Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated Kt channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (P-K/P-X) sequence K+ = Rb+ (1.0) > Cs+ (10.4) much greater than Na+, Li+, N-methyl-D-glucamine (>51). NH4+ blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 mu M), and cetiedil (IC50 79 mu M) Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 Of 74 mu M

Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis

ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2 developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibitsmammary carcinoma growth and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system:Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administeredalone, thanks to the instrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16(FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fatpad with 1 million QDcells, vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free after 7 months. Mice challenged intravenously (i.v.) developedmore than 300 lung metastasis, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 anddevelop aggressive mammary carcinomas. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year, whereas allcontrol mice developed progressive mammary carcinomas within 8 months of age. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lungnodules, whereas 73% of mice vaccinated without adjuvant and all mice vaccinated with E2SB-C001+ISA 51 were free from metastases. Furthermore, twoadministrations of ES2B-C001+ISA 51 prevented spontaneous tumor onset in Delta16 mice for more than 1 year, while untreated mice developed mammarycarcinomas by 8 months of age. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL inDelta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated byELISPot (20.7±2.9 spots/2x10^5 cells. The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2