Ingredients such as trehalose and hesperidin taken as supplements or foods reverse alterations in human T cells, reducing asbestos exposure-induced antitumor immunity.

Exposure of human immune cells to asbestos causes a reduction in antitumor immunity. The present study aimed to investigate the recovery of reduced antitumor immunity by several ingredients taken as supplements or foods, including trehalose (Treh) and glycosylated hesperidin (gHesp). Peripheral blood CD4+ cells were stimulated with IL­2, anti­CD3 and anti­CD28 antibodies for 3 days, followed by further stimulation with IL­2 for 7 days. Subsequently, cells were stimulated with IL­2 for an additional 28 days. During the 28 days, cells were cultured in the absence or presence of 50 µg/ml chrysotile asbestos fibers. In addition, cells were treated with 10 mM Treh or 10 µM gHesp. Following culture for 28 days, reverse transcription­quantitative PCR was performed to assess the expression levels of transcription factors, cytokines and specific genes, including matrix metalloproteinase­7 (MMP­7), nicotinamide nucleotide transhydrogenase (NNT) and C­X­C motif chemokine receptor 3, in unstimulated cells (fresh) and cells stimulated with PMA and ionomycin (stimuli). The results demonstrated that compared with the control group, chrysotile­exposure induced alterations in MMP­7, NNT and IL­17A expression levels were not observed in the 'Treh' and 'gHesp' groups in stimulated cells. The results suggested that Treh and gHesp may reverse asbestos exposure­induced reduced antitumor immunity in T helper cells. However, further investigation is required to confirm the efficacy of future trials involving the use of these compounds with high­risk human populations exposed to asbestos, such as workers involved in asbestos­handling activities.

Adenosine N1-Oxide Exerts Anti-inflammatory Effects through the PI3K/Akt/GSK-3ß Signaling Pathway and Promotes Osteogenic and Adipocyte Differentiation.

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3ß (GSK-3ß) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3ß phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3ß on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3ß signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.

Effects of a non-cyclodextrin cyclic carbohydrate on mouse melanoma cells: Characterization of a new type of hypopigmenting sugar.

Cyclic nigerosyl nigerose (CNN) is a cyclic tetrasaccharide that exhibits properties distinct from other conventional cyclodextrins. Herein, we demonstrate that treatment of B16 melanoma with CNN results in a dose-dependent decrease in melanin synthesis, even under conditions that stimulate melanin synthesis, without significant cytotoxity. The effects of CNN were prolonged for more than 27 days, and were gradually reversed following removal of CNN. Undigested CNN was found to accumulate within B16 cells at relatively high levels. Further, CNN showed a weak but significant direct inhibitory effect on the enzymatic activity of tyrosinase, suggesting one possible mechanism of hypopigmentation. While a slight reduction in tyrosinase expression was observed, tyrosinase expression was maintained at significant levels, processed into a mature form, and transported to late-stage melanosomes. Immunocytochemical analysis demonstrated that CNN treatment induced drastic morphological changes of Pmel17-positive and LAMP-1-positive organelles within B16 cells, suggesting that CNN is a potent organelle modulator. Colocalization of both tyrosinase-positive and LAMP-1-positive regions in CNN-treated cells indicated possible degradation of tyrosinase in LAMP-1-positive organelles; however, that possibility was ruled out by subsequent inhibition experiments. Taken together, this study opens a new paradigm of functional oligosaccharides, and offers CNN as a novel hypopigmenting molecule and organelle modulator.

In vivo leukemogenic potential of an interleukin 7 receptor α chain mutant in hematopoietic stem and progenitor cells.

