Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line

doi:10.1016/S0303-7207(98)00229-9

Molecular and Cellular Endocrinology

Volume 148, Issues 1–2, 25 February 1999, Pages 109-118

https://doi.org/10.1016/S0303-7207(98)00229-9 Get rights and content

Abstract

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) δ in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC δ isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC δ was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC δ expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC δ protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10⁻¹⁰–10⁻⁸ M estrogen for 7 days down-regulated specifically PKC δ mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC δ expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor β while the MCF-7 cells expressed predominantly estrogen receptor α and barely detectable levels of estrogen receptor β. These results suggest that the differential ability of estrogen to regulate PKC δ expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Introduction

Classical studies by Beatson in the 1890’s and studies using the rodent mammary tumor model and breast cancer cell lines have underscored the importance of hormones in the development of breast cancer (Huggins et al., 1961, Welsch, 1985, Dickson and Lippman, 1987). Estrogen plays an important role in modulating the physiology of normal breast tissue and in the development of breast cancer (Lippman et al., 1976, Soule and McGrath, 1980). The proliferative response of a tumor to estrogen is the basis of antiestrogen therapy for estrogen receptor positive6 (ER+) breast cancers. Estrogen receptor negative (ER−) or hormonally unresponsive tumors, however, grow aggressively in the absence of estrogen and are not as responsive to antiestrogen therapy (Robinson and Jordan, 1989, Harris et al., 1992). It is important to characterize the estrogen signal transduction pathway in breast cancer cells as knowledge of signaling pathways regulated by estrogen can provide information on potential targets for controlling growth.

One pathway that we have shown to be modulated by estrogen is the protein kinase C (PKC) pathway. The PKC family consists of 11 isoforms that show different tissue localization, requirements for activation and substrate specificities (Dekker and Parker, 1994), and based on various studies (Berra et al., 1993, Borner et al., 1995, Kielbassa et al., 1995, Lehel et al., 1995), is likely to have unique roles in signal transduction. Previous work in our laboratory on the PKC signaling pathway in the estrogen responsive corpus luteum of pseudopregnant rabbits and rats (Maizels et al., 1992, Cutler et al., 1994, Maizels et al., 1996 has shown that estrogen up-regulates expression specifically of the PKC δ isoform. Treatment of luteinized rat granulosa cells with estrogen in primary cell cultures also leads to specific increases in PKC δ isoform expression (Peters et al., 1998). PKC δ has been implicated in growth inhibition based on over-expression of PKC δ in various cell types (Watanabe et al., 1992, Mischak et al., 1993, Mishima et al., 1994).

The PKC signaling pathway has also been implicated in breast cancer in various studies. For example, breast cancer biopsies exhibit higher levels of total PKC activity compared to the surrounding normal tissue (O’Brian et al., 1989). Studies in breast cancer cell lines have noted differential PKC isoforms in terms of activity and protein expression between ER+ and ER− cell lines (Fabbro et al., 1986, Regazzi et al., 1986, Borner et al., 1987, Bignon et al., 1990). Selective regulation of expression of PKC isoforms has also been observed in multiple drug resistant MCF-7 cells compared to wild type cells (Lee et al., 1992, Blobe et al., 1993). Therefore, in view of the importance of PKC in the breast cancer phenotype and of the ability of estrogen to up-regulate PKC δ expression in rat and rabbit ovarian cells, we were interested to determine if there was a similar selective up-regulation of PKC δ in the estrogen responsive MCF-7 human breast cancer cell line.

Section snippets

Materials

The following materials were purchased: [α-³²P]d-CTP (specific activity 3000 Ci/mmol from Dupont-New England Nuclear (Boston, MA); [¹²⁵I]Protein A from ICN Chemical and Radioisotope Division (Irvine, CA); nytran nylon membranes from Schleicher and Schuell (Keene, NH); enhanced chemiluminescence (ECL) reagents and Hybond™-ECL nitrocellulose membranes from Amersham Life Science (Arlington Heights, IL); media components from GIBCO-BRL Life Technologies (Gaithersburg, MD); SDS-PAGE reagents and

Characterization of protein kinase C isoforms in MCF-7 cells

We initiated studies to identify the PKC isoforms expressed in our clonal ER⁺ MCF-7 human breast cancer cell line. Western blot analyses of cytosolic and Triton X-100-solubilized membrane extracts of MCF-7 cells were performed (Fig. 1). PKC α was readily detectable in both cytosolic and Triton X-100-solubilized membrane extracts. PKC β was undetectable in MCF-7 cell extracts, based on the absence of immunoreactivity at 81 kDa, however reactivity with PKC β antibody is shown in the MDA-MB 231

Discussion

The goal of the present study was to ascertain whether estrogen could regulate the expression of PKC δ in the MCF-7 ER⁺ cell line. Expression of various PKC isoforms in ER⁺ MCF-7 cells has been reported (Blobe et al., 1993, Ways et al., 1995). MCF-7 cells express PKC α, δ, ξ, ε, and η (Ways et al., 1995), however, expression of PKC β appears to vary with clonal lines of MCF-7 cells, being detectable in two reports (Lee et al., 1992, Blobe et al., 1993) and undetectable in others (Kennedy et

Acknowledgements

We gratefully acknowledge the gifts of PKC antisera from Drs S. Ohno and K. Mizuno, Yokohama City University School of Medicine, Japan; Dr K. Leach (Upjohn Co., MI); Dr D.K. Ways (East Carolina University School of Medicine, North Carolina). We would also like to thank Dr P.J. Parker (Imperial Cancer Research Fund, London) for the PKC δ cDNA and Dr Craig Jordan for providing us with ICI 164384. This work was supported by the Breast and Cervical Cancer Research Fund, Illinois Department of

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^☆: Portions of this work were presented at the 76th Annual Meeting of the Endocrine Society, Anaheim, CA, 1994, and at the 79th Annual Meeting of the Endocrine Society, Minneapolis, MN, 1997.

¹: Present address: Sugen, Inc. 351 Galveston Drive, Redwood City, CA 94063-4720.

²: Present address: Biomarkers and Prevention Research Branch, NCI, Key West Center, Room 300, 9610 Medical Center Drive, Rockville, MD 20850.

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Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line☆

Abstract

Introduction

Section snippets

Materials

Characterization of protein kinase C isoforms in MCF-7 cells

Discussion

Acknowledgements

Cell

Biochem. Biophys. Res. Comm.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

TIBS

J. Biol. Chem.

Biophys. Res. Comm.

Exp. Cell Res.

Meth. Enzymol.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Acta

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Biol. Chem.

Biochem. Biophys. Res. Comm.

Cancer Lett.

Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture

Proc. Natl. Acad. Sci.

Immunological quantitation of phospholipid/Ca2+-dependent protein kinase of human mammary carcinoma cells: inverse relationship to estrogen receptors

lnt. J. Cancer

Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins

Mol. Endocrinol.

Isolation of biologically active RNA from sources enriched in ribonuclease

Biochemistry

Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth

Cancer Res.

Delta protein kinase-C in the rat ovary: estrogen regulation and localization

Endocrinology

Estrogenic regulation of growth and polypeptide growth factor secretion in human breast carcinoma

Endocrinol. Rev.

Breast Cancer

N. Engl. J.Med.

Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization adenosine 3′,5′-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5′ genomic DNA

Mol. Endocrinol.

Mammary cancer induced by a single feeding of poly-nuclear hydrocarbons, and its suppression

Nature

Immunological quantitation of phospholipid/Ca²⁺-dependent protein kinase of human mammary carcinoma cells: inverse relationship to estrogen receptors