docetaxel induces cell death through mitotic catastrophe ... · docetaxel induces cell death...

Docetaxel induces cell death through mitoticcatastrophe in human breast cancer cells

David L. Morse,1 Heather Gray,2 Claire M. Payne,3

and Robert J. Gillies2

1Arizona Cancer Center, 2Department of Biochemistryand Molecular Biophysics, University of Arizona, and3Center for Toxicology: Microbiology and Immunology,University of Arizona, Arizona Health Sciences Center,Tucson, Arizona

AbstractApoptosis has long been considered to be the prevailingmechanism of cell death in response to chemotherapy.Currently, a more heterogeneous model of tumor responseto therapy is acknowledged wherein multiple modes ofdeath combine to generate the overall tumor response.The resulting mechanisms of cell death are likely deter-mined by the mechanism of action of the drug, the dosingregimen used, and the genetic background of the cellswithin the tumor. This study describes a nonapoptoticresponse to docetaxel therapy in human breast cancercells of increasing cancer progression (MCF-10A, MCF-7,and MDA-mb-231). Docetaxel is a microtubule-stabilizingtaxane that is being used in the clinic for the treatment ofbreast and prostate cancers and small cell carcinoma ofthe lung. The genetic backgrounds of these cells werecharacterized for the status of key pathways and geneproducts involved in drug response and cell death. Cellularresponses to docetaxel were assessed by characterizingcell viability, cell cycle checkpoint arrest, and mechanismsof cell death. Mechanisms of cell death were determinedby Annexin V binding and scoring of cytology-stained cellsby morphology and transmission electron microscopy. Theprimary mechanism of death was determined to be mitoticcatastrophe by scoring of micronucleated cells and cellsundergoing aberrant mitosis. Other, nonapoptotic modesof death were also determined. No significant changes inlevels of apoptosis were observed in response to doce-taxel. [Mol Cancer Ther 2005;4(10):1495–504]

IntroductionDocetaxel (Taxotere; ref. 1) is a microtubule-stabilizingtaxane, which has recently been approved for use in theclinic for the treatment of breast and prostate cancers andsmall cell carcinoma of the lung (2). Docetaxel hasincreased affinity for tubulin (3) and has higher antitumoractivity compared with paclitaxel (Taxol; ref. 4). Criticalcellular processes require the dynamic reorganization ofmicrotubules. Therefore, treatment of cells with docetaxelcauses the inhibition of major cellular events, such asmitotic cell division, endosomal uptake, secretion, andtransport.Despite the clear connection to mitotic mechanism,

apoptosis has generally been accepted to be the predom-inant mechanism of cell death in response to taxanechemotherapy (refs. 5–7; reviewed in ref. 8). However,recent work has indicated that other modes of cell deathmay also contribute significantly to the overall therapeuticresponse (refs. 9, 10; reviewed in refs. 11, 12). This issupported by the observation that the degree of therapeuticresponse does not correlate with apoptosis and thatantiapoptotic mutations or altered expression of genes,such as bcl-2, p21 , and p53, are not negative predictors oftherapeutic efficacy (refs. 13–15, reviewed in ref. 16). Otherforms of death include mitotic catastrophe, treatment-induced senescence, and lytic necrosis. There is consider-able merit in designing therapies to induce these alternatemodes of cell death, especially in cells that may beapoptosis deficient. Here, a nonapoptotic cell deathresponse to docetaxel is described for three human breastcancer cell lines of increasing metastatic potential.Mitotic catastrophe is characterized by the occurrence of

aberrant mitosis, or missegregation of the chromosomes,followed by cell division. Nuclear envelopes form aroundindividual chromosomes or groups of chromosomes form-ing large nonviable cells with multiple micronuclei, whichare morphologically distinguishable from apoptotic cells.Apoptotic cells have condensed chromatin with shrunkencytoplasm and fragmented nuclei, whereas micronucleatedcells are large with uncondensed chromatin and achromatin pattern that resembles that of normal-sizednuclei (11). Mitotic catastrophe can be the primary mode ofcell death following treatment with ionizing radiation(reviewed in ref. 17) and occurs in response to severalanticancer agents (10).Although apoptosis is commonly regarded as the major

mechanism of cell death in response to taxanes, bothpaclitaxel and docetaxel are observed to induce dose- andcell line–specific mixtures of apoptotic and mitotic celldeath. In HeLa cells (18) and human breast cancer cells,including MCF-7 (19), low concentrations of paclitaxelcaused a mitotic block followed by aberrant mitoses,multiple micronuclei, and tetraploidy (mitotic catastrophe),

Received 4/27/05; revised 6/28/05; accepted 7/27/05.

Grant support: NIH grants R24 CA83148, R01 CA80130, and R01CA77575.

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 toindicate this fact.

Note: Portions of this work have been published in a dissertation at theUniversity of Arizona (D.L. Morse).

Requests for reprints: Robert J. Gillies, Arizona Cancer Center, Universityof Arizona, P.O. Box 245024, Tucson, AZ 85724. Phone: 520-626-5050;Fax: 520-626-5051.

Copyright C 2005 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-05-0130

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which may be followed by apoptosis. In both non–smallcell lung carcinoma (20) and breast cancer cells, includingMCF-10 and MCF-7 (21), paclitaxel has a concentration-dependent, biphasic response. At low concentrations,mitotic catastrophe and apoptosis are observed, whereasat high concentrations terminal mitotic arrest and necrosisare observed. In a recent study, Jurkat leukemia cellsunderwent apoptosis following treatment with paclitaxel,whereas MDA-mb-231 breast cancer cells underwent aslow nonapoptotic mitotic death characterized by multi-nucleation (22). In mouse fibroblast cells, docetaxel induceddeath by a mechanism exhibiting characteristics ofboth apoptotic and necrotic death (23). Docetaxel at thez5 nmol/L dose caused human breast cancer cells (BCap3)to initiate mitotic arrest and exhibit DNA laddering andsub-G0 DNA content, which are associated with apoptosis(24); however, these cells were not analyzed for responsesassociated with mitotic catastrophe, and increases in sub-G0 DNA are also associated with mitotic catastrophe andnecrotic death. Human ovarian cancer cells treated withdocetaxel exhibited apoptotic death preceded by cells withmultifragmented nuclei (25). A recent study suggested thatdocetaxel-induced cell death is caused by c-Jun NH2-terminal kinase–mediated apoptosis in MCF-7, SK-Br-3,and MDA-mb-231 breast cancer cells (26). Dosages identicalto those used in our study were given to cells, and c-JunNH2-terminal kinase activation was observed; however, theactual mechanism of cell death was not determined.Unlike apoptosis or senescence, which are ‘‘programmed’’

responses that involve signaling, the likely mechanism fortaxane-induced mitotic catastrophe involves the disruptionof mitotic cell division by the drug’s inhibition of thedynamic reorganization of microtubules. This disruptionresults in activation of the mitotic spindle checkpoint incheckpoint-proficient tumor cells. When damage to themitotic apparatus is excessive, the checkpoint will eventu-ally release (adapt) without the mitotic machinery beingrepaired; if the checkpoint is completely or partiallydefective, the cell will