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(+).

Delta-4 expression on a stromal cell line is augmented by interleukin-6 via STAT3 activation.

OBJECTIVE: Notch receptors and their ligands are known to play an important role in hematopoietic cell fate decisions. Although expressions of Notch ligands were frequently detected in bone marrow cells or thymic epithelial cells, regulatory roles of hematopoietic cytokines on their expression are still poorly understood. In this study, we focused on a new member of Notch ligand family, Delta-4, and analyzed regulatory mechanisms of Delta-4 expression by cytokines using stromal cell lines. METHODS: For our expression study, we selected a highly Delta-4-expressing murine stromal cell line, SC9-19. Delta-4 protein expression was analyzed by Western blotting after cytokine treatment with or without various kinase inhibitors. Biologic relevance of the enhanced Delta-4 expression was examined in the coculture system using cord blood CD34(+) cells. RESULTS: When SC9-19 was treated with different cytokines, we found interleukin-6 (IL-6) was the most efficient inducer of Delta-4 protein expression. Further analysis revealed that IL-6-induced signaling for Delta-4 expression was transduced through the pathway of Janus kinase/signal transducers and activators of transcription-3 (JAK/STAT3). Other mediators such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and Src tyrosine kinase were not involved in IL-6-induced Delta-4 expression. Erythroid differentiation of CD34(+) cells was enhanced by IL-6 treatment of the Delta-4-expressing original stromal cell line but not of a Delta-4-knockdown stromal cell line. CONCLUSIONS: IL-6 augments Delta-4 expression in the stromal cell line via STAT3 activation. This study provides a novel mechanism for augmentation of Delta-4 expression by hematopoietic cytokine and suggests a role for Delta-4 in the control of hematopoiesis.

Erythropoietin induces sustained phosphorylation of STAT5 in primitive but not definitive erythrocytes generated from mouse embryonic stem cells.

OBJECTIVE: During embryonic development murine erythropoiesis occurs in two waves by producing first primitive erythroid cells (EryPs) and then definitive erythroid cells (EryDs). Erythropoietin (EPO) signaling is compared between EryPs and EryDs. METHODS: We studied the EPO signaling in EryPs and EryDs using an embryonic stem-derived culture system, which can recapitulate this in vivo development process and has thus been used as a convenient in vitro model system of erythropoiesis. RESULTS: We found that EPO induced sustained phosphorylation and nuclear translocation of signal transducer and activator of transcription 5 (STAT5) in EryPs but not EryDs. EryPs expressed dramatically higher amounts of EPO receptor compared with EryDs, indicating there was excessive signaling from the receptor upon EPO stimulation. In addition, reduced expression of tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1, and decreased total phosphatase activity in EryPs partly explain the persistent activation of STAT5. Nevertheless, Janus kinase 2 (JAK2) phosphorylation, which is essential for transduction of EPO signaling from the EPO receptor to STAT5, was observed in a transient but not a persistent manner. Inhibition of JAK activity resulted in partial suppression of transient phosphorylation of STAT5 and no suppression of sustained phosphorylation of STAT5. CONCLUSION: This study presents a unique feature of EryPs, as this is the first known example of sustained activation of STAT5 in normal cells. Our results also imply the existence of a JAK2-independent pathway of EPO signaling to induce STAT5 activation.

Delta-4 Notch ligand promotes erythroid differentiation of human umbilical cord blood CD34+ cells.

OBJECTIVE: Important roles of Notch signaling have been demonstrated in hematopoiesis. In many cases, activation of the Notch pathway leads to the inhibition of differentiation of immature precursors, suggesting a potential role in self-renewal promotion. However, the function of Notch and Notch ligands is not so straightforward because it is considerably dependent on cytokine context. In this study, we analyzed effects of one Notch ligand, Delta-4, whose function is less clear than others, such as Delta-1 and Jagged-1 and -2. METHODS: CD34(+) cells isolated from human umbilical cord blood were cocultured with a Delta-4-expressing murine stromal cell line, SC9-19, and induced to erythroid differentiation by adding stem cell factor and erythropoietin. To examine the involvement of Delta-4, we utilized stromal cell subclones expressing Delta-4 protein at higher or lower level than parental SC9-19 by plasmid transfection. Erythroid maturation was examined by surface phenotype (CD34 and glycophorin A) and cytospin morphology. Recombinant human Delta-4 protein was prepared to analyze direct effects of Delta-4. RESULTS: Under erythroid lineage-inducing conditions, we found that the increase in Delta-4 expression of SC9-19 promoted erythroid differentiation whereas the decrease in Delta-4 expression inhibited it. Morphologic examination as well as colony formation analysis supported this observation. Moreover, the experiment using recombinant Delta-4 provided direct evidence of the Delta-4 activity found in coculture system. CONCLUSIONS: By modifying Delta-4 expression of the stromal cells and using the recombinant protein, we demonstrated that Delta-4 had a differentiation promoting activity for human primitive hematopoietic cells into erythroid lineage.

