RESUMO
OBJECTIVE: To screen new candidate molecular-targeted anti-leukemia compounds with potential functions of targeted up-regulating ID4 gene expression. METHODS: Promoter region of ID4 gene including the upstream - 3000 bp sequence of transcriptional start site and message RNA sequence were fished out. Online promoter analysis tools of TESS and Genomax were used to search possible sequence of transcriptional start site and message RNA sequence were fished out. Online promoter analysis tools of TESS and Genomax were used to search possible cis-acting structure from human transcription factor database. The activity of related drugs with potential effects upon ID4 gene expression was analyzed using SAGE database. GEO database was applied to search the gene expression profiling regulated by ID4 gene. Finally, similar analysis between gene expression profiling by ID4 and genome-wide profiling regulated by 163 known drugs or active compounds was manipulated to screen the drugs and candidate compounds with similar gene expression profiling with ID4 gene. MOLT4 cell line was treated with the above candidate active compounds to investigate the ID4 gene expression by RT-PCR assay. RESULTS: ID4 gene had a type II promoter with a typical TATA box in upstream -45 bp of transcription start site. The 1300 bp-length promoter of ID4 gene contained a few cis-acting structures classified into two function types, i. e. positive regulatory type, including transcription factors Spl and c-Myb, cAMP, glucocorticoid receptor (GR) and estrogen receptor (ER), and negative regulatory type, including Wilms tumor-1 (WT1) and early growth response-2 (EGR2). The similarity of gene expression profiling was identified between cAMP and ID4 gene. ID4 gene expression was induced in MOLT4 cell line after treatment with calcium dibutyryladenosine cyclophosphate at the concentration of 0.1 mmol/L. CONCLUSION: The comprehensive bioinformatic analysis, based upon the combination of regulatory sequence prediction of promoter, similarity analysis of gene expression profiling and literature review, can be considered as a practical tool in screening the candidate drugs with the activity of targeted regulating functional genes. Calcium dibutyryladenosine cyclophosphate can induce ID4 gene expression in leukemic cells.
Assuntos
Biologia Computacional , Proteínas Inibidoras de Diferenciação/genética , Linhagem Celular Tumoral , Humanos , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Regulação para CimaRESUMO
OBJECTIVE: To investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis. METHODS: The 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. RESULTS: No 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. CONCLUSIONS: LRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.
Assuntos
Metilação de DNA , Leucemia/genética , Proteínas de Neoplasias/genética , Doença Aguda , Animais , Sequência de Bases , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Primers do DNA , Humanos , Regiões Promotoras GenéticasRESUMO
OBJECTIVE: To investigate the methylation status of LRP15 gene in acute leukemia (AL) and its role in tumorigenesis. METHODS: 73 cases of AL and 9 healthy subjects as well as COS7, K562 and HL60 cell lines were studied with methylation specific PCR (MSP). RESULTS: LRP15 was not detected in all the samples. No LRP15 methylation was detected in COS7, but LRP15 was methylated in K562 and HL60. In nearly all of the French-American-British leukemia subtypes, we found that the frequency of LRP15 methylation was 71.2% (52/73) of AL and none in the 9 healthy subjects. The difference in mean methylation for LRP15 between these two kinds of samples is statistically significant (P < 0.05). Hypermethylation of the LRP15 gene was found in 57.1% (16/28) of the newly diagnosed AL and 83.3% of the relapsed AL respectively; this is also statistically significant (P < 0.05). CONCLUSION: LRP15 methylation change is a common abnormality in leukemia and LRP15 is postulated to be a tumor suppressor gene.
