ucn-1028-c and Carcinoma--Squamous-Cell

Breast cancers from pre- vs. postmenopausal women display unique characteristics that may be related to differences in epithelial differentiation between these two populations. In addition to lobular development, lactational changes, and involution, breast epithelium can undergo metaplastic alterations, often in association with carcinoma. Because protein kinase C (PKC) regulates differentiation and proliferation in many cell types, we asked whether modulation of PKC activity could define biochemical differences in breast epithelium from pre- vs. postmenopausal women. Organ cultures of normal human breast were treated with PKC agonists and antagonists. Epithelial differentiation was evaluated based on morphologic criteria and the expression of cell-type specific proteins. Staurosporine, a nonspecific but extremely potent inhibitor of PKC, induced squamous metaplasia in eight of eight cases within 2 weeks of treatment. Other inhibitors of PKC, such as calphostin C and tamoxifen, had no effect on epithelial differentiation. Long-term treatment with phorbol esters also did not induce squamous metaplasia. However, stimulation of cAMP levels by forskolin and isobutyl-methyl-xanthene (IMX) rapidly induced squamous metaplasia, as has been previously reported. Surprisingly, squamous metaplasia occurred in 10 of 12 cultures derived from postmenopausal women in the absence of exogenous agents. Untreated cultures derived from premenopausal women never developed this type of epithelium (0 of 11). Therefore, breast epithelium from pre- and postmenopausal women responded differently to in vitro culture. Forskolin/IMX or staurosporine can reproduce these conditions, acting independent of menopausal status. Because staurosporine's action was unique among PKC inhibitors, staurosporine may induce squamous metaplasia of breast epithelium by a PKC-independent mechanism.

Topics: Adult; Aged; Anticarcinogenic Agents; Breast; Breast Neoplasms; Carcinogens; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Keratin-10; Keratinocytes; Keratins; Middle Aged; Naphthalenes; Postmenopause; Premenopause; Protein Kinase C; Staurosporine; Tamoxifen; Tetradecanoylphorbol Acetate; Thrombomodulin; Tumor Cells, Cultured; Up-Regulation

Epidermal growth factor (EGF) increases 12-lipoxygenase mRNA by about 2-fold with a lag period of 4 to 8 hr, which precedes the increase in 12-lipoxygenase activity by 2 to 4 hr in human epidermoid carcinoma A431 cells. Induction of 12-lipoxygenase expression in human erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) has been reported previously. The present report describes a study of the involvement of protein kinase C (PKC) in EGF-induced 12-lipoxygenase expression in A431 cells. EGF-induced 12-lipoxygenase expression was inhibited by methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor. Staurosporine and calphostin C, which are two PKC inhibitors, inhibited EGF-induced enzyme activity and mRNA expression of 12-lipoxygenase. 1,2-Dioctanoyl-sn-glycerol (a membrane-permeant diacylglycerol) and PMA significantly induced enzyme activity and mRNA expression. Simultaneous treatment of cells with EGF and PMA did not exhibit an additive effect, suggesting that EGF and PMA share a common biochemical pathway in 12-lipoxygenase induction. Expression of mRNA for PKC alpha, delta and zeta was detected in A431 cells, whereas no mRNA expression for PKC beta 1, gamma and epsilon was observed. Taken together, these results suggest that EGF-induced 12-lipoxygenase expression is at least in part mediated by the PKC signal transduction pathway.

Topics: Alkaloids; Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Diglycerides; Enzyme Induction; Epidermal Growth Factor; Humans; Naphthalenes; Phosphatidylinositol Phosphates; Polycyclic Compounds; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.

Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Squamous Cell; Collagenases; Enzyme Induction; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; Molecular Sequence Data; Molecular Weight; Mouth Neoplasms; Naphthalenes; Neoplasm Invasiveness; Neoplasm Proteins; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C.

Topics: Animals; Brain; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Darkness; Down-Regulation; ErbB Receptors; Humans; Kinetics; Light; Naphthalenes; Phorbol 12,13-Dibutyrate; Polycyclic Compounds; Protein Kinase C; Rats; Rats, Inbred Strains