bII-Tubulinand bIII-TubulinMediateSensitivitytoPeloruside … · PLA and laulimalide were examined...

Preclinical Development

bII-Tubulin and bIII-Tubulin Mediate Sensitivity to PelorusideA and Laulimalide, but not Paclitaxel or Vinblastine, in HumanOvarian Carcinoma Cells

Arun Kanakkanthara1,2, Peter T. Northcote1,3, and John H. Miller1,2

AbstractIncreased abundance of bII- and bIII-tubulin isotypes in cancer cells confers resistance to vinca and taxoid

site drugs; however, the role of these isotypes in the acquired resistance of cancer cells to non-vinca or non-

taxoid site binding agents has not been described. Peloruside A (PLA) and laulimalide are the only known

non-taxoid site microtubule-stabilizing agents. A human ovarian cancer cell line, 1A9-L4 (L4), previously

selected in high concentrations of laulimalide, has both a single point mutation in bI-tubulin and over-

expression of bII- and bIII-tubulin. The cells are highly resistant to PLA as well as laulimalide but show no

cross-resistance to taxoid site drugs or drugs that bind to the vinca site on b-tubulin. To understand the

functional significance of the bII- and bIII-tubulin changes in this resistant cell line, isotype-specific short

interfering RNAwas used to knock down the expression of the bII and bIII isotypes, and the cellular effects of

PLA and laulimalide were examined before and after silencing. It was found that inhibition of bII- and bIII-tubulin partially sensitized L4 cells to PLA and laulimalide, as seen by increased potency of PLA and

laulimalide for inducing growth inhibition, cellular tubulin polymerization, microtubule aberrations, and

G2-M arrest in the resistant cells. The sensitivity to paclitaxel, vinblastine, ixabepilone, and cisplatin was

unaffected by the inhibition of isotype expression. It was concluded that the increased bII- and bIII-tubulincontributed significantly to the resistance phenotype, along with the tubulin structural mutation, and that the

altered isotype effect was binding site specific. Mol Cancer Ther; 11(2); 393–404. �2011 AACR.

Introduction

The resistance of cancer cells to antimicrotubule agentsis a serious clinical problem in the successful treatment ofcancer. This resistance is considered to be a multifactorialphenomenon involving severalmechanisms such as over-expression of the P-glycoprotein (P-gp) drug efflux pump(1), structural alterations to tubulin (2–4), altered expres-sion of microtubule regulatory proteins (5), aberrantexpression of microRNAs (6), and impaired apoptoticpathways (7). Alterations in the tubulin/microtubule sys-tem, specifically in the b-tubulin subunits, are prominentmechanisms for resistance of cancer cells to antimitoticagents (2–4).In humans, b-tubulin exists as at least 7 distinct iso-

types, with the most significant differences occurring at

the carboxyl terminus region (8). These 7 isotypes havebeen characterized for their tissue-specific expressions.For example, bI (class I) and bIVb (class IVb) are consti-tutively expressed in all tissues;bII (class II), bIII (class III),and bIVa (class IVa) are expressed mainly in brain tissue;bV is expressed constitutively, but at low levels in alltissues; and bVI (class VI) is restricted to hematopoietictissues (8–10). A strong correlation between overexpres-sion of b-tubulin isotypes and drug resistance to tubulin-targeting compounds has been reported in culturedcancer cell lines and in the clinic (2, 3), although the clinicalsignificance of b-tubulin mutations is still uncertain(11, 12). Among b-tubulin isotypes, high abundance ofbII and bIII isotypes has been highlighted as a resistancemechanism of cancer cells to various tubulin-binding andDNA-damaging agents (2, 3). To date, however, there areonly a limited number of studies that have directly inves-tigated the functional significance of these isotypes inantimitotic drug resistance. Some of these studies usedcell lines that were selectedwith taxoid or vinca site drugsor that naturally overexpressedbII- orbIII-tubulin.Hence,the significance of these isotypes in cancer cell resistanceto other classes of microtubule-active agents remainslargely unknown. Understanding the role of bII- andbIII-tubulin in the resistance to a different class of tubu-lin-binding agents would help to further clarify the com-plex mechanisms of action of the b-tubulin isotypes in

Authors' Affiliations: 1Centre for Biodiscovery and Schools of 2BiologicalSciences and 3Chemical and Physical Sciences, Victoria University ofWellington, Wellington, New Zealand

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: John H. Miller, School of Biological Sciences,Victoria University of Wellington, PO Box 600, Wellington 6140, NewZealand. Phone: 64-4-463-6082; Fax: 64-4-463-5331; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0614

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 393

on November 5, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 16, 2011; DOI: 10.1158/1535-7163.MCT-11-0614

http://mct.aacrjournals.org/

contributing to survival of cancer cells in the presence ofdiverse chemotherapeutic agents.

PelorusideA (PLA) and laulimalide (Fig. 1) are 2marineorganism–derived natural products that have shownpromising anticancer activity in a panel of different mam-malian cancer cell lines (13–15). The compounds bind to asimilar or overlapping non-taxoid site on b-tubulin (16–22) and enhance tubulin polymerization. This action inhi-bits microtubule dynamics and blocks cell-cycle progres-sion at G2-M phase, promoting cell death (14, 15). Studiesin cells have shown that PLA and laulimalide retain theircytotoxicity in paclitaxel (PTX)- and epothilone-resistantcancer cell lines that have mutations in the taxoid site ofb-tubulin (16, 17). PLA and laulimalide are also poorsubstrates for the P-gp drug efflux pump, overexpressionof which significantly reduces the inhibitory activity ofPTX and vinblastine (Fig. 1) on cancer cell growth (16, 17).

We recently showed that selection of a human ovariancarcinoma cell line, 1A9-L4 (L4), in the presence of highconcentrations of laulimalide led to multiple b-tubulinalterations that included a bI-tubulin structural mutationR306H/C, in addition to increased abundance of bII- and

bIII-tubulin isotypes (22). This cell line was highly resis-tant to laulimalide and PLA but not to other tubulin-targeting drugs that bind to the taxoid, vinca, or colchicinesites on b-tubulin (22). The mutated bI-tubulin residueR306 had earlier been modeled by computer docking andshown to be an essential site for PLA and laulimalidebinding to b-tubulin (19–21), suggesting that this bI-tubu-lin mutation was likely to be one of the key resistancemechanisms of L4 cells to the 2 compounds. The impor-tance of the bII- and bIII-tubulin overexpression in theresistant cells, however, has not been directly determined.Therefore, the aim of this studywas to examine the role ofbII- and bIII-tubulin overexpression in the resistancephenotype of L4 cells by investigating the effect of shortinterfering RNA (siRNA) knockdown of these 2 isotypeson the sensitivity of L4 cells to the compounds.

