Involvement of protein kinase C in the growth regulation of human breast cancer cells.
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Breast Neoplasms↗
Biomedical subjects
C Borner
Publications and source records attributed to C Borner.
c-myc protein distribution. Neoplastic tissues of the human colon.
There is an extensive literature documenting the increased or deregulated expression of the c-myc oncogene in human malignancies. The authors have recently devised a sensitive immunocytochemical method for studying the tissue localization of c-myc protein in tissue sections of human colon. We have compared nuclear c-myc staining using a polyclonal rabbit anti-c-myc antibody and a mouse monoclonal myc antibody NCM II 274. Microscopic observation of the tissue specific pattern of c-myc protein distribution shows that nuclear staining intensity varies in normal and neoplastic crypt cell nuclei in parallel with morphologic criteria of neoplasia. These studies yield further information on the usefulness of c-myc protein as a prognostic indicator.
Cell Nucleus↗
Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts.
A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.
Animals↗
Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon.
Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC alpha and PKC epsilon. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC alpha and a concomitant decrease in PKC epsilon, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2+ in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC beta 1, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the expression of PKC alpha and PKC epsilon are mediated mainly through a PKC-independent pathway. The present results provide the first evidence that transformation of cells by an oncogene can alter the relative expression of specific isoforms of PKC. It is possible that these changes contribute to the malignant phenotype of these cells.
Animals↗
Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells.
Several forms of protein kinase C with molecular masses of 74-, 77-, and 80-kDa were detected in subcellular fractions of human breast cancer MDA-MB-231 cells which express the alpha-type protein kinase C. Several lines of evidence indicated that the 74-kDa is the precursor of the 77- and 80-kDa protein kinase C forms. (i) Pulse-labeling experiments revealed that protein kinase C is synthesized on membranes as a 74-kDa protein that can be chased into the 77- and the 80-kDa protein kinase C forms. (ii) The primary translation product of protein kinase C displayed an apparent molecular size of 74-kDa as determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells. (iii) Incubation with serine/threonine-specific protein phosphatases (potato acid phosphatase and phosphatase 1 or 2A) resulted in the complete dephosphorylation of the 77-kDa to the 74-kDa protein kinase C form. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably inactive 74-kDa form that is converted into the active 77- and 80-kDa protein kinase C by post-translational modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved by a specific, yet unidentified, "protein kinase C kinase" since the 74-kDa protein kinase C species did not undergo autophosphorylation and was neither a substrate for the purified protein kinase C, S6 kinase, phosphorylase kinase, casein kinase II, nor for the catalytic subunit of cAMP-dependent protein kinase. Except for phosphorylase kinase and the catalytic subunit of the cAMP-dependent protein kinase, phosphorylation of the 77-kDa protein kinase C form with purified protein kinase C (autophosphorylation), S6 kinase or casein kinase II shifted the molecular mass of the 77-kDa protein kinase C to 80-kDa. Prolonged exposure of MDA-MB-231 cells to phorbol 12-myristate 13-acetate not only leads to a complete down-regulation of protein kinase C activity but also to an accumulation of 74-kDa protein kinase C due to a retarded conversion of the 74-kDa into the 77- and 80-kDa protein kinase C forms in these cells. Our data indicate that tumor promoters additionally interfere with the posttranslational processing that converts the 74-kDa protein kinase C precursor into the 77- and 80-kDa forms of the enzyme.
Breast Neoplasms↗
Continuous synthesis of two protein-kinase-C-related proteins after down-regulation by phorbol esters.
The phorbol 12-myristate 13-acetate (PMA)-dependent down-regulation of immunoprecipitable protein kinase C was studied in human breast cancer cell lines that display different growth inhibitions toward the tumor promoter. PMA induces translocation of [35S]methionine-prelabeled cytosolic protein kinase C to membranes, followed by complete degradation of the enzyme (t1/2, 2 hr). PMA does not affect the protein kinase C synthesis; 20-80% of total protein kinase C of control cells was still immunoprecipitable as membrane-bound 74- and 80-kDa protein kinase C-related polypeptides if cells were allowed to incorporate [35S]methionine during PMA exposure for greater than 6 hr. These two proteins lack protein kinase activity and phorbol ester binding but reveal V8 peptide patterns identical to the active forms of protein kinase C (77/80 kDa) of PMA-untreated cells. The amounts of the immunoprecipitable membrane-bound 80-kDa protein kinase C-related polypeptide synthesized during the prolonged PMA treatment appear to inversely correlate with the extent of PMA-mediated growth inhibition of the respective human breast cancer cell line. These data suggest that after homologous down-regulation, functional protein kinase C (77/80 kDa) is replaced by a population of membrane-associated but enzymatically inactive protein kinase C-related polypeptides (74/80 kDa).
