Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia.

The SIL (SCL interrupting locus) gene was initially discovered at the site of a genomic rearrangement in a T-cell acute lymphoblastic leukemia cell line. This rearrangement, which occurs in a remarkably site-specific fashion, is present in the leukemic cells of 16 to 26% of patients with T-cell acute lymphoblastic leukemia. We have now cloned a normal SIL cDNA from a cell line which does not carry the rearrangement. The SIL cDNA has a long open reading frame of 1,287 amino acids, with a predicted molecular size of 143 kDa. The predicted protein is not homologous with any previously described protein; however, a potential eukaryotic topoisomerase I active site was identified. Cross-species hybridization using a SIL cDNA probe indicated that the SIL gene was conserved in mammals. A survey of human and murine cell lines and tissues demonstrated SIL mRNA to be ubiquitously expressed, at low levels, in hematopoietic cell lines and tissues. With the exception of 11.5-day-old mouse embryos, SIL mRNA was not detected in nonhematopoietic tissues. The genomic structure of SIL was also analyzed. The gene consists of 18 exons distributed over 70 kb, with the 5' portion of the gene demonstrating alternate exon utilization.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

mTOR Inhibition by Everolimus in Childhood Acute Lymphoblastic Leukemia Induces Caspase-Independent Cell Death

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.     

Phosphatase of regenerating liver in hematopoietic stem cells and hematological malignancies

The phosphatases of regenerating liver (PRLs), consisting PRL1, PRL2 and PRL3, are dual-specificity protein phosphatases that have been implicated as biomarkers and therapeutic targets in several solid tumors. However, their roles in hematological malignancies are largely unknown. Recent findings demonstrate that PRL2 is important for hematopoietic stem cell self-renewal and proliferation. In addition, both PRL2 and PRL3 are highly expressed in some hematological malignancies, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Moreover, PRL deficiency impairs the proliferation and survival of leukemia cells through regulating oncogenic signaling pathways. While PRLs are potential novel therapeutic targets in hematological malignancies, their exact biological function and cellular substrates remain unclear. This review will discuss how PRLs regulate hematopoietic stem cell behavior, what signaling pathways are regulated by PRLs, and how to target PRLs in hematological malignancies. An improved understanding of how PRLs function and how they are regulated may facilitate the development of PRL inhibitors that are effective in cancer treatment.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Absence of Ig V region gene somatic hypermutation in advanced Burkitt's lymphoma

Somatic hypermutation of rearranged Ig V region gene plays a major role in generating antibody diversity. Recently, V mutation has been established as a major mechanism of tumor escape from anti-Id immunotherapy. We cloned and sequenced the expressed Ig H and L chain V regions from a case of B acute lymphoblastic leukemia in order to evaluate B cell stages associated with V region mutation, and to determine which tumors would be better suited to Id directed immunotherapy. A consensus VH and V lambda sequence representing tumor at diagnosis was obtained by conventional cDNA cloning in lambda gt10 from a heterohybridoma. Primers which flanked both V regions were used in a modified polymerase chain reaction to generate multiple independent sequences from tumor cells harvested at relapse. In order to exclude mutations due to infidelity of the amplification procedure, single cDNA templates of known sequence were also amplified. The polymerase chain reaction proved to be an effective procedure to obtain multiple clones, but replication in M13 was associated with a low rate of base misincorporation. The results indicate that there is no evidence for biologically significant ongoing mutation in this t(8;14) B cell tumor when comparing sequences at diagnosis and relapse. Thus, V somatic mutation may be restricted to a discrete B cell stage whose malignant counterpart is follicular lymphoma.     

A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.     

UBC家族新成员UBF基因的组织表达谱研究

目的和方法:提取人胎肝和HL-60细胞总RNA,采用RT-PCR的方法,从中扩增UBF基因,并对扩增产物进行测序,从而证实UBF基因的天然存在性;采用原位杂交的方法研究UBF在5月龄胎儿的不同组织及HL-60细胞中的表达谱及亚细胞定位.结果:从胎肝和HL-60细胞中均扩增得到了完整的UBF基因,且其序列与注册序列完全一致;原位杂交结果显示UBF在人胚胎多种组织中表达,其中胎骨骼肌表达最强,胎肾最弱.结论:UBF基因,作为Ubc家族的一个新成员,在人胚胎的骨骼肌、肝脏、肾脏、心脏和肺中均有表达,且骨骼肌中的表达最强.     

