© 2001 by Oxford University Press
Journal of the National Cancer Institute, Vol. 93, No. 24, 1886-1888,
December 19, 2001
© 2001 Oxford University Press
BRIEF COMMUNICATION |
Genetic Analysis of the 
-Tubulin Gene, TUBB, in Non-Small-Cell Lung Cancer
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 phenol–chloroform 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 1—1F, 5`-CCCATACATACCTTGAGGCG-3`; and 1R, 5`-TTTGGACCGTTAGAAGCCC-3` (sequencing). Exon 2—2F2, 5`-GAAGCAGAGGTTGCAGTGAG-3`; 2R2, 5`-TGACAGATTCACCCAAAGGG-3`; and 2F, 5`-AGAGCGAGACTCCGTCTCAA-3` (sequencing). Exon 3—3F, 5`-TCCCTTCTGCCAGATTTCAC-3`; 3R2, 5`-CAGGACAGAATCAACCAGCTC-3`; and 3R, 5`-CCCCTACTGCCCCATAATTT-3` (sequencing). Exon 4—4F3, 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 30–40 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.
|
With the use of intronic amplification primer pairs (in which at least one oligonucleotide was located in an intron), only two of 25 NSCLC tumor cell lines had variant sequences in the coding region and adjacent splice sites. Cell line NCI-H1648 had a C-to-A substitution in the third position of codon 187, and cell line NCI-H2228 had a G-to-A substitution in the third position of codon 217; both substitutions are in exon 4. Neither of these variants altered the encoded amino acid, suggesting that they may be polymorphisms. No sequence variants were found in TUBB exon 4 from 20 NSCLC tumor samples.
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).
|
Homology searches of the draft human genome identify at least eight sequences with high sequence identity to the TUBB ribose-binding region. These sequences include previously reported
-tubulin-processed pseudogenes (9), which may be co-amplified with TUBB when two exonic primers are used. A pseudogene is a segment of genomic DNA with a high degree of sequence similarity to a true gene, but which does not encode a functional gene product (10). Some pseudogenes, such as those in the -globin gene cluster, are thought to have originated by gene duplication and retain remnant gene-like structures, including promoter regions and intron–exon boundaries. The more abundant processed pseudogenes, such as those in TUBB, are thought to have arisen by the incorporation of reverse-transcribed messenger RNA into the germline DNA and have poly-A tails but lack promoter regions and introns (2). The presence of processed pseudogenes can confound mutational analysis (11). The V180L variant detected in the NSCLC tumors is located in a known pseudogene (9). 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.
REFERENCES
1 Luduena RF. Multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol 1998;178:207–75.[Web of Science][Medline]
2 Lee MG, Lewis SA, Wilde CD, Cowan NJ. Evolutionary history of a multigene family: an expressed human beta-tubulin gene and three processed pseudogenes. Cell 1983;33:477–87.[CrossRef][Web of Science][Medline]
3
Gonzalez-Garay ML, Chang L, Blade K, Menick DR, Cabral F. A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance. J Biol Chem 1999;274:23875–82.
4
Giannakakou P, Sackett DL, Kang YK, Zhan Z, Buters JT, Fojo T, et al. Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem 1997;272:17118–25.
5
Monzo M, Rosell R, Sanchez JJ, Lee JS, O'Brate A, Gonzalez-Larriba JL, et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J Clin Oncol 1999;17:1786–93.
