Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation

Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation

Background: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of m...

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Datum:2016
Hauptverfasser: Gordiienko, I.M., Shlapatska, L.M., Kovalevska, L.M., Sidorenko, S.P.
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Sprache:English
Veröffentlicht: Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України 2016
Schriftenreihe:Experimental Oncology
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Original contributions
Online Zugang:http://dspace.nbuv.gov.ua/handle/123456789/137991
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Zitieren:Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation / I.M. Gordiienko, L.M. Shlapatska, L.M. Kovalevska, S.P. Sidorenko // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 101-107. — Бібліогр.: 35 назв. — англ.

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spelling irk-123456789-1379912018-06-18T03:05:53Z Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation Gordiienko, I.M. Shlapatska, L.M. Kovalevska, L.M. Sidorenko, S.P. Original contributions Background: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Materials and Methods: Studies were performed on normal tonsillar B-cell subpopulations, Blymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt’s lymphoma, Hodgkin’s lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. Results: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin’s lymphoma cell lines, Burkitt’s lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin’s lymphoma cell lines and Burkitt’s lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Conclusion: Malignant Bcell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform. 2016 Article Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation / I.M. Gordiienko, L.M. Shlapatska, L.M. Kovalevska, S.P. Sidorenko // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 101-107. — Бібліогр.: 35 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137991 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language English
topic Original contributions
Original contributions
spellingShingle Original contributions
Original contributions
Gordiienko, I.M.
Shlapatska, L.M.
Kovalevska, L.M.
Sidorenko, S.P.
Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
Experimental Oncology
description Background: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Materials and Methods: Studies were performed on normal tonsillar B-cell subpopulations, Blymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt’s lymphoma, Hodgkin’s lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. Results: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin’s lymphoma cell lines, Burkitt’s lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin’s lymphoma cell lines and Burkitt’s lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Conclusion: Malignant Bcell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform.
format Article
author Gordiienko, I.M.
Shlapatska, L.M.
Kovalevska, L.M.
Sidorenko, S.P.
author_facet Gordiienko, I.M.
Shlapatska, L.M.
Kovalevska, L.M.
Sidorenko, S.P.
author_sort Gordiienko, I.M.
title Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
title_short Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
title_full Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
title_fullStr Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
title_full_unstemmed Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation
title_sort differential expression of cd150/slamf1 in normal and malignant b cells on the different stages of maturation
publisher Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
publishDate 2016
topic_facet Original contributions
url http://dspace.nbuv.gov.ua/handle/123456789/137991
