Immunofluorescent Analysis of Connexin-43 Using Monoclonal Antibodies to Its Extracellular Domain

Abstract

Immunofluorescent analysis of connexin-43 was carried out on preparations of fixed and living cultures of rat and human glioma cells, HEK293 cells, and frozen sections of the rat brain with experimental glioma using monoclonal antibodies to recombinant extracellular fragment of connexin-43 (E2 second extracellular loop). These monoclonal antibodies visualized membrane and cytoplasmic pools of connexin-43 in preparations fixed with paraformaldehyde. Incubation of monoclonal antibodies to E2 extracellular loop with living cells led to visualization of only connexin hemichannels on cell membranes. No immunofluorescence characteristic of dimer connexons, organizing the gap junction, was detected. This fact indicates that antibodies to connexin-43 extracellular loop E2, obtained in our study, specifically react with target antigen solely at the stage of connexon presentation on the membrane in the form of hemichannels. These monoclonal antibodies can be used for immunophenotyping and sorting of connexin-43-positive cells in vitro and as the guide molecules in addressed delivery of diagnostic preparations and drugs to glioma cells in vivo.

Treatment of glioma by cisplatin-loaded nanogels conjugated with monoclonal antibodies against Cx43 and BSAT1

“…This assumption is also supported by rapid development of glioma in rats treated with NG/CDDP and IgG-NG/CDDP than that from CDDP group, as determined by morphometric analysis (Figure 6). To increase the efficiency of drug delivery to the glioma, nanogels were conjugated with mAbs against the targeted proteins Cx43 and BSAT1, which are highly expressed in tumor and peritumoral area (Baklaushev et al, 2009;Oliveira et al, 2005;Baklaushev et al, 2011Baklaushev et al, , 2012Yusubalieva et al, 2012). MRI data confirmed that this approach significantly decreased the tumor growth (Figure 7).…”

“…Amicon YM-30 centrifugal filters (MWCO 30 kDa), 1,2-ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), ethylenediaminetetraacetic acid (EDTA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cis-dichlorodiamminoplatinum (II) (cisplatin, CDDP) and other chemicals were from Sigma-Aldrich (St Louis, MO, USA). Anti-Cx43 and anti-BSAT1 monoclonal antibodies (Mw 150 kDa) were obtained as described earlier (Baklaushev et al, 2009. Fetal bovine serum (FBS), RPMI 1640 medium, penicillin, streptomycin, Trypsine-EDTA (0.5% trypsin, 5.3 mM EDTA tetrasodium salt), Alexa Fluor 488 goat anti-mouse IgG and other were from Invitrogen (Carlsbad, CA, USA).…”

“…Monoclonal antibodies against the E2 extracellular fragments of Cx43 and BSAT1 (MAbCx43 and MAbBSAT1) were purified from ascites of Balb/c mice using affinity chromatography on protein-A-agarose, and dialyzed against phosphate buffered saline (PBS, pH 7.4) (Baklaushev et al, 2009. Obtained mAbs were thiolated with a tenfold molar excess of Traut's reagent (0.1 M borate buffer, 5 mM EDTA, pH 8) for 45 min at r.t. After purification of thiolated mAbs (Zeba spin desalting columns, Thermo Scientific, IL, USA), they were incubated with a tenfold molar excess of MAL-PEG-NH2 overnight at 4 C in PBS.…”

“…BSAT1 is an organic anion transporter specific for cerebral microvascular endothelial cells (Li et al, 2004) that regulates transendothelial transport of thyroxine (T4) and some other amphiphilic anions through blood-brain barrier (Sugiyama et al, 2003). Previously we obtained the monoclonal antibodies (mAb) against the recombinant extracellular fragment of E2 of Cx43 interacting with the antigen in the native conformation (Baklaushev et al, 2009) and mAbs against the extracellular fragment of BSAT1 . Using obtained mAbs we have shown that protein Cx43 over-expressed in astrocytes and glioma cells in the peritumoral invasion zone as well as BSAT1 expressed in peritumoral area.…”

Antitumor Effects of Monoclonal Antibodies to Connexin 43 Extracellular Fragment in Induced Low-Differentiated Glioma

“…Hybrid cells producing MAbE2Cx43 were previously obtained by us [2]. Aliquots of hybrid cells (3-5×10 6 ) from the cryobank were defrosted and injected intraperitoneally to BALB/c mice.…”