Antibody recognition of melphalan adducts characterized using immobilized DNA: Enhanced alkylation of g-rich regions in cells compared to in vitro

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and A mp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after t he alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELIS As) indicated that in 0.01 and 0.1 M. NaOH, loss of immunoreactivity to MP5 /73 occurred with half-lives that were at least 2-fold longer than half-liv es for gain of immunoreactivity to Amp4/42. This supported previously publi shed evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. A dducts recognized by MP5/73 on RNA were considerably more stable at 100 deg reesC and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the invol vement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonuc leotides, covalently immobilized onto 96-well plates, were reacted with mel phalan and incubated for various periods with alkali, and then the levels o f adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recogn ized very weakly by Amp4/42, unlike other DNA sequences that were tested. R etention of immobilized DNA during alkali treatment was confirmed by immuno assay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligo deoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP 5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) t he different alkali stability of immunoreactive adducts and (b) previous re sults which showed that, in DNA from melphalan-treated cells, adducts recog nized by Amp4/42 formed a smaller proportion of total adducts compared to D NA alkylated in vitro. The results presented here indicate that this was ca used by a marked cellular influence on the overall sequence-dependent patte rn of DNA alkylation or repair.