Wen-Li Zhao

Metallothionein III (MT3) is a putative tumor suppressor gene that is frequently inactivated in pediatric acute myeloid leukemia by promoter hypermethylation

BackgroundAcute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear.MethodsEleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis.ResultsThe MT3 promoter was hypermethylated in leukemia cell lines. More CpG’s methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P < 0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P = 0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P < 0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1.ConclusionMT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.

Differential mRNA Expression Levels of Human Histone-Modifying Enzymes in Normal Karyotype B Cell Pediatric Acute Lymphoblastic Leukemia

Histone modification enzymes regulate gene expression by altering the accessibility of promoters to transcription factors. We sought to determine whether the genes encoding histone modification enzymes are dysregulated in pediatric acute lymphoblastic leukemia (ALL). A real-time PCR array was designed, tested and used to profile the expression of 85 genes encoding histone modification enzymes in bone marrow mononuclear cells from 30 pediatric ALL patients and 20 normal controls. The expression profile of histone-modifying genes was significantly different between normal karyotype B cell pediatric ALL and normal controls. Eleven genes were upregulated in pediatric ALL, including the histone deacetylases HDAC2 and PAK1, and seven genes were downregulated, including PRMT2 and the putative tumor suppressor EP300. Future studies will seek to determine whether these genes serve as biomarkers of pediatric ALL. Ingenuity Pathway Analysis revealed that Gene Expression and Organ Morphology was the highest rated network, with 13 focus molecules (significance score = 35). Ingenuity Pathway Analysis also indicated that curcumin and miR-34 are upstream regulators of histone-modifying enzymes; future studies will seek to validate these results and examine the role of curcumin and miR-34 in leukemia. This study provides new clues into the molecular mechanisms of pediatric ALL.

Molecular Targeting of the Oncoprotein PLK1 in Pediatric Acute Myeloid Leukemia: RO3280, a Novel PLK1 Inhibitor, Induces Apoptosis in Leukemia Cells

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Early B-cell factor 3 (EBF3) is a novel tumor suppressor gene with promoter hypermethylation in pediatric acute myeloid leukemia

BackgroundPediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear.MethodsTwelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis.ResultsEBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells.ConclusionIn this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details.Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.

Microarray profiling of bone marrow long non-coding RNA expression in Chinese pediatric acute myeloid leukemia patients.

Long non-coding RNA (lncRNA) plays a role in gene transcription, protein expression and epigenetic regulation; and altered expression results in cancer development. Acute myeloid leukemia (AML) is rare in children; and thus, this study profiled lncRNA expression in bone marrow samples from pediatric AML patients. Arraystar Human LncRNA Array V3.0 was used to profile differentially expressed lncRNAs in three bone marrow samples obtained from each pediatric AML patient and normal controls. Quantitative polymerase chain reaction (qRT-PCR) was performed to confirm dysregulated lncRNA expressions in 22 AML bone marrow samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to construct the lncRNA-mRNA co-expression network. A total of 372 dysregulated lncRNAs (difference ≥10-fold) were found in pediatric AML patients compared to normal controls. Fifty-one mRNA levels were significantly upregulated, while 85 mRNA levels were significantly downregulated by >10-fold in pediatric AML, compared to normal controls. GO terms and KEGG pathway annotation data revealed that cell cycle pathway-related genes were significantly associated with pediatric AML. As confirmed by qRT-PCR, expression of 24 of 97 lncRNA was altered in pediatric AML compared to normal controls. In pediatric AML, ENST00000435695 was the most upregulated lncRNA, while ENST00000415964 was the most downregulated lncRNA. Data from this study revealed dysregulated lncRNAs and mRNAs in pediatric AML versus normal controls that could form gene pathways to regulate cell cycle progression and immunoresponse. Further studies are required to determine whether these lncRNAs could serve as novel therapeutic targets and bbdiagnostic biomarkers in pediatric AML.

Clinical significance of microRNA-34b expression in pediatric acute leukemia.

