Melanoma cell sensitivity to Docetaxel-induced apoptosis is determined by class III beta-tubulin levels.
ABSTRACT We have previously shown that Docetaxel-induced variable degrees of apoptosis in melanoma. In this report, we studied the beta-tubulin repertoire of melanoma cell lines and show that class III beta-tubulin expression correlated with Docetaxel-resistance. Sensitive cells showed low levels of class III beta-tubulin with little microtubular incorporation, whereas class III beta-tubulin expression was higher in resistant cells and was incorporated into the cytoskeleton. As proof of concept, abrogation of class III by siRNA reverted Docetaxel-resistant cells to a sensitive phenotype, restoring the microtubular polymerisation response and promoting high levels of apoptosis through Bax activation. These results suggest that phenotypic expression of beta-tubulin class III in melanoma may help identify patients with melanoma that can respond to taxanes.
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ABSTRACT: Purpose: To further evaluate the use of microbeam irradiation (MBI) as a potential means of non-invasive brain tumor treatment by investigating the induction of a bystander effect in non-irradiated tissue. Methods: Adult rats were irradiated with 35 or 350 Gy at the European Synchotron Research Facility (ESRF), using homogenous (broad beam) irradiation (HI) or a high energy microbeam delivered to the right brain hemisphere only. The proteome of the frontal lobes were then analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry. Results: HI resulted in proteomic responses indicative of tumourigenesis; increased albumin, aconitase and triosphosphate isomerase (TPI), and decreased dihydrolipoyldehydrogenase (DLD). The MBI bystander effect proteomic changes were indicative of reactive oxygen species mediated apoptosis; reduced TPI, prohibitin and tubulin and increased glial fibrillary acidic protein (GFAP). These potentially anti-tumourigenic apoptotic proteomic changes are also associated with neurodegeneration. However the bystander effect also increased heat shock protein (HSP) 71 turnover. HSP 71 is known to protect against all of the neurological disorders characterized by the bystander effect proteome changes. Conclusions: These results indicate that the collective interaction of these MBI-induced bystander effect proteins and their mediation by HSP 71, may confer a protective effect which now warrants additional experimental attention.International Journal of Radiation Biology. 02/2013; 89(2).
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ABSTRACT: The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.Cell Death & Disease 01/2013; 4:e940. · 6.04 Impact Factor
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ABSTRACT: Microtubules are dynamic structures composed of alpha-beta-tubulin heterodimers that are essential in cell division and are important targets for cancer drugs. Mutations in beta-tubulin that affect microtubule polymer mass and/or drug binding are associated with resistance to tubulin-binding agents such as paclitaxel. The aberrant expression of specific beta-tubulin isotypes, in particular betaIII-tubulin, or of microtubule-regulating proteins is important clinically in tumour aggressiveness and resistance to chemotherapy. In addition, changes in actin regulation can also mediate resistance to tubulin-binding agents. Understanding the molecular mechanisms that mediate resistance to tubulin-binding agents will be vital to improve the efficacy of these agents.Nature Reviews Cancer 02/2010; 10(3):194-204. · 29.54 Impact Factor
Melanoma cell sensitivity to Docetaxel-induced apoptosis is
determined by class III b-tubulin levels
Nizar M. Mhaidata,1, Rick F. Thorneb,1, Charles Edo de Bockc, Xu Dong Zhangd, Peter Herseyd,*
aDepartment of Clinical Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan
bCancer Research Unit at the University of Newcastle, University Drive, Callaghan Newcastle, NSW 2308, Australia
cQueensland Institute of Medical Research, Herston, QLD 4006, Australia
dImmunology and Oncology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr. King & Watt Streets, Newcastle, NSW 2300, Australia
Received 25 October 2007; accepted 6 December 2007
Available online 18 December 2007
Edited by Richard Marais
variable degrees of apoptosis in melanoma. In this report, we
studied the b-tubulin repertoire of melanoma cell lines and show
that class III b-tubulin expression correlated with Docetaxel-
resistance. Sensitive cells showed low levels of class III b-tubulin
with little microtubular incorporation, whereas class III b-tubu-
lin expression was higher in resistant cells and was incorporated
into the cytoskeleton. As proof of concept, abrogation of class
III by siRNA reverted Docetaxel-resistant cells to a sensitive
phenotype, restoring the microtubular polymerisation response
and promoting high levels of apoptosis through Bax activation.
