[Expression of IGLL1 Gene and Its Clinical Significance in Pediatric T-ALL].

OBJECTIVE: To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients. METHODS: A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed. RESULTS: The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients. CONCLUSION: In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.

Adverse effect of oxidized cholesterol exposure on colitis is mediated by modulation of gut microbiota.

Both cholesterol and oxidized cholesterol (OXC) are present in human diets. The incidence of inflammatory bowel diseases (IBDs) is increasing in the world. The present study was to investigate the mechanism by which OXC promotes colitis using C57BL/6 mice as a model. Results shown that more severe colitis was developed in OXC-treated mice with the administration of dextran sulfate sodium (DSS) in water. Direct effects of short-term OXC exposure on gut barrier or inflammation were not observed in healthy mice. However, OXC exposure could cause gut microbiota dysbiosis with a decrease in the relative abundance of short-train fatty acids (SCFAs)-producing bacteria (Lachnospiraceae_NK4A136_group and Blautia) and an increase in the abundance of some potential harmful bacteria (Bacteroides). OXC-induced symptoms of colitis were eliminated when mice were administered with antibiotic cocktails, indicating the promoting effect of OXC on DSS-induced colitis was mediated by its effect on gut microbiota. Moreover, bacteria-depleted mice colonized with gut microbiome from OXC-DSS-exposed mice exhibited a severe colitis, further proving the gut dysbiosis caused by OXC exposure was the culprit in exacerbating the colitis. It was concluded that dietary OXC exposure increased the susceptibility of colitis in mice by causing gut microbiota dysbiosis.

[Analysis of Factors Influencing Overall Survival of MDS Patients Transplanted with HSCs].

OBJECTIVE: To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT). METHODS: 121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method. RESULTS: Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference. CONCLUSION: age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

[Epigenetic Regulation and Epigenomic Landscape in Normal Physiological Hematopoiesis--Editorial].

Abstract　　The physiological hematopoiesis depends on the programmed expression of a series of gene regulated by mechanisms at various levels. Currently, the epigenetic regulation has been considered as the most important mechanism during hematopoietic differentiation, resulting in a specific epigenomic landscape in the hematopoietic stem/progenitor cells. We try to concisely review the epigenetic mechanisms, including the genomic methylation, the histone modifications and the expression profiles of noncoding RNA, illustrating briefly the differentiation from the hematopoietic stem/progenitor cells to to the erythroid, myeloid and lymphoid cells.

Methylation level of Rap1GAP and the clinical significance in MDS.

Previous studies on the pathogenesis of myelodysplastic syndrome (MDS) have identified multiple associated gene mutations, including mutations of tetmethylcytosinedioxygenase 2, isocitrate dehydrogenase [NADP(+)] 1 cytosolic, isocitrate dehydrogenase [NADP(+)] 2 mitochondrial and additional sex combs like 1 transcriptional regulator, all of which may be considered epigenetic regulators. Furthermore, mutations of RAS type GTPase family genes have been identified in 10-15% patients with MDS. The authors' previous study on the gene expression profile of cluster of differentiation 34+ cells using microarray analysis identified elevated expression of RAP1GTPase activating protein 1 (Rap1GAP) in patients with MDS compared with that in non-malignant blood diseases (NM) control group. To further investigate the mechanism of increased Rap1GAP expression, the methylation pattern of the promoter of this gene was determined in 86 patients with MDS (n=29), acute myeloid leukemia (AML) (n=31) or NM (n=26) using bisulfite-specific polymerase chain reaction and DNA sequencing. The results demonstrated that the methylation of Rap1GAP occurred in all 29 patients with MDS at multiple CpG sites. The methylation level of Rap1GAP in patients with MDS was decreased compared with that in patients with NM. Significant differences at 4CpG sites (5,7,8 and 12) of Rap1GAP promoter were identified between MDS and NM. Furthermore, based on the present clinical records of the patient cohort, the methylation status of Rap1GAP promoter did not appear to be associated with the clinicopathological characteristics of patients with MDS, including age, gender and International Prognosis Score System. The difference in methylation level at CpG site 8 of Rap1GAP promoter was identified to be significantly increased in patients with MDS-refractory anemia with ring sideroblasts compared with that in the MDS-refractory cytopenia with multilineage dysplasia or MDS-unclassified groups. The results of the present study suggest that patients with MDS exhibit a lower overall methylation level within Rap1GAP promoter compared with patients with NM or AML. In addition, the methylation level at the four identified CpG sites can distinguish between MDS and NM.