Progenitor analysis of primitive erythropoiesis generated from in vitro culture of embryonic stem cells.

OBJECTIVE: A variety of hematopoietic lineage cells have been produced from embryonic stem (ES) cells, but their differentiation processes have not been elucidated well, especially from the point of view of progenitor analysis. In this study, we utilized our coculture system, in which ES-derived Flk-1+ cells differentiated into TER-119+ primitive erythroid (EryP) cells on OP9 cells, and looked for progenitors in primitive erythropoiesis. MATERIALS AND METHODS: We studied the kinetics of TER-119+ erythroblast generation from Flk-1+ cells by monitoring the expression of TER-119, CD41, VE-cadherin, CD34, and c-kit antigens. Multicolor analysis was performed to detect CD41+TER-119+ cells and the stained cells were sorted to examine their morphology and EryP-producing potential in colony formation. RESULTS: Kinetic studies showed that the CD41+ population appeared early in the coculture and its expression pattern implied a role as an immediate progenitor of TER-119+ EryP cells. Multicolor analysis and colony-formation study supported this notion. Other progenitor markers such as VE-cadherin, CD34, and c-kit did not seem to define an immediate progenitor of EryP cells. One interesting observation is the detection of unique populations, CD41dim and CD41bright, detectable after 48 hours of the coculture. Majority of the CD41dim population progressed to the EryP lineage, whereas the CD41bright population seemingly advanced on a pathway distinct from the CD41dim population. CONCLUSIONS: CD41 expression was a useful marker to trace hematopoietic progenitors in ES-derived differentiation system. In particular, the CD41dim but not CD41bright population could serve as immediate precursors of EryP cells.

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.

Erythroblasts derived in vitro from embryonic stem cells in the presence of erythropoietin do not express the TER-119 antigen.

OBJECTIVE: In this study, we analyzed murine primitive erythropoiesis by coculturing Flk-1+ ES-derived cells with OP9 to find efficient culture conditions for erythroid cell induction. We utilized a nonserum culture system and EPO (erythropoietin) and found that this cytokine had unique properties. MATERIALS AND METHODS: ES cells (E14.1) were first differentiated to Flk-1+ cells and then cocultured with OP9 stromal cells. BIT9500 was used as a serum replacement. The erythroid morphology, hemoglobin types, and TER-119 expression levels were analyzed. RESULTS: Primitive erythroid cells with embryonic hemoglobin were generated very efficiently when the serum-containing culture was converted to the nonserum system. In this serum-free culture, TER-119+ erythroblasts appeared first on day 2 and maturation proceeded until day 7. When EPO was added to this coculture, the number of induced floating cells increased twofold to threefold. Unexpectedly, the erythroid-specific antigen TER-119 expression of these cells was drastically reduced. Since reduced TER-119 expression is usually interpreted as maturation arrest, we examined the phenotypic features of the EPO-treated cells. We found, however, no evidence of maturation arrest in the aspects of morphology and hemoglobin content. EPO did not suppress TER-119 expression of erythroblasts derived from fetal liver or adult bone marrow. CONCLUSIONS: Our results showed that EPO had the unusual property of inducing TER-119- erythroblasts in ES-derived primitive erythropoiesis. It is likely that this effect is unique to primitive erythropoiesis.

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.

Establishment of novel B-cell precursor leukemia sister cell lines NALM-36 and NALM-37: shift of immunoglobulin phenotype to double light chain positive B-cell.

