Isolevuglandins as a gauge of lipid peroxidation in human tumors.

The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.

Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol estrogens: comparison of wild-type and variant COMT isoforms.

The oxidative metabolism of 17beta-estradiol (E2) and estrone (E1) to catechol estrogens (2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1) and estrogen quinones has been postulated to be a factor in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the methylation of catechol estrogens to methoxy estrogens, which simultaneously lowers the potential for DNA damage and increases the concentration of 2-methoxyestradiol (2-MeOE2), an antiproliferative metabolite. We expressed two recombinant forms of COMT, the wild-type (108Val) and a common variant (108Met), to determine whether their catalytic efficiencies differ with respect to catechol estrogen inactivation. The His-tagged proteins were purified by nickel-nitrilo-triacetic acid chromatography and analyzed by electrophoresis and Western immunoblot. COMT activity was assessed by determining the methylation of 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1, using gas chromatography/mass spectrometry for quantitation of the respective methoxy products. In the case of 2-OHE2 and 2-OHE1, methylation occurred at 2-OH and 3-OH groups, resulting in the formation of 2-MeOE2 and 2-OH-3-MeOE2, and 2-MeOE1 and 2-OH-3-MeOE1, respectively. In contrast, in the case of 4-OHE2 and 4-OHE1, methylation occurred only at the 4-OH group, yielding 4-MeOE2 and 4-MeOE1, respectively. Individual and competition experiments revealed the following order of product formation: 4-MeOE2 > 4-MeOE1 >> 2-MeOE2 > 2-MeOE1 > 2-OH-3-MeOE1 > 2-OH-3-MeOE2. The variant isoform differed from wild-type COMT by being thermolabile, leading to 2-3-fold lower levels of product formation. MCF-7 breast cancer cells with the variant COMT 108Met/Met genotype also displayed 2-3-fold lower catalytic activity than ZR-75 breast cancer cells with the wild-type COMT 108Val/Val genotype. Thus, inherited alterations in COMT catalytic activity are associated with significant differences in catechol estrogen and methoxy estrogen levels and, thereby, may contribute to interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.

Identification of a novel membrane protein, HP59, with therapeutic potential as a target of tumor angiogenesis.

CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological angiogenesis and inhibits tumor growth in mice and humans. CM101 also targets neonatal lung and adult sheep lung endothelial cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA library. The gene, termed sp55, encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101. The corresponding human gene, hp59, was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59 protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon, and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55 peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.

Identification and characterization of a protein containing formin homology (FH1/FH2) domains.

A novel member of the Formin/Diaphanous family of proteins was cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (Formin Homologue Overexpressed in Spleen) contains a 3414bp open reading frame and encodes for an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the Formin Homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392.

Decreased bone mineral density in Prader-Willi syndrome: comparison with obese subjects.

Bone density, anthropometric data, and markers of bone turnover were collected on 21 subjects diagnosed with Prader-Willi syndrome (PWS) and compared with 9 subjects with obesity of unknown cause. In addition, urinary N-telopeptide levels were obtained in all subjects. N-telopeptides are the peptide fragments of type I collagen, the major bone matrix material. During periods of active bone degradation or high bone turnover, high levels of N-telopeptides are excreted in the urine. However, no significant difference was detected in the urinary N-telopeptide levels when corrected for creatinine excretion (raw or transformed data) between our subjects with obesity or PWS and the observed effect size of the between-group difference was small. Although N-telopeptide levels were higher but not significantly different in the subjects with PWS compared with obese controls, the subjects with PWS had significantly decreased total bone and spine mineral density and total bone mineral content (all P < 0.001). No differences in N-telopeptide levels or bone mineral density were observed between subjects with PWS and chromosome 15q deletion or maternal disomy. Thus, decreased bone mineral density in subjects with PWS may relate to the lack of depositing bone mineral during growth when bones are becoming more dense (e.g., during adolescence), possibly because of decreased production of sex or growth hormones and/or long-standing hypotonia. It may not be caused by loss, or active degradation, of bone matrix measurable by the methods described in this study further supporting the possible need for hormone therapy during adolescence.

Production of a single chain anti-CEA antibody from the hybridoma cell line T84.66 using a modified colony-lift selection procedure to detect antigen-positive ScFv bacterial clones.