These results suggest that phenotypic expression of b-tubulin
class III in melanoma may help identify patients with melanoma
that can respond to taxanes.
? ? 2007 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
We have previously shown that Docetaxel-induced
Keywords: Melanoma; Docetaxel; Class III b-tubulin;
Given the pivotal importance of microtubules in many cellu-
lar functions, they have been the targets for anticancer drugs
such as taxanes and vinca alkaloids. By interfering with the
agents exert profound effects on cellular processes such as gene
expression, cell cycle arrest, and apoptosis [1,2]. Taxanes rep-
resent an important class of anticancer agents that have anti-
cancer effects in vitro and in vivo against cancers of lung, ova-
ries, breast, and leukemia . Recently, we have extended these
findings showing that Docetaxel induces caspase-dependent
apoptosis of some but not all human melanoma cell lines .
Resistance of cancer cells to taxanes has been attributed to
various mechanisms including over-expression of (multi-drug
resistance phenotype) P-glycoprotein that induces efflux of
the drug [5,6]. Other studies have reported that taxanes may
inappropriately induce activation of pro-survival signaling
pathways such as Ras–Raf–MEK–ERK pathway leading to
cancer cell resistance [7–9]. Studies have also shown that alter-
ation in the composition and mutations of b-tubulin isotypes,
can lead to resistance to taxanes [10,11]. Expression of mutant
tubulin or differential expression of certain tubulin isotypes has
been shown to correlate with the resistance profile of taxane-
resistant cells [10–12]. In some cases, the induced expression
of mutant tubulins  and b-tubulin isotypes classes III and
V [14,15] were shown to confer resistance to paclitaxel. Purified
mutant tubulins also have altered polymerisation characteris-
tics  and increased expression of classes I, III, and IVa iso-
types was reported in paclitaxel-resistant cells .
In the present study, we have explored the relation between
tubulin isotypes and sensitivity of melanoma cells to Docetaxel
and report that high level of class III b-tubulin can confer
resistance to Docetaxel-induced apoptosis. Moreover, a mech-
anism is suggested by the increased incorporation of class III
b-tubulin into the microtubular network in Docetaxel-resistant
2. Materials and methods
2.1. Cell lines
The panel of human melanoma cell lines used have been described
2.2. Antibodies and other reagents
Docetaxel (Taxotere), kindly provided by Aventis Pharma S.A
(France), was stored as a 100 mM solution in absolute ethanol at
?80 ?C and diluted immediately prior to use. The polyclonal anti-
Bax antibody was purchased from Upstate Biotechnology (Lake Pla-
cid, NY). The 107.3 mouse IgG1 antibody was purchased from
PharMingen (San Diego, CA). Monoclonal and polyclonal anti-a-
tubulin, classes I, III, and IV b-tubulin and b-actin were purchased
from Sigma–Aldrich (Castle Hill, NSW, Australia).
Quantitation of apoptotic cells by measurement of sub-G1 DNA
content using the PI method was carried out as described elsewhere
 under conditions established from previous work .
2.4. Measurement of tubulin polymerisation
Cytosolic and polymerised fractions of tubulin were separated by
differential centrifugation as previously described . Briefly cells
were lysed in microtubule-stabilizing buffer (20 mM Tris–HCl (pH
6.8), 0.14 M NaCl, 0.5% NP40, 1 mM MgCl2, 2 mM EGTA, and
10 ll/ml protease inhibitor cocktail (Sigma), with 4 lg/ml Docetaxel).
The polymerised fraction was obtained by collecting the supernatant
Abbreviation: MMP, mitochondrial membrane potential
*Corresponding author. Fax: +61 2 49236184.
E-mail address: Peter.Hersey@newcastle.edu.au (P. Hersey).