Assuntos
Metilação de DNA , Leucemia/genética , Proteínas/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células COS , Feminino , Células HL-60 , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Proteínas de NeoplasiasRESUMO
OBJECTIVE: To observe the early outcome of imatinib in the treatment of chronic myeloid leukemia (CML). METHODS: There were 54 CML patients including 17 patients in chronic phase (CP), 13 patients in accelerated phase (AP) and 24 patients in blast crisis (BC). The dosage of imatinib is 400 mg/day for CP patients and 600 mg/day for AP and BC patients. The prescribed dose was administrated orally, once daily after a meal with a large glass of water. Before starting the imatinib administration, all the patients had thorough physical examination as well as blood and bone marrow examination, Ph chromosome and or the bcr/abl fusion-gene detection. Blood picture was examined twice a week in the first month of administration and thereafter weekly or fortnightly in the following days. Hepatic and renal function was tested fortnightly or every four weeks. Patients had bone marrow examination, Ph chromosome and or the bcr/abl fusion-gene detection again when they achieved a hematological response (CR). Dose was adjusted from time to time according to the results of blood examination and the patients tolerance to the drug. RESULTS: After a median 5-month period of follow-up, all the 17 CP patients achieved complete hematological response, including 6 cases (35.2%) whose Ph chromosome turned negative after treatment. 8 (61.5%) out of the 13 AP patients and 9 (37.5%) out of the 24 BC patients returned to chronic phase respectively. Adverse events included myelosuppression, periorbital edema or edema of the lower limbs, myalgia and muscle cramps, nausea, vomiting, low fever, tetter and bilirubin increasing. CONCLUSIONS: imatinib has good early efficacy in the treatment of CML and the therapeutic result is the best in the CP patients, next in the AP patients and relatively poor in the BP patients. The long term outcome of imatinib needs further observation. Patients have a good tolerance to imatinib.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adolescente , Adulto , Idoso , Benzamidas , Medula Óssea/efeitos dos fármacos , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Pirimidinas/efeitos adversosRESUMO
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.
Assuntos
Metilação de DNA , Inativação Gênica , Proteínas WT1/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Decitabina , Células HL-60 , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Células K562 , Regiões Promotoras Genéticas , Células U937RESUMO
BACKGROUND AND OBJECTIVE: LRP16 is a human novel gene linked to leukemia identified recently. However, its biological function is not fully clarified so far. This study was to investigate the biological function of human LRP16 gene by database-aided bioinformatics analysis. METHODS: The structures and functions of LRP16 gene promoter and its coding protein were analyzed using bioinformatics prediction, and further experimental testing was performed. The recombinants of pGL3-basic and LRP16 promoter subclones were constructed for luciferase activity analysis. The recombinant of LRP16 open reading frame coding sequence and pcDNA3.1 eukaryotic expression vector was established and transfected into HL-60 and K562 cell lines. DNA damage of HL-60 cells after ultraviolet irradiation was evaluated using single cell gel electrophoresis. Cell cycle of K562 cells was analyzed by flow cytometry. RESULTS: LRP16 promoter was a typical class II eukaryotic promoter and its core regulation sequence was located within upstream -600 bp of transcriptional start site. In addition, seven cis-acting elements, which may be implicated in cell cycle, hematopoiesis regulation, cell proliferation and repair of DNA damage, were identified. Long type LRP16 coding protein contained homologous sequences of hismacro, COG2110, and A1pp with human histone H2A1C between 148 and 315 amino acid residue. The number of comet cells and the length of comet tail in HL-60 cells irradiated were significantly decreased and the number of living cell was significantly increased in LRP16-overexpression group compared with empty plasmid control group. The proliferation rate and ratio or quantity of G2/M and S phases were significantly increased in LRP16-overexpression K562 group compared with empty plasmid control group. LRP16-overexpression in K562 cells promoted the transition of G1 to S phase and plateau phase of cell proliferation was advanced. CONCLUSIONS: Promoter regulation prediction and protein domain analysis based on bioinformatics contribute to the study of gene function. LRP16 may play an important role in leukemia progression by promoting cell proliferation, regulating cell cycle, and antagonizing radiation-induced DNA damage.
Assuntos
Neoplasias da Mama/patologia , Ciclo Celular , Proliferação de Células , Proteínas de Neoplasias , Neoplasias da Mama/metabolismo , Hidrolases de Éster Carboxílico , Linhagem Celular Tumoral , Biologia Computacional , Dano ao DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Células K562 , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Regiões Promotoras Genéticas , TransfecçãoRESUMO
The study was aimed to investigate the promotive effect of LRP16 gene on K562 cell proliferation. Open reading frame of LRP16 gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and ligated to pGEM-T plasmid to construct LRP16 ORF-pGEM-T recombinant vector. Then, LRP16 ORF identified by sequencing was inserted into pcDNA3.1+ plasmid to construct LRP16 ORF-pcDNA3.1+ recombinant expression plasmid which was transfected into K562 cell lines to make overexpression of LRP16 gene in K562 cells. Survival of cells was determined by MTT assay and growth curve of cells was drawn, the cell cycle was detected by flow cytometry. The results showed that LRP16 ORF was successfully amplified, then the LRP16 ORF-pcDNA3.1+ recombinant plasmid was constructed. The K562 cell line with overexpression of LRP16 gene was established. The promotive effect of LRP16 gene overexpression on proliferation of K562 cells was observed and the effect partially related to the enhancement of cells from G0 to S phase induced by LRP16 gene. It is concluded that LRP16 gene overexpression shows a promotive effect on proliferation of K562 cells.
