OBJECTIVETo investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis.

METHODSThe methylation of LRP15 promoter and first exon of bone marrow mononuclear cells in 73 patients with AL, 10 with chronic leukemia (CL), 9 with hematological benign diseases, and 20 healthy transplantation donors was analyzed by using methylation specific polymerase chain reaction. The methylation of LRP15 gene promoter and first exon in COS7, K562, and HL60 cell lines was also assayed.

RESULTSNo LRP15 gene promoter methylation was detected in COS7 cell line. LRP15 gene promoter was methylated in K562 and HL60 cell lines. No deletion of LRP15 gene was detected in all samples. In nearly all French-American-British leukemia subtypes, we found that frequency of LRP15 methylation in adult patients with AL was 71.23% (52/73). There was no detectable methylation in any of the 20 healthy donors and 8 chronic myeloid leukemia patients. The difference in frequency of LRP15 methylation between AL patients and healthy donors or CL patients (10.00%, 1/10) was significant (P < 0.01). Hypermethylation of LRP15 gene was found in 57.14% (16/28) of newly diagnosed AL patients, 83.33% of relapsed AL patients respectively, which was significantly different (P < 0.05). We also demonstrated LRP15 methylation in 55.56% (5/9) adults with benign hematological diseases.

CONCLUSIONSLRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.

Acute Disease ; Animals ; Base Sequence ; COS Cells ; Cell Line, Tumor ; Cercopithecus aethiops ; DNA Methylation ; DNA Primers ; Humans ; Leukemia ; genetics ; Neoplasm Proteins ; genetics ; Promoter Regions, Genetic

This study was aimed to investigate the methylation status of WT1 gene in leukemia cell lines and its relation with expression of WT1 gene. The WT1 gene was silenced by DNA methylation or histone deacetylation, and the expression of WT1 gene was induced by using HDAC inhibitor and/or demethylation agent of DNA. Some leukemia cell lines (U937, HL-60, K562, KG1) were detected by RT-PCR, MS-PCR, restriction analysis, and DNA sequencing. U937 leukemic cells without WT1 mRNA expression were incubated with HDAC inhibitor Trichostatin A (TSA) and/or demethylation agent decitabine. The results showed that the U937 cells did not express WT1 gene, but HL-60, K562 and KG1 cells highly expressed WT1 gene; WT1 gene was unmethylated in HL-60 cells, but methylated in K562 and U937 cells. WT1 expression could be reactivated by co-incubation with TSA and decitabine, but not was observed by using single drug. It is concluded that WT1 promoter is methylated in some leukemia cells, however, the methylation can not affect its expression. DNA methylation and deacetylation of histones are synergistic to inhibit the expression of WT1 in leukemic U937 cells, the combination of TSA with decitabine can induce expression of WT1 gene.
Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; Gene Silencing ; HL-60 Cells ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; Promoter Regions, Genetic ; U937 Cells ; WT1 Proteins ; genetics

