Increased protein kinase C delta in mammary tumor cells: relationship to transformtion and metastatic progression.

Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed alpha, delta, epsilon and zeta PKCs; however, PKC delta levels were relatively increased in the highly metastatic cells. To determine if increased PKC delta could contribute to promotion/progression, we overexpressed PKC delta in the low and moderately metastatic cell lines. PKC delta overexpression had no significant effect on growth of adherent cells, but significantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKC delta (RD delta), a putative PKC delta inhibitory fragment, inhibited anchorage-independent growth. The efficacy of RD delta as a PKC delta inhibitor was demonstrated by showing that RD delta selectively interfered with PKC delta subcellular location and significantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKC delta activity and phenotypic changes in cytoskeletal-dependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth effects of expressing PKC delta and RD delta as gain and loss of function constructs, respectively, provide strong evidence that PKC delta regulates processes important for anchorage-independent growth in these mammary tumor cells.

Protein kinase C delta involvement in mammary tumor cell metastasis.

Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKCdelta levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKCdelta is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKCdelta (RDdelta) under the control of a tetracycline-inducible promoter. RDdelta expression attenuated endogenous PKCdelta activity, as demonstrated by decreased phosphorylation of the PKCdelta substrate adducin in migrating cells. Thus, in MT cells, RDdelta appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RDdelta expression influenced metastatic potential in vivo, MTLn3/RDdelta cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RDdelta expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RDdelta significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKCdelta activity by expressing the inhibitory RDdelta fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.

Tetradecanoyl phorbol acetate-induced microtubule reorganization is required for sustained mitogen-activated protein kinase activation and morphological differentiation of U937 cells.

Investigation of 12-tetradecanoyl phorbol 13-acetate (TPA)-resistant U937 cell clones has demonstrated that the normal sustained p42 mitogen-activated protein kinase (p42MAPK) activation produced by TPA treatment is absent. This is shown to be due to the inability of TPA to maintain activation of MAP/extracellular signal-regulated kinase kinase (MEK) and cRaf1. A direct relationship between sustained p42MAPK activation and differentiation is provided by the demonstration that blockade of MEK activation by PD098059 prevents TPA-induced morphological differentiation of wild type U937 cells. Using TPA-resistant clones, an involvement of microtubule reorganization and granule release is demonstrated by the ability of the microtubule depolymerizing agent nocodazole, to promote sustained p42MAPK activation in the presence of TPA. This response correlates with the lack of TPA-induced microtubule reorganization in these clones and the ability of nocodazole to partially bypass resistance to TPA. The results demonstrate a causal link between protein kinase C-dependent microtubule reorganization, sustained p42MAPK activation, and the induction of differentiation in U937 cells.

Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.

Methylselenocysteine (MSC), an organic selenium compound is an effective chemopreventive agent against mammary cell growth both in vivo and in vitro but its mechanism of action is still not understood. We have previously demonstrated that MSC is able to inhibit growth in a synchronized TM6 mouse mammary epithelial tumor cell line at 16 h time point followed by apoptosis at 48 h. The decrease in cdk2 kinase activity was coincident with prolonged arrest of cells in S-phase. The present set of experiments showed that cdk2 phosphorylation was reduced by 72% in the MSC-treated cells at 16 h time point. Expression for gadd34, 45 and 153 was elevated 2.5 to 7 fold following MSC treatment only after 16 h time point. In order to investigate a possible upstream target for MSC, we analyzed protein kinase C (PKC) in this model. Total PKC activity was reduced in TM6 cells by MSC (50 microM) within 30 min of treatment, both in cytosolic (55.4 and 77.6%) and membrane (35.2 and 34.1%) fractions for calcium-dependent and independent PKCs, respectively. PMA significantly elevated the PKC activity in membrane fraction (P < 0.01) and MSC inhibited this activation by more than 57%. The effect of MSC was selenium specific as selenomethionine and sulfurmethyl-L-cysteine (SMC) did not alter PKC activity either in cytosolic or membrane fraction. Immunoblot analysis showed that PKC-alpha was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-delta was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-e (in cytosolic and membrane fractions) and PKC-zeta (cytosolic fractions). The data presented herein suggest that PKC is a potential upstream target for MSC that may trigger one or all of the downstream effects; i.e. the decrease of cdk2 kinase activity, decreased DNA synthesis, elevation of gadd gene expression and finally apoptosis.

