Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation

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European Journal of Cell Biology 78, 497-510 (1999, July) . © Urban & Fischer Verlag· Jena http://www.urbanfischer.de/journals/ejcb

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Alterations in the expression and localization of protein kinase C isoforms during mammary gland diHerentiation Patricia A. Masso-Welch, Gordana Verstovsek, Margot M. Ipl) Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY/uSA Received October 28, 1998 Received in revised version February 11, 1999 Accepted February 11, 1999

PKC - mammary - breast - pregnancy -lactation Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs IX, b, E and ~ were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC IX, b, E and ~ proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs IX, b, E and ~ were stained in the luminal epithelium and myoepithelium, in varying isoformand developmental stage-specific locations. PKC IX was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC E was present in the nucleus, and PKC ~ was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs IX, b, E, and ~ was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific

Abbreviations. DAPI 4',6-Diamidino-2-phenylindole. - ECM Extracellular matrix. - ER Estrogen receptor. - FAK Focal adhesion kinase. - HRP Horseradish peroxidase. - MEO Mammary epithelial organoid~. - PBS Phosphate buffered saline. - PKC Protein kinase C. - PMA Phorbol12-myristate 13-acetate. - PRF Phenol red free. - PVDF Polyvinylidene difluoride. - SDS Sodium dodecyl sulfate. - TBS Tris-buffered saline.

I) Dr. Margot M. Ip, Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Carlton and Elm Streets, Buffalo, NY 142631USA, e-mail: mip @SC3101.med.buffalo.edu, Fax: + + 716 845 5865.

fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs IX, b, E, and ~. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs E and ~) or more intensely (PKCs IX and b), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.

Introduction Protein kinase C (PKC) is a family of serine/threonine kinases, of which 12 isoforms are currently recognized (reviewed in [43]). PKC is involved in signaling through a wide variety of receptors, including integrins, growth factor and hormone receptors (reviewed in [13]). The PKC family is of particular interest to mammary gland and breast cancer biologists, because its activation in normal versus transformed mammary cells and cell lines results in different responses. For example, activation of PKC by phorbol esters results in increased proliferation and decreased functional differentiation of normal, nontransformed MEC grown as explants, or in or on extracellular matrix [4, 56, 59, 61, 64]. In contrast, many transformed mammary epithelial cell lines show growth inhibition and partial secretory differentiation when treated with phorbol esters [18, 52, 63]. PKC activity has been shown to be elevated both in breast tumors [24, 50], as well as during pregnancy and lactation [11, 20]. However, these previous studies compared whole mammary glands from pregnancy or lactation, or breast tumors, with resting mammary glands [11, 20, 24, 50], which differ greatly in their stromal and epithelial cell composition. Specif0171-9335/99/78/07-497 $12.00/0

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498 P. A.Mosso-Welch, G. Verslovsek, M. M.lp

ically, both the epithelium and vasculature progressively increase during pregnancy, coincident with a decline in mature adipocytes and interstitial stroma; this is significant because stromal cells possess multiple isoforms of PKC [5]. Despite the potential for specific PKC isoforms as regulatory targets for breast cancer therapies, the differential expression of specific PKC isoforms in the mammary epithelium during normal mammary gland development has not been previously characterized. Because experimental alteration in the expression or activity of specific PKC isoforms results in coordinated changes in other isoforms, which in turn affect cell differentiation , proliferation, or transformation [1, 6, 39,45,68], we examined multiple PKC isoforms during different stages of postnatal development, when the mammary epithelium can be expected to undergo extensive proliferation and invasion (puberty and early pregnancy), functional differentiation (late pregnancy and lactation), and regression (involution). It is postulated that examining the expression and subcellular location of PKC isoforms during these tightly regulated cycles of proliferation, invasion, differentiation and regression may provide insight into the dysregulation which occurs during carcinogenesis. This current study focuses on mammary epithelium by examining mammary gland in situ, as well as by using stromal cell-depleted mammary epithelial organoids isolated at various time points to quantify PKC isoform expression by immunoblot analysis. This study demonstrates that PKCs n, 6, E and ~ are altered in the luminal epithelium and myoepithelium in an isoform- and developmental stage-specific manner.

Materials and methods Materials

Pepstatin, aprotinin, PMSF, benzamidine, trypsin, and diaminobenzidine were purchased from Sigma (St. Louis, MO). ECeM (enhanced chemiluminescence substrate) was purchased from Amersham (Arlington Heights, IL). Immobilon-P membrane was a product of Millipore (Bedford, MA). Nitex filters were purchased from Tetko Inc. (Depew, NY). Leupeptin and powdered dispase were purchased from Boehringer-Mannheim (Indianapolis, IN). Triton X-100 and 2-methyl butane were purchased from Fisher (Fair Lawn , NJ), and OCT TIssue Tech mounting medium was a product of Miles (Elkhart, IN) . Collagenase was purchased from Worthington Biochemical (Freehold , NJ) . Newborn calf serum was a product of Hyclone (Logan , UT). SDS was purchased from Bio-Rad (Hercules, CA), and aqueous Poly-Mount media from Polysciences (Warrington, PA). Ceramic coverslip holders were obtained from Thomas Scientific (Swedesboro, NJ).

Antibodies Mouse monoclonal antibody recognizing PKC a (#05-014) was purchased from UBI (Lake Placid, NY) . Rabbit polyclonal antibody against PKC 6 peptide (#AS-2443G , amino acids 662-673) was purchased from R&D (Berkeley, CA). The polyclonal rabbit antibodies to peptides from PKC E (#13226-014 , amino acids 725-737) and PKC ~ (#3199SA, amino acids 577-592) were purchased from GIBCO (Grand Island, NY). DAP! , mouse monoclonal antibody to vimentin (clone V9) , and mouse monoclonal antibody 1A4, raised against smooth muscle actin, and conjugated to fluorescein, were purchased from Sigma (St. Louis, MO) . Monoclonal antibody to cytokeratin 14 was purchased from DAKO (Carpinteri a, CA). Mouse IgGl isotype control antibody was purchased from Becton Dickinson (San lose , CA). PKC E and PKC ~ staining results obtained with commercial antisera were additionally confirmed using affinity-purified rabbit anti-

PKC E and rabbit anti-PKC ~ peptide antibodies, donated graciously by Dr. Susan laken (w. Alton lones Cell Science Center, Lake Placid , NY). Donkey anti-rabbit immunoglobulin, goat anti-mouse immunoglobulin, and donkey anti-goat immunoglobulin, conjugated to Texas red, were purchased from Accurate (Westbury, NY). All secondary antisera were preabsorbed, by the manufacturer, against rat proteins. Goat anti-mouse immunoglobulin and goat anti-rabbit immunoglobulin , conjugated to HRP, were products of Boehringer-Mannheim (Wilmington , MA) .