Coexistence of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia: A report of 4 cases.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17-25% of patients with acute lymphocytic leukemia and 0.9-3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-ß-myosin heavy chain 11 (CBFß-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFß-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFß-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFß-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFß-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFß-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

[Effect of AML1-ETO Fusion Protein on Transcriptional Regulation of p14ARF].

OBJECTIVE: To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14ARF. METHODS: P14ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14ARF promoter in AML1-ETO positive leukemia cell line. And the p14ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza. RESULTS: AML1-ETO-expressing cell subclone displayed low level of p14ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14ARF promoter. 5-Aza could increase the expression of p14ARF. CONCLUSION: P14ARF is a possible target gene of AML1-ETO. The p14ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M2b subtype of acute myeloid leukemia.

[Differentiation of K562 Cells Induced by Pulsatilla Saponin A into Erythroid Lineage].

OBJECTIVE: To explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells. METHODS: Pulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells. RESULTS: K562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated. CONCLUSION: Pulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.

High expression of S100A8 gene is associated with drug resistance to etoposide and poor prognosis in acute myeloid leukemia through influencing the apoptosis pathway.

S100A8 has been increasingly recognized as a biomarker in multiple solid tumors and has played pivotal roles in hematological malignancies. S100A8 is potentially an indicator for poor survival in acute myeloid leukemia (AML) in retrospective studies. However, the mechanisms of S100A8 are diverse in cancers. In this study, we investigated the correlation of S100A8 at the transcription level with clinical parameters in 91 de novo AML patients and explored its mechanisms of chemoresistance to etoposide in vitro. The transcription level of S100A8 was significantly lower at initial and relapse stages of AML samples than at complete remission (P<0.001) and than in the control group (P=0.0078), while no significant difference could be found between initial and relapse stages (P=0.257). Patients with high transcription levels of S100A8 exhibited a shorter overall survival (P=0.0012). HL-60 cells transfected with S100A8 showed resistance to etoposide with a higher level IC50 value and lower apoptosis rate compared with HL-60 cells transfected with empty vector. Thirty-six genes were significantly downregulated and 12 genes were significantly upregulated in S100A8 overexpression group compared with control group in which 360 genes involved in apoptotic genes array were performed by real-time reverse transcriptase polymerase chain reaction. Among them, the caspase-3, Bcl-2, and Bax were verified by Western blot analysis which indicated that the role of S100A8 in resistance to chemotherapy was closely related with antiapoptosis. In conclusion, critical S100A8 provided useful clinical information in predicting the outcome of AML. The main mechanism of S100A8 which promoted chemoresistance was antiapoptosis.

[Autophagy Activity of CD34+ Cells in MDS Patients and Its Clinical Significance].

OBJECTIVE: To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance. METHODS: The activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM). RESULTS: The autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P<0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) . CONCLUSION: The autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.

