Tamoxifen restores the E-cadherin function in human breast cancer MCF-7/6 cells and suppresses their invasive phenotype

Tamoxifen is an antiestrogen used in adjuvant therapy of breast carcinoma and could potentially prevent the development of mammary cancer. While it is widely clinically used, its exact mechanisms of action are not yet fully elucidated. MCF-7/6 cells are estrogen receptor-positive invasive human breast cancer cells with a functionally inactive cell surface E-cadherin. In this study, we report that tamoxifen, and to a lesser extent its metabolites 4-OH-tamoxifen and N-desmethyltamoxifen, restore the function of E-cadherin in MCF-7/6 cells. In an aggregation assay, 10(-6) M tamoxifen significantly increases the aggregation of MCF-7/6 cells. This effect is abrogated by a monoclonal antibody against E-cadherin (HECD-1), is fast (within 30 min), and does not require de novo protein synthesis. Tamoxifen was also found to inhibit the invasion of MCF-7/6 cells in organ culture. Our data is the first demonstration that tamoxifen can activate the function of an invasion suppressor molecule and suggest that the restoration of E-cadherin function may contribute to the therapeutic benefit of tamoxifen in breast cancer patients.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Sensitization to doxorubicin resistance in breast cancer cell lines by tamoxifen and megestrol acetate

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.     

Regulation of parathyroid hormone-related protein expression in MCF-7 breast carcinoma cells by estrogen and antiestrogens

Expression of parathyroid hormone-related protein (PTHrP) in breast carcinoma is a frequent cause of the paraneoplastic syndrome of hypercalcemia. In response to treatment with estrogen or tamoxifen, some breast cancer patients also develop a transient hypercalcemia. Therefore, the effect of 17beta-estradiol (E2), tamoxifen, or its more potent metabolite, 4-hydroxytamoxifen (OH-tamoxifen), on PTHrP expression in an estrogen receptor (ER)-positive breast carcinoma cell line (MCF-7) was evaluated. E2 increased PTHrP mRNA levels in MCF-7 cells and stimulated PTHrP(1-86) release in a dose-dependent fashion (10(-10)-10(-6) M). Tamoxifen and OH-tamoxifen also stimulated PTHrP release in a concentration-dependent fashion that paralleled their relative ER binding affinities (10(-6) or 10(-8)-10(-6) M, respectively). Combined treatment with the partial estrogen agonist, OH-tamoxifen, and E2 decreased E2-stimulated PTHrP secretion in MCF-7 cells to the levels seen with OH-tamoxifen treatment alone. These results suggest that transient estrogen- or tamoxifen-induced hypercalcemia in patients with breast carcinoma may be a PTHrP-mediated effect that is a marker of ER positivity.     

Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity

We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3 cells; endogenous levels of the other 6 PKC isozymes are low or undetectable. We have overexpressed PKC-delta and -epsilon in these cells to observe activation/translocation of these two isozymes and the biological consequences of overexpression. Both PKC-delta and -epsilon, but not PKC-alpha, are partially associated with the insoluble fraction even in the absence of phorbol 12-myristate 13-acetate (PMA). Upon PMA stimulation, both PKC-delta and -epsilon translocate to the insoluble fraction of cell homogenates, as can be observed with the endogenous PKC-alpha. Overexpression of PKC-delta induces significant changes in morphology and causes the cells to grow more slowly and to a decreased cell density in confluent cultures. These changes are accentuated by treatment with PMA. Overexpression of PKC-epsilon does not lead to morphological changes, but causes increased growth rates and higher cell densities in monolayers. None of the PKC-delta overexpressers grow in soft agar with or without PMA, but all the cell lines that overexpress PKC-epsilon grow in soft agar in the absence of PMA, but not in its presence. NIH 3T3 cells that overexpress PKC-epsilon also form tumors in nude mice with 100% incidence. This indicates that high expression of PKC-epsilon contributes to neoplastic transformation.     

Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

Effects of tamoxifen and interferon-beta or the combination on tumor-induced angiogenesis

Inhibition of angiogenesis by anti-tumor agents may play a role in tumor growth arrest. Tamoxifen and interferon-alpha/beta (IFN-alpha/beta) exhibit potentiated anti-proliferative activity against tumor cells. However, additional host-mediated effects such as modulation of angiogenesis may also inhibit tumor growth in vivo. The effect of tamoxifen and IFN-beta on angiogenesis induced by 2 human tumors, MCF-7 breast carcinoma (estradiol dependent) and NIH-OVCAR-3 ovarian carcinoma (estradiol independent), was assessed. Treatment of nude mice bearing MCF-7 tumors with tamoxifen resulted in a 68% decrease in the number of vessels at the tumor periphery. Treatment with IFN-beta yielded a 33% reduction. Treatment of nude mice bearing NIH-OVCAR-3 tumors with tamoxifen resulted in a 73% decrease in the number of vessels. Treatment with IFN-beta yielded a 57% reduction. Combination treatment resulted in augmented anti-angiogenic effects. As single agents, both tamoxifen and IFN-beta inhibited xenograft tumor growth. Ten weeks of tamoxifen treatment resulted in growth inhibition of MCF-7 and NIH-OVCAR-3 carcinomas by 85% and 66%, respectively. Ten weeks of IFN-beta treatment resulted in inhibition of growth of MCF-7 and NIH-OVCAR-3 carcinomas by 67% and 88%, respectively. The combination of tamoxifen and IFN-beta completely prevented growth of MCF-7 and NIH-OVCAR-3 carcinomas. The anti-angiogenic effects of tamoxifen and IFN-beta were additive. Inhibition of angiogenesis was detectable before measurable effects on tumor volume in both MCF-7 and NIH-OVCAR-3 tumors. Potentiation of anti-angiogenic effects by tamoxifen and IFN-beta, possibly resulting from enhanced IFN-induced gene expression, may contribute to anti-tumor activity in both estradiol-dependent and estradiol-independent tumors in vivo.     