Somatic gain-of-function mutations in interleukin 7 receptor α chain (IL7Rα) have been described in pediatric T and B acute lymphoblastic leukemias (T/B-ALLs). Most of these mutations are in-frame insertions in the extracellular juxtamembrane-transmembrane region. By using a similar mutant, a heterozygous in-frame transmembrane insertional mutation (INS), we validated leukemogenic potential in murine hematopoietic stem/progenitor cells, using a syngeneic transplantation model. We found that ectopic expression of INS alone in hematopoietic stem/progenitor cells caused myeloproliferative disorders, whereas expression of INS in combination with a Notch1 mutant led to the development of much more aggressive T-ALL than with wild-type IL7Rα. Furthermore, forced expression of INS in common lymphoid progenitors led to the development of mature B-cell ALL/lymphoma. These results demonstrated that INS has significant in vivo leukemogenic activity and that the lineage of the resulting leukemia depends on the developmental stage in which INS occurs, and/or concurrent mutations.

miR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg.

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4(+)CD8(+) phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3(+) natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.

A thymus-specific noncoding RNA, Thy-ncR1, is a cytoplasmic riboregulator of MFAP4 mRNA in immature T-cell lines.

BACKGROUND: Postgenomic transcriptome analyses have identified large numbers of noncoding (nc)RNAs in mammalian cells. However, the biological function of long ncRNAs in mammalian cells remains largely unknown. Our recent expression profiling of selected human long ncRNAs revealed that a majority were expressed in an organ-specific manner, suggesting their function was linked to specific physiological phenomena in each organ. We investigated the characteristics and function of ncRNAs that were specifically expressed in the thymus, the site of T-cell selection and maturation. RESULTS: Expression profiling of 10 thymus-specific ncRNAs in 17 T-cell leukemia cell lines derived from various stages of T-cell maturation revealed that HIT14168 ncRNA, named Thy-ncR1, was specifically expressed in cell lines derived from stage III immature T cells in which the neighbouring CD1 gene cluster is also specifically activated. The Thy-ncR1 precursor exhibited complex alternative splicing patterns and differential usage of the 5' terminus leading to the production of an estimated 24 isoforms, which were predominantly located in the cytoplasm. Selective RNAi knockdown of each Thy-ncR1 isoform demonstrated that microfibril-associated glycoprotein 4 (MFAP4) mRNA was negatively regulated by two major Thy-ncR1 isoforms. Intriguingly, the MFAP4 mRNA level was controlled by a hUPF1-dependent mRNA degradation pathway in the cytoplasm distinct from nonsense-mediated decay. CONCLUSIONS: This study identified Thy-ncR1 ncRNA to be specifically expressed in stage III immature T cells in which the neighbouring CD1 gene cluster was activated. Complex alternative splicing produces multiple Thy-ncR1 isoforms. Two major Thy-ncR1 isoforms are cytoplasmic riboregulators that suppress the expression of MFAP4 mRNA, which is degraded by an uncharacterized hUPF1-dependent pathway.

Novel mechanisms of suppressor activity exhibited by cytotoxic regulatory T cell lines, HOZOT.

OBJECTIVE: Regulatory T (Treg) cells, which play a central role in maintaining immune tolerance, can be grouped into different subtypes, such as naturally occurring Treg, type-1 T regulatory, and Th3. The suppressor activities of Treg cells are mediated through several molecular mechanisms, including immunosuppressive cytokines, cell surface molecules, and cytolytic molecules. In a previous report, we described a novel regulatory human T-cell line (termed HOZOT). The line was established by cocultivating human umbilical cord blood cells with mouse stromal cells in the absence of exogenous cytokines. In this study, we investigated the mechanism of HOZOT's suppressor activity. MATERIALS AND METHODS: Suppressor activity of HOZOT was evaluated in vitro by assessing their inhibition of allogeneic mixed lymphocyte reaction, in which CD4+CD25(-) responder T cells were stimulated by dendritic cells (DCs). Responder T cells as well as DCs were prepared from umbilical cord blood using magnetic-activated cell sorting separation system. DNA microarray analysis was performed to search for specific molecules involved in HOZOT's suppressor mechanisms. RESULTS: We confirmed that suppressing effects were observed in all three subpopulations of CD4/CD8 phenotype. We ruled out possible involvement of HOZOT's cytotoxic activity as well as participation of surface molecules, including cytotoxic T-lymphocyte-associated protein-4, programmed death-1, and glucocorticoid-inducible tumor necrosis factor receptor in suppressor. The supernatant obtained from HOZOT and DC coculture revealed mixed lymphocyte reaction inhibitory activity, indicating the presence of a soluble factor, which mediates suppressor function. Blocking experiments demonstrated that interleukin-10 and transforming growth factor-beta were not responsible factors. CONCLUSIONS: HOZOT exerted suppressor activity in the absence of cell contact mechanisms, which are distinct from those of naturally occurring Treg, type-1 T regulatory, and Th3.

