MUTAGENESIS BY THE ANTINEOPLASTIC MUSTARDS

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This project will determine the frequency and molecular structure of the mutations induced in human lymphocytes by the nitrogen mustard group of antineoplastic alkylating agents which are well-established as carcinogens in humans. Patients treated with these drugs have an increased risk of second malignancies particularly acute leukemia. Many of the induced leukemias are associated with characteristic chromosomal deletions and rearrangements. In spite of their wide use and established carcinogenicity, relatively less is known about the mechanism of mutagenicity for the nitrogen mustards than for many environmental carcinogens. Two complementary systems will be used to study mutations induced by these DNA-DNA cross-linking agents. In both systems the genetic locus to be studied is the human hypoxanthine-guanine- phosphoribosyltranferase (HPRT) locus. In the first system, the human lymphoblastoid cell line WIL-2 NS will be exposed in vitro to some of the clinically useful alkylating agents, primarily the nitrogen mustards, and the frequency of mutations induced at various levels of cytoxicity will be determined. The polymerase chain reaction, molecular cloning and DNA sequencing will be used to identify hprt mutations as base changes, deletions or rearrangements. Preliminary studies indicate that nitrogen mustard (mechlorethamine) induces a high frequency of large gene deletions at the HPRT locus. Moreover, within the HPRT gene, a "hotspot" for mechlorethamine-induced deletions has been identified in the region between exon4 and exon9, with 16/17 of such deletions comprising the 3' end of the gene. The nature of the sequence(s) that determines the increased frequency and unidirectionality for deletions in this region will be determined by cloning and sequencing of the mutant HPRT fragments containing the breakpoint. This sequence information will allow us to test various hypotheses as to the mechanism by which such deletions occur. In the second system, lymphocytes will be obtained from patients receiving therapy with cyclophosphamide for multiple sclerosis or chlorambucil for non-Hodgkin's lymphoma. In preliminary studies, we have identified an increased mutation frequency at the HPRT locus in lymphocytes of cyclophosphamide-treated patients. The molecular structure (base substitutions, deletion endpoints, etc.) of the mutations induced in vivo by cyclophosphamide, chlorambucil and melphalan will be analyzed. These complementary systems allow study of the frequency and structure of HPRT mutations under conditions of either in vitro or in vivo exposure. Patients, nurses, pharmacists and manufacturing workers are frequently exposed to the nitrogen mustards and to related compounds. This project should allow better characterization of the exact nature of the mutagenic events that occur in human cells exposed to the nitrogen mustards (or other environmental exposures) which may later lead to second malignancies, germ line mutations and birth defects in exposed individuals.