Immunocytochemical visualization of P185HER2 receptor using antibodies fused with dibarnase and conjugate of barstar with colloidal gold

Yu L. Ivanova, E. F. Edelweiss, O. G. Leonova, T. G. Balandin, V. I. Popenko, S. M. Deyev

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The localization of the P185HER2 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185HER2 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 P185HER2 in cancer cells, secondarily, to remove P185HER2 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185HER2 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185HER2 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 P185HER2 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185HER2. The localization of P185HER2 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. P185HER2 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, P185HER2 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.

Original languageEnglish
Pages (from-to)701-711
Number of pages11
JournalMolecular Biology
Volume47
Issue number5
DOIs
Publication statusPublished - Sep 2013
Externally publishedYes

Keywords

  • 4D5 scFv
  • barnase
  • barstar
  • immune electron microscopy
  • P185

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology

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