Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection

7-Methylguanine (7-meG) could be a useful marker of recent pastexposure to environmental methylating agents for use in epidemiologicalstudies. A method is described that is appropriate for suchan application. 7-meG was released from DNA by thermal hydrolysisunder conditions (pH 9, 70°C, 8 h) that preferentially releasedthe base from DNA rather than RNA and, following immunopurificationusing antibodies specific for this DNA adduct, quantificationwas achieved either by HPLC with electrochemical detection (ECD)or by ELISA. The detection limits of the two approaches were0.5 and 2 pmol 7-meG/DNA sample respectively. 7-meG was analysedin DNA samples contaminated with known amounts of RNA to testthe possible interference in the analysis by the minor modifiednucleoside 7-methylguanine, which is present as a normal componentof RNA. 7-meG levels measured in human pancreas and untreatedrat liver DNA were between 2 and 7 pmol 7-meG/µmol guanosineand this level could not be explained by RNA contamination.The combination of immunoaffinity purification and HPLC withECD provides a method that is sensitive and specific for 7-meGand suitable for integration into molecular epidemiologicalstudies.     

Preparation and characterization of antibodies to o-phosphotyrosine and their use for identification of phosphotyrosine-containing proteins

To facilitate the identification of phosphotyrosine (Ptyr)-containing proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P-tyr were prepared by hyperimmunizing the animals with P-tyr-conjugated bovine serum albumin or poly-L-lysine. As determined by a solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay, the antibodies reacted with P-tyr-conjugated target antigens but not with those conjugated with phosphoserine (P-ser) or phosphothreonine (P-thr). This immune reaction was strongly blocked by 2 mM P-tyr and phenylphosphate but not by P-ser or P-thr. The antibodies were capable of isolating, as the major P-tyr-containing components, a 170kd protein (most likely the EGF receptor) from EGF-stimulated, ³²P-labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)-transformed chick cell lysate which had been labelled in vitro, with γ-³²P-ATP. Immunofluorescent staining of RSV-transformed cells and A431 cells showed specific localization of P-tyr-containing proteins in the cytoplasm, plasma membrane, and nucleoluslike structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P-tyr-containing proteins.     

Characterization of monoclonal antibodies to the estrogen-regulated Mr 52,000 glycoprotein and their use in MCF7 cells

The Mr 52,000 glycoprotein is regulated by estrogen and released by breast cancer cells in culture (B. Westley and H. Rochefort, Cell, 20: 352-362, 1980). This rare protein was partially purified from 25 liters of medium conditioned by MCF7 cells and injected into Biozzi's selected mice. The spleen lymphocytes of one immunized mouse was fused with the murine myeloma P3-X63-Ag8-653. Sixteen hybridomas producing monoclonal antibodies to the Mr 52,000 protein were isolated, and seven of them were cloned and purified. The seven monoclonal antibodies were all of the immunoglobulin G1 isotype, and their dissociation constants ranged from 0.35 to 2.3 nM. The antibodies specifically recognized the secreted Mr 52,000 protein as evidenced by double immunoprecipitation and by immunoblotting after electrophoretic separation and transfer. Double-determinant immunoradiometric assay indicated that the seven purified monoclonal antibodies recognized three distinct regions of the Mr 52,000 protein, and it was used to assay the Mr 52,000 protein in biological fluids. These antibodies did not react with the external plasma membrane of MCF7 cells, as shown by immunofluorescence analysis. By contrast, the cytoplasm of MCF7 cells (but not T47D and RBA cells) was stained by the peroxidase-immunoperoxidase complex after plasma membrane permeation, indicating that the protein is secreted by exocytosis rather than shed from the plasma membrane.     