1These authors made an equal contribution towards the work.
0014-5793/$32.00 ? 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 582 (2008) 267–272
fraction after centrifuging the lysate at 10,000 · g for 20 min. The pel-
let (polymerised tubulin) was solubilised before electrophoresis in
SDS–PAGE sample buffer.
2.5. Indirect immunofluorescence and confocal microscopy
Melanoma cells seeded onto glass coverslips were fixed after the indi-
cated treatments before permeation with 0.1% Triton X-100. After
blocking with 3% bovine serum albumin, cells were immunostained
with primary antibodies before sequential detection with Alexa-488
anti-mouse IgG and or Alexa-594 anti-rabbit IgG (Invitrogen, Austra-
lia).In some experimentsnucleiwerevisualisedbyDAPI staining. After
mounting in SlowFade Gold reagent cells were examined using a Zeiss
Axioplan 2 epifluorescence microscope or Zeiss Axiovert 100 M fitted
with the LSM510 confocal system (Oberkochem, Germany).
2.6. Flow cytometry and mitochondrial membrane potential (DWm)
Flow cytometric analysis of permeabilised cells for Bax  and
mitochondrial membrane using JC-1 staining under established Doce-
taxel treatment conditions as previously described .
2.7. Western blot and protein expression analysis
Western blots were performed as described previously . Relative
expression was determined against control proteins (GAPDH or actin)
using densitometric analysis.
2.8. Small RNA interference (siRNA)
Transfections were performed for 48–72 h as previously described 
prior to each assay using either SiConTRol Non-targeting SiRNA
pool (D-001206-13-20) or the siGENOME SMARTpool for class III
b-tubulin (TUBB3) siRNA (Dharmacon, Lafayette, CO).
2.9. Cell viability assays
Cell viability assays were performed using the MTT method. After
siRNA treatment cells were seeded at 500 cells/well in 96 well plates
and allowed to adhere overnight before addition of Docetaxel. After
a further 72 h 30 ll of 5 mg/ml MTT (3-(4,5-dimethyl thiazolyl-2)-
2,5-diphenyl tetrazolium bromide; Sigma) was added to each well for
2 h. The supernatant was removed, the MTT formazan crystals dis-
solved in 100 ll of DMSO and the optical density read at 550 nm.
2.10. Statistical analysis
The statistical significance of intergroup differences was determined
using Student?s t-test. P values 60.05 and 60.001 are indicated by*
and**, respectively. Regression analyses were performed using the
3.1. Docetaxel-resistant melanoma cells have low levels of
Previous studies have shown that Docetaxel binds to the b-
tubulin subunit of the microtubules resulting in their polymer-
isation . We studied the Docetaxel-induced polymerisation
response of microtubules in the IgR3 and MM200 human mel-
anoma cell lines that have been shown previously to be differ-
entially sensitive to this agent . Lysates were prepared from
untreated and Docetaxel treated cells and the samples were
fractionated into soluble and polymerized tubulin fractions
as described in Section 2. Western blotting analysis showed
that under normal growth conditions, MM200 cells have a
lower level of polymerized tubulin compared to IgR3 cells
(Fig. 1A). Densitometric analysis showed that the percentage
of polymerized tubulin before and after treatment with Doce-
taxel was changed from 47% to 86% in IgR3 (P 6 0.05)
whereas a slight increase from 22% to 31% was seen in
Fig. 1. Docetaxel induces tubulin polymerisation in melanoma cells. (A) Soluble and polymerized fractions of tubulin were separated from cells
treated with or without Docetaxel at 20 nM for 3 h and the relative amounts were determined by Western blotting using antibodies against a-tubulin.
(B) Data collected from three individual experiments shown in (A) was analysed by densitometry (Columns, mean and bars, S.E.M.). (C) Cells were
also analysed by immunofluorescence microscopy for a-tubulin (green) and nuclear staining (DAPI; blue) under the treatment conditions described
for (A). bar = 20 lm.
