Quantification of specific DNA O-alkylation products in individual cells by monoclonal antibodies and digital imaging of intensified nuclear fluorescence

Abstract

We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.

Down-regulation of DNA repair in human CD34+progenitor cells corresponds to increased drug sensitivity and apoptotic response

“…Slides were then subjected to alkaline electrophoresis in the same buffer (20 minutes; 4°C; 4 V/cm), neutralized, and stained with ethidium bromide (2 g/mL). lmmunofluorescence staining lmmunofluorescence staining for adducts in the nuclear DNA of single cells was performed essentially as described 14,15 with the following modifications. 11 Cells were suspended in PBS containing 25% "HAES-steril" solution (Fresenius, Bad Homburg, Germany) and spread onto microscopic slides precoated with 2% 3-aminopropyl-triethoxysilan.…”

“…Cells were airdried, fixed with methanol (Ϫ20°C; overnight) and rehydrated in PBS. After treatment with RNases, 14 DNA was partly denatured by alkali treatment (NaOH, 70 mM in 140 mM NaCl, 40% methanol for 5 minutes, 0°C). Cells were treated with pepsin (200 g/mL in 20 mM HCl; 37°C; 5 minutes) and proteinase K (2 g/mL in 20 mM Tris/HCl, 2 mM CaCl 2 , pH 7.5; 37°C; 5 minutes), blocked with PBS/1% casein (30 minutes; room temperature), and immunostained with anti-cisplatin-DNA mAb "R-C18" (0.2 g/mL) or with anti-melphalan-DNA mAb "Amp4/42" diluted 1:50, both in PBS/1% casein (12 hours; 4°C).…”

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

“…These mechanisms comprise antioxidant systems for the deactivation of ROS molecules and efficient repair proteins for the elimination of oxidative DNA damage (19)(20)(21)(22)(23) (24)(25)(26)(27). These techniques can be applied for the determination of any DNA modification for which an appropriate monoclonal or polyclonal antibody is available.…”

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level

“…This is mainly because of the lack of sensitive and reliable assays to quantify specific DNA lesions in small samples of cells from cancer patients. In the present study, we have applied a recently established MAb-based immunoanalytical assay for the quantification of specific lesions in the nuclear DNA of individual cells (Seiler et al, 1993) to measure directly the kinetics of elimination (repair) of 06-AlkGua residues from DNA in human peripheral lymphocytes and leukaemic blasts. Among the lymphocytes or blast cells isolated from individual donors, comparatively uniform intercellular formation and repair of 06-EtGua in DNA were observed after pulse exposure to EtNU in vitro.…”

“…in biopsy material, Scherer et al, 1989;Wani et al, 1993) and throughout genomic DNA (Bohr, 1991;Le Doux et al, 1991;. However, only very recently, sufficiently sensitive analytical methods have become available which permit us to quantify specific drug-induced DNA lesions in defined gene sequences (Hochleitner et al, 1991;Zhen et al, 1992) and in single cells (Frankfurt et al, 1990;Van Delft et al, 1991;Seiler et al, 1993). In the present study, we have applied a newly developed, monoclonal antibody (MAb)-based immunocytological assay (ICA; Seiler et al, 1993) to measure the repair kinetics of 06-AlkGua in the nuclear DNA of individual human lymphocytes and leukaemic blasts after pulse exposure to N-alkyl-N-nitrosoureas.…”