Materials and Methods

Cell cultureHuman 1A9 ovarian carcinoma cells, derived from the

A2780 cell line, were obtained fromNIH. The laulimalide/

O

O

O

OHOO

O

OHO O

O

O

OH

H

NH

O

PTX (Taxol)

O

O

OH

HHO

OO

OH

LAU

O

OMe

OH

O

OOMe

HO

HO HO

MeO

HO

PLA

VBL

HN

S

N

O

O OH O

OH

IXA (Ixempra)

CPN

Figure 1. Chemical structures of thecompounds. LAU, laulimalide; IXA,ixabepilone; VBL, vinblastine;CPN, cisplatin.

Kanakkanthara et al.

Mol Cancer Ther; 11(2) February 2012 Molecular Cancer Therapeutics394




PLA-resistant cell line, L4, was a gift from Dr. ParaskeviGiannakakou of Weill Medical College of Cornell Univer-sity, NY. Neither cell linewas directly authenticated in ourlaboratory, but both 1A9 and L4 cells retained their epithe-lial phenotype throughout the study. The derivation of theL4 cell line was as previously described (22). Cells werecultured in RPMI-1640 medium (Invitrogen) supplemen-ted with 10% fetal calf serum (Invitrogen), 0.25 units/mLinsulin (SigmaChemical Co.), 100 units/mLpenicillin, and100 units/mL streptomycin (Invitrogen). The cells weremaintained in a humidified incubator in a 5% CO2 in airatmosphere at 37�C.

siRNA transfectionsiRNAs designed to target bII- or bIII-tubulin were

purchased from Dharmacon (ON-Target plus SMART-pool reagent; Supplementary Table S1). An siRNA nega-tive control (MISSION; Sigma) that has no specificity toany human genes was used as the negative transfectioncontrol throughout the experiments. L4 cells were trans-fected with the siRNAs using Lipofectamine 2000 reagent(Invitrogen) following the manufacturer’s instructions.Briefly, the cells were seeded in 35-mm dishes andallowed to attach for 24 hours, then transfected with bIIor bIII siRNA for 48 hours. Themedium in the dishes wasreplacedafter 48hourswith complete cell culturemediumto remove any free siRNAs. The cells were then processedfor mRNA and protein quantification. To select the opti-mal siRNA concentration that gave high target knock-down, but caused minimal toxicity to the cells, eachsiRNA was titrated for its knockdown effect by transfect-ing L4 cells with 2-fold serial dilutions of siRNA. Forsimultaneous silencing of both bII- and bIII-tubulinexpression, multiplexes of siRNAs were transfected inthe same way as singleplex transfection.

DrugsPLA and laulimalide were isolated from the marine

spongesMycale hentscheli (New Zealand) and Cacospongiamycofijiensis (Tonga), respectively, and stored as a1 mmol/L stock solution in absolute ethanol at �80�C.PTX and vinblastine were purchased from Sigma Chem-ical Company. Cisplatin was purchased from EBEWEPharma, and ixabepilone was purchased from Bristol-Myers Squibb (Fig. 1).

Quantitative real-time PCRTheeffects ofbII- andbIII-tubulin siRNAson themRNA

expression of the isotypes were examined by quantitativereal-time PCR (qRT-PCR) after 48 hours of siRNA trans-fection using methodology and specific primers asdescribed previously (22).

Western blotTotal cellular proteins were extracted at 72 hours or

144 hours posttransfection, and Western blotting wasdone as previously described (22). Mouse monoclonalbII-tubulin (1:1,000, MMS-422P, clone-7B9; Covance),

bIII-tubulin (3:1,000, T8660, clone-SDL.3D10; Sigma),b-tubulin (3:1,000, T4026, clone-TUB 2.1; Sigma) andb-actin (1:3,000, A2228, clone-AC74, Sigma) primary anti-bodies and a Cy5-conjugated anti-mouse secondary anti-body (1:2,500, PA45010; GE Healthcare) were used todetect the protein bands using a Fujifilm FLA-5100 imag-ing system (Fuji Photo Film Co. Ltd). The densities of theprotein bands were quantified with ImageJ software(NIH) and normalized to the b-actin band density.

Cytotoxicity assayAfter 48 hours siRNA transfection, the cells were har-

vested and transferred to a 96-well plate (5,000 cells perwell) and cultured for 24 hours. The cells were thentreated with microtubule-stabilizing and microtubule-destabilizing agents for 72 hours, and cell proliferationwas assessed by colorimetric reduction of the dyeMTT byviable cells. The IC50 of the drugs was determined from aconcentration–response curve using Sigma Plot softwareversion 8 (Systat Software Inc.).

Intracellular tubulin polymerization assayAfter 72 hours siRNA transfection, cells were treated

with various concentrations of PLA or laulimalide for 16hours, and the polymerized and nonpolymerized tubu-lins were extracted as described elsewhere (23). The rel-ative intracellular levels of soluble (nonpolymerized) andpelleted (polymerized) tubulin fractionswere assessed byimmunoblotting against a-tubulin. A rabbit polyclonalprimary antibody to a-tubulin (1:1,000, ab18251; Abcam)in conjunction with a Cy5-conjugated goat antirabbitsecondary antibody (1:2,500, PA45011V; GE Healthcare)was used to detect the protein bands.

ImmunocytochemistryAfter 48 hours of siRNA transfection, cells were har-

vested and plated onto glass coverslips in a 35-mm dish,and the cells were allowed to attach for another 24 hours.The cells were then treatedwith various concentrations ofPLA for 16 hours and fixed with ice-cold methanol-ace-tone (1:1) for 5minutes.Nonspecific antibodybindingwasblockedwith 5%bovine serumalbumin in 0.25%TritonX-100 in PBS. The cells were then incubated at room tem-perature for 1 hour with rabbit polyclonal primary anti-body to a-tubulin (1:1,000, ab18251; Abcam). After wash-ing 3 timeswith Triton-PBS, the cells were incubatedwithAlexa Fluor 594–conjugated antirabbit secondary anti-body (1:1,000, A11012; Invitrogen) for 1 hour in the dark.After washing 3 more times, the cells were costainedwith mouse monoclonal primary antibodies to eitherbII-tubulin (1:1,000, MMS-422P, clone-7B9; Covance) orbIII-tubulin (1:1,000, T8660, clone-SDL.3D10; Sigma) andanAlexa Fluor 488–conjugated antimouse secondary anti-body (1:1,000, A11008; Invitrogen). After washing withPBS 3 times, the coverslipsweremounted onto glass slidesin Prolong Gold Antifade with 40,6-diamidino-2-pheny-lindole (DAPI) to stain the nuclei (Invitrogen). Fluores-cent staining was examined with an Olympus FluoView

Tubulin Isotype Role in Resistance

www.aacrjournals.org Mol Cancer Ther; 11(2) February 2012 395




FV1000 confocal laser scanning microscope (invertedmodel IX81) using a 60� or 100� oil-immersion objectivewith the following settings: filter, Dichrome; wavelengthrange, DAPI (425–465 nm), Alexa Fluor 488 (485–545 nm),Alexa Fluor 594 (575–620 nm); imaging mode, sequential.