N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272
MM200 cells (Fig. 1B). These results were confirmed by immu-
nofluorescent staining showing in IgR3 cells that Docetaxel
treatment greatly intensified the microtubular network. In con-
trast, the microtubular network of MM200 cells was largely
unchanged with Docetaxel treatment (Fig. 1C).
3.2. Human melanoma cells have variable expression of classes
III and IV b-tubulin isotypes
The ability of taxanes to bind and polymerize tubulin was re-
ported to depend upon the expression of specific b-tubulin iso-
types [21,22]. The expression of various classes of b-tubulin
was examined in a panel of melanoma cell lines by immuno-
blotting. Fig. 2A shows that melanoma cells express classes
I, III, and IV b-tubulin. Expression of class I b-tubulin was
similar between different melanoma cells whereas classes III
and IV b-tubulin varied in expression levels (Fig. 2A and B).
Since we have shown previously that Docetaxel induces var-
iable degrees of apoptosis in melanoma cells  we examined
whether this variable expression of classes III and IV b-tubulin
tubulin levels correlated with sensitivity to Docetaxel. Fig. 2C
indicates that the expression of class III b-tubulin was inver-
sely correlated with the degree of Docetaxel-induced apopto-
sis. No significant correlations were seen between expression
of class IV b-tubulin and Docetaxel-induced apoptosis
(R2= 0.1699) or with an index considering the combined ef-
fects of class III plus class IV b-tubulin expression versus
apoptosis (data not shown).
Next, we examined the cellular distribution of class III b-
tubulin in both the sensitive IgR3 and the resistant MM200
cells before and after treatment with Docetaxel (Fig. 3). Before
treatment, the staining for class III b-tubulin in the IgR3 cells
revealed a largely cytoplasmic distribution whereas a propor-
tion of class III b-tubulin was clearly incorporated into the
microtubular network in the MM200 cells. Treatment with
Docetaxel caused little alteration in MM200 cells with much
of the class III b-tubulin retained in microtubules. In contrast,
Docetaxel caused significant disruption of IgR3 cells with class
III b-tubulin localised in membranous blebs and it did not ap-
pear to contribute to the microtubular network. The incorpo-
ration of class III b-tubulin into the microtubular network
therefore appears to phenotypically correlate to the mecha-
nism of melanoma resistance to Docetaxel.
3.3. Silencing of class III b-tubulin sensitized melanoma cells to
The results suggested that resistance of melanoma cells to
Docetaxel-induced apoptosis was due to high expression of
class III b-tubulin. We therefore examined whether modula-
tion of expression levels of class III b-tubulin altered Doce-
taxel-resistance in melanoma cells. As shown in Fig. 4A,
silencing of class III b-tubulin using siRNA inhibited expres-
sion by 79% in MM200 cells compared to the cells transfected
with a control siRNA. Expression levels of classes I and IV b-
tubulins were not affected emphasising the specificity of the
class III b-tubulin siRNA. Knockdown of class III b-tubulin
expression significantly enhanced microtubule polymerisation
(P 6 0.05) and this was accompanied by significant increases
in apoptosis induced by Docetaxel (Fig. 4B). This was partic-
ularly dramatic in the MM200 cells reverting these resistant
cells to a sensitive phenotype (P 6 0.001). The increased Doce-
taxel-induced apoptosis observed after class III b-tubulin
silencing was also accompanied by enhanced Bax activation
and increases in mitochondrial membrane potential (MMP)
changes (Fig. 4C; P 6 0.001). Furthermore, class III b-tubulin
siRNA increased the anti-proliferative effects of Docetaxel in
both MM200 and IgR3 cells, reducing the IC50 from ?30 to
10.5 nM and 9 to 2 nM respectively (Fig. 4D).
In order to determine if class III b-tubulin transfection could
increase the levels of resistance of IgR3 cells to Docetaxel, we
derived permanent transfectants of these cells over-expressing
class III b-tubulin (Suppl. Fig. 1A). Although these cells dis-
played expression of class III b-tubulin at levels similar to
Fig. 2. Expression of different b-tubulin isotypes in melanoma cells. (A) Total cellular protein from a panel of melanoma cells was subjected to
Western blots for classes I, III, and IV b-tubulins. (B) The relative expression of each class was determined by dividing the densitometric value against
a b-actin control. Columns, mean of three individual experiments; bars, S.E.. (C) Correlation between the relative expression of class III b-tubulin and
the levels of apoptosis induced by 20 nM Docetaxel after 48 h.
