The human salivary gland (HSG) epithelial cell line can differentiate when cultured on extracellular matrix preparations. We previously identified > 30 genes upregulated by adhesion of HSG cells to extracellular matrix. In the current studies, we examined the role of one of these genes, the polyamine pathway biosynthetic enzyme S-adenosylmethionine decarboxylase (SAM-DC) and the related enzyme, ornithine decarboxylase (ODC), on HSG cell differentiation during culture on extracellular matrix. HSG cells cultured on fibronectin-, collagen I gel-, and Matrigel-coated substrates for 12-24 h upregulated SAM-DC and ODC mRNA expression and enzyme activity compared to cells cultured on non-precoated substrates. After 3-5 days, HSG cells grown on Matrigel- or collagen I gel-coated substrates acquired a differentiated phenotype: the cells showed changes in culture morphology and increased expression of salivary gland differentiation markers (vimentin, SN-cystatin, and alpha-amylase). Further, culturing the cells on substrates precoated with an anti-beta 1-integrin-antibody promoted differentiation-like changes. HSG cells cultured on collagen I-or Matrigel-coated substrates rapidly entered the cell cycle but showed decreased cell proliferation at longer times. In contrast, cell proliferation was enhanced on fibronectin-coated substrates compared to cells on non-precoated substrates. Treatment with the polyamine synthesis inhibitors, difluoromethylornithine (DFMO), and methylglyoxal bis-(guanylhydrazone) (MGBG), inhibited cell proliferation and delayed H-3-thymidine incorporation in HSG cells cultured on all of the substrates. Further, inclusion of DFMO and MGBG inhibited or delayed acquisition of the differentiated phenotype in HSG cells cultured on Matrigelor collagen I gel-coated substrates. This suggests that the adhesion-dependent expression of SAM-DC and ODC contributes to extracellular matrix-dependent HSG cell differentiation. J. Cell. Physiol. 203: 353-361, 2005. (c) 2004 Wiley-Liss, Inc.

Epidermal growth factor receptor (EGFR) and HER-2/ErbB2 are members of the Erb family of signaling receptors. ErbB2 is overexpressed in many different cancers and has been linked to enhanced malignancy of tumors. We have examined the cellular translocation of Raf-1 during EGF-dependent signal transduction in two breast tumor cell lines, BT20 and SKBR3. Treatment of BT-20 breast cancer cells, which express EGFR, with EGF resulted in rapid (5 minutes) accumulation of EGFR and Raf-1 into plasma membrane–associated endocytotic vesicles. However, at later time points (30 minutes) only EGFR was endocytosed and Raf-1 dissociated from the plasma membrane and was found in the cytosol. In SKBR3 breast cancer cells, which express high levels of EGFR and ErbB2, treatment with EGF also resulted in rapid accumulation of EGFR and Raf-1 into endocytotic vesicles, but EGFR endocytosis was inhibited and Raf-1 remained associated with the plasma membrane for a prolonged period. The role of ErbB2 in the retention of Raf-1 at the plasma membrane was confirmed in BT-20 cells transfected with ErbB2. BT-20 cells expressing ErbB2 and treated with EGF retained Raf-1 at the plasma membrane for prolonged periods, whereas Raf-1 rapidly dissociated from the plasma membrane in EGF-stimulated cells transfected with a control vector. The presence of Raf-1 at the plasma membrane correlated with activation of Raf-1 and MAP kinase. Cells that expressed ErbB2 and treated with EGF showed prolonged activation of Raf-1 and MAP kinase compared with cells that expressed low levels of ErbB2. These results suggest that expression of ErbB2 promoted retention of Raf-1 in the plasma membrane, resulting in prolonged activation of the MAP kinase cascade, which may contribute to enhanced malignancy in ErbB2-expressing cancers.

MAP kinase can be activated by integrin-dependent adhesion in a FAK-dependent manner. Cell-cell contact inhibition is continuously active in controlling cell growth and the loss of cell-cell contact inhibition is correlated with the malignant characteristics of cancer cells. In this study we showed that cell adhesion to fibronectin for 1 h activated MAP kinase phosphorylation. However, when non-tumorigenic HSG cells, MCF-10A cells, or 293 cells were plated on fibronectin-coated substrates for 1 h at high cell density (which favors cell-cell contact), MAP kinase phosphorylation was not enhanced. Tumorigenic breast cancer cells, BT474, Cama, MCF-7, MDA-MB-231 and SKBR3, did not show inhibition of MAP kinase phosphorylation but rather enhanced MAP kinase phosphorylation when cultured at high density on fibronectin-coated substrates. Adhesion of HSG cells to fibronectin also increased FAK phosphorylation and this FAK phosphorylation was partially inhibited when cells were cultured at high density. Expression of Raf-1 catalytic domain-GFP in HSG cells could overcome the cell density-dependent inhibition of MAP kinase phosphorylation and FAK phosphorylation. The expression of Raf-1-catalytic domain-GFP also upregulated the expression of alphav integrin and promoted cell-cell adhesion in HSG cells. These results suggest that the active form of Raf-1 may interrupt cell-cell contact inhibition by promoting alphav integrin expression, which has been implicated in cell aggregation.

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin‐ or collagen I gel‐coated substrates, mediated by β1 integrins, has been shown to upregulate the expression of more than 30 genes within 3–6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8–2.3‐fold. HSG cells expressed PKC‐α, γ, δ, ε, μ, and ζ. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC‐γ and PKC‐δ. Cytoplasmic PKC‐γ and PKC‐δ became membrane‐associated, and immunoprecipitated PKC‐γ and PKC‐δ kinase activities were enhanced 2.5–4.0‐fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin‐coated beads to HSG monolayers co‐aggregated β1 integrin and PKC‐γ and PKC‐δ but not other PKC isoforms. Thus, integrin‐dependent adhesion of HSG cells to fibronectin or collagen I activated PKC‐γ and PKC‐δ. The role of this PKC upregulation on adhesion‐responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non‐precoated, fibronectin‐ or collagen I‐coated substrates, and analyzed for changes in adhesion‐responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion‐responsive genes including calnexin, decorin, S‐adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion‐responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion‐responsive genes. © 2001 Wiley‐Liss, Inc.