Alterations of β-tubulin isotypes in breast cancer cells resistant to docetaxel

2005 FASEB The FASEB Journal express article 10.1096/fj.04-3178fje. Published online June 9, 2005. Alterations of β-tubulin isotypes in breast cancer cells resistant to docetaxel Kawan Shalli,* Iain Brown,* Steven D. Heys,* and Andrew C. Schofield*, Schools of *Medicine and Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, Medical School, Foresterhill, Aberdeen AB25 2ZD, United Kingdom Corresponding author: Dr. Andrew C. Schofield, School of Medicine, College of Life Sciences and Medicine, University of Aberdeen, Medical School, Foresterhill, Aberdeen AB25 2ZD, UK. E-mail: a.schofield@abdn.ac.uk ABSTRACT Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the β-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in β- tubulin isotypes in vitro and has sequenced the β-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231, (oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of β-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the β-tubulin gene. At the mrna level, class I, II, III, and IVa β-tubulin mrna isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI β-tubulin mrna isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa β-tubulin mrna. However, there were increased expressions in class II, IVb, and VI β-tubulin mrna isotypes in resistant cells. Western analysis has confirmed corresponding increases in β-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III β-tubulin. This study demonstrates that altered expression of mrna β-tubulin isotypes and modulation of β-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel. Key words: drug resistance MCF-7 and MDA-MB-231 cells A n important advance in the treatment of breast cancer has been the use of the taxane docetaxel either as a single agent or as part of a combination chemotherapeutic regimen (1, 2). In terms of activity, anthracyclines and docetaxel are the most active agents against breast cancer, although two studies have suggested that docetaxel is more active than doxorubicin (3, 4). However, although up to 50% of patients treated with docetaxel will demonstrate a clinical response (5), the tumors of these patients may have either an inherent Page 1 of 15

resistance to docetaxel or may develop resistance subsequently, having been initially sensitive to this agent (6, 7). The mechanisms of action of docetaxel remain to be fully elucidated, but of particular importance is its interaction with the cellular microtubule system. The microtubular system consists of heterodimers made up of α-tubulin and β-tubulin subunits in equal proportions. This system is involved in numerous fundamentally important cellular processes (8, 9), which include maintenance of cellular shape, intracellular vesicle transport, and cell division (10). These are all of crucial importance with respect to the malignant cell. Electron crystallographic data have demonstrated that docetaxel binds to the β-tubulin subunits of the microtubule (11), which stabilizes them, resulting in their inability to depolymerize. This disruption of normal mitotic spindle formation results in an inhibition of cell division at the G 2 -M phase of the cell cycle (12). Although the molecular mechanisms by which cells either have, or acquire, resistance to docetaxel still require elucidation, on the basis of what is currently understood it is apparent that the β-tubulin gene and its resultant proteins may be important in resistance to docetaxel. Furthermore, β-tubulin exists as six different isotypes in mammalian cells. Class I β-tubulin is the most common form and is constitutively expressed, whereas the other five isotypes (class II, III, IVa, IVb, and VI) are mainly expressed in neural cells (13). In addition, these different isotypes can be variably and differentially expressed in malignant cells (14). Preliminary studies have suggested that the differential expression of β-tubulin isotypes may affect the ability of chemotherapeutic agents to bind to the microtubular system. In particular, it has been suggested that an increased expression of class III β-tubulin isotype can affect the stability of the microtubular system and may impair the stabilizing effects of taxanes in general, thus preventing its anti-tumor effects (15). To indicate further the potential importance of this as a mechanism of resistance to chemotherapy, previous studies have shown that in breast, prostate, and lung cancer cells in vitro an increased expression of class III β-tubulin is associated with resistance to paclitaxel (14, 16, 17). The role of differences in β-tubulin isotype expression in resistance to docetaxel in breast cancer is unclear. A preliminary study in patients with breast cancer did suggest that increased class III β-tubulin isotype mrna expression may also indicate patients who were unlikely to respond to docetaxel (15). However, the expressions of the other isotypes and their corresponding protein expression were not evaluated nor was the gene for β-tubulin sequenced to determine whether or not there were mutations present. This is important because mutations in the β-tubulin gene have been shown to be associated with resistance in patients receiving the taxane paclitaxel and may have been a potential mechanism of resistance in these studies (18). In trying to understand further the mechanisms of acquired resistance to docetaxel, this study, therefore, has investigated the relationship of β-tubulin isotypes and gene mutations to docetaxel resistance in breast cancer cells. In particular, the expression of all the different β-tubulin isotype mrnas and, importantly, the proteins expressed from these genes in breast cancer cells, which we have made resistant to docetaxel, have been determined. This has been compared with those demonstrated in the docetaxel-sensitive parental cells. In addition, the DNA coding region of the β-tubulin gene has been sequenced to determine whether gene mutations may have been present, which could also represent a mechanism of resistance. Page 2 of 15