The present study aimed to explore the function of miR‑34b promoter methylation in cell proliferation in childrens acute leukemia. Quantitative PCR and methylation‑specific PCR were performed to measure the levels of miR‑34b and its promoter methylation in normal cells, eight leukemia cell lines as well as primary leukemic cells isolated from patients newly diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lymphocytic lymphoma. miR‑34b levels in leukemia cell lines and primary leukemic cells were significantly lower than those in normal cells. The miR‑34b promoter was found to be methylated in all leukemia cell lines, 24 of 31 ALL patients and 8 of 19 AML patients, but not in the 23 normal controls. miR‑34b expression and methylation of its promoter were not associated with most clinical parameters assessed; however, miR‑34b levels in prednisone‑sensitive ALL were significantly different from those in insensitive ALL. A cell counting kit‑8 assay showed that transfection of miR‑34b mimics into K562 cells inhibited their proliferation. Furthermore, treatment with the demethylating agent 5‑aza‑2‑deoxycytidine significantly enhanced miR‑34b expression levels and decreased the methylation status of its promoter in HL‑60 and K562 cells. In conclusion, the results of the present study indicated that in pediatric leukemia cells and leukemia cell lines, the expression of miR‑34b is inhibited by methylation of its promoter, which impairs the restraining effects of miR‑34b on cell proliferation. It was also indicated that the expression of miR‑34b in ALL patients may affect their response to early treatments.

Analyzing the gene expression profile of anaplastic histology Wilms' tumor with real-time polymerase chain reaction arrays.

BackgroundWilms’ tumor (WT) is one of the most common malignant neoplasms of the urinary tract in children. Anaplastic histology (unfavorable histology) accounts for about 10% of whole WTs, and it is the single most important histologic predictor of treatment response and survival in patients with WT; however, until now the molecular basis of this phenotype is not very clearly.MethodsA real-time polymerase chain reaction (PCR) array was designed and tested. Next, the gene expression profile of pediatric anaplastic histology WT and normal adjacent tissues were analyzed. These expression data were anlyzed with Multi Experiment View (MEV) cluster software further. Datasets representing genes with altered expression profiles derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool (IPA).Results88 real-time PCR primer pairs for quantitative gene expression analysis of key genes involved in pediatric anaplastic histology WT were designed and tested. The gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal controls; we identified 15 genes that are up-regulated and 16 genes that are down-regulated in the former. To investigate biological interactions of these differently regulated genes, datasets representing genes with altered expression profiles were imported into the IPA for further analysis, which revealed three significant networks: Cancer, Hematological Disease, and Gene Expression, which included 27 focus molecules and a significance score of 43. The IPA analysis also grouped the differentially expressed genes into biological mechanisms related to Cell Death and Survival 1.15E−12, Cellular Development 2.84E−11, Cellular Growth and Proliferation 2.84E-11, Gene Expression 4.43E−10, and DNA Replication, Recombination, and Repair 1.39E−07. The important upstream regulators of pediatric anaplastic histology WT were TP53 and TGFβ1 signaling (P = 1.15E−14 and 3.79E−13, respectively).ConclusionsOur study demonstrates that the gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal tissues with real-time PCR array. We identified some genes that are dysregulated in pediatric anaplastic histology WT for the first time, such as HDAC7, and IPA analysis showed the most important pathways for pediatric anaplastic histology WT are TP53 and TGFβ1 signaling. This work may provide new clues into the molecular mechanisms behind pediatric anaplastic histology WT.

Zinc finger protein 382 is downregulated by promoter hypermethylation in pediatric acute myeloid leukemia patients

Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are characteristic of AML. Zinc finger protein 382 (ZNF382) has been suggested to be a tumor suppressor gene possibly regulated by promoter hypermethylation in various types of human cancer. However, ZNF382 expression and methylation status in pediatric AML is unknown. In the present study, ZNF382 transcription levels were evaluated by quantitative reverse-transcription PCR. Methylation status was investigated by methylation-specific (MSP) PCR and bisulfate genomic sequencing (BGS). The prognostic significance of ZNF382 expression and promoter methylation was assessed in 105 cases of pediatric AML. The array data suggested that the ZNF382 promoter was hypermethylated in the AML cases examined. MSP PCR and BGS analysis revealed that ZNF382 was hypermethylated in leukemia cell lines. Furthermore, treatment with 5-aza-2′-deoxycytidine (5-Aza) upregulated ZNF382 expression in the selected leukemia cell lines. The aberrant methylation of ZNF382 was observed in 10% (2/20) of the control samples compared with 26.7% (28/105) of the AML samples. ZNF382 expression was significantly decreased in the 105 AML patients compared with the controls. Patients with ZNF382 methylation showed lower ZNF382 transcript levels compared with patients exhibiting no methylation. There were no significant differences in clinical characteristics or cytogenetic analysis between the patients with or without ZNF382 methylation. ZNF382 methylation correlated with minimal residual disease (MRD). Kaplan-Meier survival analysis revealed similar survival times in the samples with ZNF382 methylation, and multivariate analysis revealed that ZNF382 methylation was not an independent prognostic factor in pediatric AML. The epigenetic inactivation of ZNF382 by promoter hypermethylation can be observed in AML cell lines and pediatric AML samples. Therefore, our study suggests that ZNF382 may be considered a putative tumor suppressor gene in pediatric AML. However, further studies focusing on the mechanisms responsible for ZNF382 downregulation in pediatric leukemia are required.

Clinical Features and Prognostic Significance of TCF3-PBX1 Fusion Gene in Chinese Children with Acute Lymphoblastic Leukemia by Using a Modified ALL-BFM-95 Protocol

For children with precursor B (pre-B) acute lymphoblastic leukemia (ALL) with TCF3-PBX1 fusion gene, their prognosis has been a controversial topic. From January 2008 to December 2012 in our hospital, 450 patients were diagnosed as ALL. Clinical characteristics of 20 patients with TCF3-PBX1 fusion gene were analyzed retrospectively, which were classified to the intermediate-risk (IR) group according to Chinese Children Leukemia Group-2008 (CCLG-2008) risk-stratification criteria and protocol based on the backbone of BFM 95 trails. Eighty five cases without TCF3-PBX1 in the same IR group were regarded as the comparison group. There were no differences in age, gender, initial white blood cell (WBC) count, status of central nerves system (CNS) at diagnosis and complete remission (CR) rates of bone marrow (BM) between the two groups (P > .05). The 5-year probability of event-free survival (EFS) rates were 84.4 ± 15.6% and 73.5 ± 15.6% in the TCF3-PBX1 group and the comparison group (P = .35), respectively. The 5-year probability of overall survival (OS) rates were 86.0 ± 17.6% and 81.8 ± 17.6% (P = .46), respectively. Relapse rates were 10.5% and 12.9% (P = 1.00), respectively. There were not cases with CNS relapse in the TCF3-PBX1 group. When intensive chemotherapy was used, the TCF3-PBX1 was associated with a favorable outcome in childhood pre-B ALL.

Pediatric T-cell acute lymphoblastic leukemia with transient pure red cell aplasia

To the Editor: T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid malignancy, while pure red cell aplasia (PRCA) is a hematological disorder, characterized by isolated failure of erythropoiesis. We herein report a case of childhood transient PRCA followed by T-ALL. A 4-year-old male presented with a 2-month history of progressive pallor, fatigue, and erythrocytopenia. Physical examination revealed significant skin and mucosal pallor. Complete blood counts (CBC) showed erythrocytes of 1.87 10/L, hemoglobin of 5.7 g/dl, mean corpuscular volume of 86 fl/cell, mean corpuscular hemoglobin of 28 pg/cell, platelets of 115 10/L, leukocytes of 4.05 10/L, and 0.14% reticulocytes. Bone marrow aspiration revealed 1% erythroblasts. Myeloblasts and promyelocytes represented 1% and 2%, respectively. Lymphocytes were 35%, and megakaryocytes were in normal number (Fig. 1A). Cytogenetic analysis showed karyotype was 46,XY. Serum virology showed that the cytomegalovirus, Epstein–Barr virus, and parvovirus B19 were all negative. Ferritin level was within normal limits. CD55 and CD59 detected on flow cytometry were normal. Autoimmune antibody and hemolytic anemia testing were all negative. A CT scan of the chest and abdomen were normal, without enlargement of the antero-posterior mediastinum nor any hepatoslenomagaly. According to clinical symptoms, positive physical sign of severe anemia, typical laboratory evidences of normocytic, normochromic anemia, reticulocytopenia, and a paucity of erythroid precursors in the marrow, the diagnosis of PRCA