The acid phosphatase (acP) isoenzymes from the blast cells of 102 cases of acute myeloid leukaemia were separated by isoelectric focusing on horizontal polyacrylamide gels. The cases were classified on the basis of the FAB cooperative group criteria. Several single bands were combined into groups (I-IV). An increase in the number of acP isoenzymes was noted which paralleled the assumed maturation along the granulocytic cell lineage from FAB M1 to FAB M3 and along the monocytic cell lineage from FAB M4 to FAB M5. One isoenzyme which was resistant to tartrate inhibition was found in 40% of the monocytic variants FAB M4 and M5, but not in the nonmonocytic cases FAB M1-M3 and M6. This particular isoenzyme, which has been described as being characteristic for hairy cell leukaemia, also appears to be a marker of the monocyte/macrophage system and the respective neoplastic counterparts. The FAB M4 and M5 patients expressed a characteristic profile of group I isoenzymes which allows for the discrimination between monocytic and nonmonocytic cells.

DNA synthesis in polyoma virus-infected mouse embryo cultures was studied by means of autoradiography and correlated cytological and immunofluorescent analyses. In confirmation of previous reports concerning the "transformation" of infected cultures, continuously-growing cell populations were obtained. Nevertheless, these cell populations were found to be merely "polyoma virus carriers," capable of producing parotid tumors after inoculation into newborn mice, but not transplantable as neoplastic cells. The autoradiographic results obtained indicated that de novo synthesis of viral DNA takes place. Increased DNA synthesis in polyoma virus infection was concluded to be a primary response caused by active synthesis of viral DNA. In addition, it was evident that synthesis of host-cell DNA also continued to a certain extent.

The ML cell lines (ML-1, -2, and -3) were derived from the cells of a patient with T-cell malignant lyntphoma who developed acute myeloblastic leukemia and whose cells showed a primary chromosome change at band 1Iq24. Surface marker studies of the ML cells showed that they had both myeloid (MCS-1, MCS-2, and OKM-1) and some T-lymphocyte (3Al/Leu-9 and OKT-4/Leu-3a) characteristics. Molecular studies on these lines were performed in order to determine the possible involvement of the Hu-ets-l gene, since it is located at band 1Iq23—nj24.All ML cell lines showed half the intensity of the Hu-ets-l DNA bands as compared to those of controls (karyotypically normal B-cell lines). In contrast, DNAs from leukemia cells with t(4;llXq21;q23) or t(l;llXq21;q23 or q24) showed no rearrangement, deletion, or amplification of the etx-l gene. 1'hese findings indicate that a chromosome region (Ilq24—»qter), including the Hu-ets-l gene, of the ML cells is deleted as a result of the primary cytogenetic change and that heterogeneity is present in the mechanism of human leukemia involving the Ilq23—»q24 region.

In 1951 at Johns Hopkins University (Baltimore, Maryland, USA), Gey et al. established the first continuously growing human cell line (HeLa) from uterine cervix carcinoma [1]. The HeLa cell line and most other human cell lines subsequently established from various solid tumors adhere to the culture vessel growing in monolayers. In 1963 at the University of Ibadan, Nigeria, Pulvertaft established the first continuous human hematopoietic cell lines, a series of cell lines derived from Nigerian patients with Burkitt lymphoma in a suspension-type cell culture: RAJI is the best known cell line of this panel [2] (Table 1). In suspension cultures, these cells are free-floating, singly or in clusters, in the nutrient medium. Electron microscopic analysis of these and subsequent Burkitt lymphomaderived cell lines led to the identification of herpes-type virus particles which were later designated Epstein-Barr virus (EBV) [4,5]. The first leukemiaderived cell line was thought to be RPMI 6410, established from an American patient with acute myeloid leukemia (AML) containing similar herpes-type virus (EBV) particles in the cells [3]; however, it was shown later that this cell line was derived from normal bystander B-cells immortalized spontaneously by EBV infection and not from the leukemia cells. The etiological significance of EBV for lymphomagenesis was questioned by several findings. Several hundred lymphoblastoid cell lines (LCLs) were established from the peripheral blood of patients with leukemias, lymphomas, other malignant tumors, and even from many healthy individuals [7– 9,60]. However, EBV was detected in every cell line irrespective of the blood 1

