Rottlerin Induces a Transformed Phenotype in Human Keratinocytes Cornelia Dietrich, 1 Nicolas Gumpert, Isabelle Heit, 2 Monika Borchert-Stuhltra ¨ ger, Franz Oesch, and Raimund Wieser 3 Institute of Toxicology, Johannes Gutenberg-University, Obere Zahlbacherstrasse 67, 55131 Mainz, Germany Received February 12, 2001 PKC plays a fundamental role in cell cycle control. Consistent with its proposed tumour suppressor func- tion, ras transfection of the human keratinocyte cell line HaCaT results in a loss of PKC expression medi- ated by TGF (Exp. Cell Res., 219, 299, 1995). To get more insight into the role of PKC in keratinocytes, we investigated the effects of Rottlerin, a specific in- hibitor of protein kinase C, in HaCaT cells. After Rottlerin treatment, HaCaT cells lost their cobble- stone morphology and displayed a spindle-shaped, fi- broblastic phenotype. Additionally, the establishment of cell– cell contacts was prevented. This was caused by an internalization of E-cadherin and -catenin as assessed by immunofluorescence. A similar phenotype was observed in the presence of a neutralizing anti-E- cadherin antibody. Rottlerin-treated HaCaT cells pro- liferated like transformed cells in a three-dimensional cell culture system. We therefore conclude that PKC is involved in mediating cell– cell contacts via E-cadherin and hence regulates differentiation in HaCaT cells. © 2001 Academic Press Key Words: human keratinocytes; cell– cell contacts; dedifferentiation; PKC. Proteinkinase C (PKC) was initially identified by Nishizuka and coworkers as a cytoplasmic, calcium- activated, phospholipid-dependent serine/threonine- specific kinase (1). Molecular cloning and biochemical studies have revealed that PKC is a family of at least 11 isoforms which can be subdivided into three main classes: (i) the conventional cPKCs (PKC, I, II, ) which require calcium, diacylglycerol, or phorbolesters, and negatively charged phospholipids for optimal acti- vation; (ii) the novel nPKCs (PKC , , , /L) which lack the calcium binding region and are thus activated as the cPKCs but independently of calcium; (iii) the atypical aPKCs (PKC/ and ) which require only neg- atively charged phospholipids for activation and do not respond to phorbolesters [for Review see (2)]. Another isoform, PKC (PKD) possibly forms a distinct sub- group: PKC is activated similarly to the novel PKCs but is structurally different to them (3). One of the best studied isoforms so far is PKC, which has been described to act as a tumour suppres- sor in several cell lines [for Review see (4)]. Consistent with a role as a tumour suppressor, PKC is downregu- lated after oncogenic transformation in cell culture (5, 6) and during carcinogenesis in vivo (7). Vice versa, restoring PKC activity induces a reversal of the trans- formed phenotype in colonic epithelial cells (8) and delays the development of preneoplastic lesions in rat hepatocarcinogenesis (9). However, the physiological function of PKC still remains to be elucidated. In keratinocytes, PKC is involved in terminal dif- ferentiation and cell cycle control. For example, the concentration of PKC increases dramatically in mu- rine epidermis between day 7 and 14 after birth corre- sponding with a decrease in proliferation and an in- crease in keratin expression (10). It has also been shown that PKC regulates involucrin expression, a protein which is involved in cornified envelope forma- tion (11). Consistent with a role in differentiation, over- expression of PKC in keratinocytes results in cell cycle arrest (12). Interestingly, oncogenic ras transfor- mation of the human keratinocyte cell line HaCaT leads to a decrease in PKC protein levels mediated by TGF (13), and downregulation of PKC has also been observed during skin carcinogenesis in the mouse re- sulting in a delay of terminal differentiation (14). Ac- cordingly, in transgenic mice overexpressing PKC epidermal tumour development in the initiation/pro- motion model is significantly reduced (15). These ob- servations are in agreement with the proposed tumour suppressor function of PKC. 1 To whom correspondence should be addressed at Institute of Toxicology, Obere Zahlbacherstrasse 67, 55131 Mainz, Germany. Fax: +49/6131/230506. E-mail: cdietric@mail.uni-mainz.de. 2 Present address: Byk Gulden, Byk Guldenstrasse 2, 78467 Kon- stanz, Germany. 3 Present address: InnoTides GmbH, its, Arnold-Sommerfeld-Ring 2, 52499 Baesweiler, Germany. Biochemical and Biophysical Research Communications 282, 575–579 (2001) doi:10.1006/bbrc.2001.4530, available online at http://www.idealibrary.com on 575 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.