Chromosomal band 11q23 is involved in reciprocal translocations in de novo acute lymphoblastic leukemia and acute myeloid leukemia (AML) of infants and young children and in epipodophyllotoxin-associated secondary AML. Perturbation of the topoisomerase II-DNA interaction by epipodophyllotoxins and resultant double-stranded breaks in DNA are cytotoxic to cancer cells. The DNA topoisomerase II inhibitors interfere specifically with topoisomerase II by stabilizing a covalent enzyme-DNA intermediate. Secondary AML with reciprocal translocations at chromosomal band 11q23 is a well-established epidemiologic consequence of treatment with these drugs. The strong association of secondary AML with chemotherapy targeted to this enzyme leads to the hypothesis that the 11q23 chromosomal breakpoints are related to drug-induced cleavage of the DNA by topoisomerase II. The 11q23 translocations also may reflect attempts at repair of the abnormal cleavage. The objective of this proposal is to bridge the gap between the well-described cause and effect relationship between topoisomerase II inhibitor chemotherapy and secondary AML, and the molecular requirements for translocations specifically involving chromosomal band 11q23.
The first aim i s to use PCR based methods to clone, sequence, align and compare twenty secondary AML 11q23 genomic breakpoints and the parental for homologues from which they were derived for consistencies either at the breakpoints or nearby that suggest consensus sequences for epipodophyllotoxin-induced topoisomerase II DNA cleavage sites. Examination of parental homologues is essential because the translocations may cause alterations in the normal sequence.
The second aim i s to use in vitro topoisomerase II DNA cleavage assays to investigate drug-stabilized topoisomerase II cleavage of parental DNAs at sites predicted by the sequencing of secondary AML genomic breakpoints.
The third aim i s to study epipodophyllotoxin-induced topoisomerase II DNA cleavage in the parental homologues of 11q23 genomic breakpoint sequences in human hematopoietic cells in tissue culture. Since perinatal toxic exposures are associated with leukemia in infants, the final aim is to clone, sequence and align twenty de novo 11q23 leukemia translocation breakpoints and their parental homologues to investigate the specificity of the mechanism for the secondary cases. Because epipodophyllotoxins are highly effective in many cancers of children and adults, the frequency of topoisomerase II inhibitor-associated secondary AML has increased. The combined analyses of 11q23 leukemia genomic breakpoint sequences from patients exposed to topoisomerase Il inhibitors, and DNA exposed to topoisomerase Il inhibitors in vitro and in tissue culture will establish the relationship between DNA topoisomerase II and the translocation breakpoints. Study of these leukemias provides a unique opportunity to link etiology and pathogenesis.
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