The accessory gene, nef, is conserved among HIV-1, HIV-2 strains and SIV [1,2]. Nef, encoded by nef, located in the 3’ portion of the HIV-1 genome overlapping the 3'-long terminal repeat (LTR), is translated at particularly high levels, often accounting for more than 75% of the total viral early proteins [3]. Nef was first found to be released from a human T cell line infected with nef-recombinant vaccinia virus into the culture supernatants [4], although the release of Nef from yeast cells [5] and HIV-1-infected cells [6] has been reported. Extracellular Nef has been shown to bind to the surface of CD4 T cells [7–9] and activated murine T lymphocytes [10]. Nef binding to the cell surface is essential for its biological activities. These data suggest that some Nef receptor (Ner) may localize on the surface of T cells. It has been reported that some proteins were associated with Nef. For example, the 64 000 and 75 000 Mr proteins [11] were identified by an in-vitro kinase assay as phosphoproteins, termed Nef-associated kinases (NAKs); however, these proteins were dispensable for Nef-mediated enhancement of infectivity [12] and CD4 cell downregulation [13], which are representative biological effects of Nef. The unknown 32–35 000 Mr cellular protein was first reported to be associated with HIV-1 Nef by Greenway et al. [14], then also with SIV0mac239 Nef by Flaherty et al.[15]. The 32–35 000 Mr protein has not been well investigated. We cloned the 35 000 Mr human neoprotein (GenBank accession number AB015434) [16], which reacts with 305 monoclonal antibody (MAb). Using FACS analysis, 305 MAb strongly stained the cell surface of T lymphocytes and monocytes. Although we preliminarily tested the cross-reaction between 305 antigen and CD11a (anti-LFA-1), CD21 (anti-B cells), and CD11b (anti-Mac) MAb, no significant cross-reaction was observed (data not shown). The most probable interpretation is that the 35 000 Mr protein specifically presents on the cell surface of T lymphocytes as well as of macrophages. We determined the number of CD4+/305+ cells among peripheral blood mononuclear cells (PBMC) of healthy donors (n = 11) as well as HIV-1-infected patients (n = 14) by FACS. All patients and healthy donors contained 13.3 ± 1.2% of 305+CD4+ and 27.4 ± 1.2% of 305+CD8+ cells, and 16.9 ± 1.9% of 305+CD4+ and 16.2 ± 1.7% 305+CD8+ cells, respectively. The ratio of 305+/CD4+ to 305+/CD8+ cells in patients (0.51 ± 0.05) was reduced significantly (P < 0.0005, Student's t-test) compared with healthy donors (1.09 ± 0.1). To investigate further the lineage of decreasing 305+ cells, we next used a triple-colour staining method for surface antigens and intracellular cytokines. After surface staining with 305 MAb and PerCP-labelled anti-CD4 or anti-CD8, PBMC from healthy donors and patients were treated with phycoerythrin-labelled anti-IL-2, anti-IFN-γ and anti-IL-4. The population of 305+CD4+ T cells did not overlap significantly the population of IL-2, IFN-γ, or IL-4 producing quadrants (data not shown). To investigate whether 305+/CD4+ cell subsets contain CD45RO+ putative memory T cell fractions, which are known to be susceptible to HIV-1 infection, 305+/CD4+ and 305+/CD8+ cells were gated by anti-CD45RO MAb. The 305+/CD4+ and 305+/CD8+ subsets from healthy donors cross-distributed into the CD45ROhigh and CD45RO+ cell populations, respectively (Fig. 1). Both the subsets, however, were not cross-distributed into CD45RA-naive T cell populations (data not shown). The 305+/CD4+/CD45ROhigh cells were relatively diminished by Nef-priming, whereas the 305+/CD8+/CD45RO+ population was not decreased remarkably (Fig. 1). These results indicate that extracellular Nef may target the T helper/memory cell population, and the 305+/CD4+/CD45RO+ cell may be a phenotype susceptible to Nef.Fig. 1.: CD45RO phenotype expression in the 305+/CD4+ and 305+/CD8+ subsets. The non-stimulation or Nef-prestimulated peripheral blood mononuclear cells (PBMC) from a healthy donor and PBMC from a patient were also stained with 305 monoclonal antibody (MAb) plus CD4-PerCP or plus CD8-PerCP, and CD45RO-phycoerythrin. The 305 MAb was probed with FITC-conjugated anti-mouse μAb. Prestimulation with 5 ng/ml of RP Nef (U26080) was performed for 1 h at 37°C and the prestimulated PBMC were washed, then used for FACS analysis. Three representative experiments are shown with 5000 events in dot plots, gated on the CD8− (CD4+) and (CD8+) CD4− populations. The numbers in the corners of the dot plots represent the net percentages of positive cells in the appropriate quadrants.It has been reported that human [17] and murine [10] CD4 T lymphocytes are susceptible to the cell-free Nef. Cytopathogenicity by intracellular Nef expression is associated with the blockade of IL-2 production from the predicted human CD4 T helper type 1 populations [18] and the cytostatic effects of intracellular Nef are also restricted to some T cell types [19]. Experiments involving nef-transgenic mice have also shown that the expression of Nef elicits the depletion of a certain subpopulation of murine peripheral and lymphatic CD4 T cells, especially thymocytes [20], and that nef is critical for severe AIDS-like pathologies such as the loss of CD4 T cells in mice, independently of HIV-1 proliferation [21]. Recently, DNA vaccination targeting for LAI Nef in HIV-1-infected patients was shown to increase CD45RO+ memory T cells and induce a cytotoxic T cell response [22]. The scavenging of Nef biological activity via Ner may thus preserve CD4 memory T cells and may provide great benefit for infected individuals through an improvement of their quality of life. Acknowledgements The authors are indebted to Drs I.M. Jones, N.L. Michael and M. Stevenson for critical reading and comments on the manuscript. They would also like to thank Drs E. Nakayama, T. Shioda and A. Iwamoto for their help in the preparation of PBMC from patients. K. Otake is supported by a research fellowship from the Japan Society for the Promotion of Science for young scientists. Kaori Otakea Michio Ohtaa Jun Minowadab Shinichi Hatamac Eiji Takahashic Atushi Ikemotod Harumi Okuyamad Yoichi Fujiid