Clonal origin and evolution of myelodysplastic syndrome analyzed by dysplastic morphology and fluorescence in situ hybridization.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem/progenitor cells. As bone marrow cells are extremely diverse in these disorders, the origin and evolution of MDS clones are difficult to identify and trace. Cellular dysplasia is a distinct morphologic feature; however, whether the dysplastic cells represent abnormal clones or only nonspecific superficial phenomena remains to be clarified. To address this question, 97 patients were examined for dysplasia features, among them bone marrow slides of 16 patients with chromosomal abnormalities were subjected to fluorescence in situ hybridization (FISH) to determine the karyotype of these dysplastic cells. Furthermore, the emerging frequencies of abnormal karyotypes in various differentiated stages of each lineage were also evaluated by a combination of morphological evaluation and FISH karyotyping. Our results indicate that the overall percentage of dysplastic cells does not differ significantly among the WHO subtypes, while the megakaryoid lineage presents the most frequent dysplasia in all subtypes. A positive correlation between dysplastic cells and FISH-detectable abnormal clones was observed, but the dysplastic morphology was not a specific feature of FISH-detectable abnormal clones. FISH-detectable abnormal clones can differentiate into mature granulocytes and erythrocytes, in coexistence with cells originating from the normal clones.

[Frequently ABL kinase domain G:C→A:T mutation and uracil DNA glycosylase abnormal expression in TKI-resistant acute lymphoblastic leukemia of Chinese population].

Most Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients often show rapid recurrence and development of ABL kinase domain (KD) mutation after tyrosine kinase inhibitor (TKI) treatment. To further investigate the mechanism of Ph(+) ALL fast relapse after TKI treatment, ABL KD mutation in 35 Chinese Ph(+) ALL with TKI resistance was detected by direct sequencing. The results showed that 77.1% (27/35) Ph(+) ALL patients with TKI resistance had ABL KD mutation and 55.6% (15/27) Ph(+) ALL patients with ABL KD mutation had T315I. Interestingly, 77.8% (21/27) Ph(+)ALL showed ABL mutation G: C→A:T, including T315I, E255K and E459K. Furthermore, all the Ph(+) ALL patients with two or more ABL KD mutations collaborated with complex chromosome abnormality and all the TKI-resistant Ph(+) ALL patients, whose karyotype progressed from simple t (9;22) into complex, developed ABL KD mutation. Moreover, the expression level of uracil-DNA glycosylase UNG2, which inhibits G:C→A:T transition in genomic DNA, decreased in Ph(+) ALL with TKI-resistance compared to that in newly diagnosis Ph(+) ALL. It is concluded that there is a high frequent ABL KD G:C→A:T mutation and a high genomic instability in Chinese TKI-resistant Ph(+) ALL. In addition, the decreased UNG2 expression in TKI-resistant Ph(+) ALL probably contributes to their high rate of ABL KD G:C→A:T mutation.

[Effect of AML1-ETO fusion protein on the expression of BCL-2].

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.

[Expression of autophagy related gene Beclin1 in myelodysplastic syndrome patients and its significance].

This study was aimed to explore the contribution of autophagy associated gene Beclin1 in the prognosis of myelodysplastic syndrome (MDS) by detecting the expression level of Beclin1 in bone marrow mononuclear cells (BMNC) from 40 MDS patients, 14 non-malignant anemia patients and 25 AML patients. The expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). At the same time, the Western blot was used to analyze the expression of Beclin1 proteins. The results showed that the expression of Beclin1 in low risk MDS patients and non-malignant anemia patients was both significantly higher than that in acute myeloid leukemia patients (P < 0.01). And more interestingly, the Beclin1 mRNA expression in MDS group was negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r = -0.495). It is concluded that the expression of Beclin1 in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores. Beclin1 is a potential biomarker for predicting prognosis of the patients with MDS.

Direct contact with bone marrow stromal cells promotes the invasions of SHI-1 leukemia cells.

