Objective To evaluate tear ferning changes of conjunctivochalasis.Design Prospective case study series.Partici- pants 30 patients(60 eyes)of conjunctivochalasis and normal subjects were selected.Methods The subjects were observed with gen- eral ophthalmic examination and tear fern test(TFT).Tear ferning was classified into 4 types.TypeⅠand TypeⅡare normal.TypeⅢand TypeⅣare abnormal.Main Outcome Measures The type of tear feming.Results TFT showed that tear ferning was de- creased in conjunctivochalasis group(TypeⅢand TypeⅣoccupied 61.7%).The difference between conjunctivoehalasis and normal control group was significant(P

Objective To elucidate the interaction between osteoblast of bone marrow microenvironment and leukemia cells,and to investigate the role of osteoblast in the leukemia cells survival and apoptosis and the influence of leukemia cells on the osteoblast.Methods Leukemia cells from AML1-ETO9a-Rac1 mouse leukemia model and osteoblast cells were used.The ratio of GFP+ leukemia cells that co-cultured with or without osteoblast was detected by FACS.In addition,the apoptosis level of leukemia cells was detected by flow cytometry by PI and Annexin Ⅴ labeling.Activation level of PARP was determined by Western-blot.Real-time PCR (RT-PCR) was utilized to detect the mRNA level of TPO,N-cadherin,OPN and Ang1 in osteoblast which was separated from leukemic mice.Results The ratio of GFP+ cells in AE9a-Rac1 leukemia cells co-cultured with osteoblast cell was significantly higher than that of AE9a-Rac1 leukemia cells cultured alone.The apoptotic level of AE9a-Rac 1 leukemia cells cultured alone was significant higher than that of AE9a-Rac 1 leukemia cells in co-culture system.Western blot showed that activated level of PARP in AE9a-Rac1 leukemia cells co-cultured with osteoblast was lower than that cultured alone.RT-PCR result showed that TPO and N-cadherin mRNA levels in primary osteoblast separated from leukemic mice were higher than that from normal mice.Ang1 and OPN mRNA levels of osteoblast from leukemia mice were lower.Conclusion Osteoblast cell can support the survival and inhibit the apoptosis of leukemia cells.Leukemia cells can influence the functions of osteoblast by microenvironment associated cytokines production.

Objective To detect the proteins levels of RhoA and CDC42 in bone marrow mononucleated cells (BMMC) of patients with primary acute leukemia,and further determine the role of abnormal interactions between hematopoietic progenitor and bone marrow microenvironment on abnormal behaviors of leukemia cells. Methods BMMC samples were separated from 54 primary acute leukemia patients and 22 normal donors and the cell lysis samples were prepared. RhoA and CDC42 proteins were determined by Western blotting. Independent pair T test was conducted to evaluate whether the differences in RhoA and CDC42 expression were statistically significant between leukemia patients and normal donors. Spearman was applied in analyzing the correlation between expression of RhoA and CDC42 proteins and clinical characters of patients. Results RhoA and CDC42 proteins level of primary acute leukemia patients was significantly higher than that of normal samples. Especially, patients with M2,M3 and M5 subtypes exhibited significant higher RhoA proteins levels and M3 subtype exhibited significant higher CDC42 protein levels. Conclusion RhoA and CDC42 protein levels of primary acute leukemia patients are significantly higher than that of normal donors. This result suggests that RhoA and CDC42 associated efficient migration of leukemia cells could be implicated in abnormal interaction of leukemic cell with bone marrow microenvironment.

