Journal Article

Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells

Dominik Stuhlmann, Niloofar Ale-Agha, Roland Reinehr, Holger Steinbrenner, Maria C. Ramos, Helmut Sies and Peter Brenneisen

in Carcinogenesis

Volume 24, issue 11, pages 1737-1748
Published in print November 2003 | ISSN: 0143-3334
Published online November 2003 | e-ISSN: 1460-2180 | DOI:
Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells

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Loss of gap junctional intercellular communication (GJIC) is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52–70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20–30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by >90%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of connexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca2+) might mediate the transduction of this effect. The involvement of Ca2+ in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor–stroma interaction of skin cells.

Keywords: CaM, calmodulin; CCD, charge coupled device; CK, cytokeratin; Cx, connexin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GJIC, gap junctional intercellular communication; HDF, human dermal fibroblasts; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HRP, horseradish peroxidase; MEK1/2, mitogen-activated protein kinase 1/2; MMP, matrix metalloproteinase; MWCO, molecular weight cut-off; NGS, normal goat serum; NHEK, normal human epidermal keratinocytes; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma; SN(2d) HDF, serum-free conditioned medium harvested after 2 days; SN(2d) SCL-1, serum-free supernatants of SCL-1 cells harvested after 2 days; TPA, 12-O-tetradecanoylphorbol-13-acetate

Journal Article.  8993 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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