An Image-Based, High-Throughput Screening Assay for Molecules that Induce Excess DNA Replication in Human Cancer Cells

Abstract

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer. Mol Cancer Res; 9(3); 294-310. Ó2011 AACR.

Replication and re-replication: Different implications of the same mechanism

“…In literature, abnormal DNA replication which leads to an increase in the total DNA content is called excessive DNA replication (EDR) [18,34]. EDR is an effect of pre-RCs re-assembly on fragments of DNA strands, which have already been replicated, and then immediate licensing and initiation of DNA replication [3].…”

“…Re-replication leads to DNA damage and stalled replication forks [18]. A number of factors inducing re-replication have been examined, for example: geminin depletion, Cdt1 overexpression [2,8] and deregulation of CDK levels [4].…”

“…Phosphorylation of geminin in mitosis, by interaction with Aurora-A (serineethreonine kinase), causes its activation and thus prevents re-replication [28]. Re-replication due to depleted level of geminin occurs only in cancer cells while normal cells possess three pathways to inactivate Cdt1, Cdc6 and ORC and therefore the absence of geminin is insufficient to trigger DNA re-replication [18,34]. It has been proven that Cdt1 overexpression plays a crucial role in inducing re-replication, but alone is not sufficient to trigger it.…”

“…Cdc28-Clb5 prevent re-replication in yeast by inhibition of pre-RC assembly, however osmotic stress activates Hog1PP which in turn downregulates Cdc28-Clb5 [17]. Zhu et al [18] have provided a broad range screening of compounds which trigger excessive DNA replication including re-replication for both normal and cancer cells. High-throughput screening has revealed a number of chemicals which induce re-replication, such as podophyllotoxin and other compounds which impact microtubule dynamics (i.e.…”

“…Re-replication can be triggered and/or enhanced by various factors such as the ratio of Cdt1 to geminin or overexpression of Cdt1/Cdc6 [3], osmotic stress [17], and also vincristine, taxol, tetraethylthiuram disulfide [18]. Additionally, sequences susceptible to replication re-initiation have been observed in yeast origin [19].…”