Flow cytometryL4 cells were seeded at a density of 5� 104 cells perwell

in a 24-well plate and transfected with different siRNAs.Seventy-two hours after transfection, the cells were trea-ted with various concentrations of PLA and laulimalidefor 16 hours, harvested, and stained with propidiumiodide to analyze their cell-cycle distribution by flowcytometry, as previously described (22).

Results

Isotype-specific siRNAs silencebII- andbIII-tubulinmRNA and protein expression in L4 cells

Todetermine the role of bII- and bIII-tubulin isotypes inthe chemoresistance to PLA and laulimalide, isotype-specific siRNAs were used to silence the expression ofbII- and/or bIII-tubulin in L4 cells. The isotype mRNAexpression levels in L4 cells have been reported previ-ously to be 55% of total b-tubulin for bII isotype and 19%for bIII isotype, compared with 7% each for both bII andbIII in parental 1A9 cells (22). The siRNAknockdownwasoptimized by transfecting the cells with 2-fold serialdilutions of siRNA. A final concentration of 25 nmol/L,which gave the highest bII- and bIII-tubulin mRNA andprotein knockdown and showed the least adverse effecton cell proliferation was selected for further experiments(Supplementary Fig. S1). Equivalent concentrations of anonsilencing siRNA control (negative control) or Lipofec-tamine-only control (mock control) were also pairedwith each transfection experiment. The cells were trans-fected with either singleplex or multiplexes in a 1:1(25:25 nmol/L) ratio of bII- and bIII-tubulin siRNAs.Withsingleplex transfection, bII- and bIII-tubulinmRNA levelswere decreased by 80% compared with the negativecontrol- or the mock-transfected L4 cells (Fig. 2A). Theknockdown of mRNA was correlated with a decrease inthe protein levels of the isotypes (Fig. 2B and C), and theknockdown effect lasted at least 6 days (SupplementaryFig. S1).Withmultiplex (bIIþbIII) transfection, themRNAand protein expression levels of bII- and bIII-tubulinweredecreased by approximately 60% each (Fig. 2). This lowersilencing efficiency with multiplex transfection was pos-sibly caused by some loss in function of the siRNAs inthe multiplex mixture compared with when they areused separately. Although singleplex siRNAs are highlyfunctional, having a high affinity for the RNA-inducedsilencing complex (RISC), in a multiplex experiment, thesiRNAs may compete with each other for loading on theRISC. The mRNA expression and protein abundance ofother b-tubulin isotypes were not affected by the bII- andbIII-tubulin siRNA transfections. Total b-tubulin abun-dance decreased significantly after knockdown, and this

was correlated with the decreased expression of bII- andbIII-tubulin (Fig. 2). The decrease in total b-tubulin wasnot expected because of the tendency for tubulin levels toautoregulate in cells (24); however, tubulin autoregulationmay have reduced the magnitude of the decrease in totaltubulin in the siRNA-treated cells. Silencing bII and bIIIisotype expression in the parental 1A9 cells was notcarried out because these tubulin isotypes are notexpressed in significant amounts in the cells (22).

bII- and bIII-tubulin silencing sensitizes L4 cells toPLA and laulimalide

The effect of knockdown of bII- and bIII-tubulin on theresistance phenotype of L4 cells was examined by com-paring the IC50 values determined from the MTT assay.The IC50 values of the cells transfected with bII-, bIII-, orbIIþbIII-tubulin siRNAs were significantly less thanthose of their negative control siRNA–transfected cellIC50 values (Fig. 3). The mock control-transfected cells,as expected, were not significantly different from thenegative control-transfected cells for any of the drugs.For PLA, the fold increase in sensitivity in bII, bIII, andbIIþbIII siRNA-transfected cells was 1.18, 1.65, and 1.88,respectively (Fig. 3). Similar results were obtained forlaulimalide, with bII, bIII, and bIIþbIII siRNA-trans-fected L4 cells showing 1.19, 1.37, and 1.44-fold increasesin sensitivity, respectively (Fig. 3, Supplementary TableS2). These results indicated that siRNA-mediated silenc-ing of bII- and bIII-tubulin expression partially restoredthe sensitivity of L4 cells to PLA and laulimalide. Fullsensitivity similar to the parental 1A9 cells wouldrequire approximately an 18-fold change for PLA anda 25-fold change for laulimalide. Thus, although sensi-tivity was partially restored to the 2 compounds, thesiRNA-transfected L4 cells were still highly resistant tothe drugs. In contrast, the bioactivities of PTX, vinblas-tine, ixabepilone (an epothilone B analog), and cisplatinwere not affected at all by knockdown of the b-tubulinisotypes in L4 cells (Fig. 3). Epothilone B is anothermicrotubule-stabilizing agent that binds to the taxoidsite on b-tubulin. In a previous study, we showed thatthe IC50 for growth inhibition of L4 cells by epothilone Bwas the same as for the parental 1A9 cells (22).

Silencing bII- and bIII-tubulin expression enhancesintracellular PLA- and laulimalide-induced tubulinpolymerization

To determine whether bII- and bIII-tubulin silencingaffects tubulin polymerization in situ, relative levels ofsoluble (S) and polymerized (P) tubulin after PLA orlaulimalide treatment were evaluated using a cell-basedin situ tubulin polymerization assay. Knockdown ofbII- and bIII-tubulin resulted in a significant increasein drug-induced microtubule assembly compared withthe negative control siRNA-treated L4 cells (Fig. 4).Treatment with 100 nmol/L PLA in bII-, bIII-, andbIIþbIII-tubulin–silenced L4 cells induced 19%, 35%,and 46% polymerization, respectively (Fig. 4A). In






contrast, PLA at 100 nmol/L failed to induce compara-ble levels of polymerized tubulin in negative controlsiRNA-treated cells(only 1% polymerized tubulin; Fig.4A). Similar results were obtained for laulimalide. Innegative control siRNA–transfected cells, 100 nmol/Llaulimalide induced only 2% polymerized tubulin;whereas, in bII-, bIII-, and bIIþbIII-tubulin knock-downs, laulimalide at 100 nmol/L induced 24%, 39%,and 48% polymerized tubulin, respectively (Fig. 4B).Thus, there was a strong correlation with the cytotox-icity results (Fig. 3) in which the bII, bIII, and bIIþbIIIknockdowns increased the sensitivity of the cells to PLA

and laulimalide, compared with the negative controlsiRNA–transfected L4 cells.

