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ToxSci Advance Access published online on June 3, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh183
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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Received February 2, 2004
Accepted May 17, 2004

Systems Evaluation

The Tumor Promoter 12-O-Tetradecanoylphorbol 13-Acetate (TPA) Provokes a Prolonged Morphologic Response and ERK Activation in Tsc2-Null Renal Tumor Cells

Todd M. Kolb 1 Myrtle A. Davis 2*

1 Program in Toxicology, University of Maryland, School of Medicine, Baltimore, MD 21201; Department of Pathology, University of Maryland, School of Medicine, Baltimore, MD 21201
2 Toxicology and Drug Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, PO Box 708, Greenfield, IN 46140

* To whom correspondence should be addressed. E-mail: davisma{at}lilly.com.


  Abstract

Loss of tumor suppressor function dramatically alters the cellular response to chemicals. The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulates cell proliferation through rapid activation of protein kinase C (PKC), followed by gradual degradation of the kinase. TPA also activates the GTPase Rap1 in some cell types. The tumor suppressor protein Tsc2 has a proposed GTPase activating protein (GAP) function for Rap1, providing a common mechanistic target for Tsc2 and TPA. We compared the cellular response of Tsc2-null (ERC-18) and Tsc2-competent (NRK-52E) renal epithelial cells to TPA treatment. Treatment of ERC-18 cells with 100 ng/ml TPA for 24 hours resulted in loss of cell-cell contact, retraction of the cell periphery and rounding. These changes were reversed 1 hour after treatment in NRK-52E cells and were apparent 24 hours after treatment of ERC-18 cells. Expression of Tsc2 in ERC-18 cells abrogated the prolonged morphologic response. TPA treatment rapidly increased phosphorylation of ERK, a reported downstream effector of both PKC and Rap1, in ERC-18 cells, but induced weak Rap1 activation. TPA-induced ERK phosphorylation was prolonged in ERC-18 cells compared to NRK-52E cells and expression of Tsc2 in ERC-18 cells did not inhibit prolonged ERK activation. The selective PKC inhibitor bisindolylmaleimide VIII however, inhibited TPA-induced changes in morphology and ERK activation. These results imply that TPA-induced changes in morphology and ERK activation are mediated primarily through PKC and not Rap1 in renal epithelial cells. These data also and imply that Tsc2 expression modulates TPA-induced changes in renal epithelial cell morphology via an ERK-independent mechanism.

Key Words: Tsc2, protein kinase C, TPA .


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