Cloning and characterization of phorbol ester differentiation-resistant U937 cell variants.

Differentiation-resistant U937 cells were derived from parental U937 human promonocytic leukemia cells by selecting for a nonadherent phenotype in cell cultures continuously exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequent analysis indicated no differences between wildtype (wt) and resistant U937 cells with respect to protein kinase C (PKC) isozyme expression, activation, or down-modulation. The subcellular localization of PKCs is identical in wt and resistant cells with the exception of PKC beta 2, which no longer colocalizes with microtubules in the TPA-resistant cell lines. In contrast to wt-U937 cells, the resistant cells do not express beta 2-integrin adhesion molecules, cd11b and cd11c, on the cell surface following TPA treatment but do express cd11b and cd11c in intracellular vesicles. TPA stimulates the translocation of these vesicles to the cell surface in wt U937 cells but not in the resistant cells. These results suggest that events downstream of PKC activation may mediate cytoskeletal reorganization and beta 2-integrin transport to the cell surface in wt-U937 cells but not in the differentiation-resistant cells.

Defective microtubule reorganization in phorbol ester-resistant U937 variants: reconstitution of the normal cell phenotype with nocodazole treatment.

Phorbol ester-induced beta 2-integrin transport to the cell surface is defective in cloned 12-O-tetradecanoylphorbol-13-acetate (TPA)-resistant U937 cell variants. Failure of the integrin-containing vesicles to reach the plasma membrane effectively blocks development of all integrin-mediated responses and the formation of a functional oxidase complex. Several lines of evidence suggested that the underlying cause of this defect may be the loss of regulatory elements in the cytoskeleton, which mediate microtubule stability and organization. Diminished protein kinase C (PKC) beta 2 association with microtubules correlated with the loss of heat-soluble microtubule-associated PKC-binding proteins and the loss of TPA-inducible reorganization of the microtubule cytoskeleton in the resistant U937 variants. Treatment with the microtubule-disrupting drug, nocodazole, was sufficient to induce the modest increase in cd11b surface expression associated with the release of this preformed integrin. Furthermore, brief nocodazole treatment followed by TPA treatment completely restored susceptibility to phorbol ester-induced differentiation in the resistant cell lines. The combination of nocodazole and TPA treatment also restored NADPH oxidase activity in the TPA-resistant clones. Results from these studies suggest that TPA-induced microtubule reorganization is a prerequisite for integrin vesicle translocation in U937 cells and that vesicle translocation to the plasma membrane may be a prerequisite for the transcriptional activation of cd11b and cd11c integrin genes in the early stages of monocyte differentiation.

Protein kinase C isozymes and substrates in mammary carcinogenesis.

Protein kinase C (PKC) comprises a family of ubiquitously expressed phospholipid-dependent enzymes that regulate cell growth and differentiation. Several effectors that modify mammary cell biology work at least partially through PKC-dependent pathways. Studies with mammary epithelial cells and tissues have demonstrated probable roles for the PKCs in processes associated with carcinogenesis including proliferation, estrogen sensitivity, and apoptosis. The involvement of PKCs in this wide variety of responses may in part be explained by the expression of multiple PKCs in breast tissue and the possibility that individual PKCs selectively phosphorylate different proteins and preferentially mediate different biological responses. Further understanding of the role of individual PKCs in mammary cell growth and tumor promotion/progression is likely to lead to new insights for breast cancer diagnosis and treatment.

Identification of a major protein kinase C-binding protein and substrate in rat embryo fibroblasts. Decreased expression in transformed cells.

We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with protein kinase C (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268,6858-6861). In this paper, we report a novel sequence, clone 72, isolated according to this method. Clone 72 has a 4.8-kilobase pair open reading frame; antibodies to clone 72 recognize a >200-kDa protein in cell and tissue extracts. Clone 72 message and protein are detected in a variety of tissues. Immunoprecipitation studies demonstrate that clone 72 is the major >200-kDa binding protein described previously in REF52 fibroblasts (Hyatt, S. L., Liao, L., Aderem, A., Nairn, A., and Jaken, S. (1994) Cell Growth & Differ. 5, 495-502). Expression of clone 72 message and protein are decreased in progressively transformed REF52 cells. Since clone 72 is both a protein kinase C (PKC)-binding protein and substrate, decreased levels of clone 72 may influence both the subcellular location of endogenous PKCs as well as signaling events associated with clone 72 phosphorylation. Our results emphasize that the role of PKCs in carcinogenesis may involve several factors, including the quantity and location of the PKCs isozymes and their downstream targets.

Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation.

U937 human promonocytic leukemia cells express PKC isozymes beta 1, beta 2, epsilon and zeta. Indirect immunocytofluorescence using affinity-purified PKC-specific antibodies indicates that each of the endogenous PKC isozymes in U937 cells display a unique compartmentalization within the intact cell. PKC-beta 1 is distributed between two identifiable pools: a cytoplasmic pool which redistributes to the plasma membrane upon activation with acute phorbol ester-treatment, and a membrane-bound pool associated with intracellular vesicles containing beta 2-integrin adhesion molecules, cd11b and cd11c. The vesicle-associated PKC-beta 1 translocates with the secretory granules to the plasma membrane upon agonist-stimulated activation. PKC-beta 2 is associated with the microtubule cytoskeleton in resting cells. PKC overlay assays indicate that PKC-beta 2 binds to proteins associated with microtubules, and not directly to tubulin. PKC-epsilon is associated with filamentous structures in resting cells and redistributes to the perinuclear region upon activation with phorbol esters. In differentiated U937 cells, PKC-beta 1 remains associated with vesicles translocating from the trans-Golgi region to the plasma membrane and PKC-epsilon is primarily associated with perinuclear and plasma membranes. PKC-zeta, which does not respond to phorbol ester treatment, is primarily cytosolic in undifferentiated cells and accumulates in the nucleus of differentiated cells blocked in the G2 phase of the cell cycle. The data clearly demonstrate that individual PKCs localize to different subcellular compartments and promote the hypothesis that PKC subcellular localization is indicative of unique functions for individual PKC isozymes.

12-Deoxyphorbol-13-O-phenylacetate-20-acetate is not protein kinase C-beta isozyme-selective in vivo.

The phorbol ester, 12-deoxyphorbol-13-O-phenylacetate-20-acetate (DOPPA) has been shown to activate specifically the protein kinase C (PKC)-beta 1 isozyme in vitro (1). We have investigated the potential of DOPPA as a PKC-beta 1/2 isozyme-specific agonist in intact cells, employing U937 cells, which express beta 1/2, epsilon and zeta PKC and in Swiss 3T3 cells which lack PKC-beta 1/2 but express alpha, delta, epsilon and zeta PKC. Immunoblot analysis with isozyme-specific antibodies indicated that DOPPA can mediate the subcellular redistribution and down-modulation of all endogenous PKC isozymes (except PKC-zeta) in both U937 and Swiss 3T3 cells. Prolonged treatment (> 6 h) of cultures in down-modulation studies is complicated by the metabolism of DOPPA to 12-deoxyphorbol-13-phenylacetate (DOPP), a compound which activates all PKC isozymes tested in vitro (Ryves, W. J., et al. (1991) FEBS Lett., 288, 5-9). Nevertheless, because DOPPA induced rapid and dose-dependent phosphorylation of p80 in cells which do not express PCK-beta, p80 phosphorylation in Swiss 3T3 cells indicates that DOPPA can activate a non-beta PKC in vivo. The data suggest that DOPPA cannot be used as a PKC-beta-selective agonist in intact cell studies.

Selective redistribution of protein kinase C isozymes by thapsigargin and staurosporine.

