[Inhibiting effect of CaMKIIN up-regulation on leukemia cells growth and its mechanism].

OBJECTIVE: To investigate the inhibitory effects of CaMKIIN on acute myeloid leukemia cell line HL-60 to explore a novel therapeutic target of leukemia. METHODS: Human CaMK II N gene expression vector pcDNA3.1/hCaMKIIN or empty vector pcDNA3.1/myc-His (-) B was transfected into HL-60 cells by Lipofectamine 2000. Human CaMK II N proteins of transfected cells were detected by Western blot. Cell proliferation affected by human CaMKIIN was determined by MTT. Colony-forming assay was performed by soft agar growth system. The cells transfected with CaMKIIN were stained with Hoechst 33342 to detect the apoptotic proportion under fluorescence microscopy. Cell cycle was analyzed by flow cytometry. RESULTS: Human CaMKIIN was stably transfected into HL-60 cells, and overexpression of human CaMKIIN inhibited the proliferation of HL-60/CaMKIIN cells compared to HL-60/mock cells and HL-60 cells [(0.44 ± 0.03) vs (0.94 ± 0.05) vs (0.94 ± 0.04), P<0.01]. The colony formation of HL-60/CaMKIIN was also markedly smaller[(21.00 ± 3.05)/500] than that of mock-transfected [(111.00±4.58)/500]] and control cells [(119.00±6.09)/500] (P<0.01). After 72 hrs-culture, the apoptotic proportion in cells transfected with CaMK II N was obviously higher than of cells transfected with mock DNA or control [(22.49 ± 2.15)% vs (7.17 ± 0.72)% vs (6.40 ± 0.55)%, P<0.01]. Up to (82.97 ± 2.90)% human CaMKIIN/HL-60 cells were arrested at G0/G1 phase, which was more than mock-transfected [(40.53 ± 2.38)%] and control cells [(41.63 ± 2.27)%] (P<0.05). Human CaMKIIN could down-regulate expression of Bcl-2 in transfected cells. CONCLUSION: CaMK IIN up-regulation could inhibit proliferation and induce apoptosis of human acute myeloid leukemia cell HL-60.

[Abnormal expression of transcription factors LYL1 and LMO2 and interaction between them in myeloid leukemia].

OBJECTIVE: To explore the expression of the transcription factors LMO2 and LYL1 and the interaction between these 2 factors in myeloid leukemia cells and to analyze the significance thereof in leukemogenesis. METHODS: Samples of peripheral blood and bone marrow were collected form 51 AML patties, and 5 normal bone marrow donors to isolate mononuclear cells (MNCs) with high percentage of CD34(+) cells. Western blotting (WB) was used to detect the protein expression of LMO2 and LYL1 in the cells. Human myeloid leukemia cells of the line K562 were cultured and transfected with pcDNA3-LMO2, plasmid containing LMO2, pcDNA3-LYL1, plasmid containing LYL1, or pcDNA-GFP, blank plasmid containing green fluorescent protein. RT-PCR was used to detect the mRNA expression of LMO2 and LYL1. Co-immunoprecipitation (co-IP) and WB were used to detect the binding protein of LMO2 and LYL1. RESULTS: The MNCs of 51.1% of the patients with acute myeloblastic leukemia (AML) without remission expressed higher levels of LMO2, the MNCs of 62.2% of the AML patients expressed higher levels of LYL1, and the MNCs of 31.1% of those expressed both. The K562 cells transfected with pcDNA3-LMO2 showed higher mRNA and protein expression levels of both LMO2 and LYL1, and the K562 cells transfected with pcDNA3-LYL1 showed higher mRNA and protein expression levels of both LYL1 and LMO2 too, as indicated by RT-PCR and WB, which suggested that the expression of LMO2 and the expression of LYL1 stimulated each other in the myeloid leukemia cells. Co-IP assay detected the presence of LMO2-LYL1 complex in those cells. CONCLUSION: The abnormal expression and protein interaction of LMO2 and LYL1 may play a role in the abnormal proliferation and differentiation of myeloid hematopoietic cells.

[Expression of transcription factor LYL1 in leukemia and its possible role in leukemogenesis].

OBJECTIVE: To study the expression of transcription factor LYL1 in leukemia and its possible role in leukemogenesis. METHODS: Fluorescence real time quantitative polymerase chain reaction was used to detect the expression levels of LYL1 in leukemias. Specific siRNA was used to silence the expression of LYL1 in K562 cells. RESULTS: Compared to CD34 positive cells from normal bone marrow, the expression of LYL1 was significantly elevated in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). LYL1 expression was higher in chronic myeloid leukemia (CML) in blastic crisis than that in chronic phase (7.831 vs 1.672, P < 0.01). LYL1 expression in AML in complete remission (CR) was down-regulated as compared with that of un-remission patients (1.400 vs 9.985, P < 0.01). Down-regulation of endogenous expression of LYL1 in K562 cells by a combination of three specific siRNA could inhibit cellular growth and clonogenicity to some extent. CONCLUSION: Over-expression of LYL1 is highly associated with AML as well as ALL. RNA interference targeting specific oncogenes such as LYL1 is potentially useful in the treatment of hematological malignancies.

[Epigenetic regulation of expression of retinoic acid receptor beta gene in leukemia cell].

OBJECTIVE: To study the reactivation of retinoic acid receptor beta (RARbeta) expression in myeloid leukemia cells by a combination of all-trans retinoic acid (ATRA) with a DNA demethylating agent, decitabine (DAC), and valproic acid (VPA, a histone deacetylase inhibitor),and their effects on cell differentiation and proliferation. METHODS: Human myeloid leukemia cells of the line U937 were cultured and treated with all-trans retinoic acid (ATRA), DAC, and VPA. 72 h later cell differentiation test, cloning formation test, and chromatin immunoprecipitation test were performed. Bone marrow specimens were collected from 56 patients with acute myeloblastic leukemia (AML) were cultured and treated with and 10 bone marrow specimens were used as controls. Methylation of RARbeta promoter was detected with methylation specific polymerase chain reaction (MSP) after bisulfite treatment. Relative levels of RARbeta mRNA were assessed with real time quantitative PCR assay. Flow cytometry assay was used to detect the myeloid differentiation marker CD11b in U937 cells. Chromatin immuno-precipitation assay (ChIP) was used to analyze the acetylated histone 3 bound to the retinoic acid response element (RARE) at the promoter region of RARbeta. RESULTS: Methylation of RARbeta was positive in 36 of the 56 (64.3%) AML patients and the U937 myeloid leukemia cells, however, was negative in the marrow mononuclear cells from the 10 healthy donors. The expression of RARbeta in U937 cells was up-regulated after treatment with ATRA (1 micromol/L) plus DAC (1 micromol/L) or VPA (0.5 mmol/L) for 72 hours, especially when the three drugs were used together. ChIP assay showed that the acetylated histone 3 bound to the RARE promoter region was increased after the cells were exposed to ATRA plus DAC/VPA. The data indicated that the reactivated expression of RARbeta might be secondary to the drug-induced histone acetylation as well as DNA demethylation. Treatment of U937 cells with ATRA and DAC/VPA also resulted in increased myeloid differentiation (CD11b expression) and decreased plating efficiency. CONCLUSION: The repressed expression of RARbeta in myeloid leukemia cells is closely related to both DNA hypermethylation and histone deacetylation, and a combination of ATRA with epigenetic modulators can be beneficial in the treatment of myeloid malignancies.