Two novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) sister cell lines, designated NALM-36 and NALM-37, were established from the peripheral blood (at diagnosis) and bone marrow (at relapse) of a 37-year-old woman with ALL. Immunophenotyping showed BCP type III pre-B cell characteristics including TdT, CD10, CD19, CD22, CD79a and HLA class II. T cell and myeloid-associated antigens tested were negative except CD5 which was 100% positive for both cell lines. The surrogate light chains lambda5 and VpreB were positive for both cell lines. Cytogenetic analysis of NALM-36 revealed an abnormal karyotype with 46, XX, add(1)(q?42), -14, +mar. Southern blot analysis of the immunoglobulin (Ig) genes status of NALM-36 at 10 months after establishment showed germ line configuration of the kappa light chain gene, and rearrangement of the lambda light and mu heavy chain genes. At 16 months we detected a phenotypic shift of Ig chain protein expression from a BCP-III pre-B cell phenotype to a BCP-IV mature B cell phenotype, with kappa and lambda double Ig light chain and mu heavy chain expression, both on the cell surface and in the cytoplasm. We designated this subline as NALM-36KL. Authenticity of the NALM-36KL, NALM-36 and NALM-37 cell lines was demonstrated by DNA fingerprinting. The extensive characterization of the sister cell lines suggests that these three novel cell lines, derived from a single patient, may represent unique and relevant in vitro model systems for BCP-type leukemia cells. They may provide useful models and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.

Establishment of the T-cell large granular lymphocyte leukemia cell line MOTN-1 carrying natural killer-cell antigens.

A novel interleukin-2 (IL-2) dependent leukemia cell line MOTN-1 was established from the peripheral blood of a 63-year-old woman with T-cell large granular lymphocyte (LGL) leukemia in chronic phase. Primary peripheral blood leukemia cells were CD3+, CD5+, CD7+, CD56+, CD94+, CD161+, TcRalphabeta+, and HLA-DR+. The immunoprofile of the established cell line MOTN-1, however, showed CD3-, CD5-, CD7+, CD56+, CD94+, CD159+, CD161+, TcRalphabeta- and HLA-DR+; the MOTN-1 cells were cytoplasmatically positive for CD3varepsilon and the products of the T-cell receptor (TcR) genes beta and gamma. While the TcRbeta and TcRgamma genes were rearranged, the TcRdelta gene was found to be deleted. DNA fingerprinting and chromosome analysis identifying the t(2;6)(q?23;q?21) and t(12;18)(q13;q?22) alterations demonstrated the authenticity and the malignant nature of the cell line. The scientific significance of MOTN-1 lies in (1) the rarity of this type of leukemia cell lines, (2) the co-expression of various T- and natural killer (NK)-cell-associated markers, and (3) its unique chromosomal aberrations.

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.

Megakaryoblastic leukemia cell line MOLM-16 derived from minimally differentiated acute leukemia with myeloid/NK precursor phenotype.

The megakaryoblastic leukemia cell line MOLM-16 was established at relapse from the peripheral blood of a 77-year-old Japanese woman with minimally differentiated acute myeloid leukemia (AML-M0). Immunophenotyping of the fresh leukemic cells revealed a myeloid/NK precursor phenotype being positive for CD7, CD13, CD33, CD34, and CD56. In addition, megakaryocyte-associated antigens CD41 and CD61 were found to be positive. The established cell line designated MOLM-16 was proliferatively responsive to the treatment with various cytokines including EPO, GM-CSF, IL-3, PIXY-321, and TPO. MOLM-16 revealed characteristics of the megakaryocytic lineage in terms of immunophenotyping being positive for CD9, CD31, CD36, CD41, CD61, CD62P, CD63, CD110, CD151, thrombospondin, von Willebrand factor (vWf), and fibrinogen. Electron microscopic analysis showed positivity for ultrastructural platelet peroxidase in the nuclear envelope. The karyotype analysis of MOLM-16 revealed various numerical and structural abnormalities including t(6;8)(q21;q24.3), t(9;18)(q13;q21) and marker chromosomes. The extensive immunological, cytogenetic and functional characterization of MOLM-16 suggests that this cell line may represent a scientifically significant in vitro model which could facilitate the evaluation of megakaryocytic differentiation.

A novel L3-acute lymphoblastic leukemia cell line (BALM-27) carrying cytoplasmic immunoglobulin 8 chain but lacking expression of cell surface immunoglobulin chains.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-27, was established from the peripheral blood specimen of a patient with B-cell ALL L3 type (ALL-L3) at diagnosis. As with the original leukemia cells, the established line was negative for all cell surface immunoglobulin (Ig) chains, but carrying only cytoplasmic Ig delta heavy chain. Southern blot analysis of the various Ig chain genes demonstrated homozygous deletion of the Jkappa gene, germ line configuration of the Jlambda and rearrangement of IgJH genes. Cytogenetic analysis of both primary leukemic bone marrow and BALM-27 cells showed the der(8;15)(q10;q10) chromosomal alteration, in addition to the t(8;22)(q24;q11) abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. The established cell line BALM-27 represents a rich resource of abundant, accessible, and manipulable cell material for analyzing the unique expression of Ig chain and for investigating the pathogenesis of B-cell malignancy. The scientific significance of BALM-27 lies in (1) the rarity of this type of leukemia cell lines, and (2) its unique chromosomal aberrations.