The objective of the study is to explore the effect of Fas, FasL and Bcl-2 on the process of apoptosis induced by chemotherapeutic drugs through detecting the expression of Fas, FasL and Bcl-2 on murine lymphoma cell line RMA. Dexamethasone(DEX), etoposide (VP-16), arsenic trioxide As(2)O(3) and all trans-retinoic-acid (ATRA) were added to the RMA cells as well as to the cells preincubated with interleukin-2 (IL-2), interleukin-6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. The effect on apoptosis was observed and the expression of Fas and FasL mRNA as well as the expression of Fas and Bcl-2 antigen were measured. DEX and VP-16 could promote apoptosis of RMA cells while upregulating the expression of Fas and FasL without affecting the expression of Bcl-2. ATRA downregulated the expression of Bcl-2 without any change of Fas and FasL, and no apoptosis of RMA cells induced by ATRA was observed. Although As(2)O(3) induced apoptosis of RMA cells, it did not affect the expression of Fas, FasL and Bcl-2, which suggested that different drugs induce apoptosis of the same kind of cells by different signal transduction system and apoptosis induced by Fas system needed the coexistence of Fas and FasL. Although IL-2, IL-6 and GM-CSF upregulated the expression of Fas protein when adding to RMA cells separately, none of them induced apoptosis. Apoptosis could be induced by combination of IL-2 and IL-6 along with the upregulation of Fas and FasL. The cytokines facilitated the apoptotic action of chemotherapeutic drugs, the drug concentration for inducing apoptosis decreased and the time period of starting apoptosis shortened. Apoptosis could be observed without the expression of FasL when anti-Fas-antibody was added to RMA cells. The results demonstrated that there was synergistic effect of chemotherapeutic drugs and some cytokines for induction of apoptosis. Fas-FasL system participated in the apoptosis induced by DEX and VP-16; different drugs induce apoptosis by different pathway of signal transduction.
Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Apoptosis ; physiology ; Arsenicals ; pharmacology ; Dexamethasone ; pharmacology ; Etoposide ; pharmacology ; Fas Ligand Protein ; Gene Expression ; drug effects ; Membrane Glycoproteins ; biosynthesis ; Mice ; Oxides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Tretinoin ; pharmacology ; Tumor Cells, Cultured ; fas Receptor ; biosynthesis

To study whether gene IGSF4 was inactived by methylation in leukocytic cells, expression of IGSF4 was examined before and after treatment with demethylating agent in U937, Molt4 and HL-60 leukemia cell lines by means of RT-PCR. The methylation of promoter in U937, Molt4 and HL-60 cells as well as 21 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of IGSF4 promoter was inactived and could be reversed by treatment with a demethylating agent in U937, Molt4 and HL-60 cell. IGSF4 promoter methylation was detected in 57.1% of acute leukemia patients. There is no difference in incidence of IGSF4 promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, IGSF4 is frequently inactived in acute leukemia and is a good candidate for the leukemia suppressor gene. As a normal suppressor gene, it may play an important role in inhibiting the development of leukemia, and the methylation of gene IGSF4 may be a good index in monitoring relapse of leukemia.
Acute Disease ; Cell Adhesion Molecule-1 ; Cell Adhesion Molecules ; Cell Line, Tumor ; DNA Methylation ; Humans ; Immunoglobulins ; genetics ; Leukemia ; genetics ; Membrane Proteins ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Tumor Suppressor Proteins

In order to observe the effect of inhibitors for demethylation and histone deacetylase on the growth of leukemia cell line K562 and the expressin of tumor related genes, the K562 cells were treated with 5-aza-2' deoxycytidine (DAC) and trichostatin A (TSA) in co-culture; the growth curves were observed; the cell cycle was detected by flow cytometry (FCM); the gene expression pattern before and after drug treatment was measured with Atlas7742-1 microarray. The results showed that the combination treatment of DAC and TSA inhibited the proliferation of K562 cells, the growth of most cells were stopped in G(1)/S phases after drug treatment, the gene expression after treatment was more than before, and a few gene expression were down-regulated. In conclusion, combination treatment of DAC and TSA had an inhibitive effect on the leukemia cell line K562, combination of DAC and TSA with microarray could be used for screening candidate genes inhibiting leukemia cells.
Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; DNA Methylation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; drug effects

