Affiliation of authors: M. Monzo, M. Taron, R. Rosell, Hospital Germans Trias i Pujol, Badalona (Barcelona), Spain.
Correspondence to: Rafael Rosell, M.D., Ph.D., Medical Oncology Service, Hospital Germans Trias i Pujol, Ctra Canyet, s/n, 08916 Badalona (Barcelona), Spain (e-mail: rrosell{at}ns.hugtip.scs.es).
Fluorescence-based DNA sequencing has been used to decipher the functional importance of DNA polymorphisms and mutations. One gene that has received recent attention is the class I -tubulin isotype M40. Mutations have been identified in M40, which accounts for more than 85% of total
-tubulin RNA in cell lines used in the National Cancer Institute anti-cancer drug screen and in 12 human tumor xenografts (1). Using four overlapping sets of primers (GenBank accession number J00314), two mutationsa Phe270Val and an Ala364Thrwere identified in M40 from paclitaxel-resistant human ovarian cancer cells (1A9PTX10 and 1A9PTX22, respectively) (2). Recently, when sequencing complementary DNA (cDNA) with similar primers, a heterozygous Leu240Ile mutation was identified in vincristine-resistant CCRF-CEM human lymphoblastic T-cell leukemia cells (3). Furthermore, cDNA from Chinese hamster lung tumor cells was sequenced for several
- and
-tubulin isotypes, and multiple nucleotide changes located outside the open reading frame were identified (4).
Using the one-dye labeled primer method for the ALFexpress sequencer (thermo sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP, Amersham Pharmacia, Uppsala, Sweden), we analyzed genomic DNA from 49 non-small-cell lung cancer (NSCLC) patients. We detected numerous heterozygous single nucleotide substitutions in the phosphate, ribose, and base regions of the -tubulin exon 4 GTP-binding site [see Fig. 1 and Table 2 of our original article (5)].
In their interesting article, Kelley et al. (6) describe a worst-case scenario with negative data on -tubulin mutations. The authors state that they "were unable to examine the base-binding region because the published sequence of oligonucleotide TB4-R is not present in TUBB." The TB4 reverse primer shown in our original article (5) was written incorrectly. The correct reading should be 5`-TGAGGTCCGGCACTGTGAG-3` (GenBank accession number J00314); however, the latest sequence from GenBank (GenBank accession number AC006165) is now correct and shows that one nucleotide of this primer sequence has changed and reads 5`-TGAGTTCCGGCACTGTGAG-3` (changed nucleotide shown in bold).
One of the major limitations in the study by Kelley et al. (6) seems to be that they scanned a small region of exon 4 that did not include the base-binding region where we found -tubulin mutations [see Fig. 1 and Table 2 of our original article (5)]. Since 1996, we have used multiple alignments of the related genomic sequences (www.ncbi.nlm.nih.gov) and used DNA analysis software (Sequencher; Gene Codes Corp., Ann Arbor, MI), confirming the presence of these mutations and ruling out the possibility that the mutations might correspond to pseudogenes. We agree that Kelley et al. (6) correctly point out in their Table 1
that some missense changes found in the ribose region (codon 180) overlap several pseudogenes (shown here in Table 1
). However, except for these changes, none of the mutations from our original study (5) were pseudogenes. In a later study (7), we used a primer pair (forward primer 5`-AAGAAGATACATCCGAGGGAATTAT-3` from intronic position 2901 and a reverse primer 5`-GGCAGTTGAGTAAGACGGCTAA-3` from exonic position 4117) (GenBank accession number AC006165) to obtain a polymerase chain reaction (PCR) product of 1216 base pairs, which encompasses the entire exon 4, a region much larger than that described by Kelley et al. By fluorescence-based sequencing of PCR products, we detected multiple heterozygous single nucleotide substitutions in genomic serum DNA from 131 patients with NSCLC (7). However, on checking the sequences in BLAST (www.ncbi.nlm.nih.gov/blast), we found that a number of these substitutions corresponded to pseudogenes, including a Val-Ile change detected in four patients.
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Because the presence of pseudogenes could not be completely ruled out at the time of our original study, the relationship between mutations and survival remains unclear. On the basis of the findings by Kelley et al. (6), we cannot rule out the possibility that some of the mutations represent rare nonpathogenic variants. Conflicting data on -tubulin research should be elucidated by sequencing cDNA, obviating the cumbersome worst-case scenario of pseudogenes.
REFERENCES
1
Nicoletti MI, Valoti G, Giannakakou P, Zhan Z, Kim JH, Lucchini V, et al. Expression of beta-tubulin isotypes in human ovarian carcinoma xenografts and in a sub-panel of human cancer cell lines from the NCI-Anticancer Drug Screen: correlation with sensitivity to microtubule active agents. Clin Cancer Res 2001;7:291222.
2
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;272:1711825.
3
Kavallaris M, Tait AS, Walsh BJ, He L, Horwitz SB, Norris MD, et al. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Res 2001;61:58039.
4 Sirotnak FM, Danenberg KD, Chen J, Fritz F, Danenberg PV. Markedly decreased binding of vincristine to tubulin in vinca alkaloid-resistant Chinese hamster cells is associated with selective overexpression of alpha and beta tubulin isoforms. Biochem Biophys Res Commun 2000;269:214.[Medline]
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;17:178696.
6
Kelley MJ, Li S, Harpole DH. Genetic analysis of the beta-tubulin gene, TUBB, in non-small-cell lung cancer. J Natl Cancer Inst 2001;93:18868.
7 Rosell R, Monzo M, Sanchez J, O'Brate A, Nogales E, Guillot M, et al. Beta-tubulin mutations in circulating extracellular DNA of non-small-cell lung cancer (NSCLC) patients [abstract]. Proc ASCO 2000;19:1897.
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