Animal care

Animals were housed in accordance with the guidelines set by the NIH and the Roswell Park Cancer Institute Animal Care and Use Committee. Female Sprague-Dawley rats were purchased from Taconic (strain Tac:N(SD)ffiR) (Germantown, NY) or Charles River (strain Crl :CDBr) (Wilmington, MA). Rats were fed, ad libitum, standard Teklad chow (Madison, WI) . During pregnancy and lactation, rats were fed Teklad breeder chow ad libitum. Animals had unlimited access to water. Animal rooms were air conditioned and humidity controlled, with a light cycle of 14 hours on and 10 hours off.

Preparation of tissues

Rats were sacrificed by CO 2 at various stages of development (50-55 day old pubescent virgin , days 7, 14 and 19 pregnant, day 7 lactation, and 12 hours, 3-6 days, 9-12 days and 24 days post weaning after 21 days of lactation). Inguinal mammary glands were immediately excised and spread carefully onto cardboard mounting boards, covered with OCT TIssue Tech mounting medium and frozen in isopentane precooled with liquid nitrogen . TIssues were stored at -80°C in airtight plastic bags until cryosectioned using an AO Cryostat (Reichert lung, Rochester, NY) at -20 DC, set at 6 ~m thickness. Unfixed frozen sections were placed onto room temperature 18 x 18 mm 2 untreated glass coverslips. Sections were then fixed for 5 minutes in ice-cold acetone, and rehydrated in PBS pH 7.4. Mammary glands from at least three rats per time point were examined .

Immunofluorescence Cryosections on glass coverslips were placed on rubber stoppers in a light-tight humid chamber. Primary antibodies were diluted (v:v) as follows: mouse anti-PKC a, 1:30; rabbit anti-PKC 0, 1:50; rabbit antiPKC E, 1:10; rabbit anti-PKC~ , 1:30; mouse anti-smooth muscle actin , 1:100; mouse anti-vimentin, 1 :100, in a total volume of 60 ~I with PBS pH 7.4. DAPI was used at 1 ~g1ml , and was added concurrently with the primary antisera where indicated . Antibody binding was performed for 45 minutes at room temperature, with rocking, in a lighttight humid chamber. In between binding steps, coverslips were rinsed by rocking for 15 minutes in 200ml of PBS pH 7.4. Rabbit antiserum was detected using Texas red-conjugated donkey anti-rabbit immunoglobulin, at 1:500 dilution (v :v) in PBS . Mouse antisera (except for mouse anti-smooth muscle actin, which was directly conjugated to fluorescein) were detected using 1:100 dilution (v:v) of goat antimouse immunoglobulin, absorbed against rat proteins by the manufacturer, followed by rinsing and detection with donkey anti-goat immunoglobulin conjugated to Texas red , at 1:500 dilution (v:v) in PBS. After rinsing in PBS, sections were dip rinsed in distilled water and mounted onto slides using aqueous Poly Mount mounting medium. Slides were kept at 4 °C in light-tight slide holders until observed . Staining was visualized and recorded using an Olympus BX-40 epifluorescence microscope, with an Olympus PM-C35DX camera (Spectra Services , Rochester, NY). Antibody controls were carried out using isotype-matched IgG 1 for PKC a antiserum, or rabbit IgG, for the rabbit antibodies, diluted to the same final concentration as the corresponding specific primary antisera. For PKCs 6, E and ~, peptide competition was also performed to confirm antibody specificity, by preincubating the primary antibody with an equal concentration of the appropriate immunogenic peptide.

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PKC expression in rat mammary gland

Preparation of ceillysates In order to examine changes in the epithelial rather than stromal cell populations, mammary epithelial organoids (MEO) were isolated from inguinal mammary glands of female Sprague-Dawley rats at various time points (50 day old, day 19 of pregnancy, day 7 of lactation, 12 hours and 3 days post weaning), as previously described [41]. Inguinal mammary glands from 8 virgin, 1 pregnant, 1 lactating, or 1 rat undergoing mammary gland involution were sufficient to prepare an individual sample. Cell Iysates were prepared according to the method of Saxon et al. [58]. Briefly, MEO were pelleted and resuspended in icecold PBS containing protease inhibitors (25 Ilglmlleupeptin, 10 Ilglml pepstatin, 1 mM PMSF, and 0.5 mM benzamidine). Cells were lysed by passing through a 20 gauge needle, then sonicated 30 pulses while on ice. An aliquot was set aside for protein determination, and the remaining sample was diluted with an equal volume of 2 x Laemmli sample buffer containing 10 % (w/v) SDS. Samples were boiled for 3 minutes, sonicated and boiled for another 3 minutes, then aliquoted and stored at -20°C. Samples were prepared independently from three separate MEO isolations for each developmental time point.

Western bloHing Individual samples containing 30 Ilg of protein (as determined by the Biorad protein assay) were loaded per lane, separated by 10 % SDSPAGE, and electrotransferred to Immobilon PVDF membrane. PKC isoforms were detected using the same antisera used for immunofluorescence, and controlled for antibody specificity by preincubating primary antibody with immunogenic peptide at the same Ilglml concentration, for 1 hour at room temperature. Reactivity with mouse antibody to PKC a was compared to the staining with mouse IgG 1 isotype antibody at the same Ilglml concentration. Primary mouse and rabbit antibodies were detected using HRP-conjugated goat anti-mouse or goat anti-rabbit immunoglobulin, diluted 1:2000 (v:v). Reactive protein bands were visualized using enhanced chemiluminescent substrate, by ,exposing Kodak X-OMat X-ray film (Kodak, Rochester, NY). The relative expression levels of PKC isoforms were quantified using a Model 300A Scanning Laser Densitometer (Molecular Dynamics) using Image Quant software (Molecular Dynamics). Quantitation of Western blots from repeated experiments was normalized by setting the sum of the densities of reactive protein bands in the multiple lanes to 100 % for each PKC isoform. Statistical significance was analyzed by one way ANOVA using the Student-Newman-Keuls Test for pairwise multiple comparisons, using SigmaStat software (Jandel Scientific). P<0.05 was judged to be statistically significant.