citation_txt Differential expression of CD150/SLAMF1 in normal and malignant b cells on the different stages of maturation / I.M. Gordiienko, L.M. Shlapatska, L.M. Kovalevska, S.P. Sidorenko // Experimental Oncology. — 2016 — Т. 38, № 2. — С. 101-107. — Бібліогр.: 35 назв. — англ.
series Experimental Oncology
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fulltext Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ��� DIFFERENTIAL EXPRESSION OF CD150/SLAMF1 IN NORMAL AND MALIGNANT B CELLS ON THE DIFFERENT STAGES OF MATURATION I.M. Gordiienko, L.M. Shlapatska, L.M. Kovalevska, S.P. Sidorenko* Department of Molecular and Cell Pathobiology, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine Background: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with up- regulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Materials and Methods: Studies were performed on normal tonsillar B-cell subpopulations, B- lymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt’s lym- phoma, Hodgkin’s lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expres- sion level of CD150 isoforms was evaluated using qRT-PCR. Results: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin’s lymphoma cell lines, Burkitt’s lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin’s lymphoma cell lines and Burkitt’s lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Conclusion: Malignant B- cell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform. Key Words: CD150/SLAMF1, CD150 isoforms, B-cell subsets, B-cell lines. CD�5� �IPO�/SLAM� is m�ltif�nctional type I trans- membrane glycoprotein that belongs to signaling lym- phocytic activation molec�le (SLAM� family within the imm�noglob�lin s�perfamily of s�rface receptors [���]. It is expressed on activated T and B cells� activated monocytes� macrophages and dendritic cells [4��]. Within B-cell lineage there is a noticeable heterogeneity in CD�5� cell s�rface expression. Nearly half of B-cell progenitors in bone marrow �pro-B and pre-B cells� express CD�5� on the cell s�rface [�]. The level of CD�5� s�rface expression is dropped on immat�re B cells and increased from the stage of naïve B cells to- ward plasma cells [����]. At the same time� expression of CD�5� is rather limi ted on the s�rface of malignant B cells with the block of differentiation at the different stages of mat�ration. High level of CD�5� s�rface expression is observed in hairy cell le�kemia� classical Hodgkin’s lymphoma �HL�� s�btype of diff�se large B-cell lymphoma with activated B cell phenotype �ABC- DLBCL�� and primary c�taneo�s follic�lar centre B-cell lymphoma [�����]. Chronic lymphocytic le�kemia is characterized by heterogeneity of CD�5� expres- sion with higher s�rface CD�5� expression in m�tated CLL s�bgro�p [�4� �5]. It sho�ld be emphasized that in the part of CLL cases CD�5� expression was lost d�ring malignant transformation [�5]. The low level of CD�5� expression was also observed in mantle cell lymphoma [��]. However� DLBCL with germinal centre phenotype� small lymphocytic lymphoma� lym- phoplasmacytic lymphoma� B�rkitt’s lymphoma �BL�� pre-B ac�te lymphoblastic le�kemia �pre-B ALL�� and m�ltiple myeloma �MM� were CD�5� negative [�����]. The differential CD�5� cell s�rface expression in B-cell malignancies co�ld be explained by several mecha- nisms: aberrant translocation of CD�5� to the plasma membrane� disr�ptions in reg�lation of CD�5� expres- sion on the transcriptional and/or translational levels. CD�5� is encoded by SLAMF1 gene that is lo- calised on the long arm of chromosome � at bands �q����4 within SLAM family gene cl�ster [���]. Seve- ral alternatively spliced isoforms of CD�5� have been identified [5� ��� ��] �Fig. ��. Canonical transmembrane CD�5� isoform �mCD�5�� with two ITSM signaling mo- tifs in cytoplasmic domain is the most widely st�died isoform. All available data abo�t CD�5� mediated signaling is concerning mCD�5� isoform [�]. Expres- sion of secreted CD�5� isoform �sCD�5��� which lacks transmembrane region� was detected in activated nor- mal