Knockdown of bII- and bIII-tubulin affects theabundance of PLA-induced microtubule aberrations

A reduced ability of PLA and laulimalide to inducemicrotubule aberrations such as microtubule bundles andmultiple asters has been shown in L4 cells (22). To deter-mine whether the increased expression of bII- and bIII-tubulin was partially responsible for this altered drug–tubulin interaction, PLA-induced microtubule bundle for-mation was examined in siRNA-treated L4 cells using

βI βII βIII

βV βVI βI βII βII

IβV βV

I βI βII βIII

βV βVI

0

50

100

150A

B C

βII siRNAβIII siRNAβII+ βIII siRNA

* *

* *

mR

NA

exp

ressio

n level

(% N

eg

. co

ntr

ol siR

NA

)

Mock

contr

ol

Mock control

βII siRNA

βII+βIII siRNA

Negative control

Mock control

βIII siRNA

βII+βIII siRNA

Negative control

βII siRNA

βIII siRNA

βII+βIII siRNA

Negative control

βII s

iRNA

βII+βII

I siR

NA

Mock

contr

ol

βIII s

iRNA

βII+βII

I siR

NA

βIII s

iRNA

βII+βII

I siR

NA

0

50

100

150

βII-tubulin βIII-tubulin

*

Total β-tubulin

**

*

βII s

iRNA

*

*

*

Pro

tein

ab

un

dan

ce

(% N

eg

. co

ntr

ol siR

NA

)

βII-tubulin

β-actin

βIII-tubulin

β-actin

Total β-tubulin

β-actin

Figure 2. qRT-PCR and Western blot confirmation of bII- and bIII-tubulin knockdown in L4 cells after siRNA transfection. A, L4 cells were transfected with25 nmol/L bII- or bIII-tubulin targeting siRNAs, and bII- or bIII-tubulin mRNA expression (after 48 hours) was examined using qRT-PCR. bII, bIII, and bIIþbIIIsiRNAs specifically silenced their target mRNAs without affecting other b-tubulin isotype levels. mRNA expression is presented as the percent of thenegative control siRNA–transfected L4 cells�SEM (n¼4 independent experiments). B, representativeWestern blots of bII-, bIII-, and total b-tubulin in bII,bIII,and bIIþbIII siRNA-transfected L4 cells 72 hours after siRNA transfection. The negative control has siRNA unrelated to any human gene sequences. Themock control has Lipofectamine but lacks siRNA. The bIIþbIII–silenced cell lysates were electrophoresed on a separate gel and the blots added to the others,which had been run together on the same gel. b-Actin was used as a loading control. C, protein abundance as a percent of the negative controlsiRNA–transfected L4 cells � SEM (n ¼ 3–4 independent experiments). �, P < 0.05, Mann–Whitney test.






immunocytochemistry and confocal microscopy. Therewas no difference in themicrotubulemorphology betweenthedrug-untreatedbII-,bIII-,bIIþbIII-tubulinknockdownsand the negative control siRNA–transfected cells (Fig. 5).However, in agreement with the tubulin polymerizationresults, 100 nmol/L PLA significantly increased the for-mation of microtubule bundles in bII (31.8 � 0.5%), bIII(47.8 � 2.6%), and bIIþbIII (57.6 � 2.0%) silenced L4 cellscompared with the negative control siRNA–treated cells(19.5� 0.7%; Fig. 5, Supplementary Table S3). The negativecontrol siRNA-treated cells needed at least 500 nmol/LPLA to obtain an equivalent proportion of microtubulebundles to that seen in the knockdown cells.

Inhibition of bII- and bIII-tubulin expressionpromotes PLA- and laulimalide-induced G2-M block

To assess whether silencing of bII- and bIII-tubulininfluences antimitotic activity of PLA and laulimalide inL4 cells, cell-cycle analysis was done. Tubulin isotypesiRNA transfection itself had no effect on the percentageof cells in G2-M in the absence of drug treatment (23%–25%; Fig. 6). There were also no differences in cell-cycleeffects of the drugs between negative control siRNA–transfected and siRNA-untransfected, Lipofectamine-

treated L4 cells (data not presented). However, lowerconcentrations of PLA and laulimalide were needed toinduce G2-M block in the isotype-specific siRNA-trans-fected cells compared with negative control siRNA–trea-ted cells. Cells transfected with bII-, bIII-, or bIIþbIII-tubulin siRNAs arrested in their G2-M phase at 300nmol/L PLA or 80 nmol/L laulimalide, whereas thenegative control siRNA–transfected cells showed no sig-nificant G2-M block at these concentrations (Fig. 6). Therewas no significant G2-M arrest at the lower concentrationsof PLA and laulimalide tested. The concentrations ofdrugs used in Fig. 6 (300 nmol/L PLA and 80 nmol/Llaulimalide)were lower than the threshold concentrationsneeded in previous studies on L4 cells (500 nmol/L PLAand 200 nmol/L laulimalide; ref. 22). In bIIþbIII-tubulin–silenced cells, a greater G2-M block was seen (58% withPLA and 57%with laulimalide) comparedwith bIII-tubu-lin–silenced cells (52% with PLA and 51% with laulima-lide) and bII-tubulin–silenced cells (44% with PLA and43% with laulimalide; Fig. 6). These differences wereconsistent with the effects of silencing seen on cell growthand aberrant microtubule morphology, indicating againthat the increased abundance ofbII- andbIII-tubulin alterscell-cycle responses to PLA and laulimalide, and the

120

100

80

60

40

20

0

PLA

Mock control

Neg. control siRNA

βII siRNAβII + βIII siRNA

βIII siRNA

Mock control: 219.8 ± 9.6Neg. control: 228.3 ± 5.2

βII siRNA: 194.0 ± 3.9**

βII+βIII siRNA: 121.7 ± 3.5**βIII siRNA: 138.1 ± 6.5**


βII siRNA: 151.4 ± 2.0*

βII+βIII siRNA: 125.2 ± 6.4*βIII siRNA: 131.5 ± 3.9*


βII siRNA: 4.8 ± 0.2

βII+βIII siRNA: 4.1 ± 0.3βIII siRNA: 4.2 ± 0.5

Mock control: 3213 ± 43Neg. control: 3311 ± 24

βII siRNA: 3363 ± 17

βII+βIII siRNA: 3333 ± 23βIII siRNA: 3281 ± 29


βII siRNA: 3.1 ± 0.4

βII+βIII siRNA: 3.0 ± 0.1βIII siRNA: 2.9 ± 0.5

Neg. control: 7.0βII siRNA: 7.1

βII+βIII siRNA: 7.2βIII siRNA: 6.6

LAU PTX

VBL IXA CPN

% C

ontr

ol

120

100

80

60

40

20

% C

ontr

ol

120

100

80

60

40

20

0

% C

ontr

ol

120

100

80

60

40

20

0

% C

ontr

ol

120

100

80

60

40

20

0%

Con

trol

120

100

80

60

40

20

% C

ontr

ol

Concentration (nmol/L)

0 10 100 1,000 1 10 100 1,00010.1 10 100 1,000

Concentration (nmol/L) Concentration (nmol/L)

10.1 10 100 1 10 100 100 1,000 10,000 100,0001,000

Concentration (nmol/L) Concentration (nmol/L) Concentration (nmol/L)

Figure 3. Effect of bII- and bIII-tubulin silencing on the sensitivity of L4 cells to PLA, laulimalide, PTX, vinblastine, ixabepilone, and cisplatin. The IC50 of thecompounds in siRNA-transfected L4 cells was determined by MTT assay. Representative graphs are presented for each compound, and the average IC50

values� SEM (nmol/L) are presented on each graph (n¼ 3–5 independent experiments, with the exception of IXA (n¼ 1 experiment). �, P < 0.05; ��, P < 0.01,Mann–Whitney test comparing negative control to target siRNA knockdown. For PLA, bII siRNA versus bIII siRNA and bII siRNA versus bIIþbIII siRNA effectswere also significantly different (P < 0.01; not shown in table), and for laulimalide, bII siRNA versus bIIþbIII siRNA effects were significantly different (P < 0.05).LAU, laulimalide; VBL, vinblastine; IXA, ixabepilone; CPN, cisplatin.






effects of knockdown of the 2 isotypes are somewhatadditive.