Results PKC isoform expression is altered during mammary gland diHerentiation To gain new insight into the potential function of PKC isoform expression in the mammary gland epithelium, studies were undertaken to determine if the expression of a particular isoform was altered during the extensive proliferation that occurs during puberty and pregnancy, the functional differentiation associated with lactation, or the apoptotic involution that occurs following weaning. Mammary epithelial organoids (ME Os ) were isolated from mammary glands of rats at day 50-55 of age (virgin), day 19 of pregnancy, day 7 of lactation, 12 hours post-weaning, and day 3 of involution. Figure 1 shows the results of Western blotting for PKC isoforms from MEO lysates. PKC isoforms were observed to migrate as follows: PKC a, 82 kDa; PKC b, 77-81 kDa; PKC E, 83-85 kDa; and PKC ~, 75-80 kDa. In each case, the reactive band was shown to be specific, as indicated by loss of reactivity upon preincubation of the antibody with the corresponding immunogenic peptide (for PKCs b, E and ~), or compared to isotype control for mouse anti-PKC a (data not shown). As seen in

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Fig. 1. Immunoblot of PKC isoforms in MEOs isolated during various developmental time points. To quantify the alterations in PKC isoforms that occur during postnatal mammary gland development, MEOs were isolated from the mammary glands of rats sacrificed at 50 days of age, to obtain pubescent virgin rat mammary gland (V), day 19 pregnancy (P), day 7 of lactation (L), 12 hours of involution (ll2h), and day 3 of involution (ld3)' a, c, e, and g Representative Western blots for PKCs a, 6, E and 1;, respectively (arrowheads). b, d, rand h Corresponding quantitation from multiple blots, normalized by setting the amount of reactive protein in each blot to 100 %. Error bars indicate standard error of the mean. PKC 6 shows significant differences in expression between these groups: virgin versus pregnant, lactating, or involuting day 3; pregnant versus lactating; lactating versus 12 h involuting or day 3 involuting; and involuting 12 h versus involuting day 3, p<0.05.

Figure 1, PKCs a, band 1; were reproducibly increased during the shift from virgin mammary gland to pregnant; this increase was statistically significant for PKC b. A decrease during lactation was seen for all four isoforms examined, followed by varying degrees of increased expression, particularly of PKCs a and b at 12 hours post-weaning. This apparent decrease in PKC isoforms during lactation may be partially due to dilution by the increased secretory milk proteins, as the cell lysates were loaded with the same amount of protein per lane. At day 3 of involution, PKC b was significantly increased, while PKCs a, E and ~ were not significantly changed from 12 h postweaning.

Localization of PKC isoforms relative to smooth muscle actin in virgin rat mammary gland Preliminary studies suggested that PKC isoforms were localized, at least in part, in the basal epithelium of ducts. Experi-

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Fig. 2. PKC colocalization with the smooth muscle actin-positive myoepithelium in vivo . Frozen sections from mammary glands of virgin rats at days 50-55 of age were indirectly stained fluorescent red with antibodies to PKC b (a) and PKC € (c). The same sections were

also stained fluorescent green with antibody to smooth muscle actin conjugated to fluorescein , and DAPI, which stains the nuclei blue (b, d) . Arrows indicate ducts or ductules , arrowheads indicate alveoli . Magnification: x 230.

ments were therefore undertaken in which an antibody to a myoepithelial marker, smooth muscle actin, was utilized together with isoform-specific PKC antibodies, to determine whether PKCs were colocalized with smooth muscle actinpositive cells. To do this, sections from mammary glands from virgin rats at day 55 of age (puberty) were stained with mouse anti-smooth muscle actin FITC, followed by DAPI to visualize nuclei and rabbit antibodies to PKCs {), E or 1;, to determine the PKC isoform expression. (Mouse anti-PKCu was not used in these studies because of difficulties in separating its detection from that of mouse anti-smooth muscle actin.) Figure 2 shows fluorescent red staining of PKCs {) and E, which were localized preferentially in the myoepithelium; the myoepithelium is stained fluorescent green with antibody to smooth muscle actin (DAPI-stained nuclei are shown fluorescing blue). PKC 1; was more similar in its expression in the myoepithelium compared to the luminal epithelium at this developmental time point (data not shown). The expression of PKCs {) and E was stronger in the thick layer of myoepithelium surrounding the ducts (arrows) compared to the more stellate myoepithelial cells that can be seen surrounding the alveoli (arrowheads). The colocalization of PKCs {) and E with smooth muscle actin was isoform-specific, as PKC T] immunoreactivity has been previously shown to be associated with the luminal epithelium rather than the myoepithelium [41].

Epithelial composition of isolated MEO The observation that PKC isoforms band E were strongly expressed in the myoepithelial layer in mammary gland from virgin rats (Figure 2) led us to reexamine the epithelial composition of our MEOs, since contributions from both myoepithelial and luminal epithelial compartments could contribute to the immunoblot results seen in Figure 1. The cellular composition of the MEO purified by our isolation procedure has been previously characterized by electron microscopy, and the lack of morphologically distinguishable myoepithelial cells was noted [12]. However, to confirm the epithelial composition of MEO from virgin mammary gland, MEO were stained with antibodies to cytokeratin 14 (Figure 3a) or smooth muscle actin (Figure 3c, e). Although by phase-contrast microscopy these organoids resemble clusters of luminal epithelium organized into ducts (Figure 3b) or alveolar structures (Figure 3d), it is clear by immunofluorescence staining that there is heterogeneity in the epithelial population. Rounded cells expressing myoepithelial markers (Figure 3a-d, small arrowheads) were observed. Only in rare cases, when extremely large organoids remained intact, cells maintaining the stellate shape characteristic of alveolar myoepithelial cells were observed (Figure 3e, f, small arrowheads). Therefore, although the original characterization of MEO isolated by our purification method primarily identified luminal epithelial cells by electron and

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Fig. 3. Demonstration of MEO composition. MEOs were stained for the myoepithelial markers cytokeratin 14 (a, with corresponding phase in b) and smooth muscle actin (Sm. M. Actin) (c, e, with corresponding phase views shown in d and f, respectively). Both rounded cells (a, e, small arrowheads) and stellate cells (e, small arrowheads) clinging to the surface of MEOs were seen to stain with antibodies to myoepithe-

Iial marker proteins. Single cells staining positively were also observed (c, small arrow). Large arrowhead in (a) indicates ductal structure at a region not stained by antibody to cytokeratin 14. Large arrowhead in (b) indicates alveolar structure at a region not stained by antibody to smooth muscle actin . Magnification bar represents 50 !lm .

light microscopy, it is possible that the extracellular matrix digestion step in isolation may cause a morphologic change in myoepithelial cell shape. This may be due to digestion of extracellular matrix components, which could induce depolymerization of myoepithelial smooth muscle actin microfilaments. Because of the epithelial heterogeneity of newly isolated organoids, developmental changes in the expression of PKC isoforms were evaluated in mammary gland sections.

