Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.     

Fibronectin increases the migration induced by stromal cell-derived factor-1 alpha (SDF-1 alpha) in pre-B acute lymphoblastic leukemia cells

The chemokine, stromal cell-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR-4 (fusin, LESTR) are thought to be involved in the trafficking of hematopoietic progenitors and stem cells, as suggested by the chemotactic effect of SDF-1 alpha on these cells. Gene inactivation studies have shown that both SDF-1 alpha and CXCR-4 are essential for B lymphopoiesis. Migration of leukemic cells may also be dependent on SDF-1 alpha and CXCR-4. Fibronectin (FN) is a component of the extracellular matrix (ECM), and one of the natural supports for cell movement in their bone hematopoietic environment. In the present study, we examined the influence of FN on the chemotactic effect of SDF-1 alpha and on the CXCR-4 expression and function on human precursor-B acute lymphoblastic leukemia (pre-B ALL) cells at sequential stages of development. Fourteen children with pre-B ALL were studied. Their immunophenotypes belonged to the first three stages of B cell differentiation. Despite relatively high levels of CXCR-4 expression at all stages, the responsiveness to SDF-1 alpha, measured as the percentage of migrating cells in the transwell culture system, varied with patients and seems to be less significant for pre-B3 (and pre-B1) than for pre-B2. There was no correlation (r = 0.2) between the SDF-1 alpha induced migration (range: 2.5-39%) and the cell surface density of CXCR-4 (range: 46.5-97.5%). The extracellular matrix protein FN, either coated on the filter (for more than 18 hours) or in soluble form, enhanced the SDF-1 alpha induced migration of pre-B ALL respectively (2 fold and 1.6 fold) without influencing CXCR-4 expression in short term cultures. Therefore, we analyzed the expression of the FN receptors, VLA-4 (CD49d) and VLA-5 (CD49e), by direct immunofluorescence, on these leukemic cells. VLA-4 was strongly expressed in all stages of pre-B ALL (range: 77-97%) while VLA-5 expression was more variable (range: 14-94%), but no correlation with the FN-dependent increased SDF-1 alpha chemotactic effect was noted. In conclusion, the migratory behavior of pre-B leukemic cells in response to SDF-1 alpha partly depends upon the stage of differentiation, and partly upon unexplained patient variability. Our results suggest that several molecules from the extracellular matrix, such as FN, may be implicated in this phenomenon.     

Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach

Background

MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis.

Methodology/Principal Findings

To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO “biological process” classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development.

Conclusion/Significance

The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse.     

Tumor necrosis factor-α induces changes in the phosphorylation,cellular localization,and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine

The stress protein hsp27 is constitutively expressed in several human cells and shows a rapid phosphorylation following treatment with tumor necrosis factor-α (TNF-α). hsp27 usually displays native molecular mass ranging from 100 to 700 kDa. Here, we have analyzed the TNF-α-mediated changes in the phosphorylation, cellular localization, and structural organization of hsp27 in HeLa cells. We report that the TNF-α-mediated hsp27 phosphorylation is a long-lasting phenomenon that correlates with the cytostatic effect of this cytokine. Following TNF-α treatment, the rapid phosphorylation of hsp27 occurred concomitantly with complex changes in the intracellular distribution and structural organization of this protein. This resulted in the quantitative redistribution of hsp27 toward the soluble phase of the cytoplasm. In addition, during the first 2 h of TNF-α treatment, a transient increase in the native molecular mass of most hsp27 molecules (≤ 700 kDa) occurred. Then, by 4 h of TNF-α treatment, the native size of this stress protein drastically regressed (< 200 kDa). During this phenomenon, the phosphorylated isoforms of hsp27 remained concentrated in the small or medium-sized oligomers (< 300 kDa) of this protein. We also analyzed the properties of human hsp27 in transfected murine L929 cell lines that constitutively express this protein. In these cells, TNF-α induced modifications in the phosphorylation, intracellular distribution, and oligomerization of human hsp27 similar to those observed in HeLa cells. Moreover, the expression of hsp27 in L929 cells was found to correlate with a reduced cytotoxicity of this cytokine. Hence, the complex changes in the phosphorylation, intracellular locale and structural organization of human hsp27 may be related to the protective activity of this protein against the deleterious effects induced by TNF-α.     

Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

Background

Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.

Results

We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.

Conclusions

Our results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.     

