Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.
Humans ; U937 Cells ; RUNX1 Translocation Partner 1 Protein ; Leukemia/genetics* ; Core Binding Factor Alpha 2 Subunit/genetics* ; Oncogene Proteins, Fusion/genetics* ; Leukemia, Myeloid, Acute/genetics*

Humans ; Core Binding Factor Alpha 2 Subunit/genetics* ; Translocation, Genetic ; Leukemia, Myeloid, Acute/genetics* ; Prognosis ; High-Throughput Nucleotide Sequencing ; RUNX1 Translocation Partner 1 Protein/genetics* ; Oncogene Proteins, Fusion/genetics*

OBJECTIVETo investigate the expression of AML1/ETO9a isoform in the acute myeloid leukemia (AML)-M2 patients.

METHODSExpressions of AML1/ETO fusion gene and AML1/ETO9a isoform were detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) in leukemia patients, MDS patients, leukemia cell lines and healthy subjects. Karyotype was studied by R-banding technique.

RESULTIn 30 newly diagnosed AML-M2 patients 15 were found to express AML1/ETO9a isoform, while the rest including 20 AML-M2CR, 18 other subtypes of AML, 5 chronic myelogenous leukemia (CML), 3 myelodysplastic syndromes (MDS), 3 leukemia cell lines (NB4, KG-1, K562) and 5 healthy subjects were AML1/ETO9a negative. Among the 15 AML/ETO9a isoform expressing cases, 13 were demonstrated t(8;21) translocation and AML1/ETO expression.

CONCLUSIONIsoform AML1/ETO9a was correlated to AML/M2, and it may promote the development of leukemia in combination with the AML1/ETO fusion gene.

Adolescent ; Adult ; Aged ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein

OBJECTIVETo establish a real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) for quantitative detection of the common molecular markers that have affirmative clinical significance in the acute and chronic leukemia patients, and evaluate its significance in diagnosing leukemias and monitoring minimal residual disease (MRD).

METHODSPrimers and TaqMan probes were designed for detecting various fusion transcripts and normal abl gene was used as the internal control. The expression level of fusion transcripts in 202 newly diagnosed leukemias were determined.

RESULTSIn absolute quantity, expression level of the fusion transcripts in various leukemias was b3a2 (b2a2) 47614.63, e1a2 98847.53, AML1-ETO 300029.51, PML-RAR alpha 25506.28, respectively, while in relative quantity to abl, the levels were 1.05, 0.91, 5.33 and 0.55, respectively.

CONCLUSIONThe relative quantification of gene expression level by using RQ-RT-PCR to abl control gene is more accurate and direct viewing. Different levels of transcription of corresponding fusion genes are found in various subtypes of leukemias at diagnosis, among which the level of AML1-ETO was higher and PML-RAR alpha lower.

Biomarkers, Tumor ; genetics ; metabolism ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Leukemia ; diagnosis ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic

OBJECTIVETo observe the effects of AML1-ETO fusion gene on the transcription activity of p21WAF1/CIP1 gene. And to explore the enhancement of leukemia pathogenesis of AML1-ETO.

METHODSThe luciferase reporter plasmids of p21WAF1/CIP1 gene promoter were constructed, and co-transfected into CV-1 cells with AML1-ETO, AML1b and AML1a expression plasmids. The trans-activity of p21WAF1/CIP1 gene promoter was assayed by luminometer.

RESULTSAML1-ETO exhibited a distinct inhibition activity of p21WAF1/CIP1 gene promoter with a sequence-specificity and dosage-dependent manner. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (19 +/- 4)% compared to control group, when 1000 ng pCMV5-AML1-ETO plasmid was used. AML1b and AMLla showed less inhibition activity. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (61 +/- 16)% and (59 +/- 16)% compared to control group, respectively, when 1000 ng plasmid was used.

CONCLUSIONAML1-ETO exhibits more inhibition activity of p21WAF1/CIP1 gene promoter than AML1b and AMLla, results from recruiting transcription co-repression complex efficiently by ETO. Based on previous researches, the effects of exogenous AML1-ETO on p21WAF1/CIP1 gene promote may be dependent on the type of cell lines.

Animals ; Cell Line ; Core Binding Factor Alpha 2 Subunit ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Haplorhini ; Oncogene Proteins, Fusion ; genetics ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; RUNX1 Translocation Partner 1 Protein ; Transcription, Genetic ; Transfection

This study was aimed to investigate the expression levels of FLT3 gene in AML-M2 patients carrying AML1/ETO fusion gene, and analyze its relation with clinical and laboratorial features and prognosis. RQ-PCR method was used to detect the expression level of FLT3 in bone marrow of 21 AML-M2 patients with AML1/ETO(+). The correlation of the expression level of FLT3 with clinical features, other laboratorial examinations and disease prognosis were analyzed. The results showed that gene expression level of FLT3 (FLT3 gene/ reference gene) in patients at initial diagnosis were 1.65%-261.5%. The expression level of FLT3 over 35% was defined as high expression group (12 cases) , while the expression level below 35% was defined as low expression group (9 cases) . The proportion of patients with extramedullary infiltration in high expression group was higher than that in low expression group (25% vs 0%, P = 0.2286). The proportion of patients at initial diagnosis with white blood cell count > 10×10(9)/L in high expression group was higher than that in low expression group (66.67% vs 22.22%), but there was no statistical significance (P = 0.0805). No significant difference was observed at the age (P = 0.1369) and the rate of bone marrow blasts (P = 0.6923) between the above mentioned two groups. The differences in complete remission rate (66.67% vs 88.89%, P = 0.3383), the relapse rate (66.67% vs 22.22%,P = 0.0805) and the mortality rate (50% vs 22.22%, P = 0.3666) between the two group were not significant, but there was a clear trend that the low expression group has a higher CR rate and a lower relapse rate and mortality rate. It is concluded that FLT3 gene high expression in AML-M2 patients with AML1/ETO(+) have a higher rate of relapse and hence poor prognosis. Therefore, detection of FLT3 expression level in routine clinical practice is important for patient's risk stratification, prognostic evaluation and effective treatment selection.
Core Binding Factor Alpha 2 Subunit ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVEBy inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle.