PKC a expression throughout mammary gland development To better understand the role of PKC isoforms in the mammary gland, frozen sections from various developmental stages were examined by immunofluorescence staining. Figure 4 shows the distribution of PKC a in mammary gland obtained from virgin rats at age 50-55 days (a) , day 19 of pregnancy (b), day 7 of lactation (c), day 21 of lactation at 12 hours postwean-

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PKC expression in rat mammary gland

PKC a immunostaining throughout development. PKC a immunostaining is shown for frozen sections of mammary glands from 50 day old virgin rats (a), rats at day 19 pregnancy (b), day 7 of lactation (c), day 21 of lactation followed by 12 hours postweaning (d, and inset), day 3 involution (e) and day 9 involution (f). Vimentin staining of mammary gland from 12 hours post weaning is pictured in (g), with the corresponding phase view in (h). Large arrows indicate ducts, arrowheads indicate alveoli. Small arrowheads in inset in (e) indicate parallel fibrils crossing the luminal epithelium. Small arrows in (a) and (b) indicate apical punctate staining of the luminal epithelium. Magnification bar indicates 25 !-1m. Inset magnification, 115 x

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ing (d), day 3 of involution (e, and inset), and day 9 of involution (f). PKC a was expressed in the luminal epithelium as well as the basal myoepithelium throughout development. In the luminal epithelium, PKC a was diffusely distributed in the cytoplasm. However, during pregnancy, PKC a was additionally found concentrated in punctate regions at the apical face of the luminal alveolar epithelium (Figure 4b, small arrows). This localization was observed as early as day 7 of pregnancy (data not shown). These punctate regions were often close to, but not always coincident with the lateral boundaries of cells seen by phase-contrast microscopy (data not shown). Myoepithelial cells were stained more intensely for PKC a than the luminal epithelium throughout development. During lactation, the staining of myoepithelial cells was discontinuous at the base of distended secretory alveoli (Figure 4c, arrowhead). At 12 hours involution (Figure 4d), and day 3 involution (Figure 4e), PKC a staining varied from being basally continuous (Figure 4d, arrowhead) to attenuated and discontinuous (Figure 4e, arrowhead), a distribution pattern consistent with the attenuation of myoepithelial cells surrounding heterogeneously distended alveoli [66]. In addition to this basal staining oriented at the base of the luminal epithelium, distinctive parallel arrays of three to four PKC a-stained fibrils were observed spanning the luminal epithelium at days 3-6 of involution (Figure 4e, inset, arrowheads). These fibrils were present in approximately 10 % of involuting alveoli. The distribution of PKC a at this time was very similar to vimentin, which also displayed arrays of filaments crossing the epithelial cells (Figure 4g, arrowheads). At days 9-12 of involution, the collapsed alveoli (Figure 4f, arrowhead) were surrounded by thickened basal cells whose PKC a staining far surpassed that of the adjacent stable ductal structures (Figure 4f, arrow). This increased PKC a expression in the myoepithelial cells surrounding the regressing alveoli, but not those surrounding the ducts, suggests a role for PKC a in alveolar myoepithelial cells during late involution.

PKC b distribution throughout mammary gland development PKC b was seen consistently in the luminal epithelium during development in a diffuse, faint cytoplasmic distribution. However, like PKC a, PKC b staining was more prominent in the basal myoepithelium than the luminal epithelium. Out of the isoforms examined, PKC b staining was the most distinctly fibrillar in appearance throughout development (Figure Sa, b and insets). The staining pattern clearly outlines the stellate shape of the myoepithelial processes that encircle alveoli (Figure Sa, b, arrowheads). During lactation (Figure 5c) and early involution (12 hours after removal of the pups after 21 days of lactation) (Figure Sd), PKC b staining was discontinuous at the base of the distended secretory alveoli (Figure Sc, d,

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arrowheads). This stammg pattern contrasted sharply with days 3-6 of involution, when the collapsed alveoli were surrounded by a ring of thickened myoepithelial cells strongly expressing PKC b (Figure Se, arrowheads). Like PKC a, the myoepithelial cells at the base of ducts were more intensely stained than those surrounding alveoli throughout most of development, and like PKC a, this pattern was reversed at late involution. Thus, at days 9-12 of involution, PKC b was more intensely stained at the base of the regressing alveoli (Figure Sf, arrowhead) than at the base of ducts (Figure Sf, arrow). Unlike PKC a at 3 days of involution (Figure 4e), PKC b staining was not organized into a series of fine parallel fibrillar arrays crossing the luminal epithelium. Rather, PKC b staining was more consistently observed in a thick basal or stellate pattern (Figure Se).

PKC E expression throughout mammary gland development PKC f staining was unique from the other isoforms examined, being present in the nuclei of luminal epithelium (Figure 6c, d), in addition to being present in the myoepithelial cells. PKC f was the only isoform of PKC examined that showed this strong and distinctive nuclear localization, which was most pronounced during lactation (Figure 6c) and 12 hours postweaning (Figure 6d), but lingered throughout involution (Figure 6f, small arrow). PKC f, like PKC a, also stained parallel arrays of fibers that crossed the luminal epithelium, at days 3-6 of involution (Figure 6e, hollow arrowhead). At late involution (days 9-12) PKC f staining of myoepithelium at the base of alveoli (Figure 6f, arrowhead) was equivalent to that at the base of ducts (Figure 6f, large arrow), making its distribution pattern distinct from that of PKCs a and b. In the myoepithelium, the distribution of PKC f was similar to PKC b, and appeared to mirror the distribution of the smooth muscle actin fibers in the myoepithelium (see Figure 2c, d). However, this colocalization was not as distinct as that for PKC b, perhaps due to the presence of PKC f staining in the myoepithelial cytoplasm as well (Figure 6a, arrow). Throughout most of development, like PKCs a and b, PKC f was stained more strongly in the myoepithelial cells lining the ducts (Figure 6b, arrow). In fact, PKC f staining was often undetectable at the base of many alveoli (Figure 6b, arrowhead). PKC ~ expression throughout mammary gland development In comparison to PKCs a, b, and f, PKC ~ was expressed at higher levels in the luminal epithelium relative to the myoepithelium in pubescent, pregnant and lactating rats. PKC ~ staining of the luminal epithelium was more concentrated at the plasma membrane than in the cytosol and nuclei of the luminal epithelium, in particular outlining the apical surface of the cell (Figure 7a, b, small arrows). During lactation, PKC Sstaining was also concentrated at the apical face of the secretory alveolar epithelium, apparently in a cluster of subapical small granules or vesicles (Figure 7c, small arrow). This apical staining was distributed more diffusely over the luminal surface than the PKC a punctate staining near cell-cell contacts seen in the luminal epithelium during pregnancy. PKC ~ was distinct from PKCs band f in that it was less stained or absent in myoepithelial cells at the base of many alveoli in virgin (Figure 7a, arrowhead), pregnant (Figure 7b, arrowhead), or lactating rat mammary glands (Figure 7c,

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Fig. S. PKC [) immunostaining throughout development. PKC [) immunostaining is shown for mammary glands from 50 day old virgin rats (a), and rats at day 19 pregnancy (b), day 7 of lactation (c) , day 21 of lactation followed by 12 hours postweaning (d), day 3 of involution (e) and day 12 of involution (f) . Insets in (a) and (b) show a higher

power view (twofold more magnified than the background) ofregions of fibrillar staining. Large arrows indicate ducts or ductules (a, b,c, f) , while large arrowheads indicate alveoli (a.f) . Magnification bar indicates 25 ~m.

