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

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 f5-fold and Top2A 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.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

“…Thus, it is possible that Top1 protein reduction may disturb splicing of S region transcripts and prolong the half life of S region transcripts, leading to the formation of frequent R-loop and consequent non-B DNA structures in the S region (3,32). In support of this hypothesis, the reduction of either Top1 or ASF induces genomic instability (17,32). In fact, we observed that the single stranded fraction of the S region was augmented by Top1 knockdown.…”

“…A specific inhibitor of Top1, camptothecin (CPT) can be intercalated into this transient cleavage complex of Top1 and DNA to block Top1's catalytic function while CPT does not affect free Top1 (16). Curiously, the reduction of Top1 by RNA mediated knockdown in cultured cells causes the genome-wide instability (17). Activation-induced cytidine deaminase (AID) is essential for CSR and involved in S region cleavage, but its molecular mechanism is unknown (12,13,18,19).…”

Topoisomerase‐1 gene copy aberrations are frequent in patients with breast cancer

“…Recently a small study including 28 patients with mBC used a multi-omic molecular profile (MMP) including Topo1 to identify potential therapeutic targets and select individualized treatment. All patients were heavily pretreated with at least three prior treatment regimens (range [3][4][5][6][7][8][9][10][11][12]. Based on the MMP 14 patients were treated with irinotecan or irinotecan plus fluorouracil.…”

“…6 However, although using similar technologies, other studies have not found Top1 protein predictive of response in the metastatic setting. 7,8 Reduced levels of Top1 in BC cells have been associated to decreased sensitivity to camptothecin 9 and a single small (n 5 22) clinical study has reported increased levels of Top1 protein in primary tumors from patients with BC. 10 Furthermore, high TOP1 gene copy numbers in BC cell lines have been found to be associated with increased sensitivity to SN38, which is the active metabolite of irinotecan.…”

Characterization of the conversion between CD133+and CD133-cells in colon cancer SW620 cell line

“…The CD133 + clonal cells and the CD133 -clonal cells display differential gene expression profiles. Using Affymetrix U133-plus-2-Genechips that contain the probe sets against the transcripts of 17,942 different genes with their Human Genome Organization (HuGO) names, 13 we detected mRNA levels in the purified CD133 + clonal cells and the purified CD133 -clonal cells. A list of 326 candidate genes was produced by taking the changes common to two independent experiments ( Fig.…”