Objective of this study was to detect the expression of HtrA2 and WT1 mRNA in acute myeloid leukemia (AML) and investigate the relationship of their expression levels with clinical variates and correlation between them. The expression levels of HtrA2 and WT1 were measured by RQ-PCR in bone marrow cells in 104 newly diagnosed AML patients and leukemia cell lines (K562, HL-60, NB4, Kasumi-1, U937), and the relationship between expression level and clinical parameters (age, sex, WBC count, diagnosis and prognosis) was investigated. The results showed that (1) the expression of HtrA2 gene in newly diagnosed AML was lower than that of the normal controls (P < 0.01), while expression of WT1 gene in newly diagnosed AML was higher than that of the normal controls (P < 0.01), the expression levels of HtrA2 and WT1 genes both did not correlate with age, sex and WBC counts of patients. There were no significant difference of HtrA2 gene expression between different NCCN prognosis group, while WT1 gene expression in better-risk group was significantly lower than that in intermediate-risk group (P = 0.003). The HtrA2 expression level rose after treatment in both CR group and non-CR group (P < 0.05), while WT1 expression level significantly decreased after treatment only in CR group (P < 0.01). Negative correlation between HtrA2 and WT1 expression was also observed (r = -0.249, P = 0.011). It is concluded that the low expression of HtrA2 and high expression of WT1 are closely related with occurrence and development of acute leukemia, so up-regulating expression of HtrA2 and interfering expression of WT1 may become the targets for leukemia therapy in the future.
Adolescent ; Adult ; Aged ; Cell Line, Tumor ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Mitochondrial Proteins ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; WT1 Proteins ; genetics ; metabolism ; Young Adult

OBJECTIVETo observe the effects of AML1A and AML1B, two splicing isoforms of AML1, on the transactivation of macrophage colony-stimulating factor receptor (M-CSF-R), and explore the mechanism of hematopoietic stem cell committed differentiation and leukemogenesis.

METHODSThe expressive plasmids of AML1A and AML1B were constructed, and co-transfected into CV-1 cells with a luciferase reporter plasmid containing M-CSF-R promoter. The transactivity of M-CSF-R promoter was assayed by luminometer.

RESULTSAML1B exhibited a distinct transactivity to M-CSF-R promoter with a sequence-specificity and dosage-dependent manner. AML1A showed no any transactivity but antagonized the effect of AML1B, causing marked reduction of M-CSF-R expression.

CONCLUSIONAn intact structure of AML1 is necessary for transactivation of M-CSF-R. AML1A may interfere with the transactivation of AML1B, and play a key role in the fine regulation of committed differentiation of hematopoietic cell.

Animals ; Cell Differentiation ; genetics ; Cells, Cultured ; Core Binding Factor Alpha 2 Subunit ; genetics ; Gene Expression Regulation, Leukemic ; Genetic Vectors ; Haplorhini ; Hematopoietic Stem Cells ; cytology ; Kidney ; cytology ; Plasmids ; genetics ; Receptor, Macrophage Colony-Stimulating Factor ; genetics ; Transcriptional Activation ; Transfection

OBJECTIVETo explore the signal transduction pathway in the differentiation and apoptosis of leukemia cells induced by heat shock protein 90 (HSP90) inhibitor 17-Allyl amide-17-demethoxygeldanamycin (17AAG).

METHODSKasumi-1 cells were treated with increasing concentrations or exposure time of 17AAG. The total kit protein (CD117), phosphorylated kit protein and its downstream signaling molecules were measured by Western blot analysis. Mutated kit protein from control and 17AAG-treated Kasumi-1 cells was immunoprecipitated and immunoblotted for associated chaperones.

RESULTSTotal kit protein and kit activity were decreased in 17AAG treated cells, but c-kit mRNA level was not. Total AKT protein and phospho-AKT, as well as phospho-STAT3 were rapidly down-regulated in Kasumi-1 cell after treatment with 17AAG. There was no change in total STAT3 protein. Immunoprecipitation showed that 1 microM 17AAG treatment for 1 hour caused kit associated HSP90 decrease and HSP70 increase.

CONCLUSION17AAG-induced apoptosis of Kasumi-1 cells is associated with a decline in Asn822Lys mutated kit protein level and phosphorylated kit, and with a downregulation in its downstream activated signaling molecules involved in proliferation. AKT is a client protein of HSP90. The changes of kit associated HSP90 and HSP70 satisfy the circulation mode of molecular chaperone complex.

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Differentiation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Heat-Shock Proteins ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Cells, Cultured