Discussion

Resistance phenotypebII- and bIII-tubulin knockdown partially sensitized

resistant L4 cells to both PLA and laulimalide. Knock-

down of bII-tubulin had less of an effect than that ofbIII-tubulin, even though it was previously shown thatexpression of bII isotype in L4 cells was enhanced morethan bIII isotype (7.4- vs. 5.6-fold; ref. 22). Simultaneousknockdown of both isotypes increased the sensitivitymore than knockdown of either isotype on its own.Although the changes in sensitivity were small com-pared with the total resistance of the L4 cell line, they

Figure 4. PLA- and laulimalide-induced intracellular tubulinpolymerization in siRNA-transfectedL4 cells. L4 cells were transfectedwith the negative control or bII, bIII,andbIIþbIII siRNAs for 72hours, thentreated with PLA or laulimalide for 16hours, and the drug-induced tubulinpolymerization was determined byan intracellular tubulinpolymerization assay. Immunoblotsof PLA (n ¼ 3 independentexperiments; A) and laulimalide(n ¼ 2 independent experiments; B)are shown. The percent solubletubulin (S) and polymerized tubulin(P) are presented below each proteinband. LAU, laulimalide.






were highly significant. We showed previously thatPLA and laulimalide have a reduced ability to inducecellular tubulin polymerization, microtubule bundling,and G2-M block in L4 cells compared with the parental1A9 cells (22). Given the bI-tubulin mutation R306H/Cin L4 cells (22) and its location in the proposed bindingsite for PLA and laulimalide (19–21), it is likely thatmost of the resistance of the cell line to these drugs is aresult of this structural mutation; however, this stillneeds to be directly tested. The sensitivity of the celllines was tested using a cell proliferation assay thatmonitors growth and death of cells by changes in cellmetabolism. Use of a clonogenic assay instead mighthave enhanced the sensitivity of the assay to the silenc-ing effects of the siRNA, although given that the siRNAknockdown persisted for up to 6 days (SupplementaryFig. S1), the MTT assay used was well within thewindow of the silencing effect.

bIII-Tubulin role in resistancebIII-Tubulin overexpression has been correlated with

resistance to microtubule-stabilizing and microtubule-destabilizing drugs (2). bIII-Tubulin seems to have a

role in microtubule dynamics and in opposing theability of tubulin-binding agents to suppress spindledynamics. For example, Panda and colleagues (25)showed that microtubules assembled from purifiedabIII isotype were considerably more dynamic thanmicrotubules made from the abII or abIV isotypes orfrom unfractionated tubulin that consisted of a mixtureof a- and b-tubulin isotypes. Consistent with this, Derryand colleagues (26) showed that microtubules com-posed of purified abIII-tubulin were 7.4-fold lesssensitive to the effects of PTX than microtubules assem-bled from unfractionated tubulin. In another study,overexpression of bIII-tubulin in transfected Chinesehamster ovary (CHO) cells decreased microtubuleassembly and conferred resistance to PTX (27). Removalof bIII-tubulin from the tubulin pool led to more rapidPTX-induced tubulin assembly (28). In a PTX-resistantlung cancer cell line, A549-T24, that is 17-fold resistantto PTX and has a 4-fold increase in bIII-tubulin expres-sion compared with the parental A549 cells, siRNA-mediated inhibition of bIII expression caused a 39%increase in sensitivity to PTX (29). A more recent studyby Gan and colleagues (30) showed that bIII-tubulin

Untreated

Neg. control siRNA

βII siRNA

βIII siRNA

βII+βIII siRNA

PLA 10 nmol/L PLA 100 nmol/L

Figure 5. PLA-induced microtubulebundling in siRNA-transfected L4cells. Seventy-two hours aftersiRNA transfection, L4 cells weretreated for 16 hours with PLA andcostained with antibodies againstbII- or bIII-tubulin and a-tubulin.Nuclei were stained with DAPI. Forbetter visualization of microtubulebundles, only a-tubulin staining isshown in the image. White arrowspoint to microtubule bundles. Thefull-color fluorescence images ofthe bII-, bIII-tubulin, and nuclearstaining of the cells are presentedin Supplementary Fig. S2.






knockdown in non-small cell lung carcinoma cellsenhanced the suppression of microtubule dynamics atlow concentrations of PTX or vincristine. These resultssupport the earlier studies (26–28) showing that over-expression of bIII-tubulin reduces the ability of PTX toinhibit microtubule dynamic instability. High levels ofexpression of bIII-tubulin have been correlated withresistance to docetaxel in breast (31) and prostate (32)cancer cells as well.bIII-Tubulin is a multifunctional protein that, when

suppressed, increases the in vitro and in vivo sensitivity ofcells to tubulin-binding and DNA-damaging agents, suchas cisplatin, through enhanced apoptosis and decreasedtumorigenesis (33, 34). The involvement of bIII-tubulin inmediating the sensitivity to DNA-damaging agents (33)suggests that bIII isotype overexpression, in addition to itsdestabilizing activity, might have a role as a cellular sur-

vival factor against chemotherapy. This is supported by thestudies of Raspaglio and colleagues (35) who showed thatcellular stresses, suchashypoxia, can induce theexpressionof bIII-tubulin. Cicchillitti and colleagues (36) also showedthat bIII-tubulin overexpressionwas associatedwith adap-tation to oxidative stress and glucose deprivation. Themechanism by which bIII-tubulin might alter these cellstress pathways, however, is not known.More recently, DeDonato and colleagues (37) showed that bIII-tubulin canact as a cytoskeletal gateway for prosurvival signals.

In this study,we showed a clear decrease in the resistantphenotype after siRNA-mediated silencing ofbIII isotype,yet the silencing of bIII had no effect on the normalsensitivity to PTX, vinblastine, ixabepilone, or cisplatin.Possible reasons for not finding resistance of L4 cells toPTX, vinblastine, and cisplatin in our study are discussedbelow.