BACKGROUND: Interactions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis. CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase inducer (EMMPRIN) had reported to participate in this process. However the roles of bone marrow microenvironment in leukemic infiltration were not well investigated. METHODS: A co-culture system between SHI-1 cells and bone marrow stromal cells (BMSCs) is used to simulate the interactions of leukemic cells with their microenvironment. The trans-matrigel invasion was used to detect the capability of SHI-1 cells invasion. The BMSCs and SHI-1 cells were mixed in a ratio of 1:10 and added to the millicell chamber coated with matrigel. Either the co-culture supernatant or the functional blocking peptide of CXCR4 and EMMPRIN were added to the trans-matrigel invasion system. The expressions of EMMPRIN in SHI-1 cells and BMSCs were detected by RT-PCR. The changes of the expression of matrix metalloproteinase-2, 9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinase 2 (TIMP-2), and CXCR4 mRNA in SHI-1 cells were determined by real-time PCR. The concentration of stromal cell derived factor 1 (SDF-1) in serum free supernatant was measured by ELISA. RESULTS: Both SHI-1 cells and BMSCs express EMMPRIN. SHI-1 cells could hardly invade the matrigel membrane; the coculture supernatant did not induce the invasion of SHI-1 cells. When contacting directly with BMSCs, SHI-1 cells invaded to the lower chamber of millicell were significantly increased. The functional blocking peptide of CXCR4 and EMMPRIN could significantly inhibit the invasion triggered by BMSCs. When co-culturing with BMSCs, the expression of CXCR4, MMP-2, MMP-9 and TIMP-2 mRNA in SHI-1 cells were significantly elevated in company with a significantly higher level of SDF-1 in the co-cultured serum-free supernatant. CONCLUSION: The interactions of leukemic cells and BMSCs play important roles in leukemic cell infiltration.

[Transfection of HL-60 cells by Venus lentiviral vector].

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

[Study of the clonal origin and development of MDS by FISH analysis of dysplasia cells in bone marrow of patients with MDS].

This study was purpose to explore whether the dysplasia of myelodysplastic syndromes (MDS) is unspecific feature or results of the abnormal clone, and to provide the evaluation of abnormal clone changes in bone marrow cells of MDS patients. The dysplasia cells in bone marrow smears was analyzed by morphologic observation, the clonal origin and development in 16 cases of MDS with abnormality of chromosome karyotypes were investigated by FISH combined with morphologic observation. The results found that both the dysplastic and nondysplastic bone cells displayed abnormal clones in the erythroid and granulocytic cells. The dysplastic bone marrow cells displayed more abnormal clones than the nondysplastic bone marrow cells in most of the patients, and the abnormal clones displayed more dysplastic cells than the normal clones. Most of the dysplastic and nondysplastic megakaryocytes were derived from abnormal clones. The abnormal clone showed a decreasing trend from the primitive stage to the terminal stage of cell differentiation. It is concluded that there is a correlation between the dysplastic cells and the abnormal clones in MDS, but the dysplasia of bone marrow cells is not a specific feature. The abnormal clones can differentiate into mature granulocytes and erythrocytes, and can be in coexistence with cells originated from the normal clones.

Epigenetic silencing of Bcl-2, CEBPA and p14(ARF) by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line.

The AML1-ETO fusion transcription factor generated by the t(8;21) translocation is considered to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia by recruiting co-repressor complexes to DNA. To investigate the role of AML1-ETO in leukemogenesis, we transfected the cloned AML1-ETO cDNA and expressed the AML1-ETO protein in U937 myelomonocytic leukemia cells. By focusing on the anti-apoptotic gene Bcl-2, the key regulator gene of granulocytic differentiation CCAAT/enhancer-binding protein α (CEBPA) and the tumor suppressor gene p14(ARF), we found that both AML1-ETO-expressing cell lines and t(8;21) leukemia samples displayed low levels of these three genes. Chromatin immunoprecipitation assays demonstrated that Bcl-2, CEBPA and p14(ARF) were direct transcriptional targets of AML1-ETO. The universal binding of AML1-ETO to genomic DNA resulted in recruitment of methyl-CpG binding protein 2 (MeCP2), reduction of histone H3 or H4 acetylation and increased trimethylation of histone H3 lysine 9 as well as lysine 27 indicating that AML1-ETO induced heterochromatic silencing of Bcl-2, CEBPA and p14(ARF). These results suggested that the aberrant transcription factor AML1-ETO epigenetically silenced the function of the Bcl-2, CEBPA and p14(ARF) genes by inducing repressed chromatin configurations at their promoters through histone modifications.

