Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells
The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical u...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
2012-10-01
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| Series: | Brazilian Journal of Medical and Biological Research |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012001000005 |
| _version_ | 1818303054124941312 |
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| author | Q. Sun J. Xiong J. Lu S. Xu Y. Li X.P. Zhong G.K. Gao H.Q. Liu |
| author_facet | Q. Sun J. Xiong J. Lu S. Xu Y. Li X.P. Zhong G.K. Gao H.Q. Liu |
| author_sort | Q. Sun |
| collection | DOAJ |
| description | The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy. |
| first_indexed | 2024-12-13T05:48:42Z |
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| id | doaj.art-835da99a42cb493691cb9e246fc14764 |
| institution | Directory Open Access Journal |
| issn | 0100-879X 1414-431X |
| language | English |
| last_indexed | 2024-12-13T05:48:42Z |
| publishDate | 2012-10-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-835da99a42cb493691cb9e246fc147642022-12-21T23:57:36ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research0100-879X1414-431X2012-10-014510913920Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cellsQ. SunJ. XiongJ. LuS. XuY. LiX.P. ZhongG.K. GaoH.Q. LiuThe distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012001000005Leukemia inhibitory factorTAT-HIV1Protein transduction domainAcute myeloid leukemiaLIF receptor |
| spellingShingle | Q. Sun J. Xiong J. Lu S. Xu Y. Li X.P. Zhong G.K. Gao H.Q. Liu Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells Brazilian Journal of Medical and Biological Research Leukemia inhibitory factor TAT-HIV1 Protein transduction domain Acute myeloid leukemia LIF receptor |
| title | Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells |
| title_full | Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells |
| title_fullStr | Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells |
| title_full_unstemmed | Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells |
| title_short | Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells |
| title_sort | secretory tat peptide mediated protein transduction of lif receptor 945 chain distal cytoplasmic motifs into human myeloid hl 60 cells |
| topic | Leukemia inhibitory factor TAT-HIV1 Protein transduction domain Acute myeloid leukemia LIF receptor |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012001000005 |
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