METHODSThe small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay.

RESULTSThe transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P < 0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively.

CONCLUSIONSThe synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.

Cell Cycle ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA Interference ; RUNX1 Translocation Partner 1 Protein ; Transfection

This study was aimed to construct the standard product for detecting the aml1/eto fusion gene by real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR), monitoring minimal residual disease (MRD) in the patents with AML-M(2). Having obtained the aml1/eto fusion gene from the patients by the RT-PCR with a pair of specific primers, the RNA standard product as 10(10) copies was gained after amplifying and transcribing in vitro and was used for detecting bone marrow and peripheral blood samples from 37 patients. The results indicated that the standard product constructed above displayed a standard curve which showed a linear correlation of the Ct with the log of the RNA concentration of each standard dilution. The average relative levels of aml1/eto fusion gene in the patients at diagnosis and the patients in relapse were higher than those in patients ongoing complete remission (CR) (p < 0.05). The relative level of aml1/eto fusion gene of the follow-up patients was higher at diagnosis, and lower in patients ongoing CR, then went up again at relapse. The patients whose relative level of aml1/eto fusion gene in CR decreased by 2 log even lower than at diagnosis had a lower risk of relapse. If the relative level of aml1/eto fusion gene kept increasing, the patients had a poor prognosis. It is concluded that RQ-RT-PCR is a sensitive, specific, reliable and reproducible method for monitoring aml1/eto fusion gene. Application of RQ-RT-PCR to detect aml1/eto fusion gene for monitoring MRD in AML-M(2) is helpful to assess the response of therapy and estimate the risk of relapse, RQ-RT-PCR may become an important method to decide the time for intensified therapy and prolong CR for patients.
Core Binding Factor Alpha 2 Subunit ; genetics ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; pathology ; Neoplasm, Residual ; Oncogene Proteins, Fusion ; genetics ; RUNX1 Translocation Partner 1 Protein ; Reverse Transcriptase Polymerase Chain Reaction ; methods

This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.
Cell Transformation, Neoplastic ; drug effects ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Cyclic AMP ; analogs & derivatives ; pharmacology ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Thionucleotides ; pharmacology ; Tumor Cells, Cultured

AML-1/ETO fusion gene is the frequent genetic lesion described in FBA M(2) type acute myeloid leukemia (AML-M(2)) and is associated with a favourable prognosis. In spite of its potential clinical relevance, this subtype leukemia usually would be undetected with conventional cytology procedures, and easily confused with acute promyelocyte leukemia (APL) in morphology. In order to investigate the immunophenotypic characteristics of bone marrow cells in AML-M(2) patients with AML-ETO gene rearrangement classified by FAB, immunophenotype of bone marrow cells in 17 AML-M(2) patients with AML-1/ETO(+) confirmed by fluorescence in situ hybridization was analyzed by using flow cytometry as compared with immunophenotype in 34 APL patients with AML-1/ETO(-). The results showed that population of blast cells (15.89% - 68.53%) and population of more heterogeneous myeloid cells were detected with right-angle scatter in 17 patients with AML-1/ETO(+), i.e. AML-M(2) by FAB classification. The blast cells expressed stem cell associated antigens CD34, HLA-DR and myeloid antigens CD33, CD13, MPO. The mean fluorescent intensity of CD33 in M(2)/ETO(+) patients was significantly lower than that in APL patients (121 +/- 92 vs 845 +/- 523, P<0.001), meanwhile positive expression rates of HLA-DR, CD19 and CD34(+)CD56(+) in M(2)/ETO(+) patients were significantly higher than that in APL patients (100%, 88.24%, 100% vs 27.27%, 8.82%, 0%, P<0.001), expression rate of CD9 in M(2)/ETO(+) patients was significantly lower than that in APL patients (P<0.001). In patients with M(2)/ETO(+) (AML-M(2)), the pattern of CD15/CD11b expression was seen as granulocytic differentiation with immature events showing CD15(+)CD11b(-) and more mature CD15(+)CD11b(+) populations, the expression of mature granulocytes CD10 was negative and similar to APL in expression figure. The granulocytes expressed CD56 in 17 patients with M(2)/ETO(+) (17/17, 100%) and its expression rate was significantly higher than that in patients with M(3) (6/34, 17.56%). It is concluded that AML-M(2) with AML-1/ETO gene rearrangement was confirmed to express an exclusive immunophenotype that shows highly predictive value for the cytogenetic pattern, and the multiparametric flow cytometry with FISH provides a technical approach to easily distinguish leukemia subtype M(2)/ETO(+) from APL.
Adolescent ; Adult ; Aged ; Child ; Core Binding Factor Alpha 2 Subunit ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Leukemic ; Gene Rearrangement ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; genetics ; immunology ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; biosynthesis ; genetics ; RUNX1 Translocation Partner 1 Protein