A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and ad...A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.展开更多
Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and βmaj promoter of MEL cell line before and after induction, MEL cell has been cult...Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and βmaj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and βmaj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β-globin gene cluster participate in the regulation of developmental specificity.展开更多
Searching for more potent and less toxic HMBA related agents. Methods. Human erythroleukemia cell K562, murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which...Searching for more potent and less toxic HMBA related agents. Methods. Human erythroleukemia cell K562, murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA, then analyzed the activity of inducing differentiati on of two new designed HMBA derivatives: HMBPA [hexamethylenebi (3 pyridin) ami de] and Co HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology, c ytochemical and molecular biology techniques. Results. We found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive, B+～76%). Co HDTA can inhibit the growth of MEL DS19, but induces differentiation just in a small population (B+ 2%～4.5%). Between 0.02～5μmo l/L, HMBPA induces 3%～8% cells committed to differentiation with little inhib ition of cell proliferation. 1μmol/L HMBPA and 2mmol/L HMBA together, can obvio usly increase the percentage of differentiated cell (B+～ 72%), inhibit DNA sy nthesis and accelerate β globin transcription. Conclusion. The new HMBA derivatives may provide potential cancer differentiatio n inducers.展开更多
文摘A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.
文摘Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and βmaj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and βmaj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β-globin gene cluster participate in the regulation of developmental specificity.
文摘Searching for more potent and less toxic HMBA related agents. Methods. Human erythroleukemia cell K562, murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA, then analyzed the activity of inducing differentiati on of two new designed HMBA derivatives: HMBPA [hexamethylenebi (3 pyridin) ami de] and Co HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology, c ytochemical and molecular biology techniques. Results. We found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive, B+～76%). Co HDTA can inhibit the growth of MEL DS19, but induces differentiation just in a small population (B+ 2%～4.5%). Between 0.02～5μmo l/L, HMBPA induces 3%～8% cells committed to differentiation with little inhib ition of cell proliferation. 1μmol/L HMBPA and 2mmol/L HMBA together, can obvio usly increase the percentage of differentiated cell (B+～ 72%), inhibit DNA sy nthesis and accelerate β globin transcription. Conclusion. The new HMBA derivatives may provide potential cancer differentiatio n inducers.
