β-Tubulin mutations in ovarian cancer using single strand conformation analysis-risk of false positive results from paraffin embedded tissues

Abstract

Mutations in the β-tubulin gene have been proposed as a resistance mechanism to paclitaxel. We therefore investigated the presence of mutations in the β-tubulin M40 gene in 40 ovarian tumours (16 paraffin-embedded and 24 freshly frozen) selected for good or poor response to chemotherapy with paclitaxel or non-tubulin-affecting regimens. The presence of mutations was investigated using single strand conformation analysis followed by sequencing of the products with altered mobility. No sequence variants in the exons of the β-tubulin M40 gene were detected. Non-reproducible shifts were identified, in eight out of 16 paraffin embedded samples. This may explain some of the previously published discrepancies. In conclusion, sequence variants in the β-tubulin M40 gene are rare and are unlikely to be a clinically relevant explanation of resistance to paclitaxel.

Introduction

Ovarian cancer is a common malignancy in women and chemotherapy plays an important role in the treatment following the initial surgery. A major clinical advance was made in the early 1990s when paclitaxel (Taxol^®) in combination with a platinum derivative was introduced in the treatment of ovarian carcinoma [1]. Paclitaxel has a unique mechanism of action in that it binds to β-tubulin in tubulin heterodimers [2]. These heterodimers, consisting of one α-tubulin and one β-tubulin subunit, self-associate into polymers and cylindrical tubes that constitute the microtubule. Microtubules undergo rapid transitions between growth and shrinkage due to association and dissociation of tubulin dimers [3]. Microtubules containing paclitaxel bound tubulin are unusually stable and thus the drug suppresses the depolymerisation of microtubules [2]. The change in tubulin dynamics leads to interference with the formation of the mitotic spindle and the cells arrest at mitosis. Eventually, bcl-2 becomes hyperphosphorylated and the cells undergo apoptosis [1].

The clinical success of cancer chemotherapy is limited by the development of drug resistance. Several potential mechanisms have been proposed for paclitaxel resistance, including alterations in the cellular target tubulin such as changes in tubulin expression and mutations in the tubulin genes [3]. However, conclusive results have been difficult to obtain mainly due to the presence of multiple tubulin isoforms that are encoded by a large gene family consisting of both functional and non-functional genes with a high degree of nucleotide similarity. In humans, six different β-tubulin isoforms have been identified and are classified as follows (Roman numerals represent the protein class and Arabic numerals the gene): class I, M40; class II, β9; class III, β4; class IVa, 5β; class IVb, β2; class VI, β1. The expression of these isoforms is tissue-dependent, but classes I and IVb are ubiquitously expressed and class I (gene M40) contributes to the major fraction of the β-tubulin isoforms [4]. Altered expression of the different β-tubulin isoforms, especially classes III and IVa, has been found in paclitaxel-resistant cell lines as compared to the parental cell line [3], [5]. It has also been suggested that paclitaxel-resistant cell lines contain ‘hypostable’ mictotubules and that the tubulin equilibrium is shifted towards dimer formation [6]. This indicates that some of the paclitaxel-resistant cells contain less stable microtubule polymers, which has led to studies of the tubulin genes to identify mutations or polymorphisms that would explain the presence of tubulin with different microtubule dynamics. In resistant cell lines, point mutations have been found at several locations in the β-tubulin gene M40 as well as in Kα1-tubulin [7], [8], [9], [10].

Several research groups have studied the presence of tubulin mutations in human tumours. Monzo et al. (1999) identified β-tubulin mutations in 33% of patients with non-small-cell lung cancer [11]. Many groups have attempted to confirm this initial study; but conflicting results have been reported [12], [13], [14], [15]. Most of these studies have used fluorescence-based DNA sequencing techniques, which may ignore small subpopulations of cells with altered DNA sequences. The analysis of the β-tubulin M40 gene has also proved to be difficult due to nucleotide similarities with the other five known isoforms as well as the presence of several pseudogenes. The accuracy of the original gene sequence for β-tubulin M40 (J00314) has also been questioned as it is thought to contain several discrepancies compared to the correct gene as well as the mRNA sequence [16], [17], [18]. We therefore designed a study to evaluate the presence of β-tubulin M40 mutations in DNA isolated from ovarian tumours using single strand conformation analysis (SSCA) as mutation analysis, followed by sequencing of the shifted products, and to correlate the results with treatment effects.