The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus.

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

Comprehensive analysis of FOXP3 mRNA expression in leukemia and transformed cell lines.

Studies of FOXP3 expression have thus far focused on T cells, including both normal and malignant T cells. In particular, adult T cell leukemia/lymphoma (ATLL) cells have been studied intensively because their phenotype resembles that of normal CD4(+)CD25(+) regulatory T (Treg) cells. However, a comprehensive study of FOXP3 expression covering all hematopoietic cell lineages has not yet been performed. In this study, FOXP3 mRNA expression was examined by quantitative PCR using a large collection of human hematopoietic cell lines derived from leukemia/lymphoma or virus-transformation, including cells lines with T, B, plasmacytoid, myeloid, monocytic, megakaryocytic, erythroid, and NK lineages. Unexpectedly, we found FOXP3 mRNA expression in a number of cell lines belonging to all of the cell lineages investigated. In sharp contrast, FOXP3 protein expression was found in only three cell lines, all of which were HTLV-I-infected. Several non-T cell lines expressed higher levels of mRNA but were still negative for protein expression. The broad mRNA expression contrasts with the restricted protein expression of FOXP3 in human hematopoietic cell lines, suggesting that post-transcriptional control mechanisms may control FOXP3 protein expression.

IL-2 activation of STAT5 enhances production of IL-10 from human cytotoxic regulatory T cells, HOZOT.

OBJECTIVE: Interleukin (IL)-10 is an immunosuppressive cytokine produced by many cell types, including T cells. We previously reported that a novel type of regulatory T (Treg) cells, termed HOZOT, which possesses a FOXP3+CD4+CD8+CD25+ phenotype and dual suppressor/cytotoxic activities, produced high levels of IL-10. In this study, we examined the mechanisms of high IL-10 production by HOZOT, focusing on Janus activating kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway. MATERIALS AND METHODS: We prepared five different types of T cells, including HOZOT from human umbilical cord blood. Cytokine productions of IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were compared among these T cells after anti-CD3/CD28 antibody stimulation in the presence or absence of IL-2. Specific inhibitors for JAK/STAT, nuclear factor-kappaB (NF-kappaB), and nuclear factor for activated T cell (NFAT) were used to analyze signal transduction mechanisms. RESULTS: IL-10 production by HOZOTs was greatly enhanced by the addition of IL-2. Little or no enhancement of IFN-gamma and TNF-alpha production was observed under the same conditions. The enhancing effect of IL-2 was specific for both HOZOT and IL-10-secreting Treg cells. T helper type 2 cells, whose IL-10 production mechanisms involve GATA-3, failed to show IL-2-mediated enhancement of IL-10. Similar enhancing effects of IL-15 and IFN-alpha suggested a major role of JAK/STAT activation pathway for high IL-10 production. Further inhibitor experiments demonstrated that STAT5 rather than STAT3 was critically involved in this mechanism. CONCLUSION: Our results demonstrated that IL-2 selectively enhanced production of IL-10 in HOZOT primarily through activation of STAT5, which synergistically acts with NF-kappaB/NFAT activation, implying a novel regulatory mechanism of IL-10 production in Treg cells.

IL-2-independent generation of FOXP3(+)CD4(+)CD8(+)CD25(+) cytotoxic regulatory T cell lines from human umbilical cord blood.

