activity further using the HSP90 inhibitor Ganetespib resulted in increased therapeutic vulnerability in both colorectal and breast cancer cells in vitro. Accordingly, subcutaneously transplanted CRC cells deficient in USP22 expression displayed increased sensitivity towards Ganetespib treatment in vivo. Together, we discovered that HSP90AB1 is USP22-dependent and that cooperative targeting of USP22 and HSP90 may provide an effective approach to the treatment of colorectal and breast cancer.

Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

2020 ◽  
Vol 21 ◽  
Author(s):  
Putthiporn Khongkaew ◽  
Phanphen Wattanaarsakit ◽  
Konstantinos I. Papadopoulos ◽  
Watcharaphong Chaemsawang
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Flower Extract ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Murine Leukemia Cell ◽  
Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

scholarly journals Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

2021 ◽  
Vol 22 (11) ◽  
pp. 5798
Author(s):  
Shoko Tokumoto ◽  
Yugo Miyata ◽  
Ruslan Deviatiiarov ◽  
Takahiro G. Yamada ◽  
Yusuke Hiki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Desiccation Tolerance ◽  
Expression Profiles ◽  
Insect Cell Line ◽  
Gene Expression Profiles ◽  
Late Embryogenesis Abundant ◽  
Heat Shock Factor 1 ◽  
Genome Wide ◽  
A Genome

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Comparative Proteomic Analysis of Polarized Human THP-1 and Mouse RAW264.7 Macrophages

2021 ◽  
Vol 12 ◽  
Author(s):  
Pengfei Li ◽  
Zhifang Hao ◽  
Jingyu Wu ◽  
Chen Ma ◽  
Yintai Xu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Mouse Cell ◽  
Activated Macrophages ◽  
M1 Macrophages ◽  
Alternatively Activated Macrophages ◽  
Raw264.7 Macrophages ◽  
Protein Expression Profiles

Macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2) in the immune system, performing pro-inflammatory and anti-inflammatory functions, respectively. Human THP-1 and mouse RAW264.7 cell line models have been widely used in various macrophage-associated studies, while the similarities and differences in protein expression profiles between the two macrophage models are still largely unclear. In this study, the protein expression profiles of M1 and M2 phenotypes from both THP-1 and RAW264.7 macrophages were systematically investigated using mass spectrometry-based proteomics. By quantitatively analyzing more than 5,000 proteins among different types of macrophages (M0, M1 and M2) from both cell lines, we identified a list of proteins that were uniquely up-regulated in each macrophage type and further confirmed 43 proteins that were commonly up-regulated in M1 macrophages of both cell lines. These results revealed considerable divergences of each polarization type between THP-1 and RAW264.7 macrophages. Moreover, the mRNA and protein expression of CMPK2, RSAD2, DDX58, and DHX58 were strongly up-regulated in M1 macrophages for both macrophage models. These data can serve as important resources for further studies of macrophage-associated diseases in experimental pathology using human and mouse cell line models.

scholarly journals The multi-faceted functioning portrait of LRF/ZBTB7A

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Caterina Constantinou ◽  
Magda Spella ◽  
Vasiliki Chondrou ◽  
George P. Patrinos ◽  
Adamantia Papachatzopoulou ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Aerobic Glycolysis ◽  
Specific Cell ◽  
Regulation Of Transcription ◽  
Cancer Type ◽  
Cellular Effects ◽  
Physiological Processes ◽  
Signaling Axis

AbstractTranscription factors (TFs) consisting of zinc fingers combined with BTB (for broad-complex, tram-track, and bric-a-brac) domain (ZBTB) are a highly conserved protein family that comprises a multifunctional and heterogeneous group of TFs, mainly modulating cell developmental events and cell fate. LRF/ZBTB7A, in particular, is reported to be implicated in a wide variety of physiological and cancer-related cell events. These physiological processes include regulation of erythrocyte maturation, B/T cell differentiation, adipogenesis, and thymic insulin expression affecting consequently insulin self-tolerance. In cancer, LRF/ZBTB7A has been reported to act either as oncogenic or as oncosuppressive factor by affecting specific cell processes (proliferation, apoptosis, invasion, migration, metastasis, etc) in opposed ways, depending on cancer type and molecular interactions. The molecular mechanisms via which LRF/ZBTB7A is known to exert either physiological or cancer-related cellular effects include chromatin organization and remodeling, regulation of the Notch signaling axis, cellular response to DNA damage stimulus, epigenetic-dependent regulation of transcription, regulation of the expression and activity of NF-κB and p53, and regulation of aerobic glycolysis and oxidative phosphorylation (Warburg effect). It is a pleiotropic TF, and thus, alterations to its expression status become detrimental for cell survival. This review summarizes its implication in different cellular activities and the commonly invoked molecular mechanisms triggered by LRF/ZBTB7A’s orchestrated action.