PKC expression in rot mammary gland

arrowhead). During early involution (12 hours, Figure 7d), it was expressed in a discontinuous basal pattern that is likely to reflect association with the attenuated myoepithelial cells surrounding the engorged alveoli (Figure 7d, arrowhead). During later involution (days 3-6, Figure 7e, and days 9-12, Figure 7f), PKC 1; was more prominent in the myoepithelium than in the luminal epithelium of alveoli (arrowhead). Like PKC E, PKC 1; was expressed equivalently in the myoepithelium at the base of ductu1es and involuting alveoli at days 9-12 (Figure 7f, arrow versus arrowhead , respectively).

Discussion This report is the first to describe multiple isoform-specific changes in PKC expression and localization, both in isolated mammary epithelial cells, and in situ in whole gland, during puberty, pregnancy, lactation and involution. We found, by immunoblot analysis of isolated MEOs from different developmental stages, that PKCs a, 0 and 1; were increased to varying degrees during the transition from virgin to pregnant mammary gland. Variations in the quantity of PKC isoforms in MEOs may be partly due to variations in the myoepithelial component that is copurified with the luminal epithelia. Therefore, the quantitative contributions of the myoepithelium and luminal epithelium to the PKC isoform protein content is still unknown. However, the examination of isoform expression in epithelial organoids is able to provide more information than the utilization of whole mammary gland lysates which contain stromal as well as epithelial cells. In whole mammary gland , the stromal cells decrease relative to the epithelium during pregnancy and lactation. Therefore, the expression of multiple isoforms of PKCs by stromal cells [5] can influence changes in whole mammary gland PKC during pregnancy and lactation. Our present study focuses on the epithelial PKC expression by using MEOs that are depleted of stromal cells, thereby eliminating the effect of shifts in stromal cell contribution during mammary gland development. Cell fractionation studies were conducted on MEOs isolated at different developmental stages, to examine changes in the subcellular localization of PKCs a, b, £ and 1;; however, no consistent, reproducible pattern was seen (data not shown). This may be partially due to the fact that these isoforms are associated preferentially with the myoepithelial cells, as PKC lj, which is preferentially associated with the luminal epithelium, yielded reproducible results upon cell fractionation of MEOs from different developmental time points [41]. Previous studies analyzing PKC activity or expression using whole mammary gland lysates rather than epithelial organoids, have been conflicting. Connor and Clegg [11] analyzed crude extracts of whole mammary gland, and found PKC a to be expressed at constant levels, but increased in Ca ++ responsiveness during pregnancy and lactation. Using DEAEcellulose-purified fractions (which should remove endogenous PKC inhibitors), the expression and activity of conventional PKCs (PKCs a, ~ and y) were described as increased in pregnant, compared to virgin whole mammary gland [20]. These results are in agreement with our PKC Western blotting results. In contrast, others have shown a decrease in total PKC activity in whole mammary gland during pregnancy and lactation, compared to virgin [8]. These studies are difficult to com-

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pare directly to the present study because of their use of whole mammary gland rather than epithelial organoids. These previous studies also examined total PKC activity rather than isoform-specific expression, as in our study. However, the total PKC activity assayed may be biased, as assays for PKC activity use substrates that are poorly phosphorylated by some PKC isoforms [14]. More importantly, the PKC activity assay measures the total activatible PKCs, in the presence of optimal concentrations of cofactors (phospholipids , required for all isoforms, and Ca++ , required for cPKCs a, ~ and y). At this point in time, there is no quantitative assay for the endogenous, isoform-specific PKC activity that exists under physiological conditions within the cell itself. Related to this, although expression of individual PKC isoforms can be evaluated by immunoblotting, protein expression does not necessarily indicate endogenous PKC activity or responsiveness to signaling. However, the ability to examine changes in isoform-specific protein expression is a starting point for further investigation.

PKC isoform expression in the luminal epithelium PKC a was observed at points of cell-cell contact at the apical face of the luminal epithelium prominently during pregnancy, which may reflect association with cell junctions. This possibility is supported by the previous observation that PKC a is associated with cell-cell contacts in normal renal proximal tubule epithelial cells [17] . Although PKC a has been suggested to be involved in the response to lactogenic hormones resulting in the induction of ~ casein gene expression in HCll mouse mammary epithelial cells [40], we observed no pronounced alteration in PKC a expression or localization in the luminal epithelium during lactation. The concentration of PKC E in the nuclei of luminal epithelial cells during lactation suggests a differentiation-specific role for this isoform. One possibility is that the nuclear localization of PKC E during lactation is induced by the high concentrations of hormones, such as prolactin, that are present at high levels in the serum at this time. Although the specific involvement of the E isoform of PKC specifically in prolactin response has not been previously observed in vitro [40], it is known that prolactin stimulation of mammary cell lines or primary cultured mammary epithelial cells results in the translocation of total PKC activity from the cytosol to the membrane/ particulate fraction , followed by a mitogenic response [2] and PKC downregulation [67] . In vivo experiments examining the effects of increased prolactin (via pituitary isografts in mice) showed no effect on conventional PKC activity in crude extracts of whole mammary gland [30]; however, these experiments did not analyze the activity or expression of nonconventional PKC isoforms, such as PKC E. The presence of PKC E in the nucleus during lactation as described in our study may reflect a role for this isoform in modulating the phosphorylation of nuclear substrates such as transcription factors involved in lactogenesis. PKC 1; was expressed in the luminal epithelium in a cytosolic subapical vesicular pattern during pregnancy and lactation, which was distinct from the punctate apical staining seen for PKC a. This cytosolic apical distribution may represent a role for PKC 1; during pregnancy and lactation in the differentiating secretory luminal epithelium; this distribution is similar to that seen with endocytic vesicles. In addition, we noted a faint subapical threadlike distribution for PKC 1;, which was seen

506 P. A. Masso-Welch, G. Verstovsek, M. M.lp

Fig. 6. PKC E immunostaining throughout development. PKC E immunostaining is shown for mammary glands from 50 day old virgin rats (a), and rats at day 19 pregnancy (b), day 7 oflactation (c), day 21 of lactation followed by 12 hours postweaning (d), day 3 involution (e) and day 9 involution (f). PKC E staining was unique compared to other isoforms because it demonstrated concentrated nuclear staining that

was most pronounced during lactation and early involution (c,d, small arrows). Black arrowhead in (e) indicates parallel array of fibrils crossing luminal epithelium. Large arrows indicate ducts (a, b, c, f), while large arrowheads indicate alveoli (a-f). Magnification bar represents

25 !-tm.

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Fig. 7. PKC ~ immunostaining throughout development. PKC ~ immunostaining is shown for mammary glands from 50 day old virgin rats (a), and rats at day 19 pregnancy (b), day 7 of lactation (c), day 21 oflactation followed by 12 hours postweaning (d), day 3 involution (e) and day 9 involution (r). In mammary glands from virgin and pregnant

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rats, PKC ~ was also seen at the apical and lateral membrane, especially apparent in ductules and ducts (a, b, small arrows). Small arrow in (c) indicates PKC ~ in secretory alveoli in punctate granules just beneath the luminal surface. Large arrows indicate ducts or ductules, large arrowheads indicate alveoli. Magnification bar represents 25 /lm.

