| Objectives To explore tissue specific activities of WT1 (Wilms'tumor gene) promoter and enhancer in initiating and modulating a reporter gene expression and the role of RbAp46(retinoblastoma (Rb) suppressor associated protein 46)gene and IGFBP-rP1 (insulin-like growth factor binding protein related protein 1) gene in leukemia development Methods The promoter and enhancer of WT1 gene was digested from the plasmid pCB.7e250(-) generously provided by Dr. Fraizer GC and subcloned into the plasmid pEGFP-1 (BD Biosciences), a promoterless vector carrying EGFP used to test the activities of promoter and/or enhancer inserted at MCS by measuring the transcription of downstream EGFP gene as a reporter. The resultant construct was transformed into E. Coli. DH5αby calcium phosphate precipitation technique or electroporation. The bacterial plasmids were prepared using alkali lysis buffer and confirmed by restriction enzymes digestion. The plasmids were extracted in large amount with the EndofreeR Plasmid Maxi Kit (QIAGEN) and transfected into NB4, THP-1, SHI-1, U937 and Jurkat by electroporation and K562 , MCF-7, 293 , ECV304, SMMC7721, HT-29 and SHG44 by DMRIE-C regent or Lipofectamine (2000) regent, respectively. Among the hematopoietic cell lines being transfected, K562, NB4, THP-1, SHI-1 do express WT1, while U937and Jurkat do not. WT1–expressing nonhematopoietic cell lines include MCF-7 and 293 and WT1–nonexpressing nonhematopoietic cell lines include ECV304, SMMC7721, HT-29 and SHG44, respectively. After 2 to 4 weeks of culture in presence of appropriate concentration of G418, EGFP expression in the transfected cells was analyzed using FCS2.0 software (Coulter). Promoter and enhancer activities were converted as the mean fluorescence intensity (MFI) of EGFP. Meanwhile the dosage of EGFP and neor at DNA and RNA level in the transfectants were estimated by real-time PCR using SYBR Green I under optimal condition. The plasmid WTA, WT1 (-/-) isoform without two alternatively spliced insertions provided by Dr. Wang ZY, were previously electroporated into NB4 and U937 cells at 350v and 950 microfarads and kept in our lab. Neomycin-resistant clones were selected by growth of the transfectants in presence of appropriate concentration of G418 for 3 weeks. The monoclones of cell overexpressing WT1 (-/-) isoform were selected by Westernblot or real-time PCR. The total expression levels of WT1 and RbAp46 genes of transfectants in U937 and NB4 cells were detected by real-time PCR. The plasmid containing full length cDNA of RbAp46 provided by Dr. Wang ZY was transfected into K562 and SHG44 cells using DMRIE-C regent and Lipofectamine regent, respectively. Empty vector was also transfected into cells to serve as a control. Neomycin-resistant clones were selected in presence of 1.5mg/ml and 0.6 mg/ml G418, respectively, for 3 weeks. The monoclones of transfectant overexpressing RbAp46 gene were obtained by limited dilution with 0.5 cell per well in 96 microwell plate for K562 cells or cloning column for SHG44, respectively, and checked by real-time PCR. Growth curve and colony formation assay were performed to examine the proliferation capability of transfectants. Cell cycle arrest was determined by flow cytometry analysis. Relative genes in transfectants were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Real-time quantitative RT-PCR method was established for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemias and IGFBP-rP1 expression levels in BM cells of 127 patients with acute leukemia (AL), 24 with chronic myelogenous leukemia in chronic phase (CML-CP), 9 with CML in blast crisis (CML-BC) and 27 withnon-leukemias patients. NB4 leukemic cells treated by 0.5 μM ATRA at different times were collected and total cellular RNA was isolated using Trizol (Gibco/BRL Life Technologies). 2 μg RNA were reverse transcribed into cDNA. The expression levels of WT1, RbAp46 and IGFBP-rP1 genes were determined by real-time PCR. The expression of CD11b was simultaneously detected using flow cytometry. Results After a complete digestion of plasmid, pCB.7e250(-),with PstI and HindIII, the fragment of WT1 promoter was obtained and purified from 1.5% agrose gel using gel purifying kit (QIAGEN). The pEGFP-1 was also digested with PstI and HindIII at MCS, and subsequently ligated with WT1 promoter by T4 ligase to construct the recombinant plasmid PEWP which carries both WT1 promoter and EGFP gene. WT1 enhancer was obtained by digestion of pCB.7e250(-) with BamH I and was inserted into PEWP at three different sites, respectively. The plasmid, PEWPA, was constructed by inserting WT1 enhancer at Afl II site of PEWP. The plasmid, PEWPD, was obtained by inserting WT1 enhancer at Not I site