Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1.

Lethal 3 malignant brain tumor 1 (L3MBTL1), a homolog of the Drosophila polycomb tumor suppressor l(3)mbt, contains three tandem MBT repeats (3xMBT) that are critical for transcriptional repression. We recently reported that the 3xMBT repeats interact with mono- and dimethylated lysines in the amino termini of histones H4 and H1b to promote methylation-dependent chromatin compaction. Using a series of histone peptides, we now show that the recognition of mono- and dimethylated lysines in histones H3, H4 and H1.4 (but not their trimethylated or unmodified counterparts) by 3xMBT occurs in the context of a basic environment, requiring a conserved aspartic acid (D355) in the second MBT repeat. Despite the broad range of in vitro binding, the chromatin association of L3MBTL1 mirrors the progressive accumulation of H4K20 monomethylation during the cell cycle. Furthermore, transcriptional repression by L3MBTL1 is enhanced by the H4K20 monomethyltransferase PR-SET7 (to which it binds) but not SUV420H1 (an H4K20 trimethylase) or G9a (an H3K9 dimethylase) and knockdown of PR-SET7 decreases H4K20me1 levels and the chromatin association of L3MBTL1. Our studies identify the importance of H4K20 monomethylation and of PR-SET7 for L3MBTL1 function.

Characterization of two novel cell lines, DERL-2 (CD56+/CD3+/Tcry5+) and DERL-7 (CD56+/CD3-/TCRgammadelta-), derived from a single patient with CD56+ non-Hodgkin's lymphoma.

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.

Cyclin A-dependent phosphorylation of the ETS-related protein, MEF, restricts its activity to the G1 phase of the cell cycle.

MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.

Coexistence of two distinct cell populations (CD56(+)TcRgammadelta(+) and CD56(+)TcRgammadelta(-)) in a case of aggressive CD56(+) lymphoma/leukemia.

BACKGROUND AND OBJECTIVE: Large granular lymphocytes derive from two major lineages: one expressing the CD3 surface antigen (T-lymphocytes), and the other lacking this marker (NK-cells). Although developmental overlaps between natural killer cells and T-cells have been described, malignancies derived from these two cell types are considered as distinct lymphoid disorders. DESIGN AND METHODS: We report the case of a 30-year old man affected by a lymphoma/leukemia syndrome presenting with hepatosplenic lymphoma which rapidly transformed into aggressive NK-leukemia. Extensive flow cytometry studies and molecular analysis were repeated during the course of the disease, and showed an unexpected changing pattern. RESULTS: At diagnosis, flow cytometry analysis showed the co-existence of two cell populations, one CD56(+), CD3(+), TcRgd(+), and the other CD56(+), CD3(-) and TcRgd(-). Molecular analysis showed that the TcR genes had the same clonally rearranged pattern involving b, g and d genes in both populations. At disease relapse and during the terminal refractory phase, only CD3(-) cells were present. INTERPRETATION AND CONCLUSIONS: This is an unusual case of CD56(+) aggressive lymphoma/leukemia characterized by the clonal expansion of two phenotypically different cell populations, variably balanced during the course of the disease. The presence of the same TcR genomic rearrangement suggests the origin from a common progenitor able to differentiate along both T- and NK-pathways.

CD56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated with simultaneous all-trans-retinoic acid and chemotherapy.

