Proposal for the designation of the natural killer antigens-positive γδ T-cell subset as γδ NKT-cells: nomenclature based on immunoprofile.

Natural killer T (NKT)-cells with both T- and NK-cell antigens can be classified into αß or γδ type according to the TCR gene expression. The WHO classification of lymphoid neoplasms did not further subdivide the above-mentioned NKT-cell malignancies according to the expression of these TCR types. γδ T-cells can be stimulated and expanded by Zoledronic acid, usually carrying Vγ9 Vδ2 TCR and various NK-associated receptors (NKR) such as CD56, CD94, CD158a, CD158b, CD161, etc. In contrast, αß T-type NKT-cells are positive for Vα24 Vß11 TCR. NKR positive γδ T-cells have clearly different features than the NKT-cells with Vα24 Vß11 TCR type, αß NKT. NKT-cells carrying γδ TCR should be classified and named as γδ NKT-cells to distinguish the cells explicitly from αß NKT-cells.

Isolation of adipose tissue-derived stem cells by direct membrane migration and expansion for clinical application.

Mesenchymal stem cells (MSCs) have recently made significant progression in multiple clinical trials targeting several clinical disorders and in the modulation of immune responses. In the present study, we isolated human adipose tissue-derived stem cells (ADSCs) by direct membrane migration method without using enzymatic digestion via collagenase, and tried to extract adequate number of cells for clinical application. Hydroxyapatite-treated nonwoven fabric membrane made up of synthetic macromolecular fiber materials, polyethylene and polyester terephthalene was used. Expansion culture of ADSCs having plastic flask adherent characteristic in serum-free condition was successfully established, and adequate number of cells were obtained for clinical application. They were found to be positive for CD44, CD73, CD90 and CD105 and negative for CD11b, CD34, CD45, CD80 and HLA-DR. The resulting immunological marker profile satisfied the immunophenotype of previously reported MSCs. Also, microscopic findings demonstrated trilineage differentiation into adipogenic, osteogenic and chondrogenic cells as the characteristics of MSCs. The isolation by nonwoven fabric membrane and expanded cells under serum-free condition satisfied the criteria of MSCs, as proposed by the International Society for Cellular Therapy. Our direct membrane migration method without enzyme digestion is useful as ADSCs can be obtained from small pieces of adipose tissue and expanded under serum-free culture condition. This method was considered to be feasible for clinical application.

Multiple splicing variants of Naf1/ABIN-1 transcripts and their alterations in hematopoietic tumors.

Naf1 (Nef-associated factor 1)/TNIP1/ABIN-1 (A20-binding inhibitor of NF-kappaB activation) is a cellular protein that interacts and cooperates with the NFkappaB inhibiting protein A20. It is reported that Naf1 attenuates epidermal growth factor (EGF)/extracellular-signal-regulated kinase2 (ERK2) nuclear signaling. Naf1 also binds to Nef, which plays a key role in acquired immunodeficiency syndrome pathogenesis and HIV-1 virus replication. Naf1 mRNA consists of 18 exons and multiple splice variants have been reported; two isoforms for exon 1, deletion of exon 2 and isoforms alpha and beta for exon 18. Using specimens from 29 acute myeloid leukemia (AML) patients, we detected a high frequency of allelic loss on DNA at STS marker D5S2014 near the Naf1 gene. We therefore performed mutation and expression analyses using leukemia-lymphoma lines and 6 pairs of clinical AML samples. There was no mutation in the Naf1 coding region of any sample. As a result of expression analysis, we identified novel splice variants of the Naf1 gene; deletion of exon 16 (Naf1 alpha2, Naf1 beta2), deletion of exon 16 with an insertion (Naf1 alpha3, Naf1 beta3) and deletion of exons 16 and 17 (Naf1 alpha4). Naf1 alpha3 and beta3 showed premature termination. In peripheral blood mononucleocytes (PBMNCs) from healthy adults, almost no expression of full-length Naf1 (Naf1FL), Naf1 alpha3 and beta3 were observed. In contrast, their expression was clear in AML blasts and in the majority of leukemia-lymphoma lines investigated. Naf1 alpha2 was widely expressed in PBMNCs from healthy adults, AML blasts and cell lines, suggesting it is the main transcript of the Naf1 gene. Luciferase assay revealed that Naf1 alpha2 had equal NF-kappaB inhibitory effect to that of Naf1FL, while Naf1 alpha4 was less effective. In clinical AML patients, the expression of Naf1 alpha3 was much higher at diagnosis than on remission after chemotherapy, suggesting the possible dominant negative effect of Naf1 alpha3.

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.

Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia.

OBJECTIVE: In previous studies, we demonstrated overexpression of the dominant-negative isoform of the transcription factor Ikaros, Ik-6, in patients with blast crisis of chronic myelogenous leukemia and B-cell acute lymphoblastic leukemia. In the present study, we analyzed expression of the Ikaros family genes Ikaros, Aiolos, and Helios in a panel of human T-cell leukemia/lymphoma cell lines and bone marrow samples of patients with T-cell acute lymphoblastic leukemia. MATERIALS AND METHODS: We performed reverse transcriptase polymerase chain reaction, sequencing analysis, immunoblotting, and Southern blotting. RESULTS: We found overexpression of novel short isoforms of Helios (Hel-5 and Hel-6) in the HD-Mar cell line. Southern blot analysis suggested that there might be a small deletion in the Helios locus of HD-Mar. In addition, we observed decreased expression of more than one Ikaros family gene in 3 of 9 patients with T-cell acute lymphoblastic leukemia. Moreover, one of the patients overexpressed novel short isoforms of Helios (Hel-7 and Hel-8). CONCLUSION: This study provides the first evidence of an Ikaros family member (other than Ikaros) of which novel short isoforms become overexpressed in human leukemia.