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 explore the role and mechanism of AML1a in abnormal hematopoiesis in mice. Plasmids pMSCV-FLAG-AML1a-IRES-YFP and pMSCV-IRES-YFP together with envelope-encoding plasmid pECO and packaging plasmid pGP were respectively transfected into 293T cells by using a method of calcium phosphate precipitation to produce retrovirus. Bone marrow mononuclear cells (BMMNC) from male C57BL/6J mice were transfected with the retroviral vector MSCV expressing FLAG-AML1a fusion protein and yellow fluorescent protein (YFP). The cells were cultured in M3434 semi-solid medium for colony formation assay and in M5300 fluid medium containing murine IL-3 (mIL-3), IL-6 (mIL-6) and SCF (mSCF) for long-term culture. The results showed that transfection of AML1a into BMMNC enhanced colony formation, colony size of the AML1a group was significantly larger than that of the control group, and the colonies were mainly composed of CFU-E and CFU-GEMM. In the long-term culture, AML1a-transfected BMMNC showed differentiation block, while the control cells were in a more mature stage. It is concluded that AML1a may block the normal hematopoiesis at the stage of primitive progenitors. At the same time, AML1a also enhances the proliferation activity of primitive progenitor cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Colony-Forming Units Assay ; Core Binding Factor Alpha 2 Subunit ; genetics ; Genetic Vectors ; Hematopoietic Stem Cells ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Retroviridae ; genetics ; Transfection

iASPP can prompt the cell proliferation and inhibit the apoptosis of many cells. There are putative binding sites of transcription factor GATA-2 upstream of iASPP transcription start site. GATA-2 plays an important role in the proliferation and differentiation of hematopoietic stem cells (HSC) and progenitors. This study was aimed to explore the role of GATA-2 protein in iASPP gene transcription. Firstly, the expression of iASPP and GATA-2 protein in some leukemia cell lines was detected by Western blot. Second, The expressive vector of pCMV5-GATA2 and the luciferase reporter vectors containing possible binding sites of GATA-2 were constructed and co-transfected into HEK293 and CV-1 cells. Then the luciferase activity was assayed by luminometer. Also, ChIP assays were performed to further confirm the specific binding of GATA-2 to iASPP promoter. The results showed that GATA-2 was overexpressed in most cell lines with high level of iASPP. GATA-2 exhibited a significant effect on luciferase activity of reporter gene iASPP and in a dose-dependant manner. The relative luciferase activity was up-regulated to about two-fold of the empty vector control when the transfection dose of pCMV5-GATA2 plasmid was increased to 100 ng. While the effect was more significant in CV-1 cells and showed a 6.7-fold increase. The ChIP assay demonstrated the in vivo specific binding of GATA-2 to iASPP. The binding sites of GATA2 were located between nt -361 ∼ -334 in upstream of iASPP gene transcription start site. It is concluded that transcription factor GATA-2 can bind with the cis-regulatory region of the iASPP promoter and up-regulate iASPP expression.
Animals ; Cell Line ; Cercopithecus aethiops ; GATA2 Transcription Factor ; genetics ; Gene Expression Regulation, Leukemic ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; K562 Cells ; Repressor Proteins ; genetics ; Transcription, Genetic ; Transcriptional Activation ; Transfection

OBJECTIVETo explore the effect of Imatinib mesylate on proliferation, differentiation and apoptosis of leukemic Kasumi-1 cells bearing c-kit mutation.

METHODSKasumi-1 cells were treated with Imatinib at different concentrations in culture. Cell proliferation was assayed by MTT assay, expressions of c-kit antigen, surface myeloid antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining and agarose gel electrophoresis. Western blot was used to analyze the level of c-kit protein tyrosine phosphorylation.

RESULTSImatinib treatment caused a time- and dose-dependent inhibition of the cell proliferation, with a 72 h IC50 of 4.45 micromol/L. Imatinib treatment induced a decrease in the mean fluorescence value of c-kit antigen, a progressive decline in S-phase cell fraction and an increase in G0/G1 cells. Treatment with 5.00 micromol/L of imatinib for 72 h induced an increase in expression of myeloid surface protein CD11, CD13 and CD15, and for 24 h induced an increase in early apoptosis cells [from 9.04% to 86.84% (P < 0.05)]. The apoptosis ladder was observed on agarose gel electrophoresis on 5-day treatment. Tyrosine phosphorylation level of c-kit protein was decreased by Imatinib treatment.

CONCLUSIONTyrosine kinase inhibitor Imatinib mesylate treatment could inhibit proliferation of Kasumi-1 cells which bear a c-kit mutation, induce differentiation, apoptosis and G0/G1 cells accumulation.

Apoptosis ; drug effects ; genetics ; Benzamides ; Cell Differentiation ; drug effects ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Imatinib Mesylate ; Mutation ; Piperazines ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Pyrimidines ; pharmacology