Search

Overexpression of the P185(HER2) protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185(HER2) in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185(HER2) domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185(HER2) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185(HER2) occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185(HER2) caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185(HER2) was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar-gold complex can be used as a new immunodetection system for exploring the P185(HER2) distribution.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

The distribution of the putative protein products of gene hcs2 in giant command neurons of the parietal ganglia of the terrestrial snail Helix lucorum has been studied using light- and electron-microscopic immunocytochemistry. The product of the hcs2 gene is a hybrid protein belonging to the EF-hand family of Ca2+-binding proteins and is a precursor of several neuropeptides. Polyclonal antibodies to neuropeptides CNP3 and CNP4 and the C-terminal Ca2+-binding domain of the precursor protein have been used to determine their intracellular localization. The targets for all three types of antibodies have been found in cytoplasmic secretory granules. The label (colloidal gold) density in the secretory granules is two times higher in the case of neuropeptides CNP3 and CNP4 than in the case of the Ca2+-binding domain. Thus, a specific association between the putative products of the hcs2 gene and the cell secretory apparatus has been demonstrated. This agrees with the earlier hypothesis that hcs2 products may serve as neurotransmitters or neuromodulators.

Background RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells. Methodology/Principal Findings In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 µM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 µM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. Conclusions/Significance These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.