of PEWP. While the plasmid, PEWPE, was constructed by inserting WT1 enhancer at BamHI site in MCS of PEWP. The capacitance for electroporation for each cell line was 950 μFD, and the voltages were 400v for SHI-1, 350v for NB4 and U937, 300v for Jurkat and 250v for THP-1, respectively. DNA-lipofectamine complexes were formulated by mixing 4 μg of each plasmid with 12μl Lipofectamine for the transfection of MCF-7, ECV304 and SMMC7721 cells, 10μl Lipofectamine2000 for HT-29 and 293 transfection and 15μl DMRIE-C for K562, respectively. After 2 to 4 weeks the cells selected in presence of 0.6-1.5 mg/ml G418 were examined for the mean fluorescence intensity (MFI) of GFP by flow cytometry. The vector, PEWP, induced 1.75±0.40, 1.74±0.03, 7.29±1.03, 9.46±1.10, 2.37±0.6, 16.54±5.45, 2.63±0.44, 0.84±0.11, 1.15±0.03, 2.96±0.47, 2.09±0.30 and 0.74±0.03 times EGFP expression in THP-1, HT-29, SHG44, MCF-7, SMMC7721, ECV304,293, SHI-1, U937, K562, NB4 and Jurkat cells, respectively, compared to that by pEGFP-1 vector. Among them PEWP induced the highest EGFP expression in ECV304 which did not express WT1 while the lowest EGFP in SHI-1 which did express WT1. Accordingly, the activity of WT1 promoter seemed neither corelated to the expression level of WT1 nor to hematopoiesis. PEWPE, with insertion of WT1 enhancer at MCS of PEWP, increased basal transcription levels of the WT1 promoter only in SMMC7721 and HT-29 cells by about 2.0 fold, respectively. PEWPD, with WT1 enhancer inserted at NotI site near the stop code of EGFP, had no ability to increase basal transcription levels of the WT1 promoter when it is transfected into MCF-7, 293 and K562 cells randomly. PEWPA, with WT1 enhancer inserted at AflII site near SV40polyA, increased basal transcription levels of the WT1 promoter in HT-29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01 fold, respectively. Some cell lines in which WT1 enhancer increased the basal transcription level of WT1 promoter were neither hematopoietic cells nor those which express WT1, suggesting the intronic enhancer doesn't only function in a hematopoietic-specific way. K562/PEWP cells in which EGFP can or can not be detected were sorted with BDFACS AriaTM and DNA from the two subpopulations was extracted by DNA extracting kit (GENTRA). The integration levels of neor and EGFP of the plasmids in the genomic DNA were determined by real-time PCR using SYBR Green I. The relative levels of neor and EGFP were 28.70 vs 38.50 and 1.36 vs 3.62 for K562/PEWP cells with or without EGFP, respectively, suggesting the plasmid PEWP constantly integrated in the cells in which EGFP could not be detected As far as the integration level of plasmids was concerned, it was not even among plasmids transfected into single cell line or different cell lines. For example, EGFPN in NB4/ PEWPA was 174.00 and 4.63 in NB4/PEGFP-1. As is the same for MCF-7, EGFPN in MCF-7/PEGFP-1 and MCF-7/ PEWPE were 701.83 and 549.24,and 40.97 and 115.80 in MCF-7/ PEWPD and MCF-7/ PEWP/NotI, respectively. These results might be caused by transfection using physical techniques including lipofectamine mediatedtransfection and eletroporation. The transcription level of EGFP was also uneven among different cell lines or single cell line transfected with different plasmids. The data showed that the expression level of EGFP was positively correlated to the integration level of plasmids detected by RQ-PCR. The expression level of EGFP depended on the amount of transfected plasmids, not too much or too small. The different degree of EGFP gene silencing was influenced by epigenetic modification. The expression level of WT1 and RbAp46 in U937 transfected with WTA and empty vector was 1.25×10-1±2×10-2 vs 5.57×10-3±1.04×10-3 for WT1 and 5.46±0.59 vs 4.96±0.46 for RbAp46, respectively. The expression level of WT1 and RbAp46 in NB4 cells transfected with WTA and empty vector was 3.2±0.18 vs1.14±0.08 for WT1 and 0.45±0.05 vs 0.22±0.02 for RbAp46, respectively. Although the expression level of WT1 in U937/WTA cells was 21-folds higher than that in control, the expression level of RbAp46 in U937/WTA cells was not upregulated consistently. The expression level of WT1 in NB4/WTA cells was 2-folds higher than that in control, however, the expression level of RbAp46 in NB4/WTA cells was upregulated 2 times. These results suggested that the upregulation of RbAp46 by WT1 also exists in leukemia cells with a cell type-specific manner. Both K562 and SHG44 subclones overexpressing RbAp46 grew more slowly and formed less colonies than the control cells transfected with empty vector. G0/G1 cell population was higher in K562 and SHG44 cells transfected by RbAp46 than that transfected by control vector. IGFBP-rP1 was the