Qing Rao

Expression and role of DJ-1 in leukemia

DJ-1 is a multifunctional protein that has been implicated in pathogenesis of some solid tumors. In this study, we found that DJ-1 was overexpressed in acute leukemia (AL) patient samples and leukemia cell lines, which gave the first clue that DJ-1 overexpression might be involved in leukemogenesis and/or disease progression of AL. Inactivation of DJ-1 by RNA-mediated interference (RNAi) in leukemia cell lines K562 and HL60 resulted in inhibition of the proliferation potential and enhancement of the sensitivity of leukemia cells to chemotherapeutic drug etoposide. Further investigation of DJ-1 activity revealed that phosphatase and tensin homolog (PTEN), as well as some proliferation and apoptosis-related genes, was regulated by DJ-1. Thus, DJ-1 might be involved in leukemogesis through regulating cell growth, proliferation, and apoptosis. It could be a potential therapeutic target for leukemia.

IL-18 increases invasiveness of HL-60 myeloid leukemia cells: up-regulation of matrix metalloproteinases-9 (MMP-9) expression.

Similar to matrix metalloproteinases (MMP-9/-2), IL-18 was overexpressed in some hematologic malignancies such as acute myeloid leukemia (AML), which is associated with a poor clinical outcome. To establish a possible functional relationship between IL-18 and MMPs in myeloid leukemia, we used semi-quantitative PCR and zymographic analysis to examine whether IL-18 stimulates human myeloid leukemia cell line HL-60 to produce MMPs and/or specific tissue inhibitors (TIMPs), and to degrade extracellular matrix (ECM) gel in vitro. In the ECM invasion assay IL-18 significantly up-regulated transmigration of HL-60 cells, which in turn was inhibited by a synthetic MMP inhibitor: O-phenanthroline (o-PE), anti-MMP-9, anti-MMP-2 as well as anti-IL-18 monoclonal antibody (McAb), respectively, suggesting that induction of gelatinases by IL-18 leads to ECM degradation by these cells. Moreover, IL-18 could significantly increase MMP-9 but not MMP-2 production at both mRNA and/or protein level, slightly up-regulate TIMP-1 mRNA, and clearly induce TIMP-2 mRNA secretion. We postulate that IL-18 may in part play a role in the clinical aggressiveness of human myeloid leukemia by stimulating MMP-9 production.

Membrane-bound macrophage colony-stimulating factor and its receptor play adhesion molecule-like roles in leukemic cells

Membrane-bound macrophage colony-stimulating factor (m-M-CSF) is the membrane form M-CSF by alternative splicing. J6-1 leukemic cell line spontaneously forms cell clusters, whose growth depends on the auto-juxtacrine mediated by m-M-CSF and its receptor (M-CSFR). In this study, M-CSFR isolated from J6-1 cells and recombinant human M-CSF soluble receptor (rh-M-CSFsR) were used to study their effects on J6-1 cells. Both receptors inhibited cell proliferation. Use of M-CSFR monoclonal antibodies, M-CSFR or rh-M-CSFsR to block either M-CSFR or m-M-CSF on cell surface inhibited the cluster forming process, while both receptors stimulated cells adhering to culture plate. Furthermore, M-CSFR and/or rh-M-CSFsR caused multiple cellular changes including cytoplasmic pH, multinuclear cell ratio, antigen expression and cell diameter. A [Ca(2+)] rise was induced within 90 s by both receptors. Western blot experiments showed that rh-M-CSFsR caused tyrosine phosphorylation on multiple cytoplasmic proteins of 45 kDa and 55-90 kDa, which could be blocked by H7. These observations suggested that m-M-CSF and M-CSFR mediate J6-1 cell intercellular adhesion with bi-directional signal transduction, and Ca(2+), protein tyrosine kinases, PKC and/or other H7 sensitive kinase(s) involve in the counter-directional signal transduction.

Icaritin induces AML cell apoptosis via the MAPK/ERK and PI3K/AKT signal pathways

Icaritin, a hydrolytic product of icaritin, is isolated from the traditional Chinese medicinal herb epimedium. Icaritin inhibits the proliferation of several tumor cell lines, but its effect on acute myeloid leukemia (AML) and underlying mechanisms remain to be identified. In the present study, we demonstrated that icaritin inhibits the proliferation of human AML cell lines NB4, HL60, and U937, in a dose- and time-dependent manner. Importantly, icaritin showed anti-leukemia activity on bone marrow mononuclear cells from 15 newly diagnosed AML patients. Flow cytometry analyses indicated that icaritin induces AML cells apoptosis. Icaritin induced activation of caspase-9, -3, -7 and the cleavage of PARP as measured by Western blotting. Icaritin downregulates p-ERK and p-AKT and inhibits the expression of c-myc. These results suggest that icaritin is a promising candidate drug for the treatment of AML. The underlying mechanisms of icaritin anti-AML activity are associated with inhibition of the MAPK/ERK and PI3K/AKT signals and downregulation of c-myc.

Aberrant expression of TSC2 gene in the newly diagnosed acute leukemia

The tuberous sclerosis (TSC) genes, TSC1 and TSC2, encode hamartin and tuberin, respectively, and are putative tumor suppressor genes that were originally identified due to their involvement in the inherited autosomal dominant disorder tuberous sclerosis. It has been elucidated that the two proteins form an intracellular heterodimer participating in signaling pathway of the mammalian Target of Rapamycin (mTOR). Recent studies showed that mTOR pathway was frequently activated in blasts from acute myeloid leukemia (AML) patient and associated with proliferation, survival, and drug-resistance of these cells. These phenomena led us to hypothesize that TSC gene might be involved in acute leukemia (AL). In this study, we investigated the TSC1 and TSC2 mRNA expression in 104 newly diagnosed AL patients and 29 healthy controls using real-time quantitative PCR (RQ-PCR) and explored the potential mechanisms of the aberrant expression through methylation-specific PCR (MSP). The results showed that the expression of TSC2 was downregulated in AL patients and the TSC2 promoter was hypermethylated which might be an important mechanism for the downregulation of TSC2 expression.