MATERIALS AND METHODS Cell culture Human breast cancer cell lines, MCF-7 (oestrogen-receptor positive) and MDA-MB-231 (oestrogen-receptor negative), were cultured in RPMI-1640 medium, supplemented with 10% (v:v) fetal calf serum, 0.2% (w:v) sodium bicarbonate, and 1% (v:v) penicillin-streptomycin at 37 C in a humidified atmosphere containing 5% carbon dioxide. The cells were made resistant to docetaxel by short-term in vitro exposure to docetaxel (a gift from Aventis Pharma, West Malling, Kent, UK), as described previously (19). MCF-7 docetaxel-resistant sublines (MCF-7 TAX30) demonstrated a 666-fold greater resistance to docetaxel than MCF-7 cells. Furthermore, MDA-MB-231 docetaxel-resistant sublines (MDA-MB-231 TAX30) showed a 1375-fold greater resistance than MDA-MB-231 cells (19). Flow cytometry The DNA content of the cells was analyzed by flow cytometry to ensure the cells were in the same cell cycle phase. MCF-7 and MDA-MB-231 cells and their docetaxel-resistant sublines were cultured until the cells reached the exponential phase. A total of 1 10 6 cells was centrifuged at 400 g for 5 min and resuspended in phosphate-buffered saline (PBS). Flow cytometric analysis, with propidium iodide as a fluorochrome, was performed based on the technique of Vindelov and colleagues (20). Cells were resuspended with 0.04% (w:v) propidium iodide/detergent solution (Sigma, Gillingham, Dorset, UK) and analyzed using a CytoronAbsolute TM flow cytometer (Ortho Diagnostic Systems). The excitation source was an argon-ion laser emitting a 639 nm beam at 15 mw. Data were analyzed using the program ORTHO Immuno Count II (Ortho Diagnostic Systems). Reverse-transcription PCR (RT-PCR) Between 3 and 6 μg total RNA were reverse-transcribed, as described previously (19). Specific oligonucleotide primers (14), concentrations of magnesium chloride, and number of PCR cycles were used to amplify each β-tubulin isotype (Table 1). In addition, β 2 -microglobulin was amplified (expected amplification size of 120 bp) with each β-tubulin primer to act as an internal loading control to normalize between lanes during densitometric analysis. The oligonucleotide primer sequences for β 2 -microglobulin were as follows: forward primer 5 ACC CCC ACT GAA AAA GAT GA 3 and reverse primer 5 ATC TTC AAA CCT CCA TGA TG 3. After an initial denaturation step at 94 C for 10 min, PCR was carried out for the relevant number of cycles for class I, II, III IVa, IVb, and VI β-tubulin isotypes using the following conditions: 94 C for 30 s, appropriate annealing temperature (Table 1) for 30 s, 72 C for 90 s, proceeded by an extra 10 min extension step at 72 C. PCR products were electrophoresed through a 2% (w:v) agarose gel. Gel images were captured by Gene snap software (Syngene, Cambridge, UK), and band densities were calculated using a Fluor-S phosphoimager (Biorad, Hemel Hempstead, Hertfordshire, UK). The experiments were repeated in triplicate with RNA isolated from three independent extractions. Western analysis Proteins were extracted from the cell lines and separated by electrophoresis through 12% polyacrylamide gels (Cambrex Bioscience, Nottingham, UK), as described previously (19). Each Page 3 of 15