Breast Neoplasms↗
Role of protein kinase C (PKC) in short- and long-term cellular responses: inhibition of agonist-mediated calcium transients and down-regulation of PKC.
Active tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or membrane-diffusible synthetic diacylglycerols such as 1,2-dioctanoyl-sn-glycerol (DiC8), which specifically activate protein kinase C (PKC), inhibited the agonist-mediated rise in cytosolic calcium [(Ca2+)i] in a mast cell line (PB-3c) and human platelets. TPA inhibition of agonist-mediated calcium transient in platelets was readily reversed by the PKC inhibitor staurosporine. In contrast to DiCs, only active tumor promoters induced a time- and dose-dependent translocation of cytosolic PKC to membranes as determined both enzymatically or by immunoblotting. However, the concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the intracellular rise in (Ca2+)i. Thus, activation of PKC seems not to be exclusively coupled to its translocation to membranes, suggesting that translocation of PKC is mainly involved in the down-regulation of PKC. Down-regulation of immunoprecipitable PKC was studied in various human breast cancer cell lines that display differential growth inhibitory responses toward the tumor promoter. TPA induced translocation of [35S]methionine-prelabeled cytosolic 80 kDa PKC to membranes followed by complete degradation of the enzyme (t1/2 = 2 h) without affecting PKC synthesis. During prolonged TPA exposure, 20-80% of total 80 kDa PKC of control cells was still synthetized as a membrane-bound 74/80 kDa PKC doublet. Although both proteins lacked PKC activity and phorbol ester binding, they revealed structural similarity with the active 80 kDa PKC form of untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium↗
Immunological quantitation of phospholipid/Ca2+-dependent protein kinase of human mammary carcinoma cells: inverse relationship to estrogen receptors.
The amounts of phospholipid- and Ca2+-dependent protein kinase (PKC) of various human mammary tumor cells containing (ER+) or lacking (ER-) estrogen receptors were estimated by quantitative immunoblotting. According to several criteria the polyclonal anti-PKC antibody raised in rabbits against porcine brain PKC specifically recognizes an 80-kDa polypeptide on immunoblots. This 80-kDa PKC presumably represents the autophosphorylated form of the holoenzyme. Immunological quantitation of PKC revealed that the levels of immunodetectable PKC varied widely among the various human mammary carcinoma cell lines but closely matched the amounts determined by enzyme activity and phorbol ester binding in the respective cell line. The largest amounts of immunodetectable PKC were found in the ER- human mammary tumor cells (0.5 to 1.5 micrograms PKC/mg of cytosolic protein). These data indicate that ER- human mammary carcinoma cell lines express significantly higher levels of PKC than their estrogen-receptor-containing counterparts.
Breast Neoplasms↗
Translocation of protein kinase C is not required to inhibit the antigen-induced increase of cytosolic calcium in a mast cell line.
Cross-linking of receptor bound IgE antibodies by multivalent antigen (DNP8-BSA) on PB-3c cells leads to an increase of cytosolic calcium ((Ca2+)i). Active tumor promoting phorbol esters and teleocidin which specifically activate the phospholipid Ca2+-sensitive protein kinase (PKC), inhibited the antigen-mediated rise in (Ca2+)i and induced a time and dose-dependent translocation of cytosolic PKC to membranes of the PB-3c cells as determined by enzyme activity or immunoblotting using a polyclonal anti-PKC antibody. This TPA concentration did not affect the subcellular distribution of PKC, although 1 nM of 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited to 50% the antigen-mediated increase in (Ca2+)i. The concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the antigen-mediated increase in (Ca2+)i. These data demonstrate that the TPA-dependent activation of PKC is not directly coupled to its translocation to membranes.
Antigen-Antibody Complex↗
Altered protein kinase C in a mast cell variant defective in exocytosis.
The murine mast cell line PB-3c is dependent on interleukin 3 (IL-3) with respect to survival and proliferation. These cells also require IL-3 to display antigen-mediated serotonin release, which is coupled to a transient increase of cytosolic free calcium ([Ca2+]i). The antigen-mediated exocytosis is inhibited by phorbol 12-tetradecanoate 13-acetate (PTA), an activator of phospholipid/Ca2+-sensitive protein kinase. In contrast, the malignant mast cell variant PB-1 is IL-3 independent with respect to proliferation but is unable to undergo antigen-mediated exocytosis. Yet this cell line exhibits basal levels of [Ca2+]i, serotonin content, and numbers of IgE receptors comparable to those of PB-3c cells. Subcellular distribution studies revealed that the specific activity of cytosolic protein kinase C of PB-1 cells was only 40% of that found in PB-3c cells. Furthermore, the PB-1 cells showed a significantly higher specific activity of membrane-bound protein kinase C than PB-3c cells. Scatchard plot analysis of [3H]-phorbol 12,13-dibutyrate binding to intact PB-1 cells demonstrated the presence of 20% high-affinity (Kd = 6 nM) and 80% low-affinity (Kd = 60 nM) phorbol ester receptors, whereas PB-3c cells displayed only the low-affinity phorbol ester binding. Immunological characterization of protein kinase C from both cell lines revealed the presence of a normal 77-kDa protein kinase C holoenzyme in both cell lines. In addition, a 72-kDa protein kinase C-related protein band was found mainly in the membrane fraction of the PB-1 variant. It is suggested that this altered and membrane-bound form of protein kinase C may be involved in the blockage of the antigen-mediated exocytosis of PB-1 cells.