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.     

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.     

A model to describe how a point mutation of the estrogen receptor alters the structure-function relationship of antiestrogens

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2- diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17 beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (ML alpha 2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 microM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and ML alpha 2H. The ML alpha 2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin

Protein kinase C-epsilon (PKC-epsilon) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-epsilon and actin. Full-length recombinant PKC-epsilon, but not PKC-betaII, -delta, -eta, or -zeta, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-epsilon amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-epsilon or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-epsilon colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-epsilon phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-epsilon sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-epsilon in a catalytically active conformation.     

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.     

Differential modulation of protein kinase C isozymes in rat parotid acinar cells. Relation to amylase secretion

We investigated the expression, distribution, and activation parameters of protein kinase C (PKC) isozymes in isolated rat parotid acinar cells. By analyzing cellular extracts by western blot analysis and for isozyme-specific RNA, the Ca(2+)-independent PKC-delta, -epsilon, and -zeta were detected in the cytosolic, particulate (plasma membrane), and nuclear fractions of unstimulated cells, whereas the Ca(2+)-dependent PKC-alpha was confined to the cytosolic and particulate fractions. The expressed isozymes showed distinct responses to phorbol 12-myristate 13-acetate (PMA), thymeleatoxin, and cell surface receptor agonists with respect to translocation from cytosol to particulate fraction and nucleus, as well as sensitivity to down-regulation caused by prolonged exposure to PMA (3-20 hr). The marked susceptibility to down-regulation displayed by PKC-alpha and -delta was accompanied by an enhanced secretory response to norepinephrine as compared with control cells. Further, the selective PKC inhibitors Ro 31-8220 and CGP 41,251 also produced a concentration-dependent enhancement of norepinephrine-induced amylase secretion. Our findings suggest that PKC-alpha or -delta plays a negative modulatory role, rather than an obligatory role, in amylase secretion. Also, the localization and redistribution of PKC-epsilon and -delta to the nucleus by PKC activators imply that one or both of these isozymes may regulate such processes as cellular proliferation and/or differentiation.     

Bryostatin 1 affects P-glycoprotein phosphorylation but not function in multidrug-resistant human breast cancer cells

The function of P-glycoprotein (Pgp), which confers multidrug resistance by active efflux of drug, is thought to be dependent on phosphorylation. Previous studies have suggested that protein kinase C (PKC) plays an important role in Pgp phosphorylation. We report here the effects of bryostatin 1, a unique PKC activator and inhibitor, on Pgp function in a multidrug-resistant MCF-7 human breast cancer subline which overexpresses PKC-alpha. Bryostatin 1 (100 nM) decreased Pgp phosphorylation after 24 h of treatment. In contrast, it did not affect Pgp function as demonstrated by the accumulation of [3H]vinblastine and rhodamine 123. We compared the effect of bryostatin 1 treatment on PKC-alpha with that of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM). 12-O-tetradecanoylphorbol-13-acetate caused translocation of PKC-alpha from the cytosol to the cell membrane after a 10-min treatment and its down-regulation after 24 h of treatment. Likewise, bryostatin 1 (100 nM) caused translocation, but only after longer treatment (1 h), and it caused down-regulation of PKC-alpha at 24 h of treatment. Thus, while the MCF-7TH cells overexpress the PKC-alpha isoform, and its down-regulation by bryostatin 1 is associated with decreased Pgp phosphorylation, these alterations do not modulate drug transport. We conclude that, while bryostatin 1 may be useful clinically because of its ability to inhibit PKC, it is not able to reverse Pgp-mediated multidrug resistance.     

Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.     

Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3

Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of CD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increase. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-beta, PMA caused a strong increase in PKC-delta and a weak increase in PKC-alpha, PKC-epsilon, and PKC-zeta expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.     

Thrombin receptor (PAR-1) antagonists. Heterocycle-based peptidomimetics of the SFLLR agonist motif

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.     

Visual evoked potentials in Alzheimer''s disease: investigations in a PET-defined collective

Evidence indicates that the lipoxygenase (LO) pathway of arachidonic acid is a key mediator of angiotensin II (AII)-induced aldosterone synthesis in adrenal glomerulosa cells. Although protein kinase C (PKC) may play a role in AII action, the precise PKC isoforms involved and whether LO products can activate PKC is not clear. We therefore evaluated the effect of AII and LO products such as 12- and 15-hydroxyeicosatetraenoic acids (HETEs) on PKC activation in isolated rat adrenal glomerulosa cells. PKC activity was measured by the phosphorylation of a PKC specific peptide while the PKC isoforms were identified by Western immunoblotting using antibodies that recognize the alpha, beta, gamma or epsilon isoforms of PKC. Treatment of the cells for 15 min with AII (10[-8]M) or the LO products 12- or 15-HETE caused a marked increase in PKC activity in membrane fractions with reciprocal decreases in the cytosolic PKC activity. Rat glomerulosa cells expressed only the alpha, and epsilon isoforms of PKC. AII increased membrane bound levels of both PKC-alpha and -epsilon (1.9- and 1.5-fold, respectively), whereas the LO products predominantly activated PKC-epsilon. Reciprocal decreases in immunoreactive cytosolic PKC levels were seen. AII-induced aldosterone synthesis was blocked by H-7 and retinal as well as by a PKC-specific pseudosubstrate inhibitor, PKC(19-36). These results suggest that AII and LO pathway-induced actions in the adrenal glomerulosa may be mediated by specific PKC isoforms.     

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.