B and T cells� mat�re dendritic cells� cases of clas- sical HL and HL cell lines� in blood ser�m of healthy individ�als and patients with rhe�matoid arthritis [�� ��� �����]. Since CD�5� is a self-ligand� sCD�5� iso- form may interact with transmembrane CD�5� in local microenvironment and initiate signaling pathways that Submitted: April 27, 2016. *Correspondence: Fax: +380442581656; E-mail: svitasyd@yahoo.com Abbreviation used: B-LCL — B-lymphoblastoid cell line; BL — Burkitt’s lymphoma; EBV — Epstein — Barr virus; HL — Hodgkin’s lym- phoma; MM — multiple myeloma; pre-B ALL — pre-B acute lymphoblas- tic leukemia; qRT-PCR — quantitative reverse transcription polymerase chain reaction; SLAM — signaling lymphocytic activation molecule. Exp Oncol ���� ��� �� ������� ��� Experimental Oncology ��� �������� ���� ���ne� leads to reg�lation of CD�5�+ cells f�nction and fate. In addition� variable membrane CD�5� �vmCD�5�� isoform with tr�ncated cytoplasmic tail and cytoplas- mic CD�5� �cCD�5�� isoform lacking leader seq�ence were predicted [5]. Recently� a novel spliced isoform of CD�5� �nCD�5�� with alternatively cytoplasmic tail was identified [��]. The nCD�5� is characterized by ��bp insert after transmembrane region. Cytoplas- mic tail of nCD�5� lacks ITSM motifs and its signaling properties are still �nknown [��]. Leader sequence Extracellular IgV-like - domain Extracellular lgC2-like - domain Transmembrane domain Cytoplasmic tail with 2 ITSM Truncated cytoplasmic tail with 1 ITSM Alternative cytoplasmic tail without any known signaling motifs NH2 NH2 NH2 NH2 NH2 LS ectodomain TM Cyt COOH COOH COOH COOH COOH mCD150 sCD150 cCD150 vmCD150 nCD150 IgV-like lgC2-like Fig. 1. Str�ct�re of CD�5� isoforms. mCD�5� isoform — canoni- cal transmembrane form of CD�5� with two ITSM motifs in cyto- plasmic tail. Sol�ble or secreted form of CD�5� �sCD�5�� lacks transmembrane domain. Cytoplasmic CD�5� isoform �cCD�5�� is devoid of the leader seq�ence� and variable CD�5� isoform �vmVD�5�� has one ITSM motif and tr�ncated cytoplasmic tail. The novel CD�5� �nCD�5�� isoform d�e to the additional insert after transmembrane region possess the alternative cytoplasmic tail witho�t any known signaling motifs The existence of alternatively spliced CD�5� iso- forms with different signaling properties indicates that they may mediate m�ltiple f�nctions by dif- ferential expression in lymphoid pop�lation at dif- ferent stages of cell mat�ration. Up to date� there is no complete information abo�t differential expres- sion of CD�5� isoforms in malignant B cells and the contrib�tion of different isoforms to CD�5�-mediated signaling. In this st�dy� we are foc�sed on eval�ation of general CD�5� protein level and mRNA expres- sion levels of CD�5� splice isoforms with conven- tional cytoplasmic tail �mCD�5��� alternatively spliced cytoplasmic tail �nCD�5�� and sol�ble secreted CD�5� �sCD�5�� in malignant B-cell lines at the dif- ferent stages of differentiation in comparison with their normal B-cell co�nterparts. To assess the expression level of CD�5� isoforms we performed q�antitative reverse transcription polymerase chain reaction �qRT- PCR� �sing primers that recognize �niq�e regions of CD�5� transcripts for each of isoforms. Up to now� qRT-PCR is the only method that allows eval�ating dif- ferential expression of CD�5� isoforms� since all known antibody recognised extracell�lar part of CD�5�� which is identical in all CD�5� isoforms. MATERIALS AND METHODS Isolation of normal B cells subpopulations. Tonsils were obtained from patients �ndergoing tonsil- lectomy. Monon�clear cell pop�lations were isolated by Lymphoprep �Axis-Shield PoCAS� Norway� density gradient centrif�gation. The obtained cells were then depleted of T cells by E-rosetting with previo�sly pre- pared sheep red blood cells. S�bpop�lations of normal B cells were isolated from total tonsillar B cells �sing direct magnetic separation. Dynabeads coated with sheep anti-mo�se