A monofunctional derivative of melphalan: preparation, DNA alkylation products, and determination of the specificity of monoclonal antibodies that recognize melphalan-DNA adducts.
Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was </=0.2%. The heat labile DNA base adducts formed by monohydroxymelphalan were shown to contain undetectable levels of cross-linked species. The ratio of adenine to guanine adducts was 0.62, similar to the equivalent ratio for melphalan. The sequence-dependent pattern of alkylation of purified DNA was indistinguishable from that of melphalan, but required a higher dose to achieve comparable extents of reaction. The specificities of two monoclonal antibodies that recognize melphalan-DNA adducts were investigated using DNA alkylated with [3H]monohydroxymelphalan. Adducts on this DNA showed similar immunoreactivities to adducts formed by melphalan. This shows clearly that neither antibody was specific for cross-linked adducts and that it is therefore possible to quantify adducts formed by both monohydroxymelphalan and melphalan with high sensitivities. The availability of monohydroxymelphalan in addition to melphalan, together with sensitive immunoassays for adducts on extracted DNA and in individual cells, constitutes a useful system for investigating cellular responses to the DNA modifications formed by a clinically relevant drug.
Leukaemia Research Fund Laboratory and Department of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K. M.J.Tilby@ncl.ac.uk
M J
MJ
Tilby
H
H
McCartney
K A
KA
Gould
C C
CC
O'Hare
J A
JA
Hartley
A G
AG
Hall
B T
BT
Golding
P D
PD
Lawley
D000478:Alkylation; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008558:Melphalan / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018906:Antineoplastic Agents, Alkylating; D018736:DNA Adducts; D004247:DNA; D008558:Melphalan