pivotal role in many cellular processes, but its effect differs from one cell type to another and remains not fully understood. the aim of this work was to investigate the dependence between the rate of hB-eGF mediated cell proliferation and activation of EGFR and ErbB4 receptors. Therefore, the effects of human recombinant sHB-EGF (rsHB-EGF) on the proliferation of cell lines with different EGFR and ErbB4 quantity and ratio, as well as activation of the marK-cascade p38 and erK1/2 (p42/44) kinases, were analyzed. For comparison, a similar study of the effect of native sHB-EGF secreted by human histiocytic lymphoma cells U937 during co-cultivation with different cell lines was performed. It was proved that cell proliferation in response to sHB-EGF depends not only on the quantity but also on the ratio of EGFR and ErbB4. It was shown that signaling through erbB4 is associated with activation of p38 kinase and signaling through eGFr associated with activation of ERK1/2 (p42/44) kinase. We assume the existence of two different mechanisms for sHB-eGF-mediated stimulation of cell proliferation, and the simultaneous launch of these mechanisms provides a maximal proliferative response. the results of this study support the feasibility of creating anti-proliferative drugs that target erbB4.

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Summary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathophysiological functions. Also, HB-EGF plays a pivotal role in progression of different tumors. So, HB-EGF seems to be a target molecule for the treatment of some cancer types. Aim: To obtain HB-EGF neutralizing polyclonal antibodies and test their anti-proliferative properties in vitro. Materials and Methods: Lab rabbits and mice were used for immunization with recombinant HB-EGF. The effect of generated polyclonal antibodies on viability and apoptosis of human epidermoid carcinoma derived A431 cell line was assessed using MTT and Annexin V-propidium iodide assays. Results: Rabbit polyclonal anti-HB-EGF serum could block binding of soluble HB-EGF to epidermal growth factor receptor/human epidermal growth factor receptor. Also, anti-HB-EGF antibodies could bind to surface of A431 cells which express abnormally high levels of membrane bound proHB-EGF and its receptor. It has been shown that immune serum with polyclonal antibodies against HB-EGF was able to block the mitogenic activation of the cells with HB-EGF and cause apoptotic cell death. Conclusion: Inhibition of HB-EGF activity with neutralizing polyclonal antibodies can effectively inhibit mitogenic activation and cause apoptosis of cancer cells with significant epidermal growth factor receptor overexpression.

Heparin-binding eGF-like growth factor (HB-eGF) is a member of the epidermal growth factor family that was proven as a potent mitogen and chemoattractant. HB-eGF mediated eGFr activation is a key event in the stimulation of gene expression, cell migration and proliferation during both normal and pathogenic physiological processes. The main goal of this research was to reveal the role of the heparin-binding domain of HB-eGF in the ligand-receptor formation and its further internalization to the cytoplasm. We used fluorescently-labeled recombinant derivative of soluble HB-EGF and its truncated form (sHB-EGF Δ84-106 ) with deletion of the heparin-binding domain. Firstly, the binding kinetics of two forms of sHB-eGF to its cell surface receptors was determined using flow cytometry. To determine how the absence of heparin-binding domain in the structure of HB-EGF affects its internalization, we analyzed the endocytosis process of EGFP-sHB-eGF Δ84-106 and eGFP-sHB-eGF complexes by confocal microscopy. It was found that the full-size form of HB-eGF is characterized by a lower intensity of translocation to the cytoplasm in comparison to HBDdeleted form. Thus, differences in the trafficking of the full-size or truncated forms of sHB-EGF in the cell cytoplasm may reflect the mechanisms of extracellular matrix influence on the biological activity of sHB-EGF. K e y w o r d s: heparin-binding eGF-like growth factor, epidermal growth factor receptor, endocytosis.