β-Tubulin mutational analysis: tantalizing findings

Abstract

Resistance to taxanes could be due to alterations in the intracellular target, tubulin. Such alterations can include decreased tubulin, point mutations, altered expression of b-tubulin isotypes and acetylation of atubulin. Acquired b-tubulin mutations in human cancer cell lines confer resistance to taxanes and epothilones by impairing their binding to microtubules [1]. We have read with great interest the comprehensive mutational analysis of the b-tubulin gene by Tsurutani et al. [2], whose findings mirror those recently reported by Kelley et al. [3]. Both studies point out that when the originally described exonic primers for b-tubulin exon 4 [4] were used, no real tubulin mutations were detected. Instead, multiple non-specific nucleotide sequences [2] or variants [3] were identified in 17 non-small-cell lung cancer (NSCLC) tumor samples and several lung cancer cell lines [2] and in 25 NSCLC cell lines and 20 NSCLC tumor samples [3]. In spite of the fact that many of these nucleotide substitutions lead to amino acid changes, mainly missense mutations [3], they were interpreted as pseudogenes. However, with cDNA [2] or intronic primers [3], such alterations were not visible, and only silent mutations were observed in 7/20 cell lines and in 4/ 22 lung tumors [2]. In the Kelley et al. [3] study, only 2/ 25 tumor cell lines were described as having either silent mutations or polymorphisms. Interestingly, the substitution at codon 217 found by Kelley et al. has been identified in several studies and was originally reported in paclitaxel-resistant Chinese hamster ovarian cell lines [5]. The Leu 217 silent polymorphism has also been described in another study [6], where no b-tubulin mutations were identified in ovarian or lung cancer tumor samples and cell lines with denaturing high pressure liquid chromatography. Recently however, in the EpoB-resistant A549 human NSCLC line, a mutation was noted at codon 292 [7]. In our recent experience, as well as in that of other investigators (Gumerlock, Gandara, unpublished data), no tubulin mutations were found in either tumor or serum DNA from NSCLC patients, regardless of disease stage. These mutational analysis were performed using a forward intronic primer at position 2901, yielding a PCR product of 1200 bp. With this same methodology, no tubulin mutations have been observed in any patients included in a prospective ongoing tailored chemotherapy trial. At the time we designed the original primers in 1996 [4], less information on tubulin genes and pseudogenes was available, and many of the originally described mutations may have been pseudogenes arising by retrotransposition. In a few instances, retrotransposition is maintained as a functional intronless gene, as is the case with CPTEN [8]. Pseudogene transcription may also modulate normal gene activity, and this phenomenon may well be the most plausible explanation for the survival differences found in our original study [4]. Further research is being pursued to elucidate this issue. The contradictory results between mutations found in cell lines and tumor tissues have no easy explanation. Cancer therapy resistance is multifactorial, including the DNA repair machinery, checkpoint kinase Chk1 [9]. In addition, the loss of p53 function makes b-tubulin mutations irreversible [10]. Current lines of tubulin research are focussing on clarifying whether up-regulation of certain b-tubulin isotypes could become a pretherapeutic determinant of the response to microtubule-damaging agents.