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.

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..

Establishment and characterization of new B-cell precursor leukemia cell line NALM-35.

A novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) cell line, NALM-35, was established from the peripheral blood of a 40-year-old woman at diagnosis of ALL. Imunophenotyping showed BCP type III characteristics including expression of TdT, CD10, CD19, CD22, CD79a and HLA class II. T-cell and myeloid-associated antigens tested were negative except CD5 and CD28. The surrogate light chains CD179a and CD179b were positive. NALM-35 cells have the morphological appearance of lymphoblasts. Cytogenetic analysis of NALM-35 revealed an abnormal karyotype with 46, XX, add(9)(p11). Southern blot analysis of the immunoglobulin genes status of NALM-35 at 10 months after establishment showed germ line configuration of the kappa and lambda light chain genes, and rearrangement of the mu heavy chain gene. DNA fingerprinting, chromosomal analysis and immunophenotyping proved that NALM-35 was clonally derived from the primary leukemia cells. The established cell line may provide a useful model system and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.

Novel B-cell acute lymphoblastic leukemia sister cell lines BALM 19-23 and BALM-26 with interclonal proliferative and phenotypic heterogeneity from a patient with hypercalcemia.

A series of human acute lymphoblastic leukemia (ALL) cell lines, BALM-19, -20, -21, -22, -23 (BALM 19-23) and BALM-26 were established from a patient with B-cell characteristics of ALL L2 type. All cell lines were derived from bone marrow specimens, BALM 19-23 from a sample taken at diagnosisand BALM-26 from one at relapse. Like the original leukemia cells, the established lines present various B-cell characteristics, being positive for cell surface immunoglobulin (Ig) chains but also for nuclear terminal deoxynucleotidyl transferase; hence the cell lines should be assigned to B-cell category B-IV. As a unique feature, the cell lines expressed the CD33 myeloid antigen in addition to the common B-cell markers. Heterogeneous antigen expression among the different cell lines was found regarding CD35, CD39, CD45RA, CD78 and CD95. The malignant nature of the cell lines was documented by negativity for the Epstein-Barr virus and by the occurrence of clonal non-random structural chromosome abnormalities. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which expressed parathyroid hormone related peptide (PTHrP) mRNA as examined by reverse transcriptase polymerase chain reaction. The established B-cell ALL sister cell lines, BALM 19-23 and BALM-26, could provide useful material for clarifying the pathogenesis of this type of B-cell malignancy. The scientific significance of this panel of cell lines lies in the availability of a series of clonally derived but phenotypically different sister cell lines established at different phases of the disease.

ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer.

ICAM-1 plays an important role in cell-cell and cell-extracellular matrix interactions, especially tumor invasion and cytotoxicity of lymphocytes. In the present study, the relationship between metastasis of gastric cancer and ICAM-1 expression by cancer cells or the serum level of s-ICAM-1 was (s-ICAM-1) was examined. ICAM-1 was detected by immunohistochemic staining in 49.0% of 108 patients with gastric cancer. The ICAM-1 expression rate was higher at a more advanced stage, based on lymph node metastasis, being 46.9% in node-negative and 56.1% in node-positive cases. In patients with liver metastasis, the rate was 90.9%, while it was 43.3% in patients without liver metastasis (p < 0.05). The serum s-ICAM-1 level was 262.1 ng/ml (median 205.5, range 176.0-271.0) in healthy subjects and 391.5 ng/ml (median 317.5, range 148.7-1,768.0) in gastric cancer patients (p < 0.001). The serum s-ICAM-1 level was significantly higher in patients with liver metastasis than in patients without liver metastasis (p < 0.0001). In addition, positive ICAM-1 expression cases had significantly higher s-ICAM-1 levels than negative ones, 408.9 +/- 188.4 and 308.1 +/- 88.1 ng/ml, respectively. These results suggested that ICAM-1 was overexpressed in cancer cells and released as s-ICAM-1, which would promote hematogenous metastasis by suppressing local anticancer immunity.