[Biocompatibility of polyethylene imine (PEI)-coated magnetic Fe3O4 nanoparticles in SHI-1 cells].

OBJECTIVE: To explore the feasibility of magnetic resonance cell imaging technology by using polyethylene imine (PEI)-coated magnetic nanoparticles of Fe4O4 (PEI-Fe4O4-MNPs) to track cell biology behavior. METHODS: Endocytic PEI-Fe4O4-MNPs in SHI-1 cells were observed by transmission electron microscopy (TEM) . Iron contents of nano-labeled cells were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and Prussian blue staining. The proliferation ability of labeled cells was detected by cell counting kit-8 (CCK-8) assay; the differentiation and colony-forming abilities were also observed. SHI-1 cells without endocytosing PEI-Fe4O4-MNPs were used as control. RESULTS: Our data showed that PEI-Fe4O4-MNPs could label SHI-1 cells. The labeling efficiency depended on the nanoparticles' concentration and the duration of cells treating. Inhibition rates of SHI-1cells labeled by 60-100 µg Fe/ml PEI-Fe4O4-MNPs were much higher than of 5-50 µg Fe/ml ones following treating by 5-100 µg Fe/ml PEI-Fe4O4-MNPs for 48 hrs. The expressions of CD11b and CD14 were (78.4±18.5)% and (18.7±2.9)% in control vs (83.3±14.2)% and (20.4±2.1)% in cells fractions treated by 30 µg Fe/ml PEI-Fe4O4-MNPs. Clony-forming rates of SHI-1 cells labeled by 0, 20 , 50 µg Fe/ml PEI-Fe4O4-MNPs were (25.20±7.22)%, (25.93±13.15)%, (23.37±9.33)%, respectively. Differentiation and colony-forming potentials of labeled cells were similar with control in the certain range of PEI-Fe4O4-MNPs concentration. CONCLUSION: SHI-1 cells were efficiently labeled by PEI-Fe4O4-MNPs with well biocompatibilities in proper range of concentration, the latter could be coupled with magnetic resonance imaging (MRI) to track cells in vivo.

[The different characteristics of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant Philadelphia chromosome-positive acute lymphoblastic leukemia and chronic myeloid leukemia].

OBJECTIVE: To identify the distribution and differentiation of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). METHODS: Bone marrow or peripheral blood samples of 112 imatinib resistant CML patients and 21 Ph(+)ALL patients were obtained from the first affiliated hospital of Soochow university according to local law. Total RNA was extracted from the mononuclear cells using a TRIzol reagent. ABL kinase domain (KD) mutation was detected by direct sequencing. RESULTS: Of the 112 imatinib resistant CML patients, 54.46%(61 cases) had ABL KD mutation. Twenty-three mutants were identified in 20 amino acid sites and 23.21% (26 cases) ABL KD mutations were in P-loop region. ABL KD mutations were also detected in 71.43% (15 cases) imatinib resistant Ph(+)ALL patients, with 10 mutations in 8 amino acid sites. The most frequent mutation was T315I (28.57%), followed by E255K/V (19.05%) and Y253F/H (14.29%). The frequency of T315I was much higher in imatinib resistant Ph(+) ALL than that in imatinib resistant CML (P = 0.001). Ph(+)ALL with additional chromosomal aberrations also had a higher rate of ABL KD mutation than that of CML (P = 0.010). Ph(+)ALL gained ABL KD mutation faster than CML (P < 0.010). CONCLUSION: Chinese imatinib resistant CML and Ph(+)ALL patients had different characteristics in ABL KD mutation. The rate of ABL KD mutation in Ph(+)ALL with additional chromosomal aberrations was much higher than that of CML with additional chromosomal aberrations.