OBJECTIVE: Since the existence of mouse naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells was demonstrated, a variety of human Treg subsets have been identified as distinct T cell populations. Here we show the establishment of novel Treg cell lines possessing unique characteristics. METHODS: Novel Treg cell lines, designated HOZOT, were generated by coculturing human umbilical cord blood cells with mouse stromal cell lines in the absence of exogenous IL-2 or other cytokines. HOZOT were characterized and compared with CD4(+)CD25(+) Treg cells in terms of the CD phenotype, FOXP3 expression, suppressor activity against allogeneic MLR, anergy property, and IL-10 production. RESULTS: HOZOT were generated and expanded as normal lymphoblastoid cells with cytotoxic activity against the cocultured stromal cells. HOZOT consisted of three subpopulations as defined by phenotype: CD4(+)CD8(+), CD4(+)CD8(dim), and CD4(-)CD8(+). All three subpopulations showed both suppressor and cytotoxic activities. While HOZOT's expression of FOXP3, CD25, GITR, and cytoplasmic CTLA-4 implied a similarity to naturally occurring CD4(+)CD25(+) Treg cells, these two Treg cells differed in IL-2 responsiveness and IL-10 production. CONCLUSIONS: Our studies introduce a new method of generating Treg cells in an IL-2-independent manner and highlight a unique Treg cell type with cytotoxic activity and a phenotype of FOXP3(+)CD4(+)CD8(+)CD25(+).

Transcription factor expression in B-cell precursor-leukemia cell lines: preferential expression of T-bet.

A number of transcription factors (TFs) have been reported that play crucial roles in hematopoiesis. However, only little is known about how these factors are involved in the mechanisms of hematopoietic development and lineage commitment. To investigate the roles of TFs in human B-cell precursors (BCPs), the present study analyzed the expression of the following 16 hematopoietic TFs: AML1, C/EBPalpha, C/EBPbeta, C/EBPgamma, C/EBPepsilon, E2A, Ets-1, GATA-1, GATA-2, GATA-3, Ikaros, IRF-1, Pax5, PU.1, T-bet and TCF-1 in 30 human BCP-leukemia cell lines. All BCP-leukemia cell lines were found to be positive for the expression of AML1, C/EBPgamma, E2A, Ets-1, IRF-1, Pax5 and PU.1 at the mRNA level. The mRNA expression of C/EBPalpha, C/EBPbeta, C/EBPepsilon, GATA-2, Ikaros, T-bet and TCF-1 was detected in 2 to 29 of the cell lines. Eight BCP-cell lines showed positivity for the dominant negative Ikaros isoform Ik6, while others were positive for expression of Ik1, 2, 3 and 4. GATA-1 and GATA-3 were universally negative. The expression of C/EBPalpha, PU.1 and T-bet was positive at the protein level in five, 29 and four out of 30 BCP-cell lines, respectively. Cell lines were stimulated with interleukin (IL)-7 and/or interferon (IFN)-gamma to investigate the regulation of TF expression. T-bet was clearly induced in the two cell lines NALM-19 and NALM-29 after stimulation. C/EBPbeta and IRF-1 were up-regulated in both cell lines and TCF-1 was down-regulated in NALM-19. No significant changes were observed for the other 12 TFs. The present report could provide useful information in the study of the role of TFs on normal and malignant human BCPs.

Acute myeloid leukemia cell lines MOLM-17 and MOLM-18 derived from patient with advanced myelodysplastic syndromes.