membrane was incubated with 1:100 dilution [in 5% (w:v) milk/tris-buffered saline with 10% (v:v) Tween 20] of either mouse monoclonal anti-human class I, III, IV β-tubulin antibodies (BioGenex); mouse monoclonal anti-human class II β-tubulin antibody (Novocastra, Newcastleupon-Tyne, UK); or 1:5000 β-actin (Abcam, Cambridge, UK) for 1 h 45 min at room temperature. It was not possible to examine the protein expression of class VI β-tubulin because no antibody was available commercially. β-actin was used as an internal loading control to normalize between lanes during densitometry. Bands were visualized using ECL+plus TM chemiluminescent detection kit (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK), according to manufacturer s instructions, and the blots were scanned with a Fluor-S PhosphorImager (Bio-Rad). The experiments were repeated in triplicate with protein isolated from three independent extractions. DNA sequencing Genomic DNA was isolated from the cell lines using QIAamp DNA mini kit (Qiagen, Crawley, West Sussex, UK), and DNA concentration was quantified by spectrophotometry. Specific oligonucleotide primers, concentrations of magnesium chloride, and annealing temperature were used to amplify the β-tubulin gene (Table 2). Primer sequences were designed using Oligo Primer Analysis Software, version 6 (Molecular Biology Insights, Cascade). Each PCR reaction contained the following components: 40 ng genomic DNA, 20 mm Tris, ph 8.4, 50 mm potassium chloride, appropriate amount of magnesium chloride (Table 2), 200 μm dntp, 10 pmol of each oligonucleotide primer, and 0.5 U Taq DNA polymerase (Roche Diagnostics, Lewes, East Sussex, UK). After an initial denaturation step at 94 C for 2 min, PCR was carried out under the following conditions for 35 cycles: 94 C for 30 s, appropriate annealing temperature (Table 2) for 30 s, 72 C for 45 s, proceeded by an extra 10 min extension step at 72 C. Excess dntp and unincorporated oligonucleotide primers were removed from the PCR products using a Montage TM PCR Centrifugal Filter Device (Millipore, Livingston, UK). DNA sequencing was performed using DYEnamic ET-Terminator cycle sequencing kit (Amersham Bioscience, Buckinghamshire, UK), according to the manufacturer s instructions. The samples were then analyzed using an automated ABI PRISM TM 377 DNA sequencing system (Applied Biosystems, Warrington, UK). Nucleotide sequences were subsequently analyzed using SeqEd TM software, version 1.0.3 (Applied Biosystems). RESULTS RNA expression of β-tubulin isotypes To determine whether an altered gene expression level of a specific β-tubulin isotype is correlated with resistance to docetaxel in breast cancer cells, mrna levels were identified by semiquantitative RT-PCR analysis. Flow cytometric analysis was performed to demonstrate cell synchrony and that cells were in the same phase of cell cycle, since β-tubulin isotypes are differentially expressed during the cell cycle (21). There were no significant differences between the percentage of cells in G 1 phase and G 2 /M phase in either MCF-7 or MDA-MB-231 breast cancer cells sensitive or resistant to docetaxel. Page 4 of 15