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most clearly in the infrequently sampled primary ducts (data not shown), suggesting an association with tight junction complexes. An association of PKC [19], and PKC ~ in particular, [16, 60] with tight junction complexes has been previously demonstrated. Expression of some specific PKC isoforms has been shown to be associated with cytostasis or differentiation. In particular, PKC a has been associated with cytostasis in breast cancer cells [9, 34, 68], and other cell types [15, 25, 27, 42, 44, 46-49]. Additionally, PKC E has also been associated with cell proliferation, as overexpression of PKC E in fibroblasts induces increased growth and anchorage independence [7, 45], although PKC E is decreased in mammary tumors compared to hyperplasias [35]. The role of PKC E in the proliferation and differentiation of normal mammary epithelial cells requires further investigation. Although PKC localization was examined during time points when the mammary epithelium should have been proliferative (day 7 of pregnancy, data not shown), we found no differences compared to PKC localization in the epithelium at days 14 and 19 of pregnancy. It is likely that the interplay between multiple PKC isoforms is what determines the balance between proliferation versus differentiation. Studies which attempt to address the role of a particular PKC isoform using transfection and other approaches are complicated by alterations in other PKC isoforms [1, 68].

Possible roles for PKC isoforms in the myoepithelium The concentrated expression of PKCs band E in the myoepithelium, demonstrated by dual staining with antibodies to smooth muscle actin, was not seen for all PKC isoforms. Like PKCs band E, PKC 1] is a novel, Ca ++ -insensitive isoform of PKC. However, its expression and distribution are distinct from PKCs a, b, E and ~, being preferentially expressed in the luminal epithelium but not the myoepithelium [41]. PKC 1] was found to be greatly upregulated during pregnancy in the alveolar and not in the ductal luminal epithelium, concentrated and secreted into milk during lactation [41]. Interesting
y, the increase in PKCs a, b, E and ~ staining in the thickened myoepithelial cells at days 3-6 involution is the opposite of the staining pattern for PKC 1], whose expression is rapidly decreased during involution [41]. Preferential expression of PKCs a, b, E and ~ in the myoepithelium suggests that these isoforms may be associated with myoepithelial specific functions, such as adhesion or basement membrane synthesis and secretion. Myoepithelial cells are involved in the synthesis of the basement membrane, which underlies them directly, conforming closely to the surface of the myoepithelial cell [65, 69]. Myoepithelial cells have been previously shown to have greater expression of integrins and adhesion-associated molecules ([22], and Masso-Welch et aI., manuscript submitted). It is of interest that the myoepithelial staining pattern for PKCs a, b, E and ~ is similar to the staining of the myoepithelium for the a6A and ~4, but not the a6B integrin subunits of the a6~1 or a6~4 laminin receptors (Masso-Welch et aI., manuscript submitted). The adhesive activity of these integrins has been shown to be modulated by PKC-mediated phosphorylation of intracellular domains [28, 29,54, 70]. The association of some specific isoforms of PKC with cell-cell and cell-matrix adhesion has been described for PKC a [17, 32, 33, 62], PKC b, [3], and PKC E [10]. The filamentous distribution of PKCs a, b, E and ~ in myoepithelial cells may reflect an interaction with smooth muscle

actin microfilaments. PKC isoforms mediate some of their effects by association with and phosphorylation of cytoskeletal elements, and an association of specific isoforms with the cytoskeleton has been shown for PKC a [17, 31-33], PKC b [3, 36,51,53], PKC E [37, 55], and PKC ~ [19, 21, 23, 38].

PKC and breast cancer The expression of PKCs a, b, E and ~ in basal myoepithelial cells is relevant to previous studies examining changes in PKC activity in breast cancer. PKC activity of DEAE-cellulosepurified material from breast tumors was shown to be increased relative to paired normal breast samples [24, 50]. However, the contribution of mammary stromal cells, or epithelium versus myoepithelial content, was not addressed. Studies that have examined changes in PKC isoform expression or activity in whole tumor compared to whole mammary gland [24, 50] are likely to reflect changes in the myoepithelial population, as well as alterations in stromal cells. Because the myoepithelium contains a disproportionate amount of the PKC a, b, and E reactivity in normal mammary gland, studies that show increased PKC in tumors in the absence of myoepithelial cells may reflect even more profound increases in PKC levels in the remaining transformed luminal epithelium. The myoepithelium is likely to be of great importance to the maintenance of normal breast morphology, because of its role in synthesizing and assembling the basement membrane and maintaining a normal morphology (the hallmark between determining hyperplasia versus neoplasia). The importance of this role of myoepitheJium is supported by the observation that myoepithelial cells are often present in benign and in situ breast tumors, whereas infiltrating breast tumors often lack myoepithelial cells [26, 57]. Acknowledgments. We would like to gratefully acknowledge the excellent technical assistance and advice of Mary Vaughan, Jane Meer, Colleen Tagliarino, Xiaoyuan Zhao, and Ed Hurley, and the helpful suggestions of Dr. Jennifer Black, Dr. Danilo Zangani, and Dr. Kathleen Darcy. This work was supported by NCI grant CA33240, and by NCI core grant P30 CA16056.

References [1] Assert, R., H. Schatz, A. Pfeiffer: Upregulation of PKC deltaand downregulation of PKC alpha-mRNA and protein by a phorbol ester in human T84 cells. FEBS Lett. 388, 195-199 (1996). [2] Banerjee, R., B. K. Vonderhaar: Prolactin-induced protein kinase C activity in a mouse mammary cell line (NOG-8). Mol. Cell. Endocrinol. 90, 61-67 (1992). [3] Barry, S. T., D. R. Critchley: The Rho A-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions. J. Cell Sci. 107, 2033-2045 (1994). [4] Birkenfeld, H. P., B. S. McIntyre, K. P. Briski, P. W. Sylvester: Protein kinase C isoenzyme expression in normal mouse mammary epithelial cells grown in primary culture. Proc. Soc. Exp. BioI. Med. 213, 65-70 (1996). [5] Borner, c., S. N. Guadagno, D. Fabbro, 1. B. Weinstein: Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters. J. BioI. Chern. 267, 12892-12899 (1992). [6] Borner, C., S. N. Guadagno, L. Hsieh, W. Hsiao, 1. B. Weinstein: Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon. Cell Growth Differ. 1,653-660 (1990).