The two acute myelomonocytic leukemia sister cell lines MOLM-17 and MOLM-18 and the Epstein-Barr-virus positive non-malignant B-lymphoblastoid cell lines (B-LCLs) B422 and B423 were established from the bone marrow sample of a 60-year-old Japanese male in the advanced leukemic phase of refractory anemia with excess of blasts, a subtype of myelodysplastic syndromes (MDS). MOLM-17/-18 are proliferatively responsive to the growth factors present in the culture supernatant of the 5637 cell line. The B-LCLs are constitutively growth factor-independent. MOLM-17 and B422 were established at eight months after the initial diagnosis, while MOLM-18 and B423 were derived from a sample one month later. Immunophenotyping of the first leukemia sample revealed a mixed lineage leukemia immunophenotype with positivity for terminal deoxynucleotidyl transferase (TdT), CD13 and CD19; the second sample revealed solely myeloid characteristics with positivity for CD13, CD41 and CD61, whereas TdT was negative. MOLM-17/-18 showed immunomarker profiles typical of the myelomonocytic lineage. The karyotype analysis of MOLM-17/-18 revealed various non-random numerical and structural abnormalities including del(5)(q?), -7, der(11)add(11)(p11.2)add(11)(q23), add(17)(p11.2), add(18)(p11.2), -20, -22 as common aberrations. Treatment with tumor necrosis factor-alpha induced pronounced cellular differentiation of both cell lines into macrophage-like cells. The overall profile of MOLM-17/-18 based on their extensive immunological, cytogenetic and functional characterization suggests that these cell lines together with the paired B-LCLs B422 and B423 may represent scientifically significant in vitro models which could facilitate investigations into the pathobiology of MDS.

5-Azacytidine supports the long-term repopulating activity of cord blood CD34(+) cells.

DNA methylation plays important roles in a wide range of biological phenomena, especially in the embryonic development and tumorigenesis. However, correlations between differentiation and DNA methylation have not been clarified well in each differentiation system. In this study, we focused our attention on regulatory roles of DNA methylation in normal hematopoietic differentiation using a demethylating reagent, 5-azacytidine (5-AzaC). As a source of hematopoietic progenitor cells, we used CD34(+) cells prepared from human umbilical cord blood and examined the effects of 5-AzaC on the colony-forming activity and the long-term culture-initiating (LTC-IC) activity of these cells. 5-AzaC treatment increased LTC-IC frequency 1.57- to 2.50-fold as compared to the nontreated control. In parallel to this, immunoblotting analysis showed that the intensity of overall DNA methylation decreased after 5-AzaC treatment. These results indicated the involvement of DNA methylation and demethylation in controlling immaturity of hematopoietic progenitor cells and the usefulness of 5-AzaC for regulating this immaturity.

Transcription factor expression in cell lines derived from natural killer-cell and natural killer-like T-cell leukemia-lymphoma.

Although a number of transcription factors (TFs) have been identified that play a pivotal role in the development of hematopoietic lineages, only little is known about factors that may influence development and lineage commitment of natural killer (NK) or NK-like T (NKT)-cells. Obviously to fully appreciate the NK- and NKT-cell differentiation process, it is important to identify and characterize the TFs effecting the NK- and NKT-cell lineage. Furthermore, these TFs may play a role in NK- or NKT-cell leukemias, in which the normal differentiation program is presumably disturbed. The present study analyzed the expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3, IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK-cell lines together with 5 malignant NKT-cell lines, 5 T-cell acute lymphoblastic leukemia (ALL) cell lines including 3 gamma/delta T-cell receptor (TCR) type and 2 alpha/beta TCR type, and 3 B-cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1, IKAROS and IRF1 were found to be positive for all cell lines tested whereas GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative for all cell lines tested except in the three positive BCP-cell lines. GATA2 was positive for 3/5 T-cell lines but negative for the other cell lines. GATA3 was positive for 7/7 NK-, 4/5 NKT-, 5/5 T- and 2/3 BCP-cell lines. TBET was positive for all NK- and NKT-cell lines and negative for all T- and BCP-cell lines except one BCP-cell line. In contrast to the expression of TBET, TCF1 was negative for all NK- and NKT-cell lines, being positive for 4/5 T- and 1/3 BCP-cell lines. Expression analysis of TFs revealed that NK- and NKT-cell lines showed identical profiles, clearly distinct from those of the other T-ALL or BCP-ALL leukemia-derived cell lines..