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.

Immunoprofiling of cell lines derived from natural killer-cell and natural killer-like T-cell leukemia-lymphoma.

T-cells and natural killer (NK)-cells can be distinguished by their immunophenotype and molecular biological studies though there is overlap in T- and NK-cell antigen expression, function, and malignant diseases. The relatively new cell type of NKT-cells (also termed NK-like T-cells) represents a subpopulation of T-cells that share some characteristics with NK-cells. T- and NKT-cells have their T-cell receptor (TCR) genes rearranged while NK-cells are identified molecularly and immunologically by the absence of TCR gene rearrangements and TCR protein and lack of certain surface antigens. Various continuous malignant cell lines have been derived from patients with T-cell, NK- and NKT-cell neoplasms. These cell lines possess several traits typical of the respective diseases. Characterization of these cell lines which was the objective of this study will facilitate future studies of cell biology and therapeutics for which cell lines are indispensable models. In view of the imprecision of morphological criteria alone, we analyzed a series of seven NK-cell, five NKT-cell and five T-cell lines using functional and immunophenotypic tools. All T-cell lines were negative for the presence of azurophilic granules, NK activity and Epstein-Barr virus (EBV). In contrast, 7/7 NK-cell and 4/5 NKT-cell lines displayed the azurophilic granules but only three of these combined twelve NK/NKT-cell lines showed significant NK activity which may be explained by the functional immaturity of the cells. EBV was found in 5/7 NK-cell and in 1/5 NKT-cell lines. As expected, T-cell lines were commonly positive for T-cell surface antigens and negative for NK-cell markers, and NK-cell lines vice versa; nevertheless, a number of immunomarkers were shared between T- and NK-cell lines. NKT-cell lines express T-cell, NK-cell and markers shared between T- and NK-cells. Sets of markers distinctive for the three types of cell lines are presented. The composite data gained on the present panels of cell lines allow for the operational definition of typical NK- and NKT-cell line profiles. Such cell lines will prove invaluable as informative models for studies of normal and neoplastic NK- and NKT-cell biology.

Malignant hematopoietic cell lines: in vitro models for the study of erythroleukemia.

A panel of leukemia cell lines has been assembled over the last 30 years representing a spectrum of erythroid cells arrested at various stages of differentiation. The oldest cell line is K-562 which is one of the most prolific in use. Most cell lines have been established from acute myeloid leukemia M6 or chronic myeloid leukemia in blast crisis and generally express immunoprofiles typically seen in immature erythroid cells. Several cell lines are constitutively growth factor-dependent, responding proliferatively to a variety of cytokines. The predominant cytogenetic abnormalities are the t(9;22)(q34;q11) found exclusively in CML-derived cell lines, and rearrangements of chromosomes 5 and 7 which occur in all disease subtypes. Ph+ve cell lines consistently displayed structural and numerical changes associated with disease evolution, including +8, -17/17p-/i(17q), and +19. It is striking that many cell lines though committed to either the erythroid or megakaryocytic lineage tend to co-express features of the other lineage, consistent with the concept of a common erythroid-megakaryocytic progenitor. Several cell lines may be induced to differentiate along the erythroid, megakaryocytic or monocytic pathway by treatment with pharmacological or physiological reagents. Notable functional features include expression of various globin chains or the complete hemoglobins as erythroid attributes. Taken together, this class of cell lines is relatively well characterized and affords useful model systems for immature erythroid cells.

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 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.

Expression of activation-induced cytidine deaminase (AID) in Burkitt lymphoma cells: rare AID-negative cell lines with the unmutated rearranged VH gene.

Activation-induced cytidine deaminase (AID) is an enzyme that catalyzes somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) gene. The expression of AID was reported to be confined to the germinal center (GC). Burkitt lymphoma (BL) cells are regarded as being derived from GC cells. BL cells have been reported to have SHM in the Ig gene with variety in terms of degree, antigen selection and ongoing mutation characteristic. We investigated the expression of AID in 15 BL cell lines by RT-PCR. In only 2 BL cell lines, Tree92 and Black93, AID expression was not detected, and the 1gVH gene of these 2 cell lines was not mutated. Tree92 expresses terminal deoxy-nucleotidyl transferase (TdT) and recombination activating gene (RAG)1, but Black93 does not, as is typical of the BL phenotype. BL cells are generally derived from GC B-cell expressing AID, but are rarely derived from the stage of B-lineage differentiation in which AID is not yet expressed, causing the absence of mutation in the IgVH.

Persistent use of false myeloma cell lines.

Multiple myeloma (MM) is a neoplasm of a terminally differentiated B-cell. Human myeloma cell lines were shown to be suitable model systems for use in various fields of the biological sciences. Within the last 20 years more than 100 cell lines have been established. So-called 'myeloma cell lines' have been previously reported and are still widely used which are in reality Epstein-Barr virus (EBV)-positive B-lymphoblastoid cell lines. The presence of the EBV-genome in residual normal B-cells provides them with a selective growth advantage after explantation. Cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. On closer examination, the use of false cell lines may be seen to invalidate a significant percentage of scientific work, or at least cast doubts on the relevance of these in vitro results to the cell type or tumor in vivo. Ultimately, use of cross-contaminated cell lines is a waste of human and material resources. Henceforth, it should be mandatory to prove the proper derivation of each new cell line by comparing DNA fingerprints or karyotypes of the patient's primary cells and the cultured cells. The availability of well characterized and authenticated bona fide MM cell lines is of great importance for the study of the biology, etiology and treatment of the disease.

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.