BP-3 alloantigen. A cell surface glycoprotein that marks early B lineage cells and mature myeloid lineage cells in mice

To explore the cell surface molecules expressed on pre-B cells we have produced a panel of alloantibodies against transformed pre-B cells from BALB/c mice by immunizing a wild mouse, Mus spretus. One of these antibodies, BP-3, recognized glycoproteins of Mr 38,000 to 48,000 on pre-B cells transformed either by the Abelson murine leukemia virus or an erb B oncogene construct. Removal of N-linked oligosaccharides from the BP-3 Ag revealed a single core protein of Mr 32,000. The Ag was expressed by bone marrow cells in all but one (A/J) of the inbred mouse strains tested and in wild mice of biochemical groups Mus-1 and Mus-2. Analysis of the tissue distribution revealed expression of the BP-3 reactive molecule on normal pre-B and B cells in the bone marrow, 35% of B cells in the circulation, 30% of the B cells in the spleen, and less than or equal to 20% of B cells in lymph nodes, peritoneal cavity, and Peyer's patches. The subpopulation of BP-3+ B cells in bone marrow and peripheral tissues displayed an immature phenotype (IgM IgD +/- ). Examination of a panel of transformed B lineage cells confirmed the early stage-specific expression of the BP-3 alloantigen. In addition, a myeloid cell line and normal myeloid cells were found to express the BP-3 alloantigen. In contrast to B lineage cells, the level of BP-3 expression increased as a function of myeloid cell differentiation. Myeloid cells in the bone marrow expressed relatively little Ag, whereas circulating neutrophils and peritoneal macrophages expressed relatively high levels of the BP-3 alloantigen with Mr 38,000, 41,000, and 46,000. The data suggest that this variably glycosylated cell surface protein could play different roles in the differentiation of B lineage and myeloid lineage cells. The BP-3 alloantigen appears to be a useful marker for virgin B cells that have recently migrated from the bone marrow to the periphery.     

Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: implications for B-cell receptor stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.     

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21^CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.     

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.     

Heat Shock Protein 27 Plays Two Distinct Roles in Controlling Human Breast Cancer Cell Migration on Laminin-5

It has recently been reported that phosphorylation of the small heat shock protein 27 (hsp27) enhances p38 MAP kinase dependent migration of bovine and human vascular endothelial cells. We have examined the role of hsp27 in controlling the constitutive migration of human breast cancer cells on the extracellular matrix molecule laminin-5. In a haptotaxis assay, anisomycin- or heat shock-induced phosphorylation of hsp27 enhances migration of MDA-MB-231 breast cancer cells constitutively overexpressing hsp27. Under these conditions, hsp27 redistributes to the nucleus. Unphosphorylated hsp27, which remains in the cytosol, induces resistance to a subset of drugs that inhibit haptotactic migration of these cells. We conclude that hsp27 plays two distinct roles in controlling migration of breast cancer cells: phosphorylated hsp27 enhances migration, while unphosphorylated hsp27 can sustain migration in the presence of inhibitory drugs.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Expression of CD9 antigen on normal activated human B cells

The expression of the CD9 pre-B acute lymphoblastic leukemia (ALL)-associated antigen was studied. CD9-positive B cells were enriched in the in vivo-activated buoyant B cell population isolated from tonsils. Small tonsil B cells activated in vitro with either PWM, phorbol 12-myristate 13-acetate (TPA), or anti-Ig plus low Mr B cell growth factor (BCGF) also demonstrated increased CD9 expression. The peak of CD9 expression (30-40% positive cells) occurred after 4-6 days of activation. The kinetics of increased CD9 expression was similar to that of the 4F2 activation antigen. CD9 antigen expression on tonsillar B cells as well as on pre-B leukemic cell lines was associated with protein kinase C activation. Two phorbols that activate protein kinase C (TPA; phorbol 12,13-dibutyrate) induced expression of the CD9 antigen whereas a phorbol analogue that does not activate C kinase (4-alpha-phorbol 12,13-didecanoate) and an analogue that is a very weak agonist (phorbol 12-myristate 13-acetate-4-0-methyl ether) were unable to induce CD9 expression on tonsil B cells or on the cell lines. The effect of the anti-CD9 monoclonal antibody, DU-ALL-1, on B cell mitogenesis was studied. Dense or buoyant tonsillar B cells were cultured in the presence or absence of DU-ALL-1 antibody plus PWM, anti-Ig, and BCGF, DU-ALL-1 antibody did not inhibit or augment the mitogenic response of resting or activated B cells. These results indicate that the CD9 pre-B ALL antigen is present on a population of normal activated tonsillar B cells and that its induction of expression is associated with protein kinase C activation.