N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272
MM200 cells (refer Fig. 2B), they were not more resistant to
Docetaxel in assays measuring apoptosis or cell viability (Sup-
pl. Fig. 1C and D). However permanent transfection of class
III b-tubulin did result in a modest increase in the expression
of a-tubulin (Suppl. Fig. 1B) that may account for this discrep-
ancy (see Section 4).
Fig. 3. Comparative distribution of a-tubulin with class III b-tubulin in IgR3 and MM200 melanoma cells before and after treatment with 20 nM
Docetaxel for 3 h. Results were recorded by dual colour confocal microscopy using monoclonal a-tubulin and class III b-tubulin polyclonal
antibodies, bar = 20 lm.
Fig. 4. Inhibition of class III b-tubulin expression sensitised melanoma cells to Docetaxel-induced apoptosis. (A) MM200 cells were transfected with
control or class III b-tubulin-specific siRNA (TUBB3) and whole cell lysates subjected to Western blot analysis for classes I, III, IV b-tubulins and a
control protein, b-actin. Data shown are representative of three individual experiments. (B) Measurements of apoptosis and tubulin polymerisation
in siRNA transfected IgR3 and MM200 cells were performed with or without 20 nM Docetaxel treatment after 48 and 3 h, respectively. (C)
Measurements of Bax activation and DWm by JC-1 staining in siRNA transfected IgR3 and MM200 cells were performed after 16–24 h with or
without 20 nM Docetaxol treatment. Data in B and C represent the means ± S.E. of three individual experiments. (D) IgR3 and MM200 cells were
treated with control or TUBB3 siRNA were subjected to the indicated dose titration of Docetaxel. After 72 h cell viability was measured using the
MTT assay. Data were averaged from triplicate determinations and normalised to the control condition, bars, S.E.M.
N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272
In the present study, we have explored for the first time the
relationship between microtubular polymerisation, the expres-
sion of tubulin isotypes and the resistance of human melanoma
cell lines to Docetaxel. We have chosen to use a relatively large
panel of cell lines that have different intrinsic properties rather
than deriving resistant tumour sublines by selection with
increasingly higher doses of drugs. The latter method is often
used to mimic events that lead to acquired drug resistance
but we believe our approach, which has been far more compre-
hensive than many of the cited studies, may be advantageous
in minimising the likelihood of in vitro artefacts.
The results showed that there was generally a low rate of
tubulin polymerisation in resistant melanoma cells and that
sensitive cells responded by increasing the microtubular mass
whereas resistant cells did not. This result appeared entirely
consistent with the known mode of action of taxanes. Exami-
nation of b-tubulin isotype expression revealed there was very
little variation in class I tubulin expression. Rather the expres-
sion of class III b-tubulin, which varied widely between differ-
ent melanoma cell lines, appeared to be a major determinant of
the degree of tubulin polymerisation and to the resistance to
Docetaxel. This concept was confirmed using an siRNA med-
iated strategy where knockdown of class III b-tubulin en-
hanced Docetaxel-induced tubulin polymerisation, activation
of Bax and downstream apoptotic events that manifested in re-
duced cell proliferation.
In contrast to the results obtained using siRNA against class
III b-tubulin, we were unable to demonstrate that high level
expression of class III b-tubulin by transfection could promote
the resistance of IgR3 melanoma cells to Docetaxel. This
somewhat unexpected result has been previously reported.
