DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

Lindsey A Bierle,Janet F Partridge,Kira L Reich,Braden E Taylor,Mary E Miller,Eliot B Blatt,Pamela K Hanson,Laura K Stultz,Sydney M Middleton,Shawnecca D Burke,Yanchang Wang

doi:10.1371/journal.pone.0138085

Abstract

Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage–including that induced by many anticancer drugs–results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

Highlights

The eukaryotic cell division cycle is a highly regulated series of events supporting DNA replication, segregation and repair in response to a variety of intracellular and extracellular stresses
We find that the KP1019-induced DNA damage response (DDR) causes a robust pre-anaphase delay in cell cycle progression that is dependent on the Rad9 checkpoint
When treated with the anticancer ruthenium complex KP1019, the budding yeast S. cerevisiae exhibits a robust dose-dependent cell cycle delay, resulting in accumulation of large budded cells that continue to increase in size, suggesting that they continue to actively metabolize [14]

Summary

Introduction

The eukaryotic cell division cycle is a highly regulated series of events supporting DNA replication, segregation and repair in response to a variety of intracellular and extracellular stresses. Agents capable of interacting with DNA can trigger the DNA damage response (DDR), which causes complex cellular signaling allowing for DNA repair and cell cycle delay [1, 2]. DNA damaging agents, including the platinum-based drug cisplatin and carboplatin, are used in the treatment of solid tumors Their clinical efficacy is hindered by doselimiting toxicity to important organ systems, such as the gastrointestinal tract and peripheral nerves [5]. One promising candidate is the ruthenium complex trans-[tetrachlorobis(1H-indazole) ruthenate(III)], known as KP1019 This drug has been shown to induce apoptosis in ovarian cancer and colon carcinoma cell lines in vitro [6, 7] and to shrink autochthonous tumors in rats [7,8,9]. The signal transduction pathways that mediate the cellular response to KP1019 have not been adequately addressed

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