1660486

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3

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.

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MEDLINE

0021-9541

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548-59

pubmed-meshheading:1660486-Blotting, Northern, pubmed-meshheading:1660486-Blotting, Southern, pubmed-meshheading:1660486-Cell Line, pubmed-meshheading:1660486-Chloramphenicol O-Acetyltransferase, pubmed-meshheading:1660486-Gene Expression Regulation, Enzymologic, pubmed-meshheading:1660486-Gene Expression Regulation, Neoplastic, pubmed-meshheading:1660486-HIV Long Terminal Repeat, pubmed-meshheading:1660486-Humans, pubmed-meshheading:1660486-Kanamycin Kinase, pubmed-meshheading:1660486-Kinetics, pubmed-meshheading:1660486-Macrophages, pubmed-meshheading:1660486-Moloney murine leukemia virus, pubmed-meshheading:1660486-Phosphotransferases, pubmed-meshheading:1660486-Plasmids, pubmed-meshheading:1660486-Recombinant Proteins, pubmed-meshheading:1660486-Teratoma, pubmed-meshheading:1660486-Tetradecanoylphorbol Acetate, pubmed-meshheading:1660486-Transcriptional Activation, pubmed-meshheading:1660486-Transfection, pubmed-meshheading:1660486-Tumor Necrosis Factor-alpha

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

Journal Article, Research Support, U.S. Gov't, P.H.S.