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Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

Laboratories of ¹ Molecular Pharmacology and ² Experimental Carcinogenesis, ³ Office of Science and Technology Partnerships, Center for Cancer Research, ⁴ Microarray Facility, Advanced Technology Center, National Cancer Institute, and ⁵ Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland

Requests for reprints: Yves Pommier, Laboratory of Molecular Pharmacology, Building 37, Room 5068, NIH, Bethesda, MD 20892-4255. Phone: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced 5-fold and Top2 compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone -H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function. [Cancer Res 2007;67(18):8752–61]

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