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies (MAbs) such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The scFv antibody (designated RK10.2) was generated using anti-CEA T84.66 hybridoma cells as a source of genetic starting material and the Pharmacia Recombinant Phage Antibody System (RPAS). Escherichia coli clones expressing antigen-positive soluble scFv were identified using a modified colony-life selection procedure and antigen-coated filters. The resultant anti-CEA scFv (designated RK10.2) had a molecular weight of approximately 33.6 kDa and an isoelectric point of 5.2 at 15 degrees C. The RK10.2 scFv interacted with LS174 T cells bearing the CEA antigen and inhibited the anti-CEA MAb/CEA antigen interaction in ELISA and the anti-CEA MAb/LS174 T cell interaction in a RIA. The modified colony-lift approach circumvented the more time-consuming phage-display approach that is normally taken to affinity select for antigen-positive scFv clones.

Production and characterization of monoclonal antibodies to ARVCF.

We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.

Radiation-guided P-selectin antibody targeted to lung cancer.

PURPOSE: P-selectin expression is significantly increased in tumor microvasculature following exposure to ionizing radiation. The purpose of this study was to image radiation-induced P-selectin expression in vivo using optical imaging and gamma camera imaging in a heterotopic lung cancer model by using ScFv antibodies to P-selectin. PROCEDURES: In vitro studies using endothelial cells were done using 3 Gy radiation and selected ScFv antibodies to P-selectin. In vivo studies were performed using Lewis lung carcinoma cells subcutaneously injected into the hind limbs of nude mice. Mice were treated with 6 Gy radiation and sham radiation 10 days post-inoculation. P-selectin expression was assessed with near-infrared imaging using Cy7 labeled antibody, and gamma camera imaging using( 111)In-DTPA labeled antibody. RESULTS: In vitro studies showed antibody binding to P-selectin in radiation treated endothelial cells. In vivo optical imaging and gamma camera imaging studies showed significant tumor-specific binding to P-selectin in irradiated tumors compared to unirradiated tumors. CONCLUSIONS: Optical imaging and gamma camera imaging are effective methods for visualizing in vivo targeting of radiation-induced P-selectin in lung tumors. This study suggests that fluorescent-labeled and radiolabeled ScFv antibodies can be used to target radiation-induced P-selectin for the tumor-specific delivery of therapeutic drugs and radionuclides in vivo.

Three-dimensional structure of pinwheel inclusions as determined by analytic geometry.

The three-dimensional ultrastructure of pinwheel inclusions found in two plant virus-host systems was determined by computer-assisted analytic geometry. Data obtained from electron micrographs of serially sectioned pinwheel inclusions were used to generate mathematical equations. The three-dimensional models described by the equations indicated that some pinwheels assume the hourglass shapes of elliptic hyperboloids. Electron micrographs obtained from thick sections of pinwheel inclusions tilted at various angles supported the elliptic hyperboloid models described by the mathematical equations.

The role of individual SH2 domains in mediating association of phospholipase C-gamma1 with the activated EGF receptor.

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.

Antigenic diversity among Helicobacter pylori vacuolating toxins.

Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 immunoglobulin G1kappa anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen.

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumor-challenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.

Endothelial localization of receptor tyrosine phosphatase, ECRTP/DEP-1, in developing and mature renal vasculature.

Developmental assembly of the renal microvasculature requires spatially and temporally coordinated migration, assembly, differentiation, and maturation of endothelial cells in the context of adjacent epithelial and mesangial cells. In this study, endothelial expression and distribution of the receptor tyrosine phosphatase ECRTP/DEP-1 were evaluated during and after developmental assembly of the renal microvasculature. Monoclonal antibodies against ECRTP/DEP-1 ectodomain epitopes localize its expression to membrane surfaces of endothelial cells in glomerular, peritubular capillary, and arterial renal sites of mature human and murine kidney. During kidney development, ECRTP/DEP-1 immunostaining is evident on a subpopulation of metanephric mesenchymal cells and on putative progenitors of glomerular capillary endothelial cells early in their recruitment to developing glomeruli. ECRTP/DEP-1 is prominently displayed on luminal membrane surfaces with punctate accumulations at inter-endothelial contacts that overlap with vascular endothelial-cadherin staining. ECRTP/DEP-1 is recruited to inter-endothelial contacts in confluent cultured human renal and dermal microvascular endothelial cells, yet experimental dissociation of vascular endothelial-cadherin from endothelial junctional complexes fails to redistribute ECRTP/DEP-1. These findings indicate that ECRTP/DEP-1 is expressed in anticipation of glomerular capillary endothelial recruitment during development, and suggest that ECRTP/DEP-1 ectodomain interacts with endothelial surface ligands that are engaged by cell-cell contact.