IgG �Dynal Biotech ASA� Norway� were washed twice in PBS+�% FCS and inc�bated either with mo�se anti-CD�� ��� µg/ml� �Imm�notech� France�� or anti-CD�� ��� µg/ml� �BD Biosciences Pharmingen� USA�� or anti-IgD ��� µg/ml� �Imm�no- tech� France� mAb d�ring 4� min at 4 °C with rotation. Then� CD��-beads� CD��-beads and IgD-beads were washed � times with cold PBS+�% FCS and added to s�spension of total tonsillar B cells at concentra- tion of fo�r beads per cell with next inc�bation d�ring �� min at 4 °C with rotation. Using combination of de- pletion and positive isolation� the following s�bpop�la- tions of tonsillar B cells were isolated: IgD+CD��−CD��− �naïve B cells�� CD��+CD��−IgD− �germinal centre B cells� — CD��+CD��−IgD− �memory B cells�. Cell lines. The B-lymphoblastoid cell lines �B-LCL� T5-�� MP-�� CESS� �.��; pre-B ALL cell lines REH� BLIN-�� NALM�; BL B-cell lines Ramos� Raji� B�AB� Da�di; HL B-cell lines KM-H�� L4��� and MM сell line RPMI ���� were kindly provided by Prof. Edward Clark �University of Washington� Seattle� WA� USA�. HL cell line L���� from DSMZ was kindly provided by Prof. Eva Klein �Karolinska Instit�te� Stockholm� Sweden�. Cell lines were maintained in RPMI ��4� medi�m s�pple- mented with ��% FCS� � mM L-gl�tamine and antibi- otics at �� °C in a h�midified atmosphere containing 5% CO�. Biochemical methods. Cell pellets were lysed in Triton lysis b�ffer ��5� mM NaCl� � mM EDTA� � mM EGTA� �� mM Tris pH = �.�� �% Triton Х���� con- taining cocktails of protease and phosphatase inhibi- tors �Sigma� USA�. Lysates were s�bjected to sodi�m dodecyl s�lfate polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-NC mem- branes �Millipore� USA� and blocked d�ring 5 h with 5% non fat dried milk in Tris-b�ffered saline with Tween �� �TBS-T�. Membranes were inc�bated with primary anti-CD�5� rabbit antibodies �Sino Biologicals Inc.� China� at 4 °C overnight followed by secondary goat anti-rabbit HRP-conj�gated antibodies �Santa Cr�z Biotechnology� USA� for � h at room temperat�re. Cla- rity Western ECL s�bstrate �Imm�nо-Star HRP� BioRad� USA� and medical X-ray film �AGFA� Belgi�m� were �sed for vis�alization of imm�noreactions. qRT-PCR. Total RNA isolation� cDNA synthesis and real-time PCR were performed as described ear- lier [��]. The following primers for real-time PCR were �sed: ExtrCD�5� �isoforms containing extracell�lar part of CD�5� receptor�: For 5՛-AGGCCCTCCAC- GTTATCTA-�՛� Rev 5՛-GCAAAAGCGCTGAACTGA-�՛; Cyt-n CD�5� �isoform containing alternative cyto- plasmic tail�: For 5՛-TGAGAAGAAGAGCCACCTT- GA-�՛� Rev 5՛- GGTTCGTTTTACCATGGGAAG-�՛; Cyt-m CD�5� �isoforms containing conventional cytoplasmic tail�: For 5՛-GTGTATGCTGGGCTGT- TAGG-�՛� Rev 5՛-AGAGGTAAAACGAACCATTACCA-�՛; Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ��� sol�ble� secreted CD�5�: For 5՛-AGACCCCTCAG- GTAAAACG-�՛; Rev 5՛-TCTGGACTTGGGCATAG- ATCG-�՛. As internal control gene for normalization was chosen TATA-box binding protein �TBP�: For 5՛-CCACTCACAGACTCTCACAAC-�՛; Rev 5՛-CTGCG- GTACAATCCCAGAACT-�՛. The PCR cycling condi- tions were the following: �� min at 95 °C� 4� cycles of �5 s at 95 °C and 4� s at �� °C for sCD�5� and �� min at 95 °C� 4� cycles of �5 s at 95 °C and 4� s at �4 °C for ExtrCD�5�� cyto-mCD�5� and cyto-nCD�5�. Applied Biosystems �5�� system software was �sed for data analysis. Ct val�es were determined for the internal control �TBP� and the tested isoforms at the same threshold level in the exponential phase of the PCR amplification c�rves. The threshold cycle �CT� method was �sed to calc�late expression level relative to the endogeno�s control. Res�lts were presented in relative �nits �r.�.