JOURNAL OF CELLULAR PHYSIOLOGY 172:306 – 313 (1997) Retinoic Acid Induced Growth Arrest of Human Breast Carcinoma Cells Requires Protein Kinase Ca Expression and Activity YUNHI CHO, ANN P. TIGHE, AND DAVID A. TALMAGE* Institute of Human Nutrition, Columbia University, New York, New York Retinoic acid inhibits proliferation of hormone-dependent, but not hormone- independent breast cancer cells. Retinoic acid-induced changes in cellular prolif- eration and differentiation are associated with disturbances in growth factor sig- naling and frequently with changes in protein kinase C expression. PKCd, e, and z are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKCa expression and concomitantly repressed PKCz expression. The changes in PKCa and PKCz reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKCa, but not the reduction in PKCz, resulted from selective activation of RARa. In total, these results support an important role for PKCa in mediating the anti-proliferative action of retinoids on human breast carcinoma cells. J. Cell. Physiol. 172:306–313, 1997.  1997 Wiley-Liss, Inc. Retinoic acid (RA) inhibits the proliferation of many, characterized means activate other serine/threonine protein kinases (e.g., p70 s6k , p56 Akt , and JNK/SAPK) but not all, human mammary carcinoma cell lines (Bol- (Chung et al., 1994; Franke et al., 1995). lag et al., 1992). Several cytokines also inhibit the pro- Twelve distinct PKC isozymes are encoded by a fam- liferation of human breast cancer cells (Bollag et al., ily of at least 11 genes. These isozymes are divided into 1992; Huslig et al., 1993), and there is growing evidence three groups based on sequence homology and cofactor that cytokines, such as the interferons, potentiate the requirement (Blobe et al., 1994): the conventional PKCs retinoic acid-induced inhibition of transformed epithe- (a, bI, bII, and g), the novel PKCs (d, e, m, h, and u), lial cell proliferation (Bollag et al., 1992; Bollag, 1991; and the atypical PKCs (i, l, and z). Physiologically, Hemmi et al., 1987). As a result, the combination of conventional and novel PKCs are activated by the con- retinoids and interferons is a promising chemothera- certed action of phospholipid and the second messenger peutic option in breast cancer (Bollag, 1991; Hemmi et diacylglycerol (Blobe et al., 1994). Conventional PKCs al., 1987; Sparano and O’Boyle, 1992). also require calcium released from intracellular stores RA responsiveness appears to be limited to hormone- by IP 3 (Blobe et al., 1994). Atypical PKCs are calcium dependent cells (Butler and Fontana, 1992) and corre- independent and are not activated by DAG but can be lates with expression of the estrogen receptor (Fontana activated by the novel phospholipid, PtdIns 3,4,5-P 3 ,a et al., 1990). The proliferative effect of estrogen on product of PtdIns 3-kinase (Nakanishi et al., 1993). mammary cells has been linked to the production of These observations support a model in which PKC iso- mitogenic growth factors and increased expression of zymes are differentially activated in response to dis- surface receptor tyrosine kinases (Daly et al., 1994; tinct growth factor or hormone activation of either PLC Halter et al., 1992). Growth factor binding to receptor or PtdIns 3-kinase. tyrosine kinases activates multiple, interactive signal- RA inhibits signaling between receptor tyrosine ki- ing pathways, most of which involve sequential activa- nases and the nucleus (Leid et al., 1993; Talmage and tion of serine/threonine protein kinases. Two of the tar- gets for receptor tyrosine kinases, phospholipase Cg (PLCg) and phosphatidylinositol 3-kinase (PtdIns 3-ki- nase), produce second messengers that activate multi- In this study we measured the level of seven of the PKC isozymes (a, b1/b2 together, g, d, e, and z). The other isozymes have not ple isozymes of protein kinase C (PKC). PLCg, activated been studied in these cells. by tyrosine phosphorylation, hydrolyzes phosphatidyl- Contract grant sponsor: American Cancer Society; Contract grant inositol 4,5-bisphosphate to diacylglycerol (DAG) and number: CN134; Contract grant sponsor: U.S. Department of inositol trisphosphate (IP 3 ), which in turn, activate con- Army; Contract grant number: DAMD17-94-J-4100. ventional and novel PKC isozymes. Phosphatidylin- *Correspondence to: David A. Talmage, Institute of Human Nu- ositol 3,4,5,-trisphosphate (PtdIns 3,4,5-P 3 ), produced trition, Columbia University, 701 West 168th Street 5-503, New after activation of PtdIns 3-kinase (Talmage et al., York, NY 10032. E-mail: dat1@columbia.edu 1989; Stephens et al., 1993), can directly activate atypi- cal PKCs (Nakanishi et al., 1993), and through poorly Received 2 October 1996; Accepted 26 March 1997  1997 WILEY-LISS, INC. JCP-506D / 8925$$506d 07-21-97 18:54:37 wlcpa W Liss: JCP