Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
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PMID:Decreased stability of DNA in cells treated with alkylating agents. 225 76

Lettre-Ehrlich cells were loaded with sufficient HN2 to produce about a 98% cell kill. Postincubation of the HN2-loaded cells in PBS resulted in the loss of about 40% of their HN2 without changing the cytolytic effect, supporting the proposal that only bound drug was effective. Postincubation of the HN2-loaded cells in PBS which contained 2% bovine serum albumin or in cell-free mouse ascitic fluid (1.8% protein) resulted in the same relative cellular HN2 loss as well as a 79% decrease in the cell kill. The cytolytic effect of HN2 is believed to be dependent on the degree to which the drug crosslinks DNA in 2 sequential reactions. It seems likely that such crosslinking occurred in nearly all of the PBS-postincubated cells, as they were nearly all killed. By analogy, albumin postincubation apparently blocked the competition of such crosslinking.
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PMID:In vitro inhibition of nitrogen mustard efficacy by postincubation of treated cells in serum protein. 262 Feb 91