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[7]

[8] [9]

[to) [11] [12)

[13] [l4)

[15]

[16] (17)

[18]

[19]

[20]

[21] [22] [23]

[24]

[25]

Cacace, A. M., S. N. Guadagno , R . S. Krauss, D. Fabbro , I. B. Weinstein: The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 8, 2095-2104 (1993). Caulfield, J. J., E E Bolander, Jr.: Involvement of protein kinase C in mouse mammary gland development. J. Endocrinol. 109, 29-34 (1986). Cho, Y. H ., A. P. Tighe, D . A . Talmage : Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase C alpha expression and activity. J. Cell. Physiol. 172, 306-313 (1997) . Chun , J., M. Ha, B. S.Jacobson: Differential translocation of protein kinase C epsilon during HeLa cell adhesion to a gelatin substratum. J. BioI. Chern. 271, 13008-13012 (1996). Connor, K., R. A. Clegg: Isoenzymes of protein kinase C in rat mammary tissue: changes in properties and relative amounts during pregnancy and lactation . Biochem. J. 291,817-824 (1993). Darcy, K. M., J. D. Black, H . A . Hahm, M. M. Ip: Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane. Exp. Cell Res. 196,49-65 (1991). Deacon , E. M., J. Pongracz, G. Griffiths, J . M. Lord: Isoenzymesof protein kinase C: Differential involvement in apoptosis and pathogenesis. J. Clin . Pathol. Clin . Mol. Pathol. SO, 124-131 (1997). Dekker, L. V. , P. McIntyre , P. J .Parker: Mutagenesis of the regulatory domain of rat protein kinase C-eta: A molecular basis for restricted histone kinase activity. J. BioI. Chern. 268, 19498-19504 (1993). Dlugosz, A. A., C. Cheng, E. K. Williams, A. G. Dharia, M. E Denning, S. H. Yuspa: Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-alpha. Cancer Res. 54,6413-6420 (1994). Dodane, V., B. Kachar: Identification of G proteins and PKC that colocalize with tight junctions. J . Membr. BioI. 149, 199-209 (1996). Dong, L. , J. L. Stevens , S.Jaken : Transformation-sensitive localization of alpha-protein kinase C at cell-cell contacts in rat renal proximal tubule epithelial cells . Cell Growth Differ. 4, 793-798 (1993). Fabbro, D., R. Regazzi , S. D. Costa, C. Borner, U. Eppenberger: Protein kinase C desensitization by phorbol esters and its impact on growth of human breast cancer cells. Biochem. Biophys. Res. Commun. 135, 65-72 (1986). Fasano, A., C. Fiorentini, G. Donelli , S. Uzzau, J. B. Kaper, K. Margaretten, X. Ding, S. Guandalini, L. Comstock, S. E. Goldblum : Zonula occludens toxin modulates tight junctions through protein kinase Codependent actin reorganization , in vitro. J . Clin. Invest. 96, 710-720 (1995) . Foncea, R. , S. Varela , M. Sapag-Hagar, S. Lavandero: Changes in protein kinase C activity, subcellular distribution and protein phosphorylation during the lactogenic cycle in rat mammary tissue . Res. Commun. Mol. Pathol. Pharmacol. 87, 253-268 (1995). Garcia-Rocha, M., J. Avila, J . Lozano: The zeta isoform of protein kinase C binds to tubulin through the pseudo substrate domain. Exp. Cell Res. 230, 1-8 (1997). Glukhova, M., V. Koteliansky, X . Sastre, J. Thiery: Adhesion systems in normal breast and in invasive breast carcinoma. Am. J. Pathol. 146,706-716 (1995) . Gomez, J. , A . Martinez de Aragon , P. Bonay, C. Pitton, A . Garcia, A . Silva, M. Fresno , E Alvarez, A. Rebollo: Physical association and functional relationship between protein kinase C zeta and the actin cytoskeleton . Eur. J . Immunol. 25,2673-2678 (1995). Gordge, P. c. , M. J . Hulme , R .A. Clegg, W R .Miller: Elevation of protein kinase A and protein kinase C activities in malignant as compared with normal human breast tissue. Eur. J. Cancer 32A. 2120-2126 (1996). Gruber, J. R., S. Desai, J. K. Blusztajn, R. M. Niles: Retinoic acid specifically increases nuclear PKC alpha and stimulates AP-l transcriptional activity in B16 mouse melanoma cells. Exp. Cell Res. 221, 377-384 (1995) .

PKC expression in rot mammary gland

509

[26] Guelstein, V. I., T. A. Tchypysheva, V. D. Ermilova , A. V. Ljubimov: Myoepithelial and basement membrane antigens in benign and malignant human breast tumors . Int. J. Cancer 53, 269-277 (1993). (27) Hocevar, B. A., A. P. Fields: Selective translocation of beta 11protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells. J. BioI. Chern. 266,28-33 (1991). [28] Hogervorst , E, L. G . Admiraal , C. Niessen, I. Kuikman , H . Janssen , H. Daams, A. Sonnenberg: Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit. J . Cell BioI. 121, 179-191 (1993). [29) Hogervorst, E , I. Kuikman , E . Noteboom , A . Sonnenberg : The role of phosphorylation in activation of the alpha 6A beta 1 laminin receptor. J. BioI. Chern. 268, 18427-18430 (1993). [30) Holladay, C. S., E E J .Bolander: Hormonal regulation of protein kinase C in the mouse mamm ary gland. Proc. Soc. Exp. BioI. Med. 183, 343-347 (1986). (31) Hyatt, S. L., T. Klauck , S. Jaken: Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts. Mol. Carcinog. 3,45-53 (1990). [32] Hyatt, S. L., L. Liao, C. Chapline , S. Jaken: Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells. Biochemistry 33, 1223-1228 (1994). [33) Jaken, S. , K. Leach, T. Klauck : Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts . J . Cell BioI. 109,697-704 (1989). [34] Kennedy, M. J., L. J . Prestigiacomo, G. Tyler, WS. May, N.E . Davidson: Differential effects of bryostatin 1 and phorbol ester on human breast cancer cell lines. Cancer Res. 52, 1278-1283 (1992). [35] Kiley, S. C. , J. Welsh, C . Narvaez, S.Jaken: Protein kinase C isozymes and substrates in mamm ary carcinogenesis. J . Mammary Gland BioI. Neoplasia 1,177-187 (1996). [36] Knutson , K. L., M. Hoenig : Regulation of distinct pools of protein kinase C delta in beta cells. J . Cell . Biochem. 60, 130-138 (1996) . [37] Lehel, C., Z. Olah , G . Jakab , Z . Szallasi, G. Petrovics, G . Harta, P. M. Blumberg , W B .Anderson: Protein kinase C epsilon subcellular localization domains and proteolytic degradation sites. A model for protein kin ase C conformational changes. J . BioI. Chern. 270, 19651-19658 (1995). [38] Lehrich, R. W, J. N. Forrest , Jr.: Protein kinase C zeta is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland. 1. BioI. Chern . 269,32446-32450 (1994) . (39) Manni, A. , E. Buckwalter, R. Etindi , S. Kunselman, A . Rossini, D . Mauger, D. Dabbs , L. Demers : Induction of a less aggressive breast cancer phenotype by protein kinase C-alpha and -beta overexpression. Cell Growth Differ. 7, 1187-1198 (1996) . [40] Marte, B. M., T. Meyer, S. Stabel , G . 1. Standke , S. Jaken , D. Fabbro, N. E. Hynes : Protein kinase C and mammary cell differentiation: involvement of protein kinase C alpha in the induction of beta-casein expression. Cell Growth Differ. 5, 239-247 (1994). [41) Masso-Welch, P. A., G. Verstovsek , K. Darcy, C. Tagliarino, M. M. Ip: Protein kinase C eta upregulation and secretion during postnatal rat mammary gland differentiation. Eur. J. Cell BioI. 77,48-59 (1998) . [42] McGarrity, T. J ., L. P. Peiffer, E . B .Neely, R . G. Palavarapu, W A . Kolton , P. Parker, M. K. Howett: Localization of protein kinase C alpha isoform expression in the human gastrointestinal tract. Cell Growth Differ. 7,953-959 (1996). [43] Mellor, H ., P. J . Parker: The extended protein kinase C superfamily. Biochem. J. 332, 281-292 (1998). [44] Mills, K. 1., S. B. Bocckino , D. J . Burns, C. R. Loomis, R. C. Smart: Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase 10 diacylglycerol. Carcinogenesis 13, 1113-1120 (1992).

