Antiproliferative and apoptotic effects of anti-human HB-EGF neutralizing polyclonal antibodies in vitro.

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.

[Cell model for the study of receptor and regulatory functions of human proHB-EGF].

Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation oftransfected cells with TPA (12-O-Tet-radecanoylphorbol-13-acetate), proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule - CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

[Toxin-neutralizing properties of antibodies to diphtheria toxin recombinant subunits A and B and a new method of their estimation].

Development of complications during diphtheria depends to a large extent on toxin-neutralizing antibodies level in the patient's blood. Active immunization of people with diphtheria anatoxin is widely used for diphtheria prevention and passive immunization with hyperimmune antitoxic horse serum is used for diphtheria treatment. A traditional component of anti-diphtheria vaccines--diphtheria anatoxin has a number of serious disadvantages, which are mainly associated with complicated procedure of its production. Thus, the search for new antigen substances, which can effectively stimulate protective humoral response to diphtheria toxin, is an urgent task in anti-diphtheria vaccine development. Furthermore, one of the most important objects is the development of new in vitro methods for estimation of diphtheria toxin-neutralizing polyclonal and monoclonal antibodies, which allow to avoid using active diphtheria toxin and toxin-sensitive laboratory animals. Comparative studies of toxin-neutralizing antibodies induction after immunization of laboratory animals with recombinant subunits A and B of diphtheria toxin were carried out. The new method for detection of protective antibodies in serum was proposed. This method is based on the ToBI test (Toxin Binding Inhibition test); namely on the property of anti-diphtheria antibodies to inhibit the biding of toxin subunit B fused with enhanced green fluorescent protein (EGFP) to the sensitive to diphtheria toxin Vero cells. The ability of subunit B to induce toxin-neutralizing antibodies in laboratory animals (rabbits and guinea pigs) was confirmed by the intradermal test, which is traditionally used to detect protective antitoxic antibodies in the serum, and by flow cytometry method, developed for this purpose. The results suggest that diphtheria toxin recombinant subunit B may be used for the induction of the protective immune response. The new developed approach for estimation diphtheria toxin-neutralizing antibodies is more ethical and safe and can substitute successfully the traditional methods.

[Cytotoxicity of the B subunit of diphtheria toxin to human histocytic lymphoma U937].

The B subunit of diphtheria toxin (DT) is responsible for interaction with receptor on the cell surface and translocation of the catalytically active A subunit across endosomal membrane into the cell cytosole. Receptor for DT and its B subunit is membrane-anchored precursor of heparin-binding epidermal growth factor-like growth factor (pro-HB-EGF), which under the action of metalloproteases turns into soluble form (sHB-EGF), which acts as a potent mitogen for different cell types. Since free B subunit of DT has no catalytic activity it is considered to be nontoxic. However its influence on the cells in vitro remains to be investigated. The aim of this study was to examine the influence of diphtheria toxin B subunit on viability of diphtheria-sensitive cells using B subunit recombinant analogues. It was shown that diphtheria toxin B subunit recombinant analogue at a concentration of 12.8 x 10(-7) M had a cytotoxic effect on the human histocytic lymphoma cell line U937, which expresses a large amount of sHB-EGF. Besides, the similar cytotoxic effect had a fusion protein which consisted of a B subunit and an enhanced green fluorescent protein (EGFP). However recombinant EGFP alone didnot influence the cell viability. Annexin-V-FITC/PI staining demonstrated that maximal cytotoxic effect had been elicited after 48 hours of cultivation. Cytotoxic test with trypan blue and propidium iodide staining excluded the direct influence of investigated proteins on the integrity of plasma membrane because of the ability of B subunit to pore formation. So, we offer a hypothesis that realization of cytotoxic effect of diphtheria toxin B subunit and its derivative on the U937 cell culture occurs via inhibition of mitogenic activity of sHB-EGF resulted in induction of apoptosis.

[The role of nicotine in regulation of lymphocyte proliferation].

The effect of nicotine on both the expression of nicotinic acetylcholine receptors (nAChRs) and proliferation of hybridoma cells and normal mouse lymphocytes has been investigated. By means of immunoenzyme assay, nicotine was shown to regulate the number of nAChRs in both hybridoma cells and normal rat splenocytes. According to the data of triazolyl blue inclusion and ELISpot assay, nicotine stimulated proliferation of both hybridoma cells and normal plasma cells generated in the course of immune response in vivo. The cell sensitivity to nicotine depended on the number of nAChRs expressed on the membrane, as well as on their functional activity affected, in particular, by adhesive contacts. The use of the open channel blocker benzohexonium revealed that proliferative signal through nAChR in hybridoma cells was mediated by ion channel opening. The data obtained demonstrate the proproliferative role of nicotine for B lymphocytes, and may account for the development of lymphoproliferative disorders in tobacco smokers.