6 Rosell R, Taron M, O'Brate A. Predictive molecular markers in non-small cell lung cancer. Curr Opin Oncol 2001;13:101–9.[CrossRef][Web of Science][Medline]
7 Georgiadis MS, Russell EK, Gazdar AF, Johnson BE. Paclitaxel cytotoxicity against human lung cancer cell lines increases with prolonged exposure durations. Clin Cancer Res 1997;3:449–54.[Abstract]
8 Owshalimpur D, Kelley MJ. Genomic structure of the EPHA1 receptor tyrosine kinase gene. Mol Cell Probes 1999;13:169–73.[CrossRef][Web of Science][Medline]
9 Wilde CD, Crowther CE, Cripe TP, Gwo-Shu Lee M, Cowan NJ. Evidence that a human beta-tubulin pseudogene is derived from its corresponding mRNA. Nature 1982;297:83–4.[CrossRef][Medline]
10 Mighell AJ, Smith NR, Robinson PA, Markham AF. Vertebrate pseudogenes. FEBS Lett 2000;468:109–14.[CrossRef][Web of Science][Medline]
11 Forgacs E, Biesterveld EJ, Sekido Y, Fong K, Muneer S, Wistuba II, et al. Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene 1998;17:1557–65.[CrossRef][Web of Science][Medline]
Manuscript received June 11, 2001; revised October 1, 2001; accepted October 11, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. J. Lee and S. M. Swain The Epothilones: Translating from the Laboratory to the Clinic Clin. Cancer Res., March 15, 2008; 14(6): 1618 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Newman, P. A. Foster, C. Stengel, J. M. Day, Y. T. Ho, J.-G. Judde, M. Lassalle, G. Prevost, M. P. Leese, B. V.L. Potter, et al. STX140 Is Efficacious In vitro and In vivo in Taxane-Resistant Breast Carcinoma Cells Clin. Cancer Res., January 15, 2008; 14(2): 597 - 606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sampath, L. M. Greenberger, C. Beyer, M. Hari, H. Liu, M. Baxter, S. Yang, C. Rios, and C. Discafani Preclinical Pharmacologic Evaluation of MST-997, an Orally Active Taxane with Superior In vitro and In vivo Efficacy in Paclitaxel- and Docetaxel-Resistant Tumor Models. Clin. Cancer Res., June 1, 2006; 12(11): 3459 - 3469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hari, F. Loganzo, T. Annable, X. Tan, S. Musto, D. B. Morilla, J. H. Nettles, J. P. Snyder, and L. M. Greenberger Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of {beta}-tubulin (Asp26Glu) and less stable microtubules. Mol. Cancer Ther., February 1, 2006; 5(2): 270 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W.L. Yee, A. Hagey, S. Verstovsek, J. Cortes, G. Garcia-Manero, S. M. O'Brien, S. Faderl, D. Thomas, W. Wierda, S. Kornblau, et al. Phase 1 Study of ABT-751, a Novel Microtubule Inhibitor, in Patients with Refractory Hematologic Malignancies Clin. Cancer Res., September 15, 2005; 11(18): 6615 - 6624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Ferguson, C. Taylor, A. Stanley, E. Butler, A. Joyce, P. Harnden, P. M. Patel, P. J. Selby, and R. E. Banks Resistance to the Tubulin-Binding Agents in Renal Cell Carcinoma: No Mutations in the Class I {beta}-Tubulin Gene but Changes in Tubulin Isotype Protein Expression Clin. Cancer Res., May 1, 2005; 11(9): 3439 - 3445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Goodin, M. P. Kane, and E. H. Rubin Epothilones: Mechanism of Action and Biologic Activity J. Clin. Oncol., May 15, 2004; 22(10): 2015 - 2025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. McLeod Genetic Strategies to Individualize Supportive Care J. Clin. Oncol., June 15, 2002; 20(12): 2765 - 2767. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Monzo, M. Taron, and R. Rosell Re: Genetic Analysis of the {beta}-Tubulin Gene, TUBB, in Non-Small-Cell Lung Cancer J Natl Cancer Inst, May 15, 2002; 94(10): 774 - 776. [Full Text] [PDF]
|
|
|
|
|
|
S. Sale, P. J. Oefner, and B. I. Sikic Re: Genetic Analysis of the {beta}-Tubulin Gene, TUBB, in Non-Small-Cell Lung Cancer J Natl Cancer Inst, May 15, 2002; 94(10): 776 - 777. [Full Text] [PDF]
|
|
|
|
|
|
M. J. Kelley RESPONSE: Re: Genetic Analysis of the {beta}-Tubulin Gene, TUBB, in Non-Small-Cell Lung Cancer J Natl Cancer Inst, May 15, 2002; 94(10): 777 - 777. [Full Text] [PDF]
|
|
|
|
|
|
F. J. Kaye Publishing Negative Data: {beta}-Tubulin Mutations in Lung Cancer J Natl Cancer Inst, December 19, 2001; 93(24): 1832 - 1833. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||