Expression of IL-18 and its receptor in human leukemia cells

The importance of IL-18, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Here we examined the mRNA and protein for IL-18 in eight human hematopoietic cell lines representing different lineages and neoplasms including leukemia, lymphoma and others. Our results revealed that IL-18 mRNA was expressed in these cells and that the corresponding protein was found in the cytoplasm. Seven of eight cell lines were also found to express two subunits of the IL-18 receptor (IL-18R) at varied levels. Furthermore, 29 out of 51 leukemia patients tested were observed to express IL-18R with 18/29 (62%) co-expression of both receptor and ligand. By blocking the IL-18 loop using specific antisense oligodeoxynucleotide (ASON) for IL-18 mRNA or anti-human IL-18R monoclonal antibody (McAbR), we were not able to demonstrate a marked inhibition on the most leukemic cell lines growth. Moreover, the potential proliferation in vitro of primary AML cells co-expressing IL-18 and its receptor was not significantly enhanced by recombinant human IL-18, suggesting that IL-18 is not apparently implicated in the proliferation of the leukemia cells via an autocrine loop. Additionally, we also found the effective modulating effect of M-CSF, IFN-alpha and TNF-alpha on IL-18R expression, implying an important in vivo effect of cytokines on IL-18-induced reaction. Moreover, the modulation of IL-18R expression was possibly irrelevant to IFN-gamma secretion induced by these cytokines.

Production of matrix metalloproteinase-9 by cord blood CD34+ cells and its role in migration

The transmigration of hematopoietic progenitor cells is a crucial step in the homing of transplanted stem cells into bone marrow (BM) microenvironment; however, the molecular basis for this is not fully understood. Matrix metalloproteinases (MMPs), which are implicated in the migration of leukocytes, are important in degrading components of extracellular matrix molecules. In this study, using zymographic analysis and enzyme-linked immunosorbent assay (ELISA), we investigated the production of MMP-9 in CD34+ cells from cord blood (CB) and BM, compared their spontaneous migration across a reconstituted basement membrane-coated filter in transwell, and studied the role of MMP-9 in the transmigration. Zymography and ELISA showed that MMP-9 is produced by freshly isolated CD34+ stem/progenitor cells obtained from CB. CB CD34+ cells showed significantly higher migrational capacity than BM CD34+ cells (p=0.008). Furthermore, the migrational ability of CB CD34+ cells over the extracellular matrix (ECM) was significantly inhibited by the inhibitor of MMP, o-phenanthroline and anti-MMP-9 monoclonal antibody (73.3±11.8% and 37.5±10.4% inhibition, respectively). Our results strengthen the potential role of MMP-9 in the higher migrational capacity of CB CD34+ cells, which may be beneficial to homing of these cells to the BM environment.

TBLR1 fuses to retinoid acid receptor α in a variant t(3;17)(q26;q21) translocation of acute promyelocytic leukemia.

The majority of acute promyelocytic leukemia (APL) cases are characterized by the PML-RARα fusion gene. Although the PML-RARα fusion gene can be detected in >98% of APL cases, RARα is also found to be fused with other partner genes, which are also related to all-trans retinoic acid (ATRA)-dependent transcriptional activity and cell differentiation. In this study, we identified a novel RARα fusion gene, TBLR1-RARα (GenBank KF589333), in a rare case of APL with a t(3;17)(q26;q21),t(7;17)(q11.2;q21) complex chromosomal rearrangement. To our knowledge, TBLR1-RARα is the 10th RARα chimeric gene that has been reported up to now. TBLR1-RARα contained the B-F domains of RARα and exhibited a distinct subcellular localization. It could form homodimers and also heterodimers with retinoid X receptor α. As a result, TBLR1-RARα exhibited diminished transcriptional activity by recruitment of more transcriptional corepressors compared with RARα. In the presence of pharmacologic doses of ATRA, TBLR1-RARα could be degraded, and its homodimerization was abrogated. Moreover, when treated with ATRA, TBLR1-RARα could mediate the dissociation and degradation of transcriptional corepressors, consequent transactivation of RARα target genes, and cell differentiation induction in a dose- and time-dependent manner.

Enrichment of N-Cadherin and Tie2-bearing CD34 + /CD38 /CD123 + leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1-ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34(+)/CD38(-)/CD123(+) compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34(+)/CD38(-)/CD123(+) LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34(+)/CD38(-)/CD123(+) LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34(+)/CD38(-)/CD123(+) LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.

Some novel features of IDH1-mutated acute myeloid leukemia revealed in Chinese patients.

Mutations of isocitrate dehydrogenase 1 (IDH1) have recently been reported in acute myeloid leukemia (AML). However, the characteristics of IDH1-mutated AML are still not known clearly. We analyzed 416 Chinese AML patients and found 28 patients (6.7%) carried this mutation. One homozygous IDH1 mutant in AML was found. The IDH1 mutations were associated with NPM1 mutations (P=0.043) and could coexist with recurrent transcription factor aberrations including AML1-ETO (6/50), PML-RARα (3/77) and CBFβ-MYH11 (1/15). For AML with AML1-ETO fusion gene, IDH1(mut) patients may have worse disease-free survival (DFS) than IDH1(wild-type) patients.