510

P. A. Masso-Welch, G. Verstovsek, M. M.lp

[45] Mischak, H., J. A. Goodnight, W. Kolch, G. Martiny-Baron, C. Schaechtle, M. G. Kazanietz, P. M. Blumberg, J. H. Pierce, J. F. Mushinski: Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. BioI. Chern. 268, 6090-6096 (1993). [46] Mischak, H., W. Kolch, J. Goodnight, W. F. Davidson, U. Rapp, S. Rose-John, J. F. Mushinski: Expression of protein kinase C genes in hemopoietic cells is cell type- and B cell differentiation stage-specific. J. Immunol. 147,3981-3987 (1991). [47] Moreton, K., R. Turner, N. Blake, A. Paton, N. Groome, M. Rumsby: Protein expression of the alpha, gamma, delta and epsilon subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum. FEBS Lett. 372, 33-38 (1995). [48] Nakaigawa, N., S.-1. Hirai, K. Mizuno, T. Shuin, M. Hosaka, S. Ohno: Differential effects of overexpression of PKC alpha and PKC delta/epsilon on cellular E2F activity in late G1 phase. Biochern. Biophys. Res. Commun. 222,95-100 (1996). [49] Niles, R. M.: Interactions between retinoic acid and protein kinase C in induction of melanoma differentiation. Adv. Exp. Med. BioI. 354, 37-57 (1994). [50] O'Brian, C. A., V. G. Vogel, S. E. Singletary, N. E. Ward: Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res. 49, 3215-3217 (1989). [51] Ogawara, M., N. Inagaki, K. Tsujimura, Y. Takai, M. Sekimata, M. H. Ha, S. Imajoh-Ohmi, S. Hirai, S. Ohno, H. Sugiura, et al.: Differential targeting of protein kinase C and CaM kinase II signalings to vimentin. J. Cell BioI. 131, 1055-1066 (1995). [52] Osborne, C. K., B. Hamilton, M. Nover, J. Ziegler: Antagonism between epidermal growth factor and phorbol ester tumor promoters in human breast cancer cells. J. Clin. Invest. 67, 943-951 (1981). [53] Owen, P. J., G. D. Johnson, J. M. Lord: Protein kinase C-delta associates with vimentin intermediate filaments in differentiated HL60 cells. Exp. Cell Res. 225, 366-373 (1996). [54] Palmantier, R., J. D. Roberts, W. C. Glasgow, T. E. Eling, K. Olden: Regulation of the adhesion of a human breast carcinoma cell line to type IV collagen and vitronectin: Roles for lipoxygenase and protein kinase C. Cancer Res. 56, 2206-2212 (1996). [55] Prekeris, R., M. W. Mayhew, J. B. Cooper, D. M. Terrian: Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function. J. Cell BioI. 132, 77-90 (1996). [56] Rillema, J. A., M. Sluyser: Increased sensitivity of mouse mammary gland for hormones and tumor promoter 12-0tetradecanoyl-phorbol-13-acetate in tissues containing the Mtv-2 gene. Horm. Res. 19, 52-56 (1984). [57] Sappino, A. P., O. Skalli, B. Jackson, W. Schiirch, G. Gabbiani: Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int. J. Cancer 41,707-712 (1988).

EJca [58] Saxon, M. L., X. Zhao, J. D. Black: Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. J. Cell BioI. 126,747-763 (1994). [59] Starzec, A., E. Spanakis, A. Nehme, V. Salle, N. Veber, C. Mainguene, P. Planchon, A. Valette, G. Prevost, L. Israel: Proliferative effects of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast pathogenesis. J. Cell. Physiol. 161, 31-38 (1994). [60] Stuart, R. 0., S. K. Nigam: Regulated assembly of tight junctions by protein kinase C. Proc. Natl. Acad. Sci. USA 92, 6072-6076 (1995). [61] Taketani, Y., T. Oka: Tumor promoter 12-0-tetradecanoylphorbol 13-acetate, like epidermal growth factor, stimulates cell proliferation and inhibits differentiation of mouse mammary epithelial cells in culture. Proc. Natl. Acad. Sci. USA SO, 1646-1649 (1983). [62] Timar, J., B. Liu, R. Bazaz, K. V. Honn: Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells. J. Histochem. Cytochem. 44,177-182 (1996). [63] Valette, A., N. Gas, S. Jozan, M. A. Roubinet, M. A. Dupont, F. Bayard: Influence of 12-0-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events. Cancer Res. 47, 1615-1620 (1987). [64] Wada, T., K. M. Darcy, X. Guan, M. M. Ip: Phorbol12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture. J. Cell. Physiol. 158, 97-109 (1994). [65] Warburton, M. J., S. R. Dundas, B. A. Gusterson, M. J. O'Hare: Regulation of urokinase-type plasminogen activator production by rat mammary myoepithelial cells. Exp. Cell Res. 228, 76-83 (1996). . [66] Warburton, M. J., D. Mitchell, E. J. Ormerod, P. Rudland: Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland. J. Histochem. Cytochem. 30, 667-676 (1982). [67] Waters, S. B., J. A. Rillema: Role of protein kinase C in the prolactin-induced responses in mouse mammary gland explants. Mol. Cell. Endocrinol. 63, 159-166 (1989). [68] Ways, D. K., C. A. Kukoly, J. deVente, J. L. Hooker, W. O. Bryant, K. J. Posekany, D. J. Fletcher, P. P. Cook, P. J. Parker: MCF7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J. Clin. Invest. 95,1906-1915 (1995). [69] Williams, J. M., C. W. Daniel: Mammary ductal elongation: Differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev. BioI. 'Yr, 274-290 (1983). [70] Zhou, Y., E. Dziak, M. Opas: Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner. J. Cell. Physiol. 155, 14-26 (1993).