Induction of CD28 on the new myeloma cell line MOLP-8 with t(11;14)(q13;q32) expressing delta/lambda type immunoglobulin.

The novel multiple myeloma (MM) cell line MOLP-8 carrying the t(11;14) (q13;q32) was established from the peripheral blood of a 52-year-old Japanese male patient with Bence-Jones delta/lambda type MM (stage IIIA with hyperammonemia). The growth of MOLP-8 cells is constitutively independent of exogenous growth factors or feeder cells. MOLP-8 cells grow mainly as free floating single cells and slightly adherent on the bottom of the plastic culture flask. Wright-Giemsa-stained MOLP-8 cells show the typical plasma cell morphology with abundant cytoplasm, heterogeneous cell size and one to three nuclei. The immunoprofile of MOLP-8 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) delta/lambda chains, CD10, CD29, CD38, CD40, CD44, CD49b, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. CD28 became positive after co-culture of MOLP-8 cells with bone marrow adherent stromal (BST) feeder cells for a week. About 30% of MOLP-8 cells adhered strongly to the BST cells, but the cellular adhesion was clearly inhibited by addition of either anti-CD29 or anti-CD106 monoclonal antibody, suggesting a specific cellular adhesion through alpha4beta1-integrin-VCAM-1 interaction. The novel MOLP-8 cell line together with the present myeloma cell lines will present useful model systems in the investigation of the biology of MM.

A novel ALL-L3 cell line, BALM-25, expressing both immunoglobulin light chains.

A human acute lymphoblastic leukemia (ALL)-derived cell line, BALM-25, was established from the bone marrow specimen of a 59-year-old male patient with B-cell ALL L3 type (ALL-L3) at diagnosis. Immunophenotyping indicated mature B-cell characteristics including expression of cell surface and cytoplasmic immunoglobulin (Ig) chains, CD10, CD19, CD20, CD38, CD39, CD40, CD71, NU-B1 and HLA class II. T-cell and myeloid associated antigens tested were negative except CD5. BALM-25 cells have a morphological appearance typical for L3-type lymphoblasts. Regarding the expression of Ig chains, while the original leukemia cells expressed Ig lambda delta mu and hence a single light (L) chain isotype, the established line revealed double L chain expression both at the cell surface and the cytoplasmic level. Definitive double L chain expression was confirmed by flow cytometry and Western blot analysis. Southern blot analysis demonstrated rearrangement of the IgJH, the Ckappa and the Clambda genes. Cytogenetic analysis of BALM-25 revealed the following numerical and structural abnormalities: 55, X, add(X)(q12), + 2, add(3)(p21), + 5, add(7)(p13), add(11)(p11.2), add(11)(q?23), add(12)(p11.2), add(14)(q22), - 15, + 16, + 16,add(18)(11.2), + 20, + marl, + mar2, + mar3, + mar,inc. The established cell line, BALM-25, provides an unlimited supply of cell material for analyzing the unique (patho)physiology of Ig expression in general and for clarifying the pathogenesis of this type of B-cell malignancy in particular.

NF-ATc2 induces apoptosis in Burkitt's lymphoma cells through signaling via the B cell antigen receptor.

Cross-linking of the B cell antigen receptor (BCR) with an anti-IgM antibody has been shown to induce dramatic apoptosis in type I Burkitt's lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation-induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF-AT. In response to BCR cross-linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF-ATc2, a subtype of NF-AT members. Interestingly, nuclear-imported NF-ATc2 functioned pro-apoptotically in BL cells. The effect of NF-ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addition, TR3 induction during BCR cross-linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF-AT. This strongly suggests that activation of NF-ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR-mediated apoptosis. Therefore, NF-ATc2 plays a crucial role in BCR-mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling.