Assuntos
Proliferação de Células , Proteínas de Neoplasias/genética , Hidrolases de Éster Carboxílico , Vetores Genéticos , Humanos , Células K562 , Fases de Leitura Aberta , PlasmídeosRESUMO
This study was purposed to investigate lrp16 gene expression in leukemia cell lines and bone marrow cells of leukemia patients and explore the relationship between lrp16 gene expression and development of leukemia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to test the lrp16 mRNA expression in 4 leukemia cell lines, including K562 (CML), HL-60 (APL), MOLT4 (ALL) and U937 cell lines, as well as in bone marrow-derived cells from 115 patients with leukemia. The effect of lrp16 gene expression on genesis and progression of leukemia was analyzed according to clinicopathological features. The results indicated that positive expression of lrp16 mRNA was found in all 4 leukemia cell lines. For leukemia patients, the positive expression rate of lrp16 mRNA in all AML patients was 38% (16/42), in which the positive rates in AML patients with complete remission (CR) and AML patients without remission were 13% (4/30) and 100% (12/12) respectively. The positive expression rate of lrp16 mRNA in ALL patients was 38% (10/26), in which the positive rate in ALL patients with CR and ALL patients without remission were 16% (3/18) and 87% (7/8) respectively. The positive expression rate of lrp16 mRNA in CML patients was 36% (9/25), in which the positive rates in CML patients with CR and CML patients without remission were 20% (4/20) and 100% (5/5) respectively. The positive rate of lrp16 mRNA in CLL patients was 31% (7/22), in which the positive rate in CLL patients with CR and CLL patients without remission were 11% (2/17) and 100% (5/5) respectively. There was no difference of lrp16 gene expression between leukemia subtypes, but there was statistical significant difference in lrp16 gene expression between CR patients and non CR patients (p < 0.001). It is concluded that the lrp16 gene is a leukemic oncogene and closely relates to genesis and progression of leukemia, which may be an indicator for evaluating clinical efficacy of leukemia therapy.
Assuntos
Medula Óssea/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Proteínas de Neoplasias/metabolismo , Adolescente , Adulto , Idoso , Medula Óssea/patologia , Hidrolases de Éster Carboxílico , Feminino , Células HL-60 , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , Adulto JovemRESUMO
OBJECTIVE: To explore ZO-1 gene expression and methylation in leukemia cells and the involvement of ZO-1 gene in leukemogenesis. METHODS: Restriction landmark genomic scanning (RLGS) was used to identify new leukemia related gene, and methylation specific PCR (MSP) for ZO-1 methylation status. ZO-1 specific siRNA was designed and prepared by in vitro transcription and transfected into K562 cells, the transfected cells were cultured for 48 hours before harvesting. The effect of ZO-1 siRNA was monitored by Northern blot. Cellular proliferation capacity was assayed by CCK-8, cell apoptosis by Annexin V-fluorescence in isothiocyanate (FITC) assay, and cell cycle by phosphatidylinositol (PI). RESULTS: The intensified spots in RLGS gel were subjected to bioinformatics analysis and one of the candidate spots was proved to be ZO-1 gene. In fresh leukemia cells, Molt4 cells and HL-60 cells, ZO-1 was hypermethylated, causing it reduced or silenced. ZO-1 gene was highly expressed with no methylation in normal peripheral blood MNC and K562 cells. There was a good correlation between promoter methylation and the gene silence. The silenced gene can be re-activated by demethylation treatment with 5-AZA-dC in most leukemia cell lines. RNA interference for ZO-1 gene in K562 cells did not interfere with cell proliferation, cell cycle and apoptosis. CONCLUSION: ZO-1 gene methylation might be involved in the tumorigenesis of acute leukemia.