PURPOSE: Preliminary reports suggest that leukemic cell expression of CD56, a neural cell adhesion molecule, is associated with adverse clinical outcome in either acute myeloid leukemia with t(8;21) or acute promyelocytic leukemia (APL). We investigated the prognostic relevance of CD56 in a series of patients with APL who were treated homogeneously with all-trans-retinoic acid (ATRA) and chemotherapy. PATIENTS AND METHODS: Clinicobiologic presenting features and therapeutic results were analyzed in a series of 100 patients with genetically proven APL who were treated, according to the example of the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto multicenter trial, with ATRA plus idarubicin (AIDA) and for whom data on CD56 expression were available at diagnosis. RESULTS: Fifteen patients (15%) showed expression of CD56 in greater than or equal to 20% blasts at diagnosis and were considered as CD56(+). No differences were found regarding age, sex, WBC and platelet counts, incidence of coagulopathy, hemoglobin and fibrinogen levels, promyelocytic leukemia/retinoic acid receptor (PML/RAR) alpha fusion type, or complete remission (CR) rate in the comparison of the CD56(+) and CD56(-) populations. Conversely, compared with patients who were CD56(-), patients with CD56(+) APL had shorter CR duration (P =.04) and overall survival (P =.002). In the multivariate analysis, CD56 positivity and initial WBC count greater than 10 x 10(9) cells/L retained statistical significance in overall survival (P =.04 and P =.02, respectively). CONCLUSION: The expression of CD56 is significantly associated with inferior CR duration and survival in patients with APL who were treated with modern frontline treatment that included ATRA and simultaneous chemotherapy. Combined with other well-established prognostic factors such as WBC count, CD56 expression at diagnosis might be used to build prognostic scores for risk-adapted therapy in APL.

Glycosyl phosphatidylinositol (GPI)-anchored molecules and the pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of one or more clones of stem cells producing progeny of mature blood cells deficient in the plasma membrane expression of all glycosyl phosphatidylinositol (GPI)-anchored proteins (AP). This is due to somatic mutations in the X-linked gene PIGA, encoding one of the several enzymes required for GPI anchor biosynthesis. More than 20 GPI-APs are variously expressed on hematological cells. GPI-APs may function as enzymes, receptors, complement regulatory proteins or adhesion molecules; they are often involved in signal transduction. The absence of GPI-APs may well explain the main clinical findings of PNH, i.e., hemolysis and thrombosis in the venous system. Other aspects of PNH pathophysiology such as various degrees of bone marrow failure and the dominance of the PNH clone may also be linked to the biology and function of GPI-APs. Results of in vitro and in vivo experiments on embryoid bodies and mice chimeric for nonfunctional Piga have recently demonstrated that Piga inactivation confers no intrinsic advantage to the affected hematopoietic clone under physiological conditions; thus additional factors are required to allow for the expansion of the mutated cells. A close association between PNH and aplastic anemia suggests that immune system mediated bone marrow failure creates and maintains the conditions for the expansion of GPI-AP deficient cells. In this scenario, a PIGA mutation would render GPI-AP deficient cells resistant to the cytotoxic autoimmune attack, enabling them to emerge. Even though the 'survival advantage' hypothesis may explain all the various aspects of this intriguing disease, a formal proof of this theory is still lacking.

Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem-cell disorder in which the affected cells are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins. Paroxysmal nocturnal hemoglobinuria is frequently associated with aplastic anemia, although the basis of this relation is unknown. OBJECTIVE: To assess the PNH status of patients with diverse marrow failure syndromes. DESIGN: Correlation of cytofluorometric data with clinical features. SETTING: Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland. PATIENTS: 115 patients with aplastic anemia, 39 patients with myelodysplasia, 28 patients who had recently undergone bone marrow transplantation, 18 patients with cancer that was treated with chemotherapy, 13 patients with large granular lymphocytosis, 20 controls who had received renal allografts, and 21 healthy participants. INTERVENTION: Patients with aplastic anemia, myelodysplasia, or renal allografts received antithymocyte globulin. MEASUREMENTS: Flow cytometry was used to assess expression of GPI-anchored proteins on granulocytes. RESULTS: Evidence of PNH was found in 25 of 115 (22%) patients with aplastic anemia. No patient with normal GPI-anchored protein expression at presentation developed PNH after therapy (n = 16). Nine of 39 (23%) patients with myelodysplasia had GPI-anchored protein-deficient cells. Abnormal cells were not detected in patients with constitutional or other forms of bone marrow failure or in renal allograft recipients who had received antithymocyte globulin. Aplastic anemia is known to respond to immunosuppressive therapy; in myelodysplasia, the presence of a PNH population was strongly correlated with hematologic improvement after administration of antithymocyte globulin (P = 0.0015). CONCLUSIONS: Flow cytometric analysis is superior to the Ham test and permits concomitant diagnosis of PNH in about 20% of patients with myelodysplasia (a rate similar to that seen in patients with aplastic anemia). The presence of GPI-anchored protein-deficient cells in myelodysplasia predicts responsiveness to immunosuppressive therapy. Early emergence of GPI-anchored protein-deficient hematopoiesis in a patient with marrow failure may point to an underlying immune pathogenesis.