To investigate the relationship between LRP15 gene promoter region methylation and its gene expression in acute leukemia patients, the status of LRP15 gene promoter region methylation was detected by MS-PCR and the gene expression was detected by RT-PCR in bone marrow samples from leukemia patients. The results indicated that the LRP15 gene expression was 47.6% in complete remission (CR) patients and 16.7% in non-CR patients respectively, while LRP15 gene promoter region methylation was 38.1% in CR group and 72.2% in non-CR group respectively. No relationship was found between LRP15 gene promoter region methylation and its expression (P = 0.0087). It is concluded that the methylation in LRP15 gene promoter region may not be the only reason for LRP15 gene silence.
DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; pathology ; Male ; Neoplasm Proteins ; biosynthesis ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; pathology ; Promoter Regions, Genetic ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Fluorescence intraoperative cholangiography (IOC) is a potential alternative for identifying anatomical variation and preventing iatrogenic bile duct injuries by using the near-infrared probe indocyanine green (ICG).However,the dynamic process and mechanism of fluorescenceIOC have not been elucidated in previous publications.Herein,the optical properties of the complex of ICG and bile,dynamic fluorescence cholangiography and iatrogenic bile duct injuries were investigated.The emission spectrum of ICG in bile peaked at 844 nm and ICG had higher tissue penetration.Extrahepatic bile ducts could fluoresce 2 min after intravenous injection,and the fluorescence intensity reached a peak at 8 min.Inaddition,biliary dynamics were observed owing to ICG excretion from the bile ducts into the duodenum.Quantitative analysis indicated that ICG-guided fluorescence IOC possessed a high signal to noise ratio compared to the surrounding peripheral tissue and the portal vein.Fluorescence IOC was based on rapid uptake of circulating ICG in plasma by hepatic cells,excretion of ICG into the bile and then its interaction with protein molecules in the bile.Moreover,fluorescence IOC was sensitive to detect bile duct ligation and acute bile duct perforation using ICG in rat models.All of the results indicated that fluorescence IOC using ICG is a valid alternative for the cholangiography of extrahepatic bile ducts and has potential for measurement of biliary dynamics.

OBJECTIVETo analyse the WT1 expression and its DNA methylation status of its promoter domain.

METHODThe expression of WT1 gene and its DNA methylation status were assayed in leukemia cell lines and normal peripheral blood mononuclear cells (PBMNC) by RT-PCR and MS-PCR.

RESULTSWT1 was overexpressed in HL60, K562 and KG1 leukemia cell lines, but not in U937 and PBMNC. Methylation of WT1 promoter was not observed in HL60 cells.

CONCLUSIONDNA methylation of WT1 gene promotor did not inhibit its expression. Other mechanisms may appear to regulate the WT1 expression.

Cell Line, Tumor ; DNA Methylation ; Genes, Wilms Tumor ; Humans ; Leukemia ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic

LRP16 is a novel gene which was found in our laboratory by using methylation-sensitive restriction landmark genomic scanning (RLGS) technique. In order to clone the full-length cDNA of this leukemia relapse associated gene, the method of rapid amplification of cDNA end (RACE) was employed. By optimizing some procedures of RACE method, the 5'- and 3'-untranslated region of LRP16 cDNA was successfully sequenced. Then, the full length of LRP16 cDNA and open reading frame (ORF) was constructed and was registered in GenBank. The above-mentioned procedure demonstrated RACE technique is a rapid and sensitive method for cloning unknown gene. Especially, it is very useful to cloning the 5'- and 3'-untranslated region of a novel gene.

It has need to separate red blood cells (RBC) from marrow graft in ABO group unmatched BMT and auto-BMT with purging tumor cells, the separating effect of methylcellulose was observed. The mixture of 0.5% methylcellulose and bone marrow was laid up in an open transfusion system, and then sedimentation of RBC was performed in the transfusion tube. The separating results of 18 marrow grafts showed that the recovery rates of mononuclear cells and CD34(+) cells were (83.8 +/- 55.2)% and (90.3 +/- 7.2)%, respectively. RBC residual rate was (4.3 +/- 1.5)%. The yield of CFU-GM was (60.8 +/- 22.4)/2 x 10(5) MNC, and there was no difference to [(69.8 +/- 23.4)/2 x 10(5) MNC] yielded from same marrow samples, separated by Ficoll-Hypaque separation. It is concluded that this method could be used for bone marrow transplantation.
Bone Marrow Transplantation ; methods ; Cell Separation ; methods ; Erythrocytes ; immunology ; Humans ; Methylcellulose ; pharmacology