Mesenchymal Stem Cells Self-Regulate Their Differentiation To Maintain the Bone Marrow Stromal System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2335-2335
Author(s):  
Iekuni Oh ◽  
Akira Miyazato ◽  
Hiroyuki Mano ◽  
Tadashi Nagai ◽  
Kazuo Muroi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Adipocyte Differentiation ◽  
Expression Profiles ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steady Level

Abstract Mesenchymal stem cells (MSCs) account for a very small population in bone marrow stroma as a non-hematopoietic component with multipotency of differentiation into adipocytes, osteocytes and chondrocytes. MSC-derived cells are known to have hematopoiesis-supporting and immunomodulatory abilities. Although clinical applications of MSCs have already been conducted for the suppression of graft versus host disease in allogeneic stem cell transplantation and for tissue regeneration, underlying mechanisms of the biological events are still obscure. Previously, we established a differentiation model of MSCs using a mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2) (Nishikawa M et al: Blood81:1184–1192, 1993). Preadipocyte (A54) and myoblast (M1601) cell lines were cloned by treatment with 5-azacytidine. A54 cells and M1601 cells can terminally differentiate into adipocytes and myotubes, respectively, under appropriate conditions, while parent 10T1/2 cells remain undifferentiated. Moreover, A54 cells show a higher ability to support hematopoiesis compared with the other cell lines. In this study, we analyzed gene expression profiles of the three cell lines by using DNA microarray and real-time PCR to investigate molecular mechanisms for maintaining immaturity of parent 10T1/2 cells. In A54 cells, 202 genes were up-regulated, including those encoding critical factors for hematopoiesis such as SCF, Angiopoietin-1, and SDF-1 as well as genes known to be involved in adipocyte differentiation such as C/EBPα, C/EBPδ and PPAR-γ genes. These data are consistent with the hematopoiesis-supporting ability of A54 cells. During adipocyte differentiation, SCF and SDF-1 expression levels decreased in A54 cells while C/EBPα expression showed a steady level. Recently, osteoblasts have been reported to play crucial roles in “niche” for self-renewal of hematopoietic stem cells. Our results also implicate that precursor cells of non-hematopoietic components may have important roles for hematopoiesis in bone marrow. Meanwhile, in parent 10T1/2 cells, 105 genes were up-regulated, including CD90, Dlk, Wnt5α and many functionally unknown genes. Although C/EBPα expression was induced in 10T1/2 cells without differentiation under the adipocyte differentiation conditions, CD90 expression decreased, Dlk showed a steady level and Wnt5α was up-regulated. Assuming that some regulatory mechanisms are needed to keep an immature state of parent 10T1/2 cells even under the differentiation-inducible conditions, we performed following experiments. First, enforced Dlk expression in A54 cells did not inhibit terminal differentiation to adipocytes under the differentiation conditions. Second, when we cultured A54 cells in the conditioned media of parent 10T1/2 cells under the differentiation-inducible conditions, adipocyte differentiation was inhibited, suggesting that 10T1/2 cells produce some soluble molecules that can inhibit adipocyte differentiation. Since Wnt family is known to be involved in the regulation of self-renewal of several stem cells, Wnt5α may be one candidate for maintenance of “stemness” of MSCs. Taken together, the data of 10T1/2 cells suggest that MSCs can self-regulate their differentiation in the bone marrow stromal system. This concept may be important to investigate the fatty change of bone marrow in aging and in aplastic anemia.

Expression Profiles of VDR-Induced Differentiation of atRA Refractory AML Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3291-3291
Author(s):  
Aurelie Baudet ◽  
Ronan Quere ◽  
Alice Roman ◽  
Jacques Marti ◽  
Therese Commes
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Growth Arrest ◽  
Promyelocytic Leukemia ◽  
S Phase ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Induced Differentiation ◽  
Acute Leukemias