MCF-7 breast cancer cells RT-PCR analysis showed increased expression of class I (2.59±0.68-fold), II (1.64±0.15-fold), III (2.21±0.35-fold), IVa (3.55±0.96-fold), and IVb (1.10±0.04-fold) β-tubulin mrna levels in MCF-7 TAX30 cells compared with MCF-7 cells sensitive to docetaxel (Fig. 1). In contrast, class VI β-tubulin mrna expression was decreased in MCF-7 TAX30 cells (4.69±1.59-fold). MDA-MB-231 breast cancer cells In MDA-MB-231 TAX30 cells, there was an increase in class II (1.54±0.09-fold), III (1.07±0.16-fold), IVb (4.20±0.33-fold), and VI (10.53±3.29-fold) β-tubulin mrna expression. Class I (1.18±0.08-fold) and class IVa (1.78±0.30-fold) β-tubulin mrna expression was decreased in MDA-MB-231 TAX30 cells. Protein expression of β-tubulin isotypes MCF-7 breast cancer cells Levels of the β-tubulin protein isotypes were measured by Western analyses as described previously. It was observed that in MCF-7 TAX30 cells, β-tubulin isotype proteins were increased for class I (1.76±0.19-fold), class II (2.10±0.12-fold), class III (2.91±0.76-fold), and class IV (2.81±0.71-fold; Fig. 2). MDA-MB-231 breast cancer cells In MDA-MB-231 TAX30 cells, there was a decrease in levels of the β-tubulin isotypes for class I (1.28±0.27-fold), class II (1.30±0.11-fold), and class III (1.54±0.47-fold). However, there was an increased level of the class IV β-tubulin isotype (2.26±0.24-fold), when compared with the docetaxel-sensitive cells (Fig. 2). DNA sequencing of the β-tubulin gene To determine whether β-tubulin gene mutation may also represent a mechanism for docetaxel resistance, the coding region of the β-tubulin gene was sequenced comparing DNA from MCF-7 and MDA-MB-231 docetaxel-resistant sublines with that from their parental docetaxel-sensitive cells. There was no evidence of alteration to the DNA sequence of the β-tubulin gene in the docetaxel-resistant sublines. DISCUSSION This study is the first to demonstrate that an altered expression of β-tubulin isotypes is associated with acquired docetaxel resistance in two human breast cancer cell lines. The study also showed differences in the profile of these changes between oestrogen-receptor positive cells and oestrogen-receptor negative cells that were resistant to docetaxel. In MCF-7 (oestrogen-receptor positive) docetaxel-resistant cells, there was an increase protein expression of classes I, II, III, and IV β-tubulin. MDA-MB-231 (oestrogen-receptor negative) docetaxel-resistant cells predominantly exhibited decreased protein expression of classes I, II, and III β-tubulin isotypes but with an increase in class IV β-tubulin isotype. Page 5 of 15

The existence of multiple isotypes of β-tubulin, with tissue specificity in their expression, suggests that different isotypes may have functional significance (22). For example, there is an aberrant expression of specific β-tubulin isotypes, such as class III β-tubulin (neuron-specific expression), in tumor tissue of non-neuronal origin that may alter microtubular function (23). Furthermore, in breast tumors themselves, there is a predominance of class II β-tubulin (24), but the significance of this is unclear. However, it is possible that the relative compositions of the components of β-tubulin may dictate, or predict, tumor cell behavior and responses to chemotherapeutic agents acting via the microtubule system (25). To support this concept, it has been confirmed that differences in β-tubulin compositions influence the regulation of the dynamics of the microtubule system (26). Microtubules composed of class III β-tubulin dimers are more dynamic (they are less stable and have a spontaneous tendency toward depolymerization) than those composed of other β-tubulin isotypes (27). This alteration in microtubular dynamics has also been demonstrated previously to be able to affect the sensitivity of cells to chemotherapeutic agents. For example, a tubulin heterodimer composed of class III, or class IV, β-tubulin isotypes was less sensitive to the effects of paclitaxel than one from unfractionated tubulin (28). Although previous studies have not examined the relationship between the expression of β- tubulin isotypes and resistance to breast cancer cells in vitro, there is evidence to support their possible involvement in, or association with, resistance to other chemotherapeutic agents and other tumor types. In A549 lung cancer cells resistant to paclitaxel, there was found to be an increased expression of mrna for classes I, II, III, and IVa β-tubulin isotypes (14). However, this is expression of mrna and these authors only examined the corresponding protein expression for class III. This theme of alterations in expression of β-tubulin isotypes, particularly class III, has also been demonstrated in other tumor types, including breast cancer cells resistant to paclitaxel and also in pancreatic tumor cells (17, 29). A small preliminary study has evaluated β-tubulin isotypes in vivo in 11 ovarian tumor specimens from patients who had chemotherapy (the details of the treatment were unclear). They also documented that there was a significant increase in mrna expression of classes I, III, and IVa β-tubulin isotypes in tumors that appeared resistant to chemotherapy (14). There was no evaluation in these studies, however, as to whether these effects at the mrna level were translated to changes in the different β-tubulin proteins themselves, and this clearly is the key point. In our study, where the β-tubulin protein was actually examined, we noted an increased expression of class I, II, III, and IV β-tubulins in MCF-7 docetaxel-resistant breast cancer cells. Although there are some differences in the relative increases in expressions of mrna and protein for individual isotypes, they do correspond and are similar. This suggests that the changes in mrna are being translated to the protein level. It is noted that although it is possible to detect class IVa and IVb mrna expression, there are no commercially available antibodies to detect these separate proteins and this may have important relevance for the differences between these mrna changes and the corresponding protein changes. However, there was a different pattern in MDA-MB-231 docetaxel-resistant cells, where only the class IV β-tubulin protein was increased but class I, II, and III β-tubulin protein expressions were decreased. In particular, however, the major change was the increased expression of class IV Page 6 of 15