Assuntos
Metilação de DNA , Leucemia/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Animais , Células HL-60 , Humanos , Células K562 , Leucemia/patologia , Proteínas de Membrana/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
The study was aimed to identify a new leukemia related gene zo-1 from leukemia and to explore its mechanism in leukemia. Methylation specific PCR (MSP) was used for testing gene zo-1 methylation in leukemia cells. The gene zo-1 specific siRNA was designed according to its sequence, and transfected into THP-1 cell, and the cells were cultured for 48 hours before harvesting. The effect of zo-1 siRNA was monitored by RT-PCR. The cellular proliferation activity was assayed by CCK-8, the apoptosis was detected by Annexin-V-fluorescence in isothiocyanate (FITC) assay, and cell cycle was observed by propidium iodide (PI). The results indicated that the gene zo-1 in patients with acute leukemia was hypermethylated, while the gene zo-1 in healthy persons was unmethylated. The THP-1 cells with unmethylation of zo-1 gene promoter overexpressed the gene zo-1, while the Molt4 and HL-60 cells with hypermethylation of gene zo-1 promoter did not express the gene zo-1. The silenced zo-1 gene in Molt4 and HL-60 leukemia cell lines could be reactivated by demethylation treatment with 5-AZA-dC. The oligofectamine-transfected siRNA for zo-1 gene successfully inhibited the expression of gene zo-1 in THP-1 cells, but did not interfere with cell proliferation, cell cycle and apoptosis. It is concluded that gene zo-1 is a leukemia-related gene. Gene zo-1 in acute leukemia was hypermethylated, the methylation status of gene zo-1 regulates the expression of gene zo-1. Lack of gene zo-1 expression in THP-1 cells does not influence the cell proliferation, apoptosis and cell cycle, which suggests that the methylation of gene zo-1 may be involved in the genesis of acute leukemia, its mechanism is worthy to be studied.
Assuntos
Metilação de DNA , Leucemia/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Ilhas de CpG , Inativação Gênica , Células HL-60 , Humanos , RNA Interferente Pequeno/genética , Proteína da Zônula de Oclusão-1RESUMO
This study was aimed to obtain higher efficiency in gene transfection into K562 cells and to study the role of green fluorescence protein (GFP) as a reporter system. Transfection efficiencies with different methods including electroporation and lipofectamine 2000 transfection, were observed and calculated under fluorescent microscopy by using GFP as a reporter system. The results showed that the transfection efficiency with electroporation (10%) was higher than that with lipofectamine 2000 (1%). In conclusion, the electroporation is a more ideal method for introduction of foreign gene into K562 cells. GFP can be used as a reporter system for optimizing transfection of K562 cells.
Assuntos
Genes Reporter , Proteínas de Fluorescência Verde/genética , Transfecção , Eletroporação , Humanos , Células K562 , LipossomosRESUMO
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.
Assuntos
Metilação de DNA , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Low expression of ID4 gene is tightly related with carcinogenesis and high expression shows a definite anti-leukemia effect, though little expression in some leukemia cells. The main purpose of this preliminary work was to analyze the construction of ID4 gene promoter and to predict the cis elements in the ID4 promoter region by scanning the drug candidate with bioinformatics method. All these work are the primary part for finding effective drugs in the treatment of leukemia via the way of ID4 expression regulation. According to the data in GenBank and Internet platform, the 5'-untranslated sequence just upstream of ID4 ORF was virtually cloned. TESS, Genomatix and GenBank databank were used to analyze the cis elements in this area. RSA was used to find the distribution patterns for all these possible elements. SAGE and GEO datasets were used to find active substances which have the effect on the ID4 expression. The rsults indicated that ID4 had a type II promoter with a typical TATA box-45 bp upstream the transcriptional original site. There were a lot of various cis elements in the 5'-untranslated region upstream, including both positive element candidates such as Sp1, c-Myb, abaA, GR, ER, Zeste and C/EBPalpha and negative element candidates such as CCAAT-binding factor, GCF, WT1-KTS, HiNF-C and EGR2. It is concluded that estrogen, dexamethasone, thyroid hormone and follicle stimulating hormone may participate in the regulation of ID4 gene expression in both positive and negative manners.
Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas Inibidoras de Diferenciação/metabolismo , Leucemia/genética , TATA Box/genética , Regiões 5' não Traduzidas/genética , Animais , Biologia Computacional/métodos , Dexametasona/farmacologia , Sistemas de Liberação de Medicamentos , Estrogênios/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The study was aimed to analyze the characteristics of LRP16 gene promoter and its activity in order to explore the possible regulation mechanism of LRP16 gene expression. A 2.6 kb genomic DNA sequence of LRP16 5'-end was obtained from NCBI by BLAST software. The 7 target sequences between 0.2 - 2.6 kb from a healthy blood donor DNA sample were amplified by PCR, then identified by DNA sequencing and semi-nest PCR. The verified sequences were analyzed on-line. The results showed that the 7 target sequences were about 400 bp different from each other. All 7 sequences were the same to these GenBank described. At last, all 7 promoter sequences were ligated with luciferase vector, and then the luciferase activity was analyzed in HeLa cells. A known gene promoter sequence can be freely obtained from NCBI database. It is concluded that LRP16 promoter is a standard type II promoter and its activity is strongest in the region from -200 to -600 bp.
Assuntos
Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Hidrolases de Éster Carboxílico , Cromossomos Humanos Par 11 , Expressão Gênica , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Análise de Sequência de DNARESUMO
OBJECTIVE: To evaluate the possibility of Id4 gene promoter methylation as a biomarker for minimal residual disease (MRD) detection in acute leukemia. METHODS: Methylation specific-PCR technique was used to detect Id4 gene methylation in samples with different percentages of leukemia cells from leukemia cell line and bone marrows from leukemia patients in complete remission (CR). RESULTS: Id4 gene methylation could be detected in samples containing 1% or lower leukemia cells. Frequency of Id4 gene methylation in acute lymphoblastic leukemia (ALL) patients in CR was 64.3% being higher than that in acute myeloid leukemia (AML) patients in CR. In 14 ALL patients with Id4 gene methylation, 8 relapsed in 12 months, while only one relapsed in 9 patients without Id4 gene methylation. In 8 AML patients with Id4 gene methylation, 5 relapsed in 12 months, while two relapsed in 20 AML patients with Id4 gene methylation. CONCLUSION: Id4 gene promoter methylation is a candidate of biomarker for MRD detection in acute leukemias.
Assuntos
Metilação de DNA , Proteínas Inibidoras de Diferenciação/genética , Leucemia/diagnóstico , Neoplasia Residual/diagnóstico , Doença Aguda , Adolescente , Adulto , Idoso , Linhagem Celular , Feminino , Humanos , Leucemia/genética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Adulto JovemRESUMO
To study the methylation in the promoter of LRP15 gene and its relationship with gene expression and to explore the possible mechanism of regulating LRP15 gene methylation, the methylation in the promoter of LRP15 gene in K562 cell line was detected by MS-PCR. Then K562 was exposed to 5-aza-2'-deoxycytidine (CdR) and trichostatin (TSA), to determine whether the silencing of LRP15 gene by de novo methylation could be reversed. As a result, it was confirmed by MS-PCR that the promoter of LRP15 was hypermathylated in K562 cell line, and lost its transcription activity. After CdR, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Observation demonstrated that the expression of LRP15 was controlled by methylation in its promoter in K562. It is suggested that methyltransferase inhibitor and deacetylase inhibitor may be effective agents in leukemia therapy.
Assuntos
Metilação de DNA , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Células K562 , Proteínas de Neoplasias/biossíntese , Reação em Cadeia da Polimerase/métodosRESUMO
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.
Assuntos
Metilação de DNA , Imunoglobulinas/genética , Leucemia/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Doença Aguda , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Humanos , Reação em Cadeia da Polimerase , Proteínas Supressoras de TumorRESUMO
OBJECTIVE: To analyse the WT1 expression and its DNA methylation status of its promoter domain. METHOD: The 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. RESULTS: WT1 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. CONCLUSION: DNA methylation of WT1 gene promotor did not inhibit its expression. Other mechanisms may appear to regulate the WT1 expression.
Assuntos
Leucemia/genética , Linhagem Celular Tumoral , Metilação de DNA , Genes do Tumor de Wilms , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Células K562/efeitos dos fármacos , Azacitidina/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Decitabina , Humanos , Ácidos Hidroxâmicos/farmacologiaRESUMO
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.