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 thatCyp1a1 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 ∼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.
Footnotes
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↵1 Current address: Ann Arbor Laboratories, Pfizer, Inc., 2800 Plymouth Rd., Ann Arbor, MI 48105.
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↵2 Current address: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555.
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This work was supported by National Institutes of Health Grants CA34469 and ES07806 and was assisted by the services of the Cell Culture Core and the Cell Imaging and Cytometry Core, which are supported by National Institutes of Environmental Health Sciences Grant P30 ES06639.
- Abbreviations:
- AHR
- aryl hydrocarbon receptor
- ARNT
- aryl hydrocarbon receptor nuclear translocator
- CYP
- cytochrome P450
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- FACs
- fluorescence-activated cell sorter
- hnRNA
- heterogeneous nuclear RNA
- rcRNA
- recombinant RNA
- bp
- base pair
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- pRb
- retinoblastoma protein
- Received May 3, 2001.
- Accepted July 27, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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