Complete remission in acute myeloid leukaemia with t(8;21) following treatment with G-CSF: flow cytometric analysis of in vivo and in vitro effects on cell maturation.

A 75-year-old patient diagnosed as having acute myeloid leukaemia with t(8;21) received G-CSF alone as induction therapy. Complete remission was achieved following 2 weeks of treatment. Flow cytometric analysis, performed by CD45 technique modified by the introduction of preliminary gating with LDS-751, confirmed the disappearance of blast cells along with myeloid maturation. Finally, in vitro studies demonstrated that G-CSF, as compared to other differentiation inducers, was able to induce a striking effect toward neutrophilic differentiation.

In vitro exposure of acute promyelocytic leukemia cells to arsenic trioxide (As2O3) induces the solitary expression of CD66c (NCA-50/90), a member of the CEA family.

Arsenic trioxide (As2O3) is a useful drug for the treatment of acute promyelocytic leukemia (APL), acting through a complex mechanism involving the induction of apoptosis. We investigated by flow cytometry whether in vitro treatment of APL leukemic cells with As2O3 determined specific surface membrane changes. Twelve APL bone marrow aspirates were analyzed following 7 days of in vitro treatment with As2O3 (0.25, 0.5 and 2.5 microM) with regard to the expression of a series of differentiation antigens. Twelve acute myeloid leukemia (AML) samples of non-APL morphotype were analyzed as controls. Exposure of APL as well as non-APL samples to any concentration of As2O3 did not affect the expression of beta2 integrins (CD11a and CD11b), CD45 isoforms (RA, RB and R0), CD44/H-CAM, CD33 and the CEA-related antigen family members CD66ade and CD66b, thus failing to disclose any maturating effect. Of interest, in all APL samples (but not in AML) every tested dose of As2O3 determined a dramatic upregulation of CD66c display; intermediate concentration (0.5 microM) of As2O3 increased the median percentage of CD66c+ cells from 5% in control cultures (25th-75th percentile 2-12%) to 80% in drug-exposed cultures (25th-75th percentile 58-90%) (P<0.001). The induction of solitary expression of CD66c is a new finding which demonstrates As2O3 capability of generating phenotypic changes absolutely restricted to APL cells Moreover, these results provide experimental basis for considering the involvement of the newly described CD66 signalling pathway in As2O3-driven programmed cell death.

Immunophenotypic analysis enables the correct prediction of t(8;21) in acute myeloid leukaemia.

Immunophenotypic findings from 14 patients affected by acute myeloid leukaemia (AML) with t(8;21) were compared to those obtained from 79 AML patients with normal or other aberrant karyotypes. Classic lineage markers, adhesion molecules, surface enzymes, stem-cell-related antigens and HLA-DR were investigated. Following evaluation by the Mann-Whitney test, we found that t(8;21) AMLs showed a significantly higher expression of CD19, CD34, CD56, CD45RA and CD54. Conversely, blasts from patients in the control group significantly expressed higher levels of CD45RO, CD33, CD36, CD11b and CD14. In order to split the data at the best cut-off point to achieve the most homogeneous subset with regard to cytogenetic pattern, i.e. t(8;21) or not, the CART (Classification and Regression Trees) method was applied. In the univariate analysis by CART, statistically significant differences were found when CD19 was dichotomized at 10%, CD34 at 37%, CD45RA at 84%, CD54 at 21%, CD56 at 12%, CD36 at 14%, CD45RO at 25%, CD11b at 18% and CD14 at 12%. Once cut-off points were established by CART, we applied the logistic regression model to establish which combination of two or more antigens was most predictive for t(8;21). The combination CD19-CD34 at the cut-off points indicated above correctly classified 92/93 cases (98.9%). The addition of any other antigen combination to the CD19/CD34 model failed to improve the level of prediction. We conclude that AML with t(8;21) displays an exclusive immunophenotype that is highly predictive of the cytogenetic pattern.

CD66c antigen expression is myeloid restricted in normal bone marrow but is a common feature of CD10+ early-B-cell malignancies.

CD66c is a surface (and intracellular) molecule bound to the membrane by a glycosyl-phosphatidylinositol anchor. While its expression on peripheral granulocytes is well recognized, less is known about its distribution in early steps of normal and neoplastic hematopoiesis. We analyzed by flow cytometry cell surface expression of CD66c on bone marrow cells from 4 healthy subjects and on bone marrow or peripheral blood cells from 127 patients with newly diagnosed hematologic malignancies: 70 de novo acute myeloid leukemias (AML), 6 refractory anemias with excess of blasts in transformation, 3 myeloid and 3 lymphoid blastic phases of chronic myelogenous leukemia, 33 B-lineage and 6 T-lineage acute lymphoblastic leukemias (B- and T-ALL), and 3 B-cell and 3 T-cell non-Hodgkin's lymphomas in the leukemic phase. We found that in normal bone marrow CD66c expression was myeloid restricted, reaching its highest level on promyelocytes. As for de novo AML, slight expression of CD66c was found on 6/25 (24%) AML-M4 and only occasionally in other subgroups. In 9 out of 10 cases of acute promyelocytic leukemia, CD66c was totally absent, but antigen expression was easily detectable following in vitro exposure to all-trans retinoic acid. Among lymphoid malignancies, CD10+ early-B-ALL consistently expressed the molecule (20/23 cases, or 87%) whereas both CD10- early-B ALL and SmIg+ B-ALL completely lacked it. Finally, dual staining with CD66c and CD10 proved to be a suitable tool for distinguishing even low percentages of residual leukemic cells (CD10+/CD66c+) from normal regenerating early-B cells (CD10+/CD66c ) in CD10+ early-B-ALL induced into remission.

Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: a tool for measuring the extent of the PNH clone.

The membrane expression of nine glycosyl phosphatidyl inositol (GPI)-linked molecules was analyzed by flow cytometry on circulating cells from 18 patients affected by paroxysmal nocturnal hemoglobinuria (PNH). The results allowed us to select CD66b, CD14, CD59, CD24 and CD59 monoclonal antibodies as the most suitable reagents for discriminating between normal and PNH cells in PMN, monocytes, RBC and B or T lymphocytes, respectively. In order to assess whether the analysis of distinct cell populations could provide differential information on the extent of the disease, we compared the proportion of residual normal cells in RBC, monocyte and PMN populations. The mean percentage of unaffected cells was higher in RBC as compared to PMN (50.5 +/- 18.7 vs 17.7 +/- 19.7, P < 0.0001). The proportion of normal PMN was, in turn, significantly greater than that of normal monocytes (17.7 +/- 19.7 vs 8.7 +/- 11.0; P < 0.05). The percentage of CD14+ monocytes was directly related to Hb concentration and platelet (Plt) count, and inversely to percent lysis at the Ham's test. The percentage of CD66b+ PMN was directly related to Plt count and Hb level, while the percentage of CD59+ RBC was associated, in an inverse fashion, only to the Ham's test. No significant correlation was found between cell marker expression and PMN count, reticulocytosis, bilirubin and serum LDH. By dividing the patients into two groups, according to high (> 10 percent) or low (< 10 percent) percentage of CD14+ monocytes, a statistical analysis showed that the main hematological parameters were significantly different.

The PIG-A gene somatic mutation responsible for paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria is the first example of a non neoplastic human disease caused by the somatic mutation of an X-linked gene. The PIG-A gene maps to Xp22.1 and is required for the transfer of N-acetyl glucosamine to phosphoinositol, an early step in the production of the GPI anchor. A deficiency of GPI-linked proteins on the cell surface is responsible for the PNH cell defect, which can be detected by flow cytometry not only on red cells, but also on myeloid cells and in some patients even on lymphoid cells. Its location on the X-chromosome explains how a single recessive mutation can cause the appearance of the abnormal clone. A number of patients may have more than one PNH clone, suggesting that the expansion of GPI-deficient clones occurs under the pressure of a selection mechanism.