�. Dissociation c�rve analysis was performed after every r�n to check the specificity of the reaction. For all type of cells� ��5 reactions �each in triplicates� were r�n for each isoform with internal control and standard error of the mean �SEM� was calc�lated. RESULTS AND DISCUSSION CD�5� expression levels were mostly st�died �sing flow cytometry on live cells detecting its cell s�rface expression [4� �� �4� �5]. To eval�ate expression of all CD�5� isoforms on protein level we �sed western blot analysis. CD�5� was expressed at approximately the same level in tonsillar normal B-cell s�bpop�lations with slightly higher level in germinal centre B cells. The lo- west level of CD�5� was detected in memory B cells �Fig. �� I�. B-LCL �T5-�� MP-�� �.��� and CESS� and HL cell lines �KM-H�� L4�� and L����� were characte- rized by high CD�5� expression level that exceeds its in normal B cells �p to fo�r times �Fig. �� II� V and data not shown�. The germinal centre B cells are the develop mentally related normal lymphoid co�nterparts of BL B cells [��� ��]� b�t not all BL cell lines expressed CD�5� on the cell s�rface [4� ��� ��]. CD�5� protein expression was detected at the comparatively same level with germinal centre B cells only in B�AB and Raji b�t not in Ramos and Da�di BL cell lines �Fig. �� IV�. MM derives from plasma cells [�4] that have the highest level of CD�5� s�rface expression [����]. However� MM cell line RPMI-���� did not express CD�5� protein �Fig. �� VI�. Only one pre-B ALL cell line �BLIN-�� was CD�5� positive �Fig. �� III�� despite CD�5� expression was detected on the cell s�rface of nearly half of nor- mal pre-B cells [�]. Taken together� o�r data s�ggest that malignant B cells are characterized by aberrant expression of CD�5� on protein level� in comparison with their normal B cell co�nterparts. To answer the q�estion whether CD�5� is trans- cribed in CD�5� negative B-cell lines and which of the CD�5� isoforms are expressed in malignant B-cell lines we performed qRT-PCR �sing primers that specifically recognize different CD�5� domains. Primers that are specific to extracell�lar domain of CD�5� �extrCD�5�� allow to eval�ate the general mRNA expression level of all CD�5� isoforms. Primers to conventional cytoplasmic tail recognise the major transmembrane mCD�5� isoform �mCD�5�� and also minor cytoplasmic cCD�5� as well as tr�ncated vmCD�5� isoforms. Using specific primers to alterna- tive insert in cytoplasmic tail we were able to detect expression of a novel nCD�5�. For the detection of se- creted� sol�ble sCD�5� isoform we �sed primers that allow excl�ding the transmembrane domain. 0 10 20 30 40 50 Na ïve G C M em or y T5 -1 M P- 1 RE H BL IN -1 NA LM 6 Ra m os BJ AB Ra ji Da ud i KM -H 2 L1 23 6 RP M I8 22 6 Re la tiv e un its CD150 CD150 Actin I II III IV V VI Fig. 2. The expression level of CD�5� protein in s�bpop�lation of normal tonsillar B cells �I�� B-LCL �II�� pre-B-ALL cell lines �III�� BL cell lines �IV�� HL cell lines �V�� MM cell line �VI�. Western blot analysis. Actin expression level was �sed as loading control. The level of CD�5� expression was normalized against the level of actin �sing Adobe Photoshop CS5 program and presented as relative �nits CD�5� mRNA �as indicated by extCD�5�� was detected in all tested normal B-cell s�bsets �Fig. �� a�. The mCD�5� isoform was expressed at slightly lower level than the general CD�5� mRNA �Fig. �� b�. The sCD�5� mRNA expression was h�ndreds of times lower than general CD�5� mRNA level in total and germinal centre B cells. At the same time� sCD�5� isoform was practically �ndetectable in naïve and memory B-cell s�bsets �Fig. �� c�. The expression level of nCD�5� isoform was tenfold lesser� in compari- son with the expression of total CD�5� or mCD�5�. It sho�ld be noticed that maxim�m nCD�5� mRNA level was detected in memory B cells �Fig. �� d�. Th�s� the mCD�5� transmembrane isoform with conven- tional cytoplasmic tail is a predominant CD�5� isoform in s�bpop�lations of normal tonsillar B cells. Total CD�5� �extCD�5�� mRNA was expressed at the highest level in B-LCL: �p to �9.�� ± �.� r.