A

B CLAUPLA

0 20 40 60 80 100

20

40

60

80

Neg. control siRNA

βII siRNA

βIII siRNAβII+βIII siRNA*

LAU concentration (nmol/L)

0 100 200 300 400

20

40

60

80

Neg. control siRNA

βII siRNA

βIII siRNAβII+βIII siRNA*

PLA concentration (nmol/L)

% C

ells in

G2–M

% C

ells in

G2–M

Figure 6. G2-M block induced by PLA and laulimalide in siRNA-transfected L4 cells. bII, bIII, and bIIþbIII siRNA-transfected cells were treated with PLA orlaulimalide for 16 hours, and the DNA content in each phase of the cell cycle was analyzed using flow cytometry. Representative histograms are shown in A.The percentage of cells in each phase of the cell cycle is presented at the top of each histogram. A summary of the percentage of cells in G2-Mphase followingtreatment with PLA and laulimalide is given in B and C. Results are based on 4 independent experiments, bars ¼ SEM; P < 0.01 for the bIIþbIII siRNAknockdowns for both PLA and laulimalide. Single knockdowns of bII and bIII were not significant; Kruskal–Wallis test compared negative control to targetsiRNA knockdown. Using a Dunn multiple comparison test, the only significant differences were for the double knockdowns for PLA at 300 nmol/L andlaulimalide at 80 nmol/L. �, P < 0.01. LAU, laulimalide.






bII-tubulin role in resistanceThe mechanisms by which bII-tubulin overexpression

confers resistance on cells are poorly understood. Purifiedvertebrate bII-tubulin has different assembly and drug-binding properties to a mixture of b-tubulin isotypes (38).Usingmonoclonal antibodies specific for bII, Banerjee andcolleagues (39) prepared isotypically pure abII tubulindimers from bovine brain and examined their assemblyproperties in the presence of microtubule-associated pro-teins (MAP) and PTX. They found that, in the presence ofMAPs, the abII dimers assembled into microtubules con-siderably faster than unfractionated tubulin dimers.Derry and colleagues (26) measured the effects of PTXon the dynamics of microtubules composed of purifiedabII-tubulin isotypes and showed thatmicrotubules com-posed of purified abII were 1.6-fold less sensitive to theeffects of PTX than microtubules assembled from unfrac-tionated tubulin. Consistent with this, several other stud-ies in cells have reported high levels of bII-tubulin isotypein PTX-resistant ovarian (40), murine J774.2 (41), andDTX-resistant breast cancer cell lines (42). Whereas Ganand Kavallaris (43) showed that siRNA-mediated knock-down of bII-tubulin hypersensitized the lung cancer celllines NCI-H460 and Clau-6 to vinca alkaloids, no changein the sensitivity to PTX was seen in these cell linesfollowing knockdown of the bII-tubulin isotype.

In this study, we show that knockdown of bII-tubulindecreases the resistance of L4 cells to PLA and lauli-malide by about 15%, and this effect is correlated withan increased ability of the compounds to induce tubulinpolymerization, microtubule aberrations, and G2-Mblock in the cells. To our knowledge, this is the firstcellular study to directly show the involvement of bII-tubulin overexpression in limiting drug-induced tubu-lin polymerization and in conferring selective resistanceto microtubule-stabilizing agents. The only publishedstudy that has shown a role for bII-tubulin in reducingthe suppressive effect of PTX on microtubule dynamicsused in vitro purified tubulin (26).

Lack of resistance of L4 cells to PTX, vinblastine,and cisplatin

An important finding in this study was that, despiteoverexpression of bII and bIII tubulin, L4 cells retained anormal sensitivity to PTX, vinblastine, and cisplatin.Although the studies described above link overexpres-sion of these isotypes to resistance to these drugs (2, 3),some other studies have found results similar to ours. Ina phase III clinical trial with docetaxel and doxorubicinon patients with locally advanced or metastatic breastcancer, tumors with higher levels of bIII-tubulin isotypehad an increased probability of showing a response todocetaxel (44). High abundance of bIII-tubulin has alsocontributed to an increased sensitivity to epothilone B inhuman ovarian cancer cells (45). In an in vivo study,Nicolletti and colleagues (46) reported no correlationbetween isotype expression and PTX sensitivity. Inanother study using CHO cells, overexpression of bII-

tubulin had no effect on the sensitivity of the cells toPTX (47). Thus, overexpression of bII- or bIII-tubulindoes not always confer cancer cell resistance to PTX orvinblastine. Although, in vitro systems have shown amicrotubule-destabilizing property of bIII-tubulin, itsinvolvement in the resistance to vinca site drugs andDNA-damaging agents suggests that these drugs canalso implement different cellular survival pathwaysagainst specific chemotherapies. This might in partexplain the fact that in L4 cells, bII- or bIII-tubulininduced a resistance mechanism that specifically affect-ed PLA and laulimalide, but not PTX, vinblastine, orcisplatin. Recently, Wilmes and colleagues (48) showedin the human HL-60 promyelocytic leukemia cell linethat there were differences in the proteomic effects ofPLA and PTX, particularly with regard to apoptoticproteins. This suggests that PLA and PTX may activatedifferent apoptotic pathways that are differentiallyaffected by bII and bIII expression levels.

Another possible explanation for the different effects onPLA/laulimalide and PTX/vinblastine/cisplatin is thattumor cells may acquire resistance to tubulin-bindingagents by overexpressing specific isotypes to which thedrugshave a reducedbindingaffinity.Usingdigital signalprocessing, Chen and colleagues (49) modeled the bind-ing affinities of PLA, PTX, and vinblastine to 3 b-tubulinisotypes: bII, bIII, and bIV. The authors predicted that theorder of binding affinity of PTX and vinblastine wasbII>bIV>bIII and bIV�bII»bIII, respectively. They pre-dicted that PLA would have a reduced affinity for bII-and bIII-tubulin isotypes, comparedwith that for the bIV-tubulin isotype (bIV>bII¼bIII). This altereddrug-bindingaffinity is further supported by a more recent study byBegaye and colleagues (50) who showed that an A296Smutation confers 10- to 15-fold resistance to PLA in 1A9ovarian carcinoma cells. Importantly, the human bII iso-type also has a serine residue at position 296. Thus, in thecase of PLA and laulimalide, the overexpression of bIIisotypes with lesser affinities for the 2 drugs could limitthe binding of the drugs and reduce their potency in cellsoverexpressing these isotypes. For bIII-tubulin, it seemsthat its function in other cell lines that mediates changesseen in sensitivity to PTX, vinblastine, and cisplatin (2) isnot active in the L4 cells. Either the bI-tubulinmutation inL4 cells is inhibiting or compensating for this function ofthe bIII isotype, or bIII-tubulin itself is altered or mutatedin L4 cells such that it no longer affects sensitivity to PTX,vinblastine, and cisplatin but reduces the interaction ofPLA and laulimalide with tubulin. It would be interestingto sequence thebIII-tubulin inL4 cells to seewhether it hascomutated with bI-tubulin.