Animals↗
Effects of tumor promoters on growth and on cellular redistribution of phospholipid/Ca2+-dependent protein kinase in human breast cancer cells.
Active, structurally unrelated tumor promoters (12-0-tetradecanoyl-phorbol-13-acetate (TPA), teleocidin and aplysiatoxin) inhibit growth of mammary carcinoma cells (MCF7- greater than BT-20 greater than MDA-MB-231 greater than = ZR-75-1 greater than HBL-100). This efficiency in inhibiting cell growth correlates with the tumor-promoting activity of a series of phorbol ester derivatives. The phospholipid/calcium-dependent protein kinase (PKC), a target for phorbol ester action, was measured by polyacrylamide gel electrophoresis. The levels of PKC were higher (p less than 0.001) in estrogen-receptor-negative than in estrogen-receptor-positive cells. Treatment of cells with active tumor promoters results in time- and dose-dependent translocation of cytosolic PKC to membrane fractions. Less potent phorbol esters induce only partial translocation of PKC (i.e., decrease of cytosolic without increase in membrane-bound PKC), whereas inactive esters have no effect. No correlation was found between PKC concentration or the amount of PKC translocated to membranes and the sensitivity of the respective cells to TPA. It is concluded that tumor-promoter-mediated growth inhibition of breast cancer cell lines is due to mechanism(s) occurring after the translocation of PKC.
Breast Neoplasms↗
Protein kinase C desensitization by phorbol esters and its impact on growth of human breast cancer cells.
Active phorbol esters such as TPA (12-0-tetra-decanoylphorbol-13-acetate) inhibited growth of mammary carcinoma cells (MCF-7 greater than BT-20 greater than MDA-MB-231 greater than = ZR-75-1 greater than HBL-100) with the exception of T-47-D cells presumably by interacting with the phospholipid/Ca2+-dependent protein kinase (PKC). The nonresponsive T-47-D cells exhibited the lowest PKC activity. A rapid (30 min) TPA-dependent translocation of cytosolic PKC to membranes was found in the five TPA-sensitive cell without affecting cell growth. However, TPA-treatment of more than 10 hours inhibited reversibly the growth of TPA-responsive cells. This effect coincided with the complete loss of cellular PKC activity due to the proteolysis of the translocated membrane-bound PKC holoenzyme (75K) into 60K and 50K PKC fragments. Resumption of cell growth after TPA-removal was closely related to the specific reappearance of the PKC holoenzyme activity (75K) in the TPA-responsive human mammary tumor cell lines suggesting an involvement of PKC in growth regulation.
Breast Neoplasms↗
Dissection of functional domains in Bcl-2 alpha by site-directed mutagenesis.
Bcl-2 alpha is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2 alpha or Bcl-2 beta, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2 alpha is active and membrane bound, but Bcl-2 beta is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane association of Bcl-2 alpha. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overexpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2 alpha/beta splice site, as well as part of domain X in Bcl-2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Phorbol ester and epidermal growth factor receptors in human breast cancer.
Recently hormone - dependent mammary carcinoma cell lines were shown to exhibit in vitro significantly lower protein kinase C (PKC) activities and epidermal growth factor receptor (EGF-R) as compared to hormone - independent cell lines. Measurements of EGF-R levels in primary human breast cancer biopsies were determined by [125I]-EGF binding. The EGF binding correlated inversely with the estrogen (ER) (p less than 0.001), progesterone receptor (PR) (p less than 0.005) contents and with the age of the patients. In contrast, the amounts of PKC, determined by phorbol ester binding, correlated inversely only with the PR (p less than 0.001), but not with the ER (p = 0.065). There was, however, a significant inverse correlation (p less than 0.05) between phorbol ester binding and ER levels if ER positive biopsies but with a PR negative value (i.e. with a non functional estrogen receptor) were excluded from statistical analysis. These data suggest an inverse relationship between the EGF-R or the phorbol ester receptor and the steroid receptor system in human breast cancer.
Breast Neoplasms↗