Involvement of human beta-defensin-2 in intracellular signaling: in vitro study.

AIM: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. MATERIALS AND METHODS: A431cells were cultured in the presence of 1 microg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. RESULTS: 30 min incubation of A431 cells with 1 mug/ml of hBD-2 didn't influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment - in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment - in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. CONCLUSION: Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells.

Involvement of human beta-defensin-2 in proliferation of transformed cells of human cervix.

AIM: To evaluate the influence of human beta-defensin-2 (hBD-2) on viability and proliferation of cultured human epithelial cells and the patterns of hBD-2 expression in normal tissues and early-stage human cervical neoplasia in the relation to proliferative state of these cells. MATERIALS AND METHODS: The influence of recombinant hBD-2 on viability and proliferation of cultured cells of A431 and M-HeLa lines in vitro was performed by MTT-test, 3H-thymidine incorporation and cell counting techniques. Immunohistochemical analysis of expression of hBD-2 and PCNA in tissue samples (10 normal cases (control), 30 carcinomas of the cervix uteri: 15 - squamous cell carcinoma in situ (Stage 0), and 15 squamous cell carcinoma (Stage Ia)) was performed with the use of anti-hBD-2 and anti-PCNA-mAbs, respectively. RESULTS: We have revealed that hBD-2 significantly stimulated proliferation of A431 and M-HeLa cells in a concentration-dependent manner in the range of 0.1-2 microg/ml, whilst at higher concentrations (> 3-5 microg/ml) it negatively influenced cell viability. The results of immunohistochemical study have shown that malignant transformation of human cervical epithelium is accompanied by the increase of expression of hBD-2 and PCNA. However, the correlative analysis of the expression of the mentioned markers has revealed no relation between them. CONCLUSION: The effect of hBD-2 on viability and proliferation of cultured epithelial cells possesses a concentration-dependent character. Expression of hBD-2 is increased in early-stage cervical carcinoma.

A protein kinase inhibitor from A431 subline overexpressing TGF alpha possesses antimicrobial activity.

During the last decade the key role of antimicrobial peptides in innate immunity has been argued. They were found in plants and in different phylogenic groups of animals (insects, amphibia, and even in mammals). We report the production of a human peptide antibiotic that was previously characterized as an EGF receptor tyrosine kinase inhibitor in epidermoid carcinoma A431/1522 cell subline overexpressing TGF alpha. It is a 3 kDa hydrophobic cationic peptide cytotoxic for different species of Gr+ and Gr- bacteria in micromolar concentration range, and demonstrating slight fungicidal activity.

[Expression of the gene for transforming growth factor type alpha, transferred into A431 cells by a recombinant retroviral vector]. / Ekspressiia gena transformiruiushchego faktora rosta tipa alpha, perenesennogo rekombinantnym retrovirusnym vektorom v kletki A431.

Cells of human epidermoid carcinoma A431 were used for obtaining of the cell line constantly expressing TGF-alpha. Recombinant virions were obtained by introducing the proviral DNA into PA317 cells by means of electrotransfection. A protein with the EGF-competing activity was found in a conditioned media of the chosen clone A431/1522-4. The concentration of this protein was several times higher than in a conditioned media of wild type A431 cells. By means of electrophoresis, isoelectric focusing, and immunochemical analysis it was shown that the protein is TGF-alpha. Similarly to EGF, the extracted TGF-alpha entirely displaced 125I-EGF specifically bound to receptor. TGF-alpha produced by the A431/1522 cells also stimulated autophosphorylation of the EGF receptor.