Abstract
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 approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-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.
Highlights
DNA topoisomerase I (Top1) is biologically and pharmacologically important for at least two reasons related to what might be considered its classic activity, enzymatic DNA nicking-closing
Hygromycin B–resistant clones were selected, and the resulting cell lines were designated as HCT116-siTop1, MCF-7-siTop1, HCT116-siCtrl, and MCF-7-siCtrl
The main steps used to produce the shRNA vectors and small interfering RNA (siRNA) cell lines are described in Supplementary Fig. S1
Summary
DNA topoisomerase I (Top1) is biologically and pharmacologically important for at least two reasons related to what might be considered its classic activity, enzymatic DNA nicking-closing. It is essential for the removal of topological stress in DNA. Doi:10.1158/0008-5472.CAN-06-4554 associated with replication, transcription, repair, and recombination [1, 2]. It is the sole molecular target of camptothecins, which are used to treat human cancers [3,4,5]. Top1cc form with broad sequence selectivity, allowing the enzyme to act at a large number of sites in the genome
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
