The objective is to identify the major cytotoxic DNA-DNA crosslinks produced by the clinical alkylating antitumor drugs cyclophosphamide and ifosfamide as a means of elucidating their mechanism of action at the molecular level.
The specific aims are the following: (1) Design and procurement of the oligonucleotides needed to investigate likely N7-guanyl-N7-guanyl DNA interstrand and intrastrand crosslinking sites, i.e., G-C, G-X-C, G-X-X-C and G-G, G-X-G, G-X-X-G, respectively. (2) Chemical synthesis of the oligomers crosslinked (interstrand or intrastrand) with the active alkylating metabolites of cyclophosphamide and ifosfamide, i.e., phosphoramide mustard (PM) and isophosphoramide mustard (IPM), respectively. (3) Gel electrophoretic and/or HPLC purification of the appropriate guanyl-guanyl interstrand and intrastrand crosslinked products from phosphoramide mustard (PM) and isophosphoramide mustard (IPM) and characterization by Maxam- Gilbert sequencing reactions and/or by degradation to bis(2- guanylethyl)amine. (4) Incorporation of each crosslinked product into the shuttle vector pTZSV28. (5) Transfection of the modified shuttle vectors (described in specific aim 4) into human 293 cells for determination of their effect on the production of progeny plasmids in comparison to progeny plasmid production by control shuttle vectors. (6) Determine if a guanyl monoadduct or guanyl- guanyl intrastrand crosslinks (G-G, G-X-G, G-X-X-G, G-X-X-X-G) of single strand templates (or, if necessary, of their appropriate duplexes) will form upon reaction with phosphoramide mustard (PM) and isophosphoramide mustard (IPM) and, if so, if one or more will serve as blocking lesions for human DNA polymerases. The proposed investigations follow published experimental design and methodology and seek to extend earlier, analogous studies using the clinical anticancer drug nitrogen mustard (HN2, mechlorethamine). The proposed research will attempt to determine (a) whether PM and HN2 prefer identical or different sequence specificity for interstrand crosslinking because of their structural differences and (b) what effect the 7-atom crosslink derivable from IPM has on sequence specificity.
| Struck, R F; Davis Jr, R L; Berardini, M D et al. (2000) DNA guanine-guanine crosslinking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosfamide. Cancer Chemother Pharmacol 45:59-62 |
| Berardini, M; Foster, P L; Loechler, E L (1999) DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli. J Bacteriol 181:2878-82 |
| Berardini, M; Mackay, W; Loechler, E L (1997) Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard. Biochemistry 36:3506-13 |