Transforming growth factor-beta(1) augments granulocyte-macrophage colony-stimulating factor-induced proliferation of umbilical cord blood CD34(+) cells with an associated tyrosine phosphorylation of STAT5.

OBJECTIVE: Several investigators have reported that transforming growth factor (TGF)-beta(1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically support cell proliferation. However, the mechanisms involved have not been elucidated. To clarify the mechanisms of the synergistic action of TGF-beta(1) and GM-CSF, we compared the activation states of STAT5 and mitogen-activated protein kinase in CD34(+) cells and in GM-CSF-dependent hematopoietic cell lines. MATERIALS AND METHODS: Human CD34(+) cells and GM-CSF-dependent cell lines (FKH-1, YNH-1, and M-07e) were stimulated with 1.25 ng/mL GM-CSF and/or 0.25 ng/mL TGF-beta(1), and 1.25 ng/mL GM-CSF and/or 0.25 ng/mL, 0.025 ng/mL TGF-beta(1), respectively, and cell proliferation was analyzed by [3H]thymidine uptake. Expression of signal transduction proteins and their phosphorylation states were determined by Western blotting. RESULTS: TGF-beta(1) synergistically enhanced the GM-CSF-augmented growth of CD34(+) cells and FKH-1 cells, but inhibited the growth of YNH-1 and M-07e cells. Tyrosine phosphorylation of STAT5 induced by GM-CSF was enhanced by stimulation with the combination of TGF-beta(1) and GM-CSF (TGF-beta(1)/GM-CSF) compared with that induced by GM-CSF alone in CD34(+) cells and FKH-1 cells. However, combinations of TGF-beta(1)/GM-CSF caused inhibition of GM-CSF-induced tyrosine phosphorylation in M-07e cells. No significant difference was observed in mitogen-activated protein kinase activation between CD34(+) cells and FKH-1 cells stimulated with GM-CSF/TGF-beta(1) or GM-CSF alone. CONCLUSIONS: Results suggest that TGF-beta(1) may augment GM-CSF-induced proliferation of CD34(+) cells in association with enhanced tyrosine phosphorylation of STAT5. Our data suggest a novel mechanism for the synergistic enhancement of cellular growth induced by the combination of TGF-beta(1) and GM-CSF.

CD45 tyrosine phosphatase inhibits erythroid differentiation of umbilical cord blood CD34+ cells associated with selective inactivation of Lyn.

CD45 is a membrane-associated tyrosine phosphatase that dephosphorylates Src family kinases and Janus kinases (JAKs). To clarify the role of CD45 in hematopoietic differentiation, we examined the effects of anti-CD45 monoclonal antibody NU-L(PAN) on the proliferation and differentiation of umbilical cord blood CD34(+) cells. NU-L(PAN) showed a prominent inhibition of the proliferation of CD34(+) cells induced by the mouse bone marrow stromal cell line MS-5 or erythropoietin (EPO). However, NU-L(PAN) did not affect the proliferation induced by interleukin 3. NU-L(PAN) also inhibited MS-5-induced or EPO-induced erythroid differentiation of CD34(+) cells. The cells stimulated with EPO in the presence of NU-L(PAN) morphologically showed differentiation arrest at the stage of basophilic erythroblasts after 11 days of culture, whereas the cells treated with EPO without NU-L(PAN) differentiated into mature red blood cells. The Src family kinase Lyn and JAK2 were phosphorylated when erythroblasts obtained after 4 days of culture of CD34(+) cells in the presence of EPO were restimulated with EPO. Overnight NU-L(PAN) treatment before addition of EPO reduced the phosphorylation of Lyn but not that of JAK2. Simultaneously, the enhancement of Lyn kinase activity after restimulation with EPO was reduced by NU-L(PAN) treatment. These results indicate selective inactivation of Lyn by CD45 activated with NU-L(PAN) and could partly explain the inhibitory mechanism on erythropoiesis exhibited by EPO. These findings suggest that CD45 may play a pivotal role in erythropoiesis.