Measurement by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA of healthy individuals and cancer patients treated with dacarbazine and procarbazine. Human data and method development for 7-alkylguanines

A ³²P-postlabelling method was developed to measure 7-methylguaninein human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-mono-phosphate(7-me-dGMP) was isolated from normal nucleotides using stronganion-exchange column chromatography. Overall the method gave35–45% yield as measured with DNA methylated with tritiateddimethyl suffate. Total white blood cell DNA from healthy non-smokers(n = 17) contained from 2.5 7-methylguanine residues/10⁷ nucleotides,corrected for the losses in preparation. Among four patientssampled immediately after a total dose of 1050–2800 mgof dacarbazine or procarbazine, the mean adduct level was 577-methylguanine residues/10⁷ nucleotides. As further methoddevelopment, we also investigated the phosphorylation reactionby T4 polynucleotide kinase using dinucleotides containing 7-methylguanineand corresponding imidazole ring-opened products as substrates.We found that imidazole ring-opened dTpdG-Me is resistant todigestion with deoxyribonuclease I, snake venom phosphodiesteraseand prostatic acid phosphatase. It is quantitatively phosphorylatedat femtomolar levels. This method is shown to be suitable forthe detection of 7-methylguanine in DNA, and is suggested tobe the approach most suited to postlabelling large and labile7-alkylguanines in DNA.     

Dimethylnitrosamine-induced structural damage to DNA results from repair of O6-methylguanine rather than repair of 7-methylguanine

DNA isolated from the livers of rats treated with a non-necrotizing dose of [14C]dimethylnitrosamine was fractionated by chromatography of benzoylated-DEAE-cellulose. The preparations of native DNA and DNA containing single stranded regions were then hydrolysed and amounts of methylated guanine determined. Four hours after dimethylnitrosamine treatment there was no difference in the levels of 7-methylguanine and O6-methylguanine in the 2 DNA fractions. By 24 h, although there was no difference in the amount of 7-methylguanine between the DNA fractions, there was a 10-fold difference in the level of O6-methylguanine. The elimination of O6-methylguanine from the fraction of DNA containing single stranded regions is discussed in terms of differing repair processes initiated by 7-methylguanine and O6-methylguanine.     

Induction of adipocyte formation in 10T1/2 cells by 1-methylguanine and 7-methylguanine

1-Methylguanine and 7-methylguanine are naturally occurring modified purines derived from tRNA, found in elevated levels in the serum and urine of cancer patients. When C3H/10T1/2 clone-8 mouse cells are exposed to low levels of the methylated purines, they are induced to differentiate into adipocytes. Differentiation is induced in a dose-dependent manner and is similar in extent to that achieved by other inducing agents, such as 5-azacytidine. The methylated purines are not mutagenic, nor are they incorporated into DNA. They may exert their effect by modifying cellular regulatory processes, such as methylation of DNA. High levels of circulating methylated purines in cancer patients may play a role in tumor-host interactions.     

Preparation of conjugates of adriamycin with antibodies to alpha-fetoprotein and their properties

For its selective accumulation into tumor cells, the antitumor drug adriamycin (ADM) was attached to specific antibodies reactive against alpha-fetoprotein, a carcinoembryonic protein, and the antibody and antitumor activities of the resulting conjugate were investigated. Using the glutaraldehyde or carbodiimide binding methods, preparations containing 4-5 moles of ADM per mole of antibody were obtained. They retained 60-75% of the original antibody activity. Upon incubation of AH-66 ascites hepatoma cells with the conjugates, an increased number of dead cells and inhibited uptake of 3H-thymidine by the cells were observed. The conjugates were more effective against tumor cells than the free drug, and it was further presumed that the cytotoxic action of ADM on normal cells could be reduced by binding it to a macromolecular protein.     

Preparation of monoclonal antibodies against insulin and their applications

Monoclonal antibodies (MAbs) against insulin are useful for insulin assays because of their specificity and plentiful supply. We have produced four monoclonal cell strains stably secreting the monoclonal antibodies against insulin; further established subpopulation, titer, affinity; and identified the antibodies as being of subclass IgG(2b)(kappa), one strain, or IgG(1)(kappa), three strains. The smallest detectable level of human insulin by ELISA using this IgG(1) was 1.25 microg/L. The monoclonal antibodies have been used for analyzing insulin content from the sera of type 2 diabetes patients and normal subjects.     