Phosphatidylethanolamine methylase and cyclic nucleotide phosphodiesterase activities in human B lymphoid hemopathies

Phospholipid methylase and cyclic nucleotide phosphodiesterase activities were studied in human B lympho?d hemopathies (51 patients: acute lymphoblastic leukemia, B lymphoma, chronic lymphocytic leukemia, hairy cell leukemia) and compared with activities in lymphoblast?d and Burkitt lymphoma cell lines and with normal B lymphocytes: methylase activity proved to be lower in ALL and high grade lymphoma and inversely related to the percent of cells in S phase state; the A/G ratio of phosphodiesterases was low in ALL and CLL and high in hairy cell leukemia and it was related to the percent of cells in S phase state.     

Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.     

Cloning of human myeloid-associated differentiation marker (MYADM) gene whose expression was up-regulated in NB4 cells induced by all-trans retinoic acid

A full-length cDNA of 3192 bp isolated from human bone marrow cDNA library was predicted an ORF encoding 298 amino acids. The deduced protein, containing seven putative transmembrane segments and sharing 75.8% amino acid identity with mouse Myadm protein, was named as human MYADM. The results of Northern blot analysis showed that MYADM was ubiquitously expressed in 15 of 16 adult tissues tested, except thymus. To determine whether the novel human gene was involved in hematopoietic differentiation process as mouse Myadm did, we examined the mRNA expressive abundance of this gene between normal bone marrow cells and peripheral blood leukocytes, and detected the expression change in NB4 cells induced by all–trans retinoic acid at different induce time by the semi-quantitative RT-PCR. The results showed that the expression of the novel gene was not only significantly higher in peripheral blood leukocytes than in bone marrow cells, but also significantly up-regulated when the NB4 cells(derived from a patient with acute promyelocytic leukemia) were induced by all-trans retinoic acid (ATRA) for 48hr. It is suggested that human MYADM was also associated with the differentiation of hematopoietic cells or acute promyelocytic leukemia cells. In addition, MYADM was mapped to human chromosome 19q13.33-q13.4 by Radiation Hybrid mapping, and it consists of 3 exons and 2 introns and spans a 7.1-Kb genomic region.     

Generation of rac3 null mutant mice: role of Rac3 in Bcr/Abl-caused lymphoblastic leukemia

Numerous studies indirectly implicate Rac GTPases in cancer. To investigate if Rac3 contributes to normal or malignant cell function, we generated rac3 null mutants through gene targeting. These mice were viable, fertile, and lacked an obvious external phenotype. This shows Rac3 function is dispensable for embryonic development. Bcr/Abl is a deregulated tyrosine kinase that causes chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia in humans. Vav1, a hematopoiesis-specific exchange factor for Rac, was constitutively tyrosine phosphorylated in primary lymphomas from Bcr/Abl P190 transgenic mice, suggesting inappropriate Rac activation. rac3 is expressed in these malignant hematopoietic cells. Using lysates from BCR/ABL transgenic mice that express or lack rac3, we detected the presence of activated Rac3 but not Rac1 or Rac2 in the malignant precursor B-lineage lymphoblasts. In addition, in female P190 BCR/ABL transgenic mice, lack of rac3 was associated with a longer average survival. These data are the first to directly show a stimulatory role for Rac in leukemia in vivo. Moreover, our data suggest that interference with Rac3 activity, for example, by using geranyl-geranyltransferase inhibitors, may provide a positive clinical benefit for patients with Ph-positive acute lymphoblastic leukemia.     

Construction of human-mouse T cell hybrids that express human T cell-associated surface antigens and allow the chromosomal localization of these antigens

We have constructed somatic cell hybrids between the murine T cell line BW5147 and cells from patients suffering from T cell acute lymphoblastic leukemia. The obtained hybrid clones were analyzed for expression of human T cell antigens and presence of human chromosomes. T cell hybrids derived from fusion between the BW5147 cell line and bone marrow cells from a patient with pre-T acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1-/T6-/T4-/T8-/T3-) appeared to express the human T cell antigen Tp41, which can be recognized by the monoclonal antibodies 3A1 and WT1. Although this panel of hybrid cells contained all human chromosomes, no other T cell antigens were expressed. Fusion of the BW5147 cell line with peripheral blood cells from a patient with a more mature T cell acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1+/T6-/T4+/T8+/T3-) resulted in a panel of hybrid clones that expressed not only the Tp41 antigen, but also the human T cell antigens T1 and T4; two hybrids even expressed the T3 antigen. This panel of hybrids also contained the whole human genome. The two panels of human-mouse T cell hybrids allowed us to assign the genes coding for the human T cell antigens Tp41, T1, and T4 to human chromosomes 17, 11, and 12, respectively. Furthermore, these data support our previous suggestion that the expression of human lymphoid differentiation antigens in human-mouse lymphoid hybrids is influenced by the differentiation stage of the fusion partners.     

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.