Protein kinase C (PKC) is the major cellular receptor for tumor promoting phorbol esters. Phorbol esters activate alpha-, beta-, delta- and epsilon-PKCs in GH4C1 rat pituitary cells and cause their redistribution from a soluble to a particulate fraction. We have now characterized the effect of several non-phorbol ester tumor promoters on PKC isozyme distribution in GH4C1 cells. The incomplete tumor promoter mezerein caused redistribution of alpha-, beta-, delta- and epsilon-PKCs. Thus, it did not display partial agonist activity. The phosphatase inhibitor okadaic acid did not cause redistribution of any isozyme. The calcium ATPase inhibitor thapsigargin and the ser/thr kinase inhibitor staurosporine caused redistribution of epsilon-PKC and, to a lesser extent, delta-PKC. Although the mechanism of the selective effect on delta- and epsilon-PKCs is not yet known, these data clearly demonstrate that their subcellular distribution can be regulated by a pathway that does not influence alpha- and beta-PKCs. Phorbol ester activation of epsilon-PKC was associated with appearance of a more slowly migrating immunoreactive band in the particulate fraction. Both epsilon-PKC forms accumulated phosphate during phorbol ester treatment. The phosphorylated forms of epsilon-PKC were preferentially recovered in the particulate fraction. Although staurosporine caused redistribution, it prevented the phorbol dibutyrate (PDBu)-mediated appearance of the upper band of the doublet and the increased phosphorylation of both bands. The PDBu-mediated redistribution of alpha- and beta-PKCs was not inhibited by staurosporine, even though staurosporine effectively inhibited PKC catalytic activity. Therefore, catalytic activity is not required for redistribution.

Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells.

GH4C1 cells, which express Ca(2+)-dependent alpha- and beta- as well as Ca(2+)-independent gamma-, epsilon- and zeta-protein kinase C (PKC) isozymes, provide a cell culture model for studying isozyme-specific properties and functions. Hormonal activation of PKCs regulates the differentiated functions of these cells, namely secretion and synthesis of prolactin (PRL). We previously reported that thyrotropin-releasing hormone (TRH) selectively down-modulates epsilon-PKC with no effect on alpha- or beta-PKCs (Kiley, S.C., Schaap, D., Parker, P., Hsieh, L.-L., and Jaken, S. (1990) J. Biol. Chem. 265, 15704-15712). We now extend those studies to explore the relationship between TRH-stimulated diacylglycerol (DAG) levels and epsilon-PKC down-modulation. TRH stimulates three distinct DAG phases in GH cells. Phase 1 DAG peaks at 15 s, is accompanied by a 6-fold increase in intracellular Ca2+, and causes the redistribution of alpha-, beta-, delta, and epsilon-PKC isozymes from a soluble to a detergent-insoluble particulate compartment. Phase 2 DAG peaks at 10 min, is not associated with a Ca2+ signal, and does not activate PKC by any criteria tested. Phase 3 DAG peaks at 6 h and is sustained through 12 h. This novel DAG phase is not associated with increased intracellular Ca2+. The time course of phase 3 DAG formation corresponds to the time course of TRH-stimulated epsilon-PKC down-regulation; maximal effects are observed at 6-12 h for both events. Unlike alpha-, beta-, and delta-PKCs which are preferentially distributed in the soluble fraction of resting GH cells, epsilon-PKC is also distributed in the detergent-insoluble particulate fraction. The selective compartmentalization of epsilon-PKC in the particulate fraction may render this pool uniquely susceptible to proteolytic degradation. The time course of phase 3 DAG formation and epsilon-PKC down-modulation corresponds to the time course of decreasing PRL message synthesis in GH4 cells. The data suggests that loss of epsilon-PKC may be associated with the down-regulation of prolactin synthesis and that regulation of PRL gene transcription may be an epsilon-PKC-specific function in GH cells.

Large granular lymphocytes provide an accessory function in the in vitro development of influenza A virus-specific cytotoxic T cells.

We report that large granular lymphocytes (LGL) have an accessory function in the development of cytotoxic T cells (Tc) through the production of soluble factor(s). LGL and T cells were separated on Percoll gradients and the ability of the separated and of the recombined LGL and T cells to generate influenza A virus-specific Tc activity was measured. When stimulated by virus-infected, irradiated, adherent cells, neither LGL nor T cells cultured separately produced Tc activity. When they were co-cultured, however, even if separated by a 0.22-micron pore size membrane, Tc responses were readily generated from the small T cell precursors and natural killer activity was maintained in the LGL. Thus, LGL were required as accessory cells for Tc responses to occur and the effect was mediated by a soluble factor(s). alpha-Interferon (IFN) was produced in cultures containing LGL and/or stimulating adherent cells, whereas gamma-IFN was only produced in cultures containing both LGL and T cells. Therefore, neither alpha- nor gamma-IFN appeared to be the LGL produced soluble factor that mediated the accessory effect of LGL on Tc responses.