RESUMO
The Myc antagonists Mad1, Mxi1 and Rox proteins share two highly conserved domains, Sin3-interacting domain (SID) and basic helix-loop-helix leucine zipper domain (bHLHzip), which are essential for these proteins to function during molecular switching from proliferation to differentiation. In an attempt to identify mutations in Mad1, Mxi1 and Rox genes in human haematological malignancies, we screened 10 haematopoietic cell lines, bone marrow mononuclear cells (BMMNC) from 26 patients with haematological malignancies and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription-polymerase chain reaction, single strand conformation polymorphism analysis and sequencing. Mad1, Mxi1 and Rox genes were expressed in all samples. Four polymorphisms were found in cell lines BMMNC and PBMNC: two in Mad1, one in Mxi1 and one in Rox. Nine missense mutations were detected: two in Mad1 in patients, four in Mxi1 (three in patients and one in KG-1 cell line), and three in Rox in patients. No mutations were detected in PBMNC from healthy volunteers. Among six patients with acute lymphoblastic leukaemia, two had Mxi1 mutations and another two had Rox mutations. These mutations were associated with poorer clinical outcomes. This is the first report to show that Mad1, Mxi1 and Rox genes were expressed and displayed mutations in haematological malignancies.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Análise Mutacional de DNA , Leucemia/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Doença Aguda , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Células HL-60 , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/metabolismo , Células U937RESUMO
OBJECTIVE: To investigate the expression and mutation of Mad1, Mxi1 and Rox genes in leukemia cells. METHODS: Expression and mutation of Mad1, Mxi1 and Rox genes in bone marrow mononuclear cells (BMMNC) from 26 de novo acute leukemia (AL) patients, and in peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, as well as in 7 human leukemic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing. RESULTS: RT-PCR showed that all the above cells expressed Mad1, Mxi1 and Rox mRNA. SSCP revealed four polymorphisms: two in Mad1, one each in Mxi1 and Rox. DNA sequencing detected nine missense mutations: two in Mad1 in AL patients, four in Mxi1 (three in AL patients and one in KG-1 cell line), and three in Rox in AL patients. The mutations of Mad1, Mxi1 and Rox mRNA were detected in 2, 3 and 3 patients, respectively. CONCLUSION: It is for the first time to demonstrate the mutations of Mad1, Mxi1 and Rox genes in AL patients suggesting these mutated genes involve in the pathogenesis of leukemia.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Leucemia/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo Conformacional de Fita Simples , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
This study was purposed to investigate the effect of phycocyanin at different concentration on proliferation of K562 cells, to detect the changes of integrin beta1 expression and intracellular focal adhesion kinase (FAK) gene expression on the surface K562 cells treated with phycocyanin, and to explore the possible mechanism of integrin beta1 effect on phycocyanin inhibiting proliferation of K562 cells. The expression level of integrin beta1 on the surface of K562 cells was evaluated by flow cytometry (FCM); the growth of K562 cells treated with phycocyanin was measured by MTT assay; the expression level of FAK mRNA was analyzed by relatively quantitative RT-PCR after four-day culture of K562 cells with phycocyanin of 40 microg/ml, 80 microg/ml and 160 microg/ml, respectively. The results showed that integrin beta1 expression on the surface of K562 cells was significantly higher than that in bone marrow mononuclear cells (BMMNC) from normal subjects. Phycocyanin could not change the level of integrin beta1 expression. Phycocyanin could increase the expression of FAK gene on K562 cells and inhibit the proliferation of K562 cells. It is concluded that phycocyanin can inhibit the proliferation of K562 cells through enhancing the conjunction of cell stroma with integrin beta1 on K562 cell surface, up-regulating the expression level of FAK gene in K562 cells, restoring the signaling pathway of proliferation inhibition mediated by integrin beta1. The possible mechanism of phycocyanin in the proliferation inhibition of K562 cells is to increase the expression of FAK gene. The phycocyanin may be considered as a potential agent for inhibition of cancer cell proliferation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Integrina beta1/biossíntese , Ficocianina/farmacologia , Quinase 1 de Adesão Focal/biossíntese , Quinase 1 de Adesão Focal/genética , Humanos , Integrina beta1/genética , Células K562 , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.