Conclusions

This study clearly showed that knockdown of bII- andbIII-tubulin increased the sensitivity of resistant L4 cellsto PLA and laulimalide, and that this increased sensiti-vity was associated with an increase in drug–tubulin






interactions in the cells, as evidenced by intracellulartubulin polymerization, formation of microtubule bun-dles, and G2-M block. The exact mechanism bywhich bII-and bIII-tubulin affect L4 cell resistance is, however, notknown. The lack of resistance of L4 cells to PTX, vinblas-tine, and cisplatin suggests that the effect of these isotypeson resistance is specific for PLA and laulimalide com-pared with other microtubule-targeting and DNA-dam-aging agents. This difference might be related to thedistinct PLA- and laulimalide-binding site on b-tubulinand differences in the mechanisms of polymerization orkilling by these drugs.

Disclosure of Potential Conflicts of Interest

P.T. Northcote and J.H. Miller have a patent on Peloruside A.

Acknowledgments

The authors thankDr. Paraskevi Giannakakou for kindly providing the1A9 and L4 cell lines.

Grant Support

This research was supported by grants to J.H. Miller from the CancerSociety of New Zealand, the Wellington Medical Research Foundation,and the Victoria University of Wellington.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedAugust 10, 2011; revisedDecember 2, 2011; acceptedDecember7, 2011; published OnlineFirst December 16, 2011.

References1. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role

of ATP-dependent transporters. Nat Rev Cancer 2002;2:48–58.2. Kavallaris M. Microtubules and resistance to tubulin-binding agents.

Nat Rev Cancer 2010;10:194–204.3. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field

of cancer therapeutics. Nat Rev Drug Discov 2010;9:790–803.4. Yin S, Bhattacharya R, Cabral F. Human mutations that confer pac-

litaxel resistance. Mol Cancer Ther 2010;9:327–35.5. Martello LA, Verdier-Pinard P, Shen HJ, He L, Torres K, Orr GA, et al.

Elevated levels ofmicrotubule destabilizing factors in a taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation. Cancer Res2003;63:1207–13.

6. Zhou M, Liu Z, Zhao Y, Ding Y, Liu H, Xi Y, et al. MicroRNA-125bconfers the resistance of breast cancer cells to paclitaxel throughsuppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expres-sion. J Biol Chem 2010;285:21496–507.

7. Kutuk O, Letai A. Alteration of the mitochondrial apoptotic pathway iskey to acquired paclitaxel resistance and can be reversed by ABT-737.Cancer Res 2008;68:7985–94.

8. Sullivan KF, Cleveland DW. Identification of conserved isotype-defin-ing variable region sequences for four vertebrate b-tubulin polypeptideclasses. Proc Natl Acad Sci U S A 1986;83:4327–31.

9. Wang D, Villasante A, Lewis SA, Cowan NJ. The mammalian b-tubulinrepertoire: hematopoietic expressionof a novel heterologousb-tubulinisotype. J Cell Biol 1986;103:1903–10.

10. Burgoyne RD, Cambray-Deakin MA, Lewis SA, Sarkar S, Cowan NJ.Differential distribution of b-tubulin isotypes in cerebellum. EMBO J1988;7:2311–9.

11. MaenoK, ItoK,HamaY,ShinguK,KimuraM,SanoM, et al.Mutation ofthe class I b-tubulin gene does not predict response to paclitaxel forbreast cancer. Cancer Lett 2003;198:89–97.

12. Mesquita B, Veiga I, Pereira D, Tavares A, Pinto IM, Pinto C, et al. Nosignificant role for beta tubulin mutations and mismatch repair defectsin ovarian cancer resistance to paclitaxel/cisplatin. BMC Cancer2005;5:101.

13. West LM, Northcote PT, Battershill CN. Peloruside A: a potent cyto-toxic macrolide isolated from the New Zealand marine sponge Mycalesp. J Org Chem 2000;65:445–9.

14. Mooberry SL, Tien G, Hernandez AH, Plubrukarn A, Davidson BS.Laulimalide and isolaulimalide, new paclitaxel-like microtubule stabi-lizing agents. Cancer Res 1999;59:653–60.

15. Hood KA, West LM, Rouw�e B, Northcote PT, Berridge MV, WakefieldSJ, et al. Peloruside A, a novel antimitotic agent with paclitaxel-likemicrotubule stabilizing activity. Cancer Res 2002;62:3356–60.

16. Gaitanos TN, Buey RM, Díaz F, Northcote PT, Spittle PT, Andreu JM,et al. Peloruside A does not bind to the taxoid site on b-tubulin and

retains its activity in multidrug-resistant cell lines. Cancer Res2004;64:5063–7.

17. Pryor DE, O'Brate A, Bilcer G, D��az JF, Wang Y, Wang Y, et al. Themicrotubule stabilizing agent laulimalide does not bind in the taxoidsite, kills cells resistant to paclitaxel and epothilones, and maynot require its epoxide moiety for activity. Biochemistry 2002;41:9109–15.

18. Hamel E, Day BW, Miller JH, Jung MK, Northcote PT, Ghosh AK, et al.Synergistic effects of peloruside A and laulimalide with taxoid sitedrugs, but not with each other, on tubulin assembly. Mol Pharmacol2006;70:1555–64.

19. BennettMJ,BarakatK,Huzil JT, Tuszynski J, SchriemerDC.Discoveryand characterization of the laulimalide-microtubule binding mode bymass shift perturbation mapping. Chem Biol 2010;17:725–34.

20. Nguyen TL, Xu X, Gussio R, Ghosh AK, Hamel E. The assembly-inducing laulimalide/peloruside A binding site on tubulin: Molecularmodeling and biochemical studies with [3H]peloruside A. J Chem InfModel 2010;50:2019–28.

21. Khrapunovich-Baine M, Menon V, Huang Yang CP, Northcote PT,Miller JH, Hogue Angeletti R, et al. Hallmarks of molecular action ofmicrotubule stabilizing agents: Effects of epothilone B, ixabepilone,peloruside A, and laulimalide on microtubule conformation. J BiolChem 2011;286:11765–78.

22. Kanakkanthara A, Wilmes A, O'Brate A, Escuin D, Chan A, Gjyrezi A,et al. Peloruside- and laulimalide-resistant human ovarian carcinomacells have bI-tubulin mutations and altered expression of bII- and bIII-tubulin isotypes. Mol Cancer Ther 2011;10:1419–29.

23. Giannakakou P, Sackett DL, Kang YK, Zhan Z, Buters JTM, Fojo T,et al. Paclitaxel-resistant human ovarian cancer cells have mutantb-tubulins that exhibit impaired paclitaxel-driven polymerization. J BiolChem 1997;272:17118–25.

24. Gay DA, Sisodia SS, Cleveland DW. Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation. Proc NatlAcad Sci U S A 1989;86:5763–7.

25. Panda D, Miller HP, Banerjee A, Ludue~na RF, Wilson L. Microtubuledynamics in vitro are regulated by the tubulin isotype composition.Proc Natl Acad Sci U S A 1994;91:11358–62.

26. Derry WB, Wilson L, Khan IA, Luduena RF, Jordan MA. Taxol differ-entially modulates the dynamics of microtubules assembled fromunfractionated and purified beta-tubulin isotypes. Biochemistry1997;36:3554–62.