Monoclonal antibodies to sea cucumber polysaccharide and their use in a sandwich ELISA assay

Antigen was synthesized with L-SCP, a sea cucumber polysaccharide isolated from Pearsonothuria graeffei (Semper) and bovine serum albumin (BSA) as a carrier protein. Spleen cells with high titer antibody producing ability were removed and fused with myeloma cells of SP2/0-Ag14 origin. Three stable murine monoclonal antibodies (MAb ascites) producing cell lines to L-SCP were generated according to a conventional immunization protocol. Their epitope mapping and binding specificity, which was characterized by blocking and inhibition enzyme-linked immunosorbent assay (ELISA) indicated that these specific MAb ascites have similar binding patterns. A sandwich ELISA was developed on the basis of employing L-SCP specific antibodies including MAb 3G6 as capture antibody and HRP-3G6 as detection antibody. The working range for L-SCP in aqueous solution from this method was 100-10,000 ng/mL with good sensitivity, specificity, and precision (relative standard deviation ≤7.9%). Thus the developed ELISA can be used as a convenient tool for the rapid detection of L-SCP in biological examples in the future.     

Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1 (AFB1) or an analogue, 5,7-dimethoxycyclopentenon (2,3-c) coumarin, produced antibodies that bind 3H-AFB1. The specificities of the antisera with respect to aflatoxins BI, B2a, G1, G2, Q1, P1, and some other structually related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFB1-were used to analyze serum, urine, and crude extracts of corn and peanut supplemented with aflatoxin. In the foodstuffs, as little as 1 mug AFB1/kg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic aflatoxin ingestion and cancer.     

Monoclonal antibodies and rabbit antisera recognizing 4-aminobiphenyl--DNA adducts and application to immunoaffinity chromatography.

Monoclonal antibodies and rabbit antisera were produced that recognized 4-aminobiphenyl, its major DNA adducts and other metabolites. The antigens used to raise these antibodies were synthesized by coupling the aromatic amine to protein through a diazotization reaction. The goal of this immunization strategy was to induce antibodies that also cross-reacted with most 4-aminobiphenyl-derived metabolites. A total of 20 mice and four rabbits were immunized and every animal produced a strong immune response for 4-aminobiphenyl and its derivatives. Two IgG1 monoclonal antibodies, 3D6 and 2E11, were isolated from two different mouse spleen cell fusions. One of the monoclonal antibodies, 3D6, had a high recognition for the three major 4-aminobiphenyl-DNA adducts: N-(deoxyguanosine-8-yl)-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosine-N2-yl)-4-aminobiphenyl, with affinity constants between 2 and 4 x 10(9) l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 10(9) l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobiphenyl-DNA adduct-specific antibodies. Preparative immunoaffinity resins were made for each monoclonal antibody. These resins quantitatively bound 500 ng each [3H]N-acetyl-aminobiphenyl, [3H]N-aminobiphenyl and [3H]N-(deoxyguanosine-8-yl)-4-aminobiphenyl. Preliminary experiments were performed to test the applicability of the preparative monoclonal antibody immunoaffinity column to isolate [3H]4-aminobiphenyl-derived metabolites in dosed rat and dog urine. About 70% of the radioactivity in rat or dog urine could be bound to the immunoaffinity columns. The combined immunoaffinity column/HPLC analysis of the dog urine led to the identification of a novel urinary metabolite, N-formyl-aminobiphenyl. HPLC analysis of a rat urine sample tentatively found 4-aminobiphenyl, N-acetyl-4-aminobiphenyl and N-formyl-4-aminobiphenyl by co-chromatography, and these compounds accounted for 20, 6.8 and 6.5% of the total radioactivity in the chromatogram respectively. Taken together, these data show that these 4-aminobiphenyl-specific monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.     

Detection by antihapten antibodies of liver-bound compounds related to azocarcinogens or their metabolites.

The localization of known azocarcinogens and metabolites such as p-aminoazobenzene and N-methyl-p-aminoazobenzene bound to components of liver cells of rats fed single or multiple doses of 3'-methyl-p-dimethylaminoazobenzene has been determined with the use of antibodies raised against p'-azo-p-aminoazobenzene and p'-azo-N-monomethyl-p-aminoazobenzene in the indirect fluorescent antibody procedure. These 2 antisera reacted with liver cells of rats fed 3'-methyl-p-dimethylaminoazobenzene, p'-amino-p-aminoazobenzene, p'-amino-N-monomethyl-p-aminoazobenzene and N-methyl-p-aminoazobenzene. The results obtained in this study suggest that both major and minor metabolites of azocarcinogens have common antigenic determinants.     

