Weakened spindle checkpoint with reduced BubR1 expression in paclitaxel-resistant ovarian carcinoma cell line SKOV3-TR30

Abstract

Objective

Paclitaxel sensitivity has recently been associated with the spindle checkpoint. The aim of our study is to investigate the status of spindle checkpoint and the alteration of its major components in phenotype with acquired paclitaxel resistance in ovarian carcinoma.

Methods

A paclitaxel-resistant ovarian carcinoma cell line, SKOV3-TR30, with the resistant ability of 27-fold greater than its parental cell line, was derived from SKOV3 cell line. The competence of spindle checkpoint and the expression of the major spindle checkpoint proteins (BubR1 and Mad2) in SKOV3-TR30 cells were investigated.

Results

Mitotic index and flow cytometric analysis revealed that SKOV3-TR30 cells were not arrested in M phase in contrast to SKOV3, which showed a clear mitotic arrest in the presence of paclitaxel or nocodazole. The expressions of securin and cyclin B in SKOV3-TR30 cells were significantly lower than those in SKOV3 cells, indicating the premature degradation of APC substrates in SKOV3-TR30 cells in response to spindle damagers. Chromosome spread analysis showed increased rate of premature sister chromatid segregation in SKOV3-TR30 cells. The expression of spindle checkpoint protein BubR1 was evidently lower in SKOV3-TR30 cells than that in SKOV3 cells. However, there was no significant difference in Mad2 expression between SKOV3-TR30 and SKOV3 cells.

Conclusions

This study demonstrates that weakened spindle checkpoint with reduced expression of BubR1, but not Mad2, is associated with acquired paclitaxel resistance in ovarian carcinoma cells.

Introduction

Despite significant initial response rates for advanced ovarian carcinoma using paclitaxel–platinum combination chemotherapy, the vast majority of patients relapse and develop a drug-resistant disease with an overall 5-year survival of < 50% [1], [2]. Acquired resistance to paclitaxel is one of the most significant reasons for its failure in chemotherapy [3]. Previous studies have demonstrated that paclitaxel resistance is attributable to various mechanisms: increased drug efflux that results from up-regulation of membrane transporters (e.g., P-gp) [4], [5]; alterations in the expression of β-tubulin isotypes [6]; and changes in apoptotic regulatory proteins (e.g., Bcl-2) [7], [8], [9]. However, limited achievements have been obtained in the development of means to combat resistance according to the possible mechanisms. Thus, the molecular mechanism of paclitaxel resistance has yet to be precisely defined.

When paclitaxel stabilizes microtubules and interferes with normal formation of mitotic spindle, the spindle checkpoint is activated to make cells arrest at mitosis and cause cell death subsequently [10], [11]. The spindle checkpoint, also known as the mitotic spindle assembly checkpoint, is the major cell cycle control mechanism in mitosis, which ensures proper chromosome segregation during mitosis by producing inhibitors of the anaphase-promoting complex/cyclosome (APC/C) [12], [13], [14]. This E3 ubiquitin ligase complex is required for the ubiquitination and destruction of securin and cyclin B, prerequisites for anaphase onset and mitotic exit. Several evolutionary conserved proteins including Bub1, Bub3, BubR1, Mad1, Mad2 and Mps1 are required for spindle checkpoint function [12], [13], [14].

Recently, spindle checkpoint has been associated with sensitivity to spindle-damaging anticancer drugs [15], [16], [17], [18], [19], [20], [21], [22]. For example, Sudo and colleagues showed that loss of the spindle checkpoint by transient siRNA transfection in MCF-7 cells suppressed paclitaxel-induced cell death [18]. However, by inactivating the spindle checkpoint, histone deacetylase inhibitor trichostatin A (TSA) can potentiate the lethal effects of microtubule-disrupting drugs [19]. In addition, Tao et al. found that cells that can sustain prolonged (> 48 h) spindle checkpoint signaling (e.g., HT29) are less susceptible to KSPIA and taxol-mediated killing [20]. These contradictory and limited data require further study to determine whether cancers with a weakened or defective spindle checkpoint are more or less suitable for treatment with antimitotic drugs.

It is well established that spindle checkpoint is absolutely essential for viability in vertebrates and complete inactivation of spindle checkpoint in cells is lethal [23], [24]. Because cancer cells without spindle checkpoint were not able to be alive longtime too, previous studies with gene silencing technology could not address the long-term effect of spindle checkpoint on chemosensitivity. Thus, elucidating the status of spindle checkpoint in developed resistance phenotype is as important as those researches from genotype to phenotype with gene-manipulating technology. However, the status of spindle checkpoint in phenotype with acquired paclitaxel resistance remains unknown.

In this study, a paclitaxel-resistant ovarian carcinoma cell line, SKOV3-TR30, was developed by exposing parental SKOV3 cells to increased concentration of paclitaxel. The competence of spindle checkpoint and the expression of major components of spindle checkpoint (Mad2, BubR1) in SKOV3-TR30 cells were investigated.