Affiliations of authors: M. J. Kelley, S. Li (Department of Medicine), D. H. Harpole (Department of Surgery), Thoracic Oncology Program, Duke University Medical Center, Durham, NC, and Durham Veterans Affairs Hospital.
Correspondence to: Michael J. Kelley, M.D., Hematology/Oncology (111G), Durham Veterans Affairs Hospital, Rm. E3007, 508 Fulton St., Durham, NC 27705 (e-mail: kelleym{at}duke.edu).
Tubulin, the cellular target for the taxane chemotherapeutic agents, is composed of heterodimers. There are at least six human genes encoding different tubulin
subunits (1). In most epithelial tumor cells, the most highly expressed isoform of
-tubulin is
5, which is encoded by the TUBB gene, also referred to as M40 (2). Chinese hamster ovary cells (3) and an ovarian tumor cell line (4) adapted for growth in vitro in the presence of the taxane paclitaxel have been found to have mutations in TUBB. Monzó et al. (5) reported TUBB mutations in 16 (33%) of 49 tumor samples from previously untreated patients with advanced non-small-cell lung cancer (NSCLC). All of the mutations, except two, were located in exon 4, which encodes more than half of the
-tubulin protein and includes the adenosine triphosphate-binding site composed of the ribose-, phosphate-, and base-binding regions. There was also a statistically significant association between the presence of TUBB mutations and both poor treatment response to paclitaxel-containing chemotherapy and shortened overall survival (5). These associations led to the proposal to use the presence of TUBB mutations as a basis for selecting initial chemotherapy for patients with advanced NSCLC (6).
To better study the association between TUBB mutations and tumor cell growth and taxane resistance in patients with NSCLC, we selected 25 tumor cell lines (supplementary Table 1, available at the Journal's Web site http://jnci.oupjournals.org) that differed in their in vitro sensitivity to paclitaxel (7). Genomic DNA was isolated from these cell lines, from normal human peripheral blood leukocytes, and from 20 NSCLC primary tumor samples by proteinase K digestion and phenolchloroform extraction. Patients providing a tumor sample gave written informed consent to use their tumor under an institutional review board-approved human study. Oligonucleotides for polymerase chain reaction (PCR) amplification of the coding regions of TUBB were designed on the basis of the genomic sequence of TUBB from the Human Genome Project (GenBank accession number AC006165) with the use of GeneWorks version 2.45 (IntelliGenetics, Mountain View, CA). At least one oligonucleotide was required to be within an intronic sequence. Oligonucleotides used in the amplification and sequencing reactions were as follows: Exon 11F, 5`-CCCATACATACCTTGAGGCG-3`; and 1R, 5`-TTTGGACCGTTAGAAGCCC-3` (sequencing). Exon 22F2, 5`-GAAGCAGAGGTTGCAGTGAG-3`; 2R2, 5`-TGACAGATTCACCCAAAGGG-3`; and 2F, 5`-AGAGCGAGACTCCGTCTCAA-3` (sequencing). Exon 33F, 5`-TCCCTTCTGCCAGATTTCAC-3`; 3R2, 5`-CAGGACAGAATCAACCAGCTC-3`; and 3R, 5`-CCCCTACTGCCCCATAATTT-3` (sequencing). Exon 44F3, 5`-AGGTAGTGCCTACTATTGCTGG-3`; 4R4, 5`-TGAGTAAGACGGCTAAGGGAAC-3` (sequencing); 4R2, 5`-AGCCATCATGTTCTTGGCA-3` (sequencing); 4F2, 5`-AGTTGGCAGTCAACATGGTC-3` (sequencing); and 4F4, 5`-TTGAGCTTTTCTCCTGACTGC-3` (sequencing). PCR and direct DNA sequencing were performed as described previously (8). Typical amplification conditions consisted of an initial denaturation at 94 °C for 2 minutes, followed by 3040 cycles, with one cycle consisting of 94 °C for 30 seconds, annealing temperatures of 50 °C or 55 °C for 30 seconds and 72 °C for 2 minutes, and a final extension at 72 °C for 10 minutes.
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To explain the lack of detection of TUBB mutations, we sequenced portions of exon 4 in four NSCLC cell lines (NCI-H322, NCI-H533, NCI-H838, and NCI-H1373) by using the previously reported exonic PCR primers (5) (Fig. 1, A and B). In each cell line, multiple sequence variants were detected in the regions of exon 4 that encode the ribose-binding region (Table 1
) and the phosphate-binding region (data not shown). Each variant was confirmed by sequence analysis on both strands. Additional sequence variants were found in other regions of exon 4 when both amplification primers were contained within the coding region or 3` untranslated region (data not shown). Direct comparison of the sequences obtained with the intronic or exonic primers clearly showed that the sequence variants were seen only when both amplification primers were within the coding region (Fig. 1, C
). One of the variants detected with the use of the exonic, but not intronic, amplification primers was a valine-to-isoleucine missense change at codon 180 (V180L). This mutation was described in two of four NSCLC tumor samples reported to have mutations in the ribose-binding region (5). We were unable to examine the base-binding region because the published sequence of oligonucleotide TB4-R (5) is not present in TUBB. Six of 20 previously reported sequence variants were detected with the use of the TB4-R oligonucleotide (5).
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Sequence analysis of the paclitaxel-resistant cell lines 1A9PTX10 and 1A9PTX22 (supplied by Dr. Tito Fojo, National Cancer Institute, Bethesda, MD) (4) confirmed the presence of the previously published mutations (data not shown). Furthermore, both mutant cell lines were homozygous (or hemizygous) for TUBB mutations when analyzed with the intronic primer pairs, suggesting that both alleles of TUBB must be mutant for the taxane-resistant phenotype.
Thus, TUBB mutations are not common in NSCLC tumors and tumor cell lines. The previously reported TUBB mutations in NSCLC tumors (5) may be an artifact of co-amplification of pseudogenes. The known -tubulin pseudogenes are distributed to chromosomes 1, 6 (at locations distinct from TUBB), 8, and 19. Differential genetic loss or amplification of these sequences may result in the more readily apparent detection of sequence variations in tumor samples relative to normal tissue. Therefore, the apparent association of TUBB sequence variants with poor treatment response and survival may be associated with tumor aneuploidy. Further study of this phenomenon is required before TUBB "mutations" are used in selecting treatments for patients with NSCLC.
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Manuscript received June 11, 2001; revised October 1, 2001; accepted October 11, 2001.
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