Complementary functions of ATM and H2AX in development and suppression of genomic instability

Upon DNA damage, histone H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other phosphoinositide 3-kinase-related protein kinases. To elucidate further the potential overlapping and unique functions of ATM and H2AX, we asked whether they have synergistic functions in the development and maintenance of genomic stability by inactivating both genes in mouse germ line. Combined ATM/H2AX deficiency caused embryonic lethality and dramatic cellular genomic instability. Mechanistically, severe genomic instability in the double-deficient cells is associated with a requirement for H2AX to repair oxidative DNA damage resulting from ATM deficiency. We discuss these findings in the context of synergies between ATM and other repair factors.     

A targeting model of boron neutron-capture therapy to hepatoma cellsin vivo with a boronated anti-(α-fetoprotein) monoclonal antibody

We described previously that¹⁰B atoms delivered by monoclonal antibody (mAb) exerted a cytotoxic effect on AH66 cells in a dose-dependent manner upon thermal neutron irradiationin vitro. In the present study, the delivering capacity of boronated anti-(-fetoprotein) (AFP) mAb to carry¹⁰B atoms to AFP-producing tumor xenografts in nude mice was determined. Boronated mAb was prepared by conjugating 50 mM ¹⁰B compound to an anti-AFP mAb (2 mg/ml) usingN-succinimidyl-3-) (2-pyridyldithio) propionate. The number of¹⁰B atoms conjugated directly to the mAb was estimated to be 459/antibody by prompt -ray spectrometry. Boron concentrations in tumor tissue obtained 12, 24, 72, and 120 h after injection of 3.0 mg¹⁰B-conjugated anti-AFP mAb were 11.10±3.12 (SD,n=6), 29.30±5.11, 33.02±11.8, and 12.91±5.62 ppm respectively. For control¹⁰B-conjugated anti-dinitrophenol (DNP) mAb, the values were 9.59±0.99, 10.37±2.86, 10.00±2.95, and 8.83±4.71 ppm respectively. The concentrations in blood were less than 0.40±0.10 ppm with anti-AFP mAb and less than 0.51±0.15 ppm with anti-DNP mAb at each sampling time (12, 24, 72, and 120 h). The number of¹⁰B atoms delivered to the tumor cells was calculated to be 0.62×10⁹, 1.63×10⁹, 1.84×10⁹ and 0.72×10⁹ at each sampling time after injection of¹⁰B-anti-AFP mAb. The amount of¹⁰B atoms necessary for effective boron neutron-capture therapy was estimated to be 10⁹/tumor cell. We were able to carry 1.84×10^{9 10}B atoms to AH66 tumor cells by using¹⁰B-anti-AFP mAb. The accumulation reached its peak 72 h after injection. These data indicated that the¹⁰B-conjugated antitumor mAb could deliver a sufficient amount of¹⁰B atoms to the tumor cells to induce cytotoxic effects 72 h after injection upon thermal neutron irradiation.Abbreviations BNCT boron neutron-capture therapy - AFP -fetoprotein - SPDP N-succinimidyl-3-(2-pyridyldithia) propionate - DNP dinitrophenol     

Potentiation of the ligand-binding activity of integrin alpha3beta1 via association with tetraspanin CD151

CD151, one of the tetraspanins, forms a stable complex with integrin alpha3beta1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin alpha3beta1-CD151 complexes, was capable of dissociating CD151 from integrin alpha3beta1, thereby allowing us to deplete CD151 from purified integrin alpha3beta1-CD151 complexes. The CD151-free integrin alpha3beta1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin alpha3beta1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated beta1-containing integrins. These results raised the possibility that the association of integrin alpha3beta1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin alpha3beta1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin alpha3beta1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin alpha3beta1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.     

Inflammatory responses in cuff-induced atherosclerosis in rabbits

Cuff-treatment of the rabbit carotid artery produced a diffuse intimal thickening which resembled early lesions of atherosclerosis. A limited amount of focal endothelial damage occurred first (0.5 h), leukocytes infiltrated the subendothelium and extensive endothelial denudation occurred at 24 h. At 3 days, the regenerating endothelium covered the denuded area, and the media was edematous. At 7 days proliferation of intimal cells became visible. Maximum intimal thickening occurred at 3 weeks. Daily injection of dexamethasone (0.01-10 mg/kg i.m.) and ticlopidine (1-100 mg/kg i.m.) dose-dependently attenuated the intimal thickening. Indomethacin had little effect. Inflammatory exudate from zymosan-activated air pouch induced chemotaxis of rat smooth muscle cells (SMC) in vitro. Similar chemotactic activity was observed with leukotriene B4 (LTB4) but not with the other lipoxygenase products tested. The exudate contained reasonable amounts of LTB4, which would account for its chemotactic activity. Dexamethasone inhibited the chemotaxis by the exudate and proliferation of SMC. These results are discussed in relation to the mechanism of atherogenesis. It is concluded that leukocytes play a major role in cuff-induced intimal thickening, and that their products cause endothelial denudation and SMC chemotaxis. Involvement of platelet aggregation in atherogenesis is also suggested.