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Continuous human leukemia-lymphoma cell lines have become invaluable tools for hematological research as they provide an unlimited amount of cellular material. The first human lymphoma cell line Raji was established in 1963; since then several hundred leukemia-lymphoma cell lines spanning almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) have been described. The cardinal features of leukemia-lymphoma cell lines are their monoclonal origin, arrest of differentiation, and (growth factor-independent or -dependent) unlimited proliferation. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed characterization of both primary and cultured cells in absolutely necessary. New cell lines, in particular, must be adequately, characterized; while cell culture data and immunological and cytogenetic features are essential, cell lines should be described in as much detail as possible. In addition to this recommended multiparameter characterization and the obligatory immortality of the culture, authentication of the true origin of the cells, novelty, scientific significance and availability of the cell line for other investigators are of utmost importance. It is still extremely difficult to establish new leukemia-lymphoma cell lines (except for some subtypes), and most attempts fail. Paramount to the lack of our understanding as to why certain cells start to proliferate in culture and others do not (thus implying a random process), is probably the difficulty of mimicking in vitro the physiological in vivo microenvironment. Attempts to improve the efficiency of cell line establishment should focus on examining the appropriateness of the in vitro culture conditions; these conditions should emulate as closely as possible the in vivo situation. In summary, leukemia-lymphoma cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described.

We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)

Continuous human leukemia-lymphoma cell lines have become indispensable tools in hematological research since the establishment of the first human lymphoma cell line Raji in 1963. We summarize here historical landmarks in the establishment of unique leukemia-lymphoma-derived cell lines from the various cell lineages; their special importance in hematopoietic research is emphasized. The first cell lines were derived from African Burkitt lymphomas and were found to integrate the Epstein-Barr virus in their genome leading to the discovery and isolation of this virus. However, it was later recognized that not every cell line derived from a patient with leukemia-lymphoma represents a malignant cell line as residual normal B-lymphocytes can also be immortalized by EBV infection. During the following 20-30 years many other types of hematopoietic cell lines, commonly derived from hematopoietic neoplasms, were established. These panels of cell lines now span almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) and the various distinct stages of differentiation along the respective cell axes. From early on, cell lines became important tools for basic and clinical hematological research, initially mainly in the field of immunology, but later expanding to other areas also. It became apparent that leukemia-lymphoma cell lines are of monoclonal origin, are arrested at a discrete maturational stage during differentiation in each lineage, and show sustained and growth factor-independent or -dependent unlimited proliferation. Categorization of cell lines might best be based on the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, detailed characterizations of both the cell lines and the primary cells from which the cell lines originated are absolutely mandatory. In summary, the availability of large numbers of continuous leukemia-lymphoma cell lines has greatly facilitated clinical and immunobiological studies of normal and malignant hematopoiesis. Human leukemia-lymphoma cell lines will continue to provide exquisite model systems for many biomedical disciplines.

