SLAMF1/CD150 expression and topology in prostate and breast cancer cell lines.

BACKGROUND: SLAMF1/CD150 receptor is aberrantly expressed in malignant hematopoietic cells compared to ubiquitous expression in their normal analogues. However, the data about CD150 expression and function outside the hematopoietic system are limited. The aim of this pilot study was to examine the profile of mRNA expression of CD150 isoforms and the protein topology in highly and low malignant breast (BC) and prostate cancer (PC) cell lines. MATERIALS AND METHODS: The study was performed on BC T47D, MDA-MB-231, ВСС/Р and BC/ML cell lines and PC LNCap, Du-145 and PC-3 cell lines. The quantitative polymerase chain reaction was applied for study of CD150 isoforms mRNA expression and flow cytometry was used for determination of protein localization. RESULTS: Analyzed BC cell lines did not express CD150 on the cell surface membrane (csCD150-), but more than 45% of cells were positive for CD150 cytoplasmic reaction (cyCD150+). The cyCD150 expression level in T47D cells of luminal BC subtype was higher than in basal BC cell lines MDA-MB-231, ВСС/Р and BC/ML. The PC cell lines expressed CD150 both on the cell surface and in cytoplasm. The highest number of csCD150+ and cyCD150+ cells was revealed in less aggressive androgen responsive, non-metastatic LNCap cell line. All studied BC and PC cell lines expressed mRNA of canonical transmembrane mCD150 and novel nCD150 isoforms but with different pattern of prevalence. Soluble CD150 isoform was revealed at the low level only in BCC/P BC cell line and LNCap, PC-3 PC cell lines. CONCLUSIONS: We have shown that BC and PC cell lines differentially expressed multifunctional receptor CD150 at the mRNA and protein levels that may indicate its association with the degree of their malignancy.

Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium.

In this study, using new approach (laser diffraction + biological dyes), we have demonstrated the decrease of cells viability in vitro in the deuterated growth medium, whereas in the deuterium-depleted medium, there was an increase of cell viability. We have also found that not all dyes are equally sensitive to the D/H ratios in the culture medium (system) as well as to the different cell types (cancer vs normal cells).

Tissue-engineered bone for treatment of combat related limb injuries.

AIM: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. MATERIALS AND METHODS: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. RESULTS: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn't any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3-6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5-6 months post-op. CONCLUSION: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.

Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications.

AIM: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. MATERIALS AND METHODS: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. RESULTS: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. CONCLUSION: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40-200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.Key Words: regenerative medicine, neural crest, hair follicle, neural crest-derived multipotent stem cells, directed differentiation, large-scale expansion.

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

UNLABELLED: 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, B-lymphoblastoid 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 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.

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. II. receptor-mediated Akt/PKB and ERK1/2 activation and transcription factors expression profile.

BACKGROUND: X-linked lymphoproliferative disease type 1 (XLP1) belongs to genetically determined primary immunodeficiency syndromes with mutations in SH2D1A/DSHP/SAP gene. The dramatic increase of the risk of B-cell lymphoma development in XLP1 patients is linked not only to SAP deficiency of NK, NKT and T cells, but probably to the impairment of B cell differentiation. AIM: To analyze the receptor-mediated Akt/PKB and ERK1/2 phosphorylation and expression of transcription factors that are involved in B cell maturation in EBV-transformed B-lymphoblastoid cell lines (B-LCLs) from XLP1 patients (XLP B-LCLs) in comparison with conventional B-LCLs. METHODS: Studies were performed on EBV-transformed XLP B-LCLs IARC 739, SC-XLP and RP-XLP in comparison with SAP-negative B-LCL T5-1 and SAP-positive B-LCL MP-1. Western blot analysis was used for evaluation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204) phosphorylation in response to ligation of CD150, CD40, and IgM cell surface receptors. The expression levels of transcription factors IRF4, IRF8, BCL6, BLIMP1, SPIB, PU.1 and MITF were assessed using quantitative RT-PCR. RESULTS: It was shown that SAP deficiency in XLP B-LCL did not abrogate CD150-mediated Akt and ERK1/2 phosphorylation. At the same time, ligation of CD150 or IgM affects kinetics and amplitude of ERK1/2 activation. In XLP B-LCL the CD150 signaling with IgM coligation play the dominant role in both Akt and ERK1/2 phosphorylation. We found that significantly reduced IRF4, IRF8 and PU.1 expression levels are the key features of XLP B-LCLs. CONCLUSION: XLP B-LCLs and conventional B-LCLs have differences in kinetics and amplitude of Akt and ERK1/2 phosphorylation. Analysis of transcription factors profile revealed the distinguishing features of XLP B-LCLs with SAP deficiency that may impair B cell differentiation.Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, BCR, Akt/PKB, ERK1/2, transcription factors.

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. I. Cell surface phenotype and functional studies.

BACKGROUND: Mutations in SH2D1A/DSHP/SAP gene are responsible for the onset of X-linked lymphoproliferative disease type 1 (XLP1) that have increased risk for B-cell lymphoma development. In XLP1 patients SAP deficient NK, NKT and CD8(+) cytotoxic T cells are inefficient in eliminating EBV-infected proliferating B cells that may partially contribute to the lymphoma development. However, little is known about impairment of B cell characteristics in XLP1. AIM: To analyze the cell surface phenotype and functional characteristics of EBV-transformed B-lymphoblastoid cell lines from XLP1 patients (XLP B-LCLs) in comparison with conventional B-lymphoblastoid cell lines (B-LCLs). METHODS: Studies were performed on SAP-negative B-LCLs T5-1, 6.16, RPMI 1788; SAP-positive B-LCL MP-1 and XLP B-LCLs IARC 739, XLP-D, XLP-8005. Cell surface immunophenotyping was performed using flow cytometry analysis. The level of apoptotic cells (Annexin V-binding), cell viability (MTT assay), and cell proliferation (trypan blue exclusion test) were evaluated in response to ligation of CD40, CD95, CD150 and IgM cell surface receptors. RESULTS: A cell surface phenotype and functional features that distinguish XLP B-LCLs from conventional B-LCLs were revealed. XLP B-LCLs showed the upregulated level of CD20, CD38 and CD86 cell surface expression and downregulation of CD40, CD80 and CD150 expression. The major functional differences of XLP B-LCLs from conventional B-LCLs concern the modulation of CD95 apoptosis via CD40 and CD150 receptors and unresponsiveness to proliferative signals triggered by CD40 or colligation of BCR with CD150. CONCLUSION: The data suggest that the B-LCL from XLP1 patients have an intrinsic defect that affects cell activation, apoptosis, and proliferation.