only gene that has been observed to express in K562 transfection by RbAp46 gene while it was not expressed in nontransfected K562 cells or K562 cell transfected with "empty vector". This phenomenon was not seen in SHG44 cells. The M-Estimators of RbAp46N in 98 patients with newly diagnosed ALs and 5 with relapsed ALs were higher than those of 28 with ALs in complete remission(CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08 ). Statistic differences were found neither between the CR group and control group nor between the newlydiagnosed group and relapsed group. The M-Estimators of RbAp46 in patients in CML-CP is 58.27, similar to that of control, but much lower than that of CML-BC (173.24). Of the 98 newly diagnosed ALs, The M-Estimators of RbAp46N in M3 and M4 were lowest among acute leukemia subtypes. Furthermore, the RbAp46N expression levels were not correlated to expression of fusion genes of BCR/ABL, PML/RARα, and multidrug resistant gene(MDR1), while were positively correlated with WT1 expression levels and negatively correlated with AML1/ETO fusion gene expression. The M-Estimators of IGFBP-r P1N in 63 patients with newly diagnosed AMLs were higher than those of 27 with ALLs, 10 with AMLs in complete remission(CR)and 27 non-leukemic controls (193.80, 88.84, 67.82, and 81.30 respectively). IGFBP-r P1N was 101.13 in CML-BC and 168.77 in CML-CP with no statistic differences between them, and among the CML-BC, AML, ALL and control groups. The M-Estimators of IGFBP-r P1N were lowest in M4 and highest in M5 among initial acute leukemia subtypes, and there were statistic differences between M4 and M5, and between M4 and M2. IGFBP-r P1N expression levels were not correlated to expression of fusion genes of BCR/ABL and AML1/ETO, but positively correlated with PML/RARαfusion gene, WT1 and RbAp46 expression levels, with coefficiency of 0.71, 0.31 and 0.52, respectively. WT1 and IGFBPr-P1 genes expression was rapidly decreased during the differentiation of NB4 cells induced by ATRA. The WT1N level was 1.91, 1.21, 0.67, 0.44 , 0.18, 0.04, respectively, at 0, 4, 8, 12, 24 and 48 h after exposure to ATRA, and IGFBP-RP1N were 1.17, 0.80, 0.54, 0.28, 0.17 and 0.15 at same time points. While RbAp46N were decreased slowly during first 24 h, and became more markedly decreased at 48 h. RbAp46N was 2.65, 1.86, 1.40, 1.27, 1.48 and 0.49, respectively. The trend of RbAp46N in control group was similar. RbAp46N was 2.65, 2.65, 1.63, 2.4, 2.12 and 1.18 respectively. It suggested the expression level of RbAp46 was depending on the culture time. However, there was a statistic difference between the two groups at the same time. The changes of WT1 in treatment group was related to the others (γ=0.829,P=0.021,γ=1,P=0.000), and the changes of these three genes were adversely related to that of CD11b expression (γ=-1.0,P=0.000,γ=-0.829,P=0.021 andγ=-1.0, P=0.000). Conclusions WT1 promoter functions in all cell lines tested and has no tissue specificity. While WT1 enhancer has the ability to improve WT1 promoter to regulate gene expression not only in hematopoiesis but also in nonhematopoietic tissue. The transcription activities of WT1 promoter and enhancer seems not directly related to the expression level of WT1. WT1 enhancer constructed in different site of PEWP shows different activitives in increasing transcription activity of WT1 promoter in a cell type-specific manner. Transgene expression is suppressed because of multicopies of plasmids integration. The expression level of EGFP was positively correlated to the integration level of plasmids detected by RQ-PCR. The expression level of EGFP decreased when plasmids were overintegrated or lowintegrated in genomic DNA. The different degree of EGFP gene silencing was influenced by epigenetic modification. The relation between WT1 and RbAp46 in leukemia cells also displayed in a cell type-specific way. The proliferation of transfectants overexpressing RbAp46 is suppressed because DNA synthesis is inhibited. The relation between IGFBP-rP1 and RbAp46 in leukemia cells also exhibit a cell type-specific way. The expression levels of WT1, RbAp46 and IGFBP-rP1 are decreased consistently during the differentiation of NB4 cells treated with ATRA and increased in patients with acute myelogenous leukemias at first diagnosis or relapse. It appears, therefore, that RbAp46 and IGFBP-rP1 genes expression are associated with the immature cells from which leukemic cells originate just like WT1 does. We thus hypothesize that WT1, RbAp46 and IGFBP-rP1 gene axis pathway might exist in leukemia and this pathway may be abnormally activated during the development of acute myelogenous leukemia.
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