27. Hari M, Yang H, Zeng C, Canizales M, Cabral F. Expression of class IIIbeta-tubulin reduces microtubule assembly and confers resistance topaclitaxel. Cell Motil Cytoskeleton 2003;56:45–56.

28. Lu Q, Ludue~na RF. Removal of bIII isotype enhances taxol inducedmicrotubule assembly. Cell Struct Funct 1993;18:173–82.






29. Kavallaris M, Burkhart CA, Horwitz SB. Antisense oligonucleotides toclass III beta-tubulin sensitize drug-resistant cells to Taxol. Br JCancer1999;80:1020–5.

30. GanPP,McCarroll JA, Po'uha ST, Kamath K, JordanMA, KavallarisM.Microtubule dynamics, mitotic arrest, and apoptosis: Drug-induceddifferential effects of bIII-tubulin. Mol Cancer Ther 2010;9:1339–48.

31. Hasegawa S, Miyoshi Y, Egawa C, Ishitobi M, Taguchi T, Tamaki Y,et al. Prediction of response to docetaxel by quantitative analysis ofclass I and III beta-tubulin isotype mRNA expression in human breastcancers. Clin Cancer Res 2003;9:2992–7.

32. PloussardG, Terry S,Maill�eP, Allory Y, SirabN, Kheuang L, et al. ClassIII beta-tubulin expression predicts prostate tumor aggressivenessand patient response to docetaxel-based chemotherapy. Cancer Res2010;70:9253–64.

33. Gan PP, Pasquier E, Kavallaris M. Class III b-tubulin mediates sensi-tivity to chemotherapeutic drugs in non small cell lung cancer. CancerRes 2007;67:9356–63.

34. McCarroll JA, Gan PP, Liu M, Kavallaris M. bIII-tubulin is a multifunc-tional protein involved in drug sensitivity and tumorigenesis in non-small cell lung cancer. Cancer Res 2010;70:4995–5003.

35. Raspaglio G, Filippetti F, Prislei S, Penci R, De Maria I, Cicchillitti L,et al. Hypoxia induces class III beta-tubulin gene expression byHIF-1alpha binding to its 30

flanking region. Gene 2008;409:100–8.36. Cicchillitti L, Penci R, Di Michele M, Filippetti F, Rotilio D, Donati MB,

et al. Proteomic characterization of cytoskeletal and mitochondrialclass III beta-tubulin. Mol Cancer Ther 2008;7:2070–9.

37. DeDonatoM,MarianiM, Petrella L,Martinelli E, Zannoni GF, Vellone V,et al. Class III b-tubulin and the cytoskeletal gateway for drug resis-tance in ovarian cancer. J Cell Physiol 2011 Apr 25 [Epub ahead ofprint].

38. Ludue~na RF. Are tubulin isotypes functionally significant. Mol Biol Cell1993;4:445–57.

39. Banerjee A, Roach MC, Trcka P, Luduena RF. Preparation of amonoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III,and alpha beta IV tubulin dimers from bovine brain. J Biol Chem1992;267:5625–30.

40. Kavallaris M, Kuo DYS, Burkhart CA, Regl DL, Norris MD, Haber M,et al. Taxol-resistant epithelial ovarian tumors are associated with

altered expression of specific b-tubulin isotypes. J Clin Invest1997;100:1282–93.

41. Haber M, Burkhart CA, Regl DL, Madafiglio J, Norris MD, Horwitz SB.Altered expression of M beta 2, the class II beta-tubulin isotype in amurine J774.2 cell linewith a high level of taxol resistance. J Biol Chem1995;270:31269–75.

42. Shalli K, Brown I, Heys SD, Schofield AC. Alterations of b-tubulinisotypes in breast cancer cells resistant to docetaxel. FASEB J2005;19:1299–301.

43. Gan PP, Kavallaris M. Tubulin-targeted drug action: functional signif-icance of class II and class IVb beta-tubulin in vinca alkaloid sensitivity.Cancer Res 2008;68:9817–24.

44. Galmarini CM, Treilleux I, Cardoso F, Bernard-Marty C, Durbecq V,Gancberg D, et al. Class III beta-tubulin isotype predicts response inadvanced breast cancer patients randomly treated either with single-agent doxorubicin or docetaxel. Clin Cancer Res 2008;14:4511–6.

45. Mozzetti S, Iantomasi R, De Maria I, Prislei S, Mariani M, CamperchioliA, et al. Molecular mechanisms of patupilone resistance. Cancer Res2008;68:10197–204.

46. Nicoletti MI, Valoti G, GiannakakouP, Zhan Z, Kim JH, Lucchini V, et al.Expression of beta-tubulin isotypes in human ovarian carcinomaxenografts and in a sub-panel of human cancer cell lines from theNCI-Anticancer Drug Screen: correlation with sensitivity to microtu-bule active agents. Clin Cancer Res 2001;7:2912–22.

47. Blade K,Menick DR, Cabral F. Overexpression of class I, II or IVb beta-tubulin isotypes in CHO cells is insufficient to confer resistance topaclitaxel. J Cell Sci 1999;112:2213–21.

48. Wilmes A, Chan A, Rawson P, William Jordan T, Miller JH. Paclitaxeleffects on the proteome of HL-60 promyelocytic leukemic cells:comparison to peloruside A. Invest New Drugs 2010; Sep 23 [Epubahead of print].

49. Chen K, Huzil JT, Freedman H, Ramachandran P, Antoniou A, Tus-zynski JA, et al. Identification of tubulin binding sites and prediction ofrelative differences in binding affinities to tubulin isotypes using digitalsignal processing. J Mol Graph Model 2008;27:497–505.

50. Begaye A, Trostel S, Zhao Z, Taylor RE, Schriemer DC, Sackett DL.Mutations in the b-tubulin binding site for peloruside A confer resis-tance by targeting a cleft significant in side chain binding. Cell Cycle2011;10:3387–96.






2012;11:393-404. Published OnlineFirst December 16, 2011.Mol Cancer Ther Arun Kanakkanthara, Peter T. Northcote and John H. Miller Carcinoma CellsLaulimalide, but not Paclitaxel or Vinblastine, in Human Ovarian

III-Tubulin Mediate Sensitivity to Peloruside A andβII-Tubulin and β

Updated version

10.1158/1535-7163.MCT-11-0614doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2011/12/16/1535-7163.MCT-11-0614.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/11/2/393.full#ref-list-1

This article cites 48 articles, 29 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/11/2/393.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/11/2/393To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-11-0614

http://mct.aacrjournals.org/content/suppl/2011/12/16/1535-7163.MCT-11-0614.DC1

http://mct.aacrjournals.org/content/11/2/393.full#ref-list-1

http://mct.aacrjournals.org/content/11/2/393.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/11/2/393


bII-Tubulinand bIII-TubulinMediateSensitivitytoPeloruside … · PLA and laulimalide were examined...

Documents

Transcript of bII-Tubulinand bIII-TubulinMediateSensitivitytoPeloruside … · PLA and laulimalide were examined...