Abstract Whatever the success of all-trans retinoic acid (atRA) therapy in acute promyelocytic leukemia (FAB AML3), other AML are poorly responsive to atRA and don’t benefit of any differentiation therapy. Moreover, primarily responsive patients often acquire resistance. Among molecules that control normal myelopoiesis, 1,25(OH)2-vitaminD3 (VD3), through its receptor VDR, allows maturation of myelomonocytic precursors. If VD3 has low effects on AML cells differentiation, its activity can be enhanced by various molecules. So, we tested several differentiation activators in combination with the non calcemiant VDR agonist EB1089 (EB) (Leo Pharmaceutics) on two atRA-refractory cell lines. Thus, U937 cells reproduce myelomonocytic acute leukemias that are strongly sensitive to VDR-induced differentiation and with a poor atRA sensitivity. Parallel, NB4-derived LR2 model mimics promyelocytic leukemia with acquired atRA resistance and has lower response to VD3. First, we confirm VDR-sensitivity of those cell lines. Moreover, as expected, U937 cells respond to EB1089 at lower dose (1nM) than NB4-LR2 (10nM) as showed by CD11b myeloid marker level. Among tested activators, the cytokine TGFb induces the most complete differentiation for both cell lines. After a 72 hour-treatment, only 5% of cells are still clustered in S phase. In addition, all are positive for the monocyte marker CD14 which expression reaches to 10 and 6 time fold the base level for U937 and NB4-LR2 respectively. At our knowledge, it is the first model of complete differentiation of NB4-LR2. Even if this agent can’t be used in therapy, it provides a reference for comparing other potential differentiating molecules. In fact, Histones Deacetylases Inhibitors (HDI) and arsenate (ATO) both benefit to VDR-induced differentiation of atRA refractory cell lines. Specifically, ATO benefits to EB-induced differentiation, as both U937 and NB4-LR2 cells acquire monocytic phenotype (CD14 expression) even though growth arrest is weaker. Indeed there are still 10–15% of S-phase clustered cells. Concerning the association of HDI valproic acid (VPA) and trichostatine A (TSA) with EB, growth arrest is strongest for both cell lines (5–10% S phase clustered cells) at 1mM and 100nM respectively. For phenotype markers, responses vary: U937 cells only respond to TSA combination whereas NB4-LR2 cells differentiate with TSA or VPA. Moreover, only TSA-EB treated cells become positive for CD14 even if expression is low. The addition of LGD1069, agonist of the Retinoid X receptor, a obligatory partner of VDR, improves HDI effects in all measured parameters. In case of NB4-LR2 cell line, flow cytometry data were reinforced by expression profile analyses. A hundred messengers were selected from bibliography and SAGE libraries of the promyelocytic cell line NB4 (unpublished data) and profiles were established by semi-quantitative real time PCR on low density array. Manual clusterization after comparison to granulocytic and monocytic differentiation (atRA-treated NB4 and VDTGFb-treated NB4-LR2 cells respectively) allows selection of about 10 messengers sufficient to predict patients’ response and to specify differentiation. In addition, the clinical use of VDR agonist, HDI and ATO is conceivable because of low side effects.

Lack of PRDM1α Expression in Hodgkin/Reed-Sternberg Cells Is Likely Mediated by a Translational or Post-Translational Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4367-4367
Author(s):  
Wayne Tam ◽  
Leonard Tan ◽  
Mario Gomez ◽  
Shaoan Fan ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Western Blotting ◽  
Plasma Cell ◽  
Zinc Finger Protein ◽  
Cell Phenotype

Abstract Hodgkin/Reed Sternberg (H/RS) cells are the neoplastic cells in classical Hodgkin lymphoma (HL). They are thought to resemble post-germinal center (GC) B cells with expression of markers associated with late stage of B-cell differentiation, for example, interferon regulatory factor -4/multiple myeloma-1 (IRF4/MUM1) and syndecan 1 (CD138). The PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1), a transcription repressor with a master regulatory role in plasma cell differentiation, is normally co-expressed with IRF-4/MUM-1 in plasma cells and in a subset of activated GC cells committed to plasma cell fate. We studied expression of PRDM1α, the functional isoform of PRDM1, in 14 classical HL cases [including 3 positive for Epstein-Barr-virus (EBV)] and 4 HL cell lines by immunohistochemistry and Western blotting, respectively. H/RS cells in primary HL cases are negative for PRDM1α, implying a desynchrony in expression between IRF-4/MUM1 and PRDM1. While the myeloma cell line U266 expresses relatively abundant PRDM1α, it was undetectable by Western Blotting in all HL cell lines tested, except for the EBV-positive HL cell line L591 which, unlike in vivo H/RS cells, has a Type III EBV latency pattern. PRDM1α expression in L591 but not in vivo H/RS cells suggests that PRDM1 expression may be modulated by latency type-specific EBV-encoded gene products or the B-cell phenotype exhibited by the cell line. The lack of PRDM1α protein in H/RS cells is not due to impaired gene transcription, since real-time quantitative PCR revealed similarly abundant PRDM1α transcripts in the HL cell lines as U266. In the absence of mutation in the PRDM1 coding region, these results suggest that failure to accumulate PRDM1α protein in H/RS cells is likely due to abnormal translation repression or protein turnover. Loss of functional PRDM1 as a result of translational or post-translational deregulation may represent a novel molecular lesion in HL.

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