ABSTRACT
Analyses of CYP1A1 mRNA were used to monitor the responsiveness of murine hepatoma 1c1c7 and human monocytic U937 cells in different phases of the cell cycle to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Concentrations of TCDD capable of inducing CYP1A1 were not cytostatic to either cell line. Steady-state CYP1A1 mRNA contents were reduced (45-90%) in TCDD-treated cultures arrested in G2/M as a consequence of exposure to microtubule disrupters (Colcemid, estramustine, vinblastine) or the microtubule stabilizer Taxol, relative to TCDD-treated asynchronous 1c1c7 cultures. The accumulation of mRNAs corresponding to Nmo1, another TCDD-inducible gene of the Ah battery, was also reduced in TCDD-treated G2/M cultures. Quantitative reverse transcriptase-polymerase chain reaction analyses of CYP1A1 heterogeneous nuclear RNA (hnRNA) revealed that Cyp1a1 transcription was suppressed in G2/M cells. This suppression reflected neither changes in the relative content of the proteins comprising the aryl hydrocarbon receptor (AHR) complex nor a suppression of AHR activation and translocation to the nucleus. Release of 1c1c7 cultures arrested in G2/M restored TCDD responsiveness. Centrifugal elutriation of TCDD-treated asynchronously growing U937 cells was used to prepare populations of cells in specific phases of the cell cycle. Within 3 h of TCDD exposure late G1/early S phase cells had CYP1A1 mRNA contents approximately 1.4- and 3-fold higher than the contents of asynchronous/early G1 and G2/M cultures, respectively. These studies suggest that the transcriptional activation of members of the Ah battery by TCDD is cell cycle-dependent, and markedly suppressed in G2/M cells.
Subject(s)
Cytochrome P-450 CYP1A1/biosynthesis , Dioxins/pharmacology , Blotting, Western , CDC2 Protein Kinase/metabolism , Cell Cycle/drug effects , Centrifugation , Chromosomes/drug effects , Chromosomes/ultrastructure , Enzyme Activation/drug effects , Enzyme Activators/pharmacology , Flow Cytometry , Fluorescent Antibody Technique , Humans , Orotic Acid/metabolism , Polychlorinated Dibenzodioxins/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Nuclear/biosynthesis , Receptors, Aryl Hydrocarbon/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
WEHI-231 is a mouse B-cell line, which is a well-established model for studying signal transduction in B lymphocytes, normally responding to cross-linking of the B-cell receptor (BCR) complex by the rapid upregulation of protein tyrosine kinase activity, followed by increased intracellular calcium and activation of protein kinase C. In WEHI-231, activation of protein kinase C is functionally associated with downregulation of DNA synthesis, followed by the induction of apoptosis. We have found in WEHI-231, that at low and environmentally relevant exposure levels (0.1 microM) mercury is not toxic, but still interferes with signal transduction in that it attenuates the growth inhibitory effects of BCR cross-linking. The molecular target for mercury resulting in attenuation of the BCR-mediated growth inhibitory signal is likely proximal to activation of the BCR complex, as HgCl2 had no effect on the negative growth signal generated downstream by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Treatment of WEHI-231 cells with high and toxic concentrations of Hg results in a marked increase in protein tyrosine phosphorylation in a great many proteins; whereas treatment of WEHI-231 cells with 0.1 microM mercury is not toxic. Under these conditions mercury selectively perturbed BCR-mediated protein tyrosine phosphorylation of a 75 kDa protein, without grossly affecting tyrosine phosphorylation levels of most other proteins. These data suggest that low levels of mercury, which are not toxic, may still contribute to immune dysfunction by interfering with antigen-receptor-mediated and protein-kinase-dependent signal transduction in lymphocytes.
Subject(s)
DNA/biosynthesis , Mercury/metabolism , Signal Transduction , Animals , Cell Division/drug effects , Dose-Response Relationship, Drug , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Immunoglobulin M/pharmacology , Ions , Lymphoma, B-Cell , Mercaptoethanol/metabolism , Mercaptoethanol/pharmacology , Mercuric Chloride/metabolism , Mercuric Chloride/pharmacology , Mice , Mitogens/metabolism , Mitogens/pharmacology , Receptors, Antigen, B-Cell/metabolism , Tetradecanoylphorbol Acetate/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, CulturedABSTRACT
Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (
