Effects of Chk1 inhibitor or paclitaxel on cisplatin-induced cell-cycle kinetics and survival in parental and cisplatin-resistant HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci)

Chizumi Yamada ,Atsushi Kaida ,Kazuaki Okuyama ,Kiyoshi Harada ,Masahiko Miura

doi:10.15761/icst.1000120

Abstract

Cisplatin chemotherapy easily induces a resistant phenotype in tumor cells. In p53-deficient tumor cells, cisplatin causes G2 arrest, which allows cells to repair DNA damage. We attempted to visualize how cisplatin-induced G2 arrest is modified by Chk1 inhibitor or paclitaxel (PTX) and examine the effects on cell survival in both parental and cisplatin-resistant tumor cells. We used HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Cisplatin-resistant HeLa- Fucci (CR) cells were established by incubating parental HeLa cells with doses of cisplatin that increased in a stepwise manner. Time-lapse images were acquired by fluorescence microscopy, and surviving fractions were determined by colony-forming assay. Cisplatin treatment caused parental cells to accumulate green cells, representing G2 arrest, whereas combined therapy with a Chk1 inhibitor abrogated this effect, leading to a remarkable sensitization. CR cells did not exhibit G2 arrest, and Chk1 inhibitor did not influence the cell cycle, accompanied by slight reduction in survival. PTX arrested both cells, to similar extents, in mitosis with abnormal fluorescence; however, parental cells were arrested in G2 phase before entering mitosis, whereas CR cells were not, when cisplatin was used in combination. Moreover, cell survival increased in parent cells, but not in CR cells, relative to treatment with PTX alone. In conclusion, we should carefully consider G2 arrest kinetics, depending on the presence or absence of cisplatin resistance, in efforts to sensitize cells to cisplatin-based chemotherapy.

Highlights

Cisplatin is a commonly used chemotherapeutic agent that forms monoadducts, and intra-strand cross-links and inter-strand crosslinks with DNA [1]
Sakaue-Sawano et al developed the fluorescent ubiquitination-based cell cycle indicator (Fucci) methodology for visualizing cell-cycle progression using fluorescent proteins: Fucciexpressing cells in G1 and other phases emit red and green fluorescence, respectively [11], allowing us to detect changes in cell-cycle stage in live cells. Taking advantage of this system, we explored the effect of G2/M checkpoint modulation on G2 arrest and cell survival, using HeLa cells expressing the Fucci probes (HeLa-Fucci) and a cisplatin-resistant cell line derived from parental HeLa-Fucci cells (CR)
We examined the kinetics of DNA Double-Strand Breaks (DSBs) following cisplatin treatment (Figure 1D), which are formed in the course of inter-strand cross links repair [3]. 53BP1 was used as a marker of DSBs [12]

Summary

Introduction

Cisplatin is a commonly used chemotherapeutic agent that forms monoadducts, and intra-strand cross-links and inter-strand crosslinks with DNA [1]. Of these lesions, inter-strand crosslinks make the predominant contribution to cell death, even though their yield is quite low [1,2,3]. Inter-strand crosslinks make the predominant contribution to cell death, even though their yield is quite low [1,2,3] These lesions can activate cell-cycle checkpoints that mediate the cell-cycle arrest required for repair of DNA damage [4]. PTX agent arrests cells in mitosis [10]; complicated cell-cycle kinetics should be induced when PTX and cisplatin are combined

Methods

Results

Conclusion