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-gamma remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-alpha and IFN-beta were as effective as IFN-gamma in RPMI-8226 cells, but less than IFN-gamma in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-gamma. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1 beta, and tumor necrosis factor (TNF)-alpha, the former three were as effective as IFN-gamma in KG-1 cells, but only TNF-alpha stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-alpha, IFN-beta, and IFN-gamma. The effect of IFN-gamma in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-gamma in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-gamma is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-gamma is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis.

The non-antigen specific rapid cytotoxic (CT) death of immature TdT+CD4+CD8+ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as "effectors," with IFN-alpha but not IFN-gamma, or of the susceptible immature TdT+CD4+CD8+ T cells, referred to as "targets," with IFN-gamma but not IFN-alpha, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-alpha treated effector (gp120+CD8+ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-gamma treated target (CD4+CD8+ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-gamma treated effectors or IFN-alpha treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.

The mRNA encoding full-length erythropoietin (EPO) receptor (EPOR-F ) comprises exons I through VIII. Another membrane-bound EPOR (EPOR-T) isoform has a truncated cytoplasmic region and is encoded by the mRNA containing unspliced intron VII (EPOR-T mRNA). EPOR-T is believed to have a dominantly negative function against EPOR-F. We show that EPOR-T mRNA is markedly decreased in the blood cells of patients with polycythemia vera (PV). We also show that EPOR-T mRNA is not detected in erythroid/megakaryocytic leukemia cell lines, but is expressed in nonerythroid/nonmegakaryocytic lines, suggesting the presence of a cell type–specific system by which intron VII of the EPOR transcript is spliced. Deregulation of this splicing system in early hematopoietic progenitors possibly explains the profound decrease in EPOR-T mRNA and consequent pathophysiology of PV.

By infecting human leukemia cell lines in vitro with HIV-1IIIB' a number of HIV-1 carrier clones were generated. Among them, 5 of 13 CD8+ HIV-1 carrier T cell clones were shown to acquire a rapid cytotoxic activity (within 1 hour) specific to TdT+CD4+CD8+ immature T cells including normal thymocytes. This novel cytotoxic reaction, without requiring virus infection and indicating a rapid T cell precursor elimination during active lymphopoiesis, suggests a mechanism responsible for mature CD4+ T cell depletion in HIV-1 infected individuals.

A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 Kd) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag+ donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64+ monocytes. Typing of HLA-DRB1, DPA1 and DPB1 alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of HLA-DPA1*0201 and DPB1*0301 positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of HLA-DPA1*0201-DPB1*0301 gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.

To investigate whether the lymphocyte homing receptors, adhesion molecules regulating normal lymphocyte traffic, influence the dissemination of lymphoma cells, 24 lymphoma/leukemia cell lines were inoculated into SCID mice subcutaneously, and the correlation between the expression of the adhesion molecules and the metastatic potential of the cell lines was examined. Among the six adhesion molecules examined (LFA-1, ICAM-1, CLA, VLA-4, L-selectin and CD44), L-selectin increased the incidence of lymph node metastasis, and CD44 expression was related to both lymph node and organ (hematogenous) metastasis. A monoclonal antibody to the standard form of CD44 (CD44s), Hermes-3, inhibited the local growth and remote metastasis of CD44+ cell lines. Thus, it is concluded that at least CD44s expression is important in both lymphatic and hematogenous metastasis.

The human Evi-1 gene located on chromosome 3q26, encodes a zinc finger protein that functions as a transcription factor. It was frequently overexpressed in leukemias having 3q26 abnormalities such as t(3;3)(q21;q26) and inv(3)(q21 q26), and subjected to structural alteration in t(3;21)(q26;q22). In addition, recent studies indicated that several cases of leukemias without 3q26 abnormalities also expressed Evi-1 gene. In this study we present another case of structural alteration of Evi-1 gene in a case of inv(3)(q21 q26), in which Evi-1 was truncated and a shorter form of Evi-1 protein was expressed upon rearrangement of the gene. We also studied expression of the Evi-1 gene in a variety of leukemias by northern blot analysis. Evi-1 was overexpressed not only in leukemias with 3q26 abnormalities, but, in those without 3q26 abnormalities, especially in blast crisis of CML. Our result also supports an idea that Evi-1 is a relevant oncogene whose overexpression or structural changes might play a crucial role in development of human leukemias.