protein (by a factor of 2.26), the antibody for which is detecting the combined protein product of the mrna isotypes IVa (decreased by a factor of 1.78) and class IVb (increased by a factor of 4.20). The variability between mrna expression and protein expression of other β-tubulin isotypes indicates the complicated possible post-transcriptional changes that are well recognized. This, therefore, illustrates the importance of assessing protein expression with mrna expression when trying to elucidate these molecular mechanisms. Our data confirm and extend those of previous studies with regard to differences in β-tubulin mrna expression and are the first to demonstrate this in breast cancer cells resistant to docetaxel. Clearly, there are differences between the two cell lines in terms of patterns of expression of mrnas and proteins. However, one common finding was that there was an increased expression at the protein level of the class IV β-tubulin isotype. As proteins are the key effector molecules at the cellular level, this may represent a common mechanism of resistance to docetaxel in breast cancer cells. These differences, however, may also be specific to tumor and cell type, but further studies will be required to define these relationships more fully. One of the limitations of the present study may be whether an in vitro model of docetaxel resistance reflects changes that occur in vivo. Nevertheless, an important study has shown that the patterns of gene expression in breast cancer cells in vitro, exposed to chemotherapy (doxorubicin and 5-fluorouracil), were similar to those observed in patients with breast tumors treated with these drugs and mitomycin C (30). Another point to consider is that it is unclear whether the altered expression of specific β-tubulin isotypes is a consequence of the exposure of cells to docetaxel or is causative of the resistant phenotype. The changes in β-tubulin expression may arise as a direct action of docetaxel or via a secondary mechanism that remains to be clarified. However, although other mechanisms may be important in resistance to docetaxel, for example P-glycoprotein overexpression, in the case of these cells we have previously shown that inhibition of P-glycoprotein function does not fully restore sensitivity to docetaxel (31). However, the evidence as discussed above would suggest a mechanism whereby different compositions of tubulin may result in differential effects and hence variable anti-tumor activities of docetaxel (Fig. 3). Our results now require evaluation in the clinical setting. In particular, it will be important to determine if these β-tubulin isotype profiles of tumor biopsies could be used to indicate which patients receiving docetaxel would be most likely to be resistant to it before treatment commenced. This concept of molecular profiling of tumors before treatment starts is important, and in our preliminary clinical studies we showed that patients whose tumors are negative for oestrogen receptor and Bcl-2 have a higher likelihood of responding to chemotherapy (32). Furthermore, Chang and colleagues (33) have used a 92 gene expression profile to determine which tumors would be most likely to respond to docetaxel. However, this did not include an assessment of protein expression. In conclusion, we have shown for the first time that in acquired resistance there is altered expression of β-tubulin isotypes, not just at the mrna level but at the protein level, in docetaxel-resistant breast cancer cells. A specific β-tubulin expression profile may prove to be important in determining exactly how docetaxel will interact with the microtubular system and thus determine the effectivity in terms of anti-tumor actions. This requires further study, as it may be fundamentally important in understanding the molecular mechanisms of resistance to docetaxel. Page 7 of 15

ACKNOWLEDGEMENTS We would like to thank the Gates Trust, Aberdeen Royal Infirmary Breast Unit, and the Breast Cancer Campaign for financial support. In addition, we would like to acknowledge Aventis Pharma Limited for the kind gift of docetaxel. Finally, we are grateful to Susan Moir and Sarah McDonald for help with creating and confirming the docetaxel-resistant breast cancer cell lines. REFERENCES 1. Bissett, D., and Kays, S. B. (1993) Taxol and Taxotere current status and future prospects. Eur. J. Cancer 29, 1228 1231 2. Seidman, A. D., Hudis, C., and Crown, J. P. (1993) Phase II evaluation of taxotere as initial chemotherapy for metastatic breast cancer. Proc. Am. Soc. Clin. Oncol. 12A, 52 3. Chan, S., Friedrichs, K., Noel, D., Pintér, T., Belle, S. V., Vorobiof, D., Duarte, R., Gil, M. G., Bodrogi, I., Murray, E., et al. (1999) Prospective randomized trial of docetaxel versus doxorubicin in patients with metastatic breast cancer. J. Clin. Oncol. 17, 2341 2354 4. Smith, I. C., Heys, S. D., Hutcheon, A. W., Miller, I. D., Payne, S., Gilbert, F. J., Ah-See, A. K., Eremin, O., Walker, L. G., Sarkar, T. K., et al. (2002) Neoadjuvant chemotherapy in breast cancer: significantly enhanced response with docetaxel. J. Clin. Oncol. 20, 1456 1466 5. O Brien, M. E., Leonard, R. C., Barrett-Lee, P. J., Eggleton, S. P., and Bizzari, J. P. (1999) Docetaxel in the community setting: an analysis of 337 breast cancer patients treated with docetaxel (taxotere) in the UK. Annal Oncol. 10, 205 210 6. Bonneterre, J., Spielman, M., Guastalla, J. P., Marty, M., Veins, P., Chollet, P., Roché, H., Fumoleau, P., Mauriac, L., Bourgeois, H., et al. (1999) Efficacy and safety of docetaxel (taxotere) in heavily pretreated advanced breast cancer patients: the French compassionate use programme experience. Eur. J. Cancer 35, 1431 1439 7. Sjöström, J., Blomqvist, C., Mouridsen, H., Pluzanska, A., Ottosson-Lönn, S., Bengtsson, N., Østenstad, B., Mjaaland, I., Palm-Sjövall, M., Wist, E., et al. (1999) Docetaxel compared with sequential methotrexate and 5-fluorouracil in patients with advanced breast cancer after anthracycline failure: a randomised phase III study with crossover on progression by the Scandinavian breast group. Eur. J. Cancer 35, 1194 1201 8. Ringel, I., and Horwitz, S. B. (1989) A new semisynthetic analogue of taxol. J. Natl. Cancer Inst. 285, 197 203 9. Jordan, M. A., and Wilson, L. (1998) Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr. Opin. Cell Biol. 10, 123 130 10. Nogales, E. (2000) Structural insights into microtubule function. Annu. Rev. Biochem. 69, 277 302 Page 8 of 15

