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- Title
Immunocytochemical visualization of P185 receptor using antibodies fused with dibarnase and conjugate of barstar with colloidal gold.
- Authors
Ivanova, Yu.; Edelweiss, E.; Leonova, O.; Balandin, T.; Popenko, V.; Deyev, S.
- Abstract
The localization of the P185 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185 receptor is a marker of breast and ovarian tumors; it is also considered to be a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, firstly, to study the distribution of P185 in cancer cells, secondarily, to remove P185 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185 domain fused with two molecules of barnase (cytotoxic RNAase from Bacillus amyloliquefaciens) and its specific inhibitor, barstar. Fluorescent microscopy showed that the module 4D5 scFc-dibarnase:barstar is efficient for identifying P185 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185. The localization of P185 in human ovarian carcinoma cells (SKOV-3) and breast carcinoma cells (BT-474) was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185 is distributed unequally on the cell surface with preferential localization on protrusions or close to their bases and at contacts between protrusions and the cell membrane. At 37°C, P185 is internalized through coated pits and vesicles and concentrates in endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in BT-474 cells.
- Subjects
CANCER cells; CANCER treatment; FLUORESCENCE microscopy; CONFOCAL microscopy; BACILLUS amyloliquefaciens; BREAST cancer diagnosis
- Publication
Molecular Biology, 2013, Vol 47, Issue 5, p701
- ISSN
0026-8933
- Publication type
Article
- DOI
10.1134/S0026893313050063