For example, transfection of class III b-tubulin into prostate
cancer cells, which is upregulated in these cells by Paclitaxel
exposure, was unable to confer resistance to Paclitaxel and
other microtubule targeting agents . However, it was deter-
mined that stable transfection of class III b-tubulin actually
caused compensatory increases in the expression of a-tubulin
in conjunction with increases in both class II and class IV b-
tubulins. The authors of this work as well as Hari et al. 
have both flagged the difficulties encountered when using gene
transfection to assay the contribution of individual tubulin iso-
types. The mechanism of the compensatory phenomenon is not
well studied but appears to be acting through transcriptional
regulation  and this also appears to be occurring in the
IgR3 cells with a modest increase in a-tubulin expression noted
to occur. We conclude from our study that siRNA is a prefer-
able technique to demonstrate the functional effects on individ-
ual tubulin isotypes. This is probably because its effects
generally occur quite rapidly and its actions are effective
against the entire cell population under treatment.
While our study showed that class III b-tubulin appeared
important in unselected melanoma cell lines, studies in other
types of cancer cell lines have revealed that alterations in b-
tubulins occurs with resistance to microtubule-targeting drugs
although not always associated with a single isotype. For
example, the increased expression of classes III and IVa b-
tubulin has also been associated with the Paclitaxel-resistance
in non-small lung carcinoma cell lines  and in K562 eryth-
roleukemia cells . Docetaxel-resistant MCF-7 breast cancer
cells also exhibited increased expression of classes I, II, III and
IV b-tubulin isotypes whereas all isotypes examined except for
class IV were decreased in Docetaxel-resistant MB-MDA-231
breast cancer cells . Notwithstanding the differences ob-
served between various cancer cell lines, increased levels of
b-tubulins do appear to have significance for clinical outcomes
with taxanes. Samples of ovarian tumours from patients trea-
ted with Paclitaxel displayed increased levels of classes I, III
and IVa b-tubulins when compared to non-treated patients
. More recently, acquired expression of class III b-tubulin
was not seen in another study of ovarian cancer, however,
the pre-treatment level of this isotype did act as an indepen-
dent prognostic indicator of poor outcome . Similarly, in-
creased expression of classes I and III b-tubulin in breast
 and class III b-tubulin non-small cell lung cancer  also
appear to predict poor outcomes with Paclitaxel and Doce-
Questions still remain in regard to the mechanism(s) that
underlie the prognostic significance of b-tubulin isotypes.
One model proposed by Carbral and colleagues associates
unstable or dynamic microtubules with the resistance of cells
to microtubule targeting agents [29,30]. In accordance, when
class III b-tubulin is assembled into microtubules it has been
reported to result in more unstable microtubules  that are
less sensitive to Paclitaxel . In our study we observed that
Docetaxel-resistance in melanoma cells coincided with in-
creased class III b-tubulin incorporation into the microtubular
network. High levels of class III b-tubulin have also been re-
ported to reduce the assembly of microtubules  and this
is also consistent with our results measuring the percentages
of microtubular polymerisation. Together these data suggest
that the large monomeric pool of class III b-tubulin acts to
promote dynamic/unstable microtubules that in turn limits
the polymerised microtubular mass in response to Docetaxel.
Towards an understanding of how this process may be regu-
lated, we have recently reported that PKCe is associated with
pro-survival signaling through ERK in melanoma cells treated
with Docetaxel . Moreover PKCe was also shown to co-
immunoprecipitate with cytosolic class III b-tubulin suggesting
it may also be influencing the incorporation rates of class III b-
tubulin into microtubules.
To our knowledge no clinical data currently exists on the
expression of b-tubulin isotypes in melanoma in vivo. In light
of the work presented here there is now a need determine if
expression of class III b-tubulin can be correlated with tumour
responses in xenograft models and in melanoma patients trea-
ted with taxanes. It will also be important to better understand
the complex mechanisms governing the cellular regulation of
the tubulin repertoire that is clearly very important in deter-
mining how cells respond to taxanes.
Acknowledgements: This work was supported by the NSW State Can-
cer Council and National Health and Medical Research Council of
Australia. X.D. Zhang is a Cancer Institute NSW Fellow. The authors
would also like to thank Mr. Tiongsun Chia for his invaluable techni-
Appendix A. Supplementary material
Supplementary data associated with this article can be
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