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Table 1 Oligonucleotide primers used to determine β-tubulin mrna expression Primer Sequences (5-3 ) PCR Product Size (bp) PCR Cycles Isotype Tann ( C)* MgCl 2 (mm) I 55 2.5 Sense: ACC TCG CTG CTC CAG CCT CT 154 23 Antisense: CCG GCC TGG ATG TGC ACG AT II 55 3.0 Sense: CGC ATC TCC GAG CAG TTC AC 208 26 Antisense: TCG CCC TCC TCC TCC TCG A III 55 3.0 Sense: CTG CTC GCA GCT GGA GTG AG 141 27 Antisense: CAT AAA TAC TGC AGG AGG GC IVa 58 2.0 Sense: TCT CCG CCG CAT CTT CCA 272 33 Antisense: GCT CTG GGG GAC ATA ATT TCC TC IVb 58 2.5 Sense: GAG CTT GCC AGC CTC GTT CT 215 30 Antisense: CCG ATC TGG TTG CCG CAC TG VI 55 2.5 Sense: ACA GTG TGT TGG CTC ACA CC 142 28 Antisense: CCG ATC TGG TTG CCG CAC TG *Oligonucleotide annealing temperature; magnesium chloride concentration; ref. 24. Page 11 of 15

Table 2 Oligonucleotide primers used to amplify and sequence the β-tubulin gene Name Tann ( C)* MgCl 2 (mm) Primer Sequences (5-3 ) PCR Product Size (bp) Exon 1 55 3.0 Sense: TTC CTG CCG TCG CGT TTG 318 Antisense: TTG AGA GGG GGA AAT CTT GA Exon 2-3 58 3.0 Sense: GGT CAA GAG ATC TAG ACC AT 942 Antisense: CTC TAC CCT CCG TTA GAT T Exon 3 50 3.0 Sense: GTC TCC CAT TTC CAG TAT ATC 261 Antisense: CTA ACT TTT CCT GTG TCC TTG Exon 4a 55 2.5 Sense: GCG CCG AGC TGG TTG ATT 240 Antisense: CGG AGA GGG TGG CAT TGT AGG Exon 4b 55 2.5 Sense: AGG CTC TGG AAT GGG CAC TCT 314 Antisense: CAT TGA GCT GGC CAG GGA AAC Exon 4c 50 3.0 Sense: GCT GAC CAC ACC AAC CTA CGG 344 Antisense: GCA TCT GCT CAT CGA CCT CCT Exon 4d 55 2.5 Sense: CTC ACC GTG GCT GCT GTC TTC 197 Antisense: TCC TGG ATG GCT GTG CTA TTG Exon 4e 50 2.5 Sense: GGC AGT CAC CTT TCA TTG GC 289 Antisense: CGG CTA AGG GAA CTG AGA A *Oligonucleotide annealing temperature; magnesium chloride concentration; ref. 16. Page 12 of 15

Fig. 1 Figure 1. Alteration of β-tubulin mrna expression in docetaxel-resistant breast cancer cells. Total RNA extracted from each cell line was amplified using β-tubulin isotype-specific oligonucleotide primer sequences. Results are presented as the ratio between parental cell lines and their docetaxel-resistant sublines. Relative expression levels have been normalized using β 2 -microglobulin as the internal control. A value of 1 indicates no difference in mrna expression between breast cancer cells and their resistant sublines. A value greater than 1 indicates an increase in expression in docetaxel-resistant sublines. Data are the mean of 3 independent experiments ± SE. Page 13 of 15

Fig. 2 Figure 2. Alteration of β-tubulin protein expression in docetaxel-resistant breast cancer cells. Protein extracted from each cell line was separated by PAGE, transferred to nitrocellulose membranes, and probed with anti-human β-tubulin isotypespecific antibodies. Results are presented as the ratio between parental cell lines and their docetaxel-resistant sublines. Relative expression levels have been normalized using β-actin as the internal control. A value of 1 indicates no difference in protein expression between breast cancer cells and their resistant sublines. A value greater than 1 indicates an increase in expression, while a value less than 1 indicates a decrease in expression in docetaxel-resistant sublines. Data are the mean of 3 independent experiments ± SE. Page 14 of 15

Fig. 3 Figure 3. Schematic representation of the role of the microtubular system in cell division and its interactions with docetaxel and effects on apoptosis (34); it is the hypothesis of a model of acquired resistance to docetaxel. Page 15 of 15