Levels of O6-methylguanine acceptor protein in tissues of rats and their relationship to carcinogenicity and aging

N-nitroso compounds react with cellular DNA to produce various damaging adducts, one of the more important being O6-alkylguanine. DNA restoration is accomplished by transfer of the alkyl group to a cysteine residue of an acceptor protein. The levels of acceptor activity were compared in several tissues from well-fed and dietary-restricted inbred SD rats 30-1,194 days of age. Striking and consistent differences were found in the levels of acceptor activity in different tissues from both groups; these levels corresponded to their sensitivity to tumorigenesis by alkylating agents. Acceptor activity levels were highest in the liver and somewhat less in the spleen; there were significantly lower levels in brain and kidney. The random loss with time in the integrity of DNA may cause alterations in cellular function or limit cellular proliferation, thus leading to senescence and death. DNA repair processes may alter the rate of accumulation of damage, thereby affecting potential longevity. There were no significant age-associated changes in the ability of cells from either dietary group to remove DNA adducts and there was no evidence of alterations in the acceptor protein with age that would compromise its functional activity.     

Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase

Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine3'-monophosphate (7-methyl-dGMP), whkh was ring-opened in alkalito 2'-deoxy-N⁵-methyl-N⁵-formyl-2,5,6-triamino4-oxopyrimidine3'-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylatedby nuclease P₁ in contrast to normal deoxynucleotides. It wasefficiently 5'-phosphorylated by T₄ polynucleotide kinase. Whenmethylated DNA was alkali-treated and digested with mkro-coccalnuclease, spleen phospbodiesterase and nuclease P₁, ROM-dGMPwas formed and this was labeled with [-³²P]-ATP hi the presenceof polynucleotide kinase. Ring-opening and P₁ treatment appearmethods of choice for ³²P-post-labeUng of 7-alkylguanines inDNA.     

Preparation and characterization of new anti-PSMA monoclonal antibodies with potential clinical use

Monoclonal antibodies with high specificity for prostate cancer tissue are of interest for diagnostic and therapeutic applications employing targeted therapy. The prostate-specific membrane antigen (PSMA) is a protein predominantly found in epithelial cells of prostate tissue origin and its expression correlates with tumor aggressiveness. Here, we report the development and characterization of new antibodies against PSMA. Murine monoclonal antibodies (MAb) were obtained by immunizing mice with a peptide corresponding to PSMA extracellular residues 490-500 -- GKSLYESWTKK (PSMA(490-500)). The MAbs react specifically to PSMA and to the prostate cancer cell line LNCaP with an affinity for PSMA in the low nanomolar range. This study also demonstrates the potential use of these antibodies for targeted drug delivery to prostate cancer cells. Nanomolar concentrations of PSMA-specific MAb in association with a molecule with cytotoxic potential were sufficient to allow for binding and uptake by LNCaP cells within minutes, leading to complete cell death within 3 days. These MAbs have potential clinical value in the development of diagnostic and therapeutic applications for prostate cancer.     

Preparation and characterization of monoclonal antibodies to thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.     

Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts

Antisera were raised in rabbits to three peptides which correspond to sequences of amino acids present in human O6-alkylguanine-DNA alkyltransferase (residues 1-11, 8-20 and 197-207). These antisera recognized the alkyltransferase protein on Western blots of proteins from a number of Mer+ human tumor cell lines but this protein was found to be absent from Mer- tumor cell lines. The alkyltransferase protein was also detected by these antisera in some extracts from human liver samples but other human liver extracts having a high alkyltransferase activity failed to react with antibodies directed towards the carboxyl terminus of the protein, suggesting that part of this region can be removed by protease action without loss of activity or that there is genetic variability in this region. This result indicates that studies with a number of antisera or with an antibody known to be directed towards an essential, invariant region of the alkyltransferase will be needed for unequivocal detection of the alkyltransferase by antibody screening. The immunoreactive human alkyltransferase protein was absent from CHO cell lines that had previously been selected for alkyltransferase expression after transfection with human DNA. It is therefore probable that the hamster gene has been reactivated in these cells.     

Generation and characterization of monoclonal antibodies to TDRD7 protein

TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein.