Antibody Recognition of Melphalan Adducts Characterized Using Immobilized DNA:  Enhanced Alkylation of G-Rich Regions in Cells Compared to in Vitro

Abstract

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) 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-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, 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) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

p53 Elevation in Relation to Levels and Cytotoxicity of Mono- and Bifunctional Melphalan-DNA Adducts

“…2), despite its lower molar alkylating capacity, was unexpected and illustrates the importance of monitoring adduct levels. It cannot be attributed to an assay artifact (Tilby et al, 1998) or an influence of local DNA sequence on adduct recognition by antibody MP5/73 (McCartney et al, 2001), especially because melphalan and monohydroxymelphalan displayed indistinguishable patterns of sequence-dependent alkylation (Tilby et al, 1998). The higher adduct levels after exposure to monohydroxymelphalan did not result from slower adduct removal (Fig.…”

Covalent binding of nitrogen mustards to the cysteine-34 residue in human serum albumin

“…It is believed that these compounds act through inter-and intra-strand crosslinking of guanine residues in DNA, which process inhibits cell proliferation (for a review see for example Lawley and Phillips 1996). Adduct formation of nitrogen mustards with DNA has been studied extensively over the last two decades, and methods for (sensitive) detection of these adducts have been developed (Tilby et al 1995;Thulin et al 1996;McCartney et al 2001).…”