�. in T5-� B-LCL �Fig. 4� a I�. All B-LCLs �sed in o�r st�dy are Epstein — Barr vir�s �EBV� immortalized normal B cells with established type III EBV latency. It was previ- o�sly shown that infection of B cells by EBV �preg�lated cell s�rface expression of CD�5�. Moreover� expres- sion of CD�5� depends on EBV-encoded oncogene LMP� that is highly expressed in type III EBV latency [�5]. That is why elevated CD�5� mRNA and protein expression level in B-LCL� compared to normal B cells� co�ld be EBV-dependent. The total CD�5� �extCD�5�� mRNA was detected also in all tested HL cell lines and two BL cell lines �B�AB and Raji� that correlated with ��4 Experimental Oncology ��� �������� ���� ���ne� CD�5� protein expression level and did not exceeded s�ch in normal B cells. Moreover� it was expressed at the low level ��p to �.� r.�.� in cell lines NALM�� Ramos� Da�di� and RPMI ���� �Fig. 4� a�� which did not express CD�5� on protein level �see Fig. ��. 0.001 0.01 0.1 1 10 Total B cells Naïve B cells GC B cells Memory B cells Re la tiv e un its extrCD150 0.001 0.01 0.1 1 10 Total B cells Naïve B cells GC B cells Memory B cells Re la tiv e un its mCD150 0.001 0.01 0.1 1 10 Total B cells Naïve B cells GC B cells Memory B cells Re la tiv e un its sCD150 0.001 0.01 0.1 1 10 Total B cells Naïve B cells GC B cells Memory B cells Re la tiv e un its nCD150 d b c a Fig. 3. The mRNA expression level of total CD�5� isoforms extrCD�5� �a�� mCD�5� isoform �b�� sCD�5� isoform �c�� and nCD�5� �d� in total normal B cells� naïve B cells� germinal centre B cells and memory B cells. Real-time qRT-PCR. The res�lts� presented as mean of triplicates �± SEM�� are from one of three independent experiments In malignant B-cell l ines expression level of mCD�5� transmembrane isoform with conventional cytoplasmic tail repeated overall expression level for total CD�5� �Fig. 4� b�. It was shown that sCD�5� isoform was expressed in all tested B-LCL� HL cell lines and BL cell line Raji. At the same time� sCD�5� was not fo�nd in all tested pre-B ALL cell lines� BL cell lines Ramos� B�AB and Da�di� and MM cell line RPMI ���� �Fig. 4� c� II� III� V�. 0.001 0.01 0.1 1 10 100 6. 16 T5 -1 M P- 1 CE SS RE H BL IN -1 Na lm 6 Ra m os Ra ji BJ AB Da ud i KM -H 2 L4 28 L1 23 6 RP M I 8 22 6 I II III IV V Re la tiv e un its extrCD150 0.001 0.01 0.1 1 10 100 6. 16 T5 -1 M P- 1 CE SS RE H BL IN -1 Na lm 6 Ra m os Ra ji BJ AB Da ud i KM -H 2 L4 28 L1 23 6 RP M I 8 22 6 I II III IV V Re la tiv e un its mCD150 0.001 0.01 0.1 1 10 100 6. 16 T5 -1 M P- 1 CE SS RE H BL IN -1 Na lm 6 Ra m os Ra ji BJ AB Da ud i KM -H 2 L4 28 L1 23 6 RP M I 8 22 6 I II III IV V Re la tiv e un its nCD150 0.001 0.01 0.1 1 10 100 6. 16 T5 -1 M P- 1 CE SS RE H BL IN -1 Na lm 6 Ra m os Ra ji BJ AB Da ud i KM -H 2 L4 28 L1 23 6 RP M I 8 22 6 I II III IV V Re la tiv e un its sCD150 d b c a Fig. 4. mRNA expression level of total CD�5� isoforms extrCD�5� �a�� mCD�5� isoform �b�� sCD�5� isoform �c�� and nCD�5� �d� in B-LCLs �I�� pre-B ALL cell lines �II�� BL cell lines �III�� HL cell lines �IV�� MM cell line �V�. Real-time qRT-PCR. The res�lts presented as mean of tripli- cates �± SEM� are from one of three independent experiments. Red lines show the expression level of corresponding CD�5� isoforms in total tonsillar B cells Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ��5 On the other hand� the ele vated over normal B cells level of sCD�5� isoform was observed in �.�� and CESS B-LCLs �Fig. 4� d� I�. The nCD�5� mRNA was revealed in the majority of tested cell lines with the exception of pre-B ALL cell line REH and BL cell line Da�di �Fig. 4� d�. Similarly to normal B-cell s�bsets� in malignant B- cell lines expression level of nCD�5� was lower� com- pared to the expression of mCD�5� isoform �Fig. 4� d�. It was s�rprising that in pre-B-ALL cell line NALM-� the nCD�5� was a major CD�5� isoform �Fig. 4� d, II�. O�r res�lts demonstrated that CD�5� isoforms are differentially expressed in st�died B-cell lines with predominant expression of mCD�5� transmembrane splice isoform with conventional cytoplasmic tail. 9��95% of h�man lymphoma/le�kemia in ad�lts are of B cell origin. CD�5� is expressed at different stages of B cell mat�ration from pro-B to plasma cells with �preg�lation of its expression d�ring differentia- tion [�� 9]. However� CD�5� cell s�rface expression is rather limited on malignant B cells [��� ��� ��]. In o�r st�dy� we revealed heterogeneity of CD�5� expres- sion in malignant B-cell lines �s�mmarized in Table� that may reflect the different stages of differentiation of normal co�nterparts of malignant cells. Table. Expression of CD150 on the protein and mRNA level in normal B-cell subsets and B-cell lines Subpopulation of normal B cells and cell lines Cell surface expression General ex- pression level (Western blot analysis) mRNA expression ex trC D1 50 m CD 15 0 sC D1 50 nC D1 50 Naïve B cells + [9] + +++ ++ − ++ GC B cells + [9] + +++ +++ + ++ Memory B cells + [9] + ++ +++ − +++ 6.16 EBV+ +++ [4] ++ +++ +++ + + T5-1 EBV+ +++ [34] +++ +++ +++ + ++ MP-1 EBV+ +++ [34] +++ +++ +++ + + CESS EBV+ +++ [4] ++ +++ +++ + ++ REH EBV− − [4] − − − − − BLIN-1 EBV− ++ (unpubl.) + ++ + − + Nalm6 EBV− − [4] − ++ + − ++ Ramos EBV− − [4] − + + − + Raji EBV+ ++ [4] + ++ + + + BJAB EBV− ++ [4] + ++ + − + Daudi EBV+ − [8] − + + − − KM-H2 EBV− +++ [20, 35] ++ ++ ++ + + L428 EBV− +++ [20, 35] ++ ++ + + + L1236 EBV− +++ [20, 35] ++ +++ +++ + ++ RPMI 8226 EBV− − [4] − ++ ++ − + Note: +++ high expression level; ++ medium expression level; + low expres- sion level; — expression was not detected. It co�ld be tr�e for pre-B-cell lines� since abo�t half of normal pro-B and pre-B cells express CD�5�. Only in one pre-B-cell line REH CD�5� was absent both on mRNA and protein level. In NALM� CD�5� was ex- pressed only on mRNA level� and in BLIN-� CD�5� pro- tein was also detected. Probably� these pre-B-cell lines represent their normal co�nterparts at the seq�ential stages of differentiation. As to BL cell lines� the sit�- ation is different. Two of fo�r BL cell lines express CD�5� protein independently of EBV presence. How- ever� all tested BL cell lines express CD�5� mRNA that did not exceed the level in normal B cells. At the same time� germinal centre B cells express CD�5� both on mRNA and protein level. Contrary to BL cell lines� in st�died HL cell lines� which derived from crippled� pre-apoptotic germinal centre B cells� we fo�nd the correlation between CD�5� expression on mRNA and protein levels. Despite the high level of CD�5� expres- sion on the s�rface of normal plasma cells� MM cell line RPMI ���� expressed CD�5� only on mRNA level. Th�s� we fo�nd that in malignant B cell lines CD�5� expression on mRNA level is m�ch broader than on protein level. 95% of m�lti-exonic mammalian genes �ndergo alternative splicing that leads to proteomic diversi- ties [��]. Expression of different transcript variants and protein isoforms often occ�rs in cell type/tiss�e spe- cific and developmental stage specific manner [��]. In the past decade� it was acc�m�lated a lot of data abo�t altering in splicing patterns of n�mero�s genes �e.g. TP53, BCL2L1, TNR6, BARD1, AR, PI3K etc.� in cancer development and progression [��]. That is why� st�dying isoform expression profile is important for more comprehensive characterization of cancer heterogeneity and may reveal novel therape�tic tar- gets. In c�rrent work� we showed that mRNA expres- sion level of all CD�5� isoforms in malignant B-cell lines did not exceed that in normal B cells or even was lower. Up to now� signaling properties of CD�5� are associ- ated with paired ITSM� which are present in mCD�5�� sCD�5� and predicted cCD�5� isoform. The primers that were �sed in o�r st�dy discriminate between sCD�5� isoform and mCD�5� + cCD�5� isoforms. The cCD�5� isoform d�e to the lack of leader seq�ence co�ld not be translocated to the endoplasmic retic�l�m for f�rther translation and post-translation modifica- tions. So� cCD�5� translates into non-glycosylated protein core with molec�lar weight near 4� kDa. According to the western blot analysis of CD�5� ex- pression� we did not observe any band in the area of 4��5� kDa. This fact indirectly s�ggests that cCD�5� is missing or expressed as a minor isoform in st�died normal and malignant B cells and do not significantly affect the res�lts obtained with primers to conventional cytoplasmic tail of CD�5�. Expres- sion of sCD�5� in HL cell lines� B-LCL and BL cell line Raji may indicate the role of secreted CD�5� isoform in constit�tive activation of CD�5�-mediated signaling pathways. Discovery of a novel CD�5� isoform pre- dominant expression in glial t�mors raised the q�es- tion whether expression level of nCD�5� is higher than mCD�5� in cells of imm�ne system that co�ld change conception of CD�5� signaling properties. However� in o�r st�dy� increased nCD�5� expression level was revealed only in one st�died cell line — pre-B-cell line NALM�� where CD�5� protein was not detected. Taken together� the mCD�5� with signaling properties is the main CD�5� isoform in normal B-cell s�bsets� EBV-transformed B-LCLs and malignant B-cells lines. Moreover� we did not find any significant differences in q�antitative ratio of mRNA expression level of st�- died CD�5� splice isoforms between malignant B-cell lines and normal B-cell s�btypes. Mechanisms that �nderlie fail�re of CD�5� protein expression d�ring malignant transformation are c�r- ��� Experimental Oncology ��� �������� ���� ���ne� rently �nknown. M�ltilevel reg�lation of gene expres- sion embracing transcription� RNA processing� trans- lation and post-translational level� allow cells strictly coordinate changes in profile of gene expression in re- sponse to internal and external stim�li [�9]. The ins�f- ficiency of CD�5� protein expression d�ring malignant transformation of B cells is probably associated with deviation at the post-transcriptional level of gene reg�- lation. Post-transcriptional gene reg�lation incl�des processing of mRNA� export of mRNA to the cytoplasm and localization� mRNA stability and translation [��]. The key negative reg�lators of translation are short non-coding RNA — miRNAs. The miRNA can red�ce target gene expression via ind�ction of mRNA degrada- tion or by inhibiting mRNA translation witho�t affecting of mRNA level [��]. According to the miRNAs database “miRTarBase” �http://mirtarbase.mbc.nct�.ed�.tw/ php/search.php?org=hsa&kw=SLAMF�&opt=target� near twenty miRNAs may potentially target CD�5�. However� no one p�tative miRNA for CD�5� was vali- dated by strong experimental approaches. Translation initiation co�ld depend on different cis- element present in 5՛-UTR and/or �՛-UTR. Upstream open reading frames ��ORF� in 5՛-UTR — seq�ences franked by stop and start codons localised �pstream of the main ORF� act as major reg�lator of translation initiation. Approximately 5�% of h�man mRNAs contain �ORF. The n�mber of �ORF in mRNA correlates with significantly red�ced protein expression level [��]. Re- cently� it was shown that CD�5� has at least two isoforms based on their 5՛-UTR str�ct�re [��]. Long isoform of SLAMF1 mRNA contains fo�r �ORF at 5՛-UTR and is characterised by 5�� times decreases its translation activity� in comparison with SLAMF1 mRNA short isoform witho�t �ORF in 5՛-UTR [��]. It is possible that in malig- nant B-cell lines lacking expression of CD�5� protein transcription may switch to the long mRNA isoforms le ading to less efficient translation� as indicated by pre- vailing mRNA over protein expression. Taken together� malignant B-cell lines at the diffe- rent stages of mat�ration only partially resemble their normal co�nterparts by CD�5� expression. Mostly� neoplastic B cells attempt to escape CD�5� protein expression pres�mably �sing diverse mechanisms. ACKNOWLEDGEMENTS A�thors thank to Prof. Eva Klein �Karolinska Instit�te� Stockholm� Sweden� and Prof. E.A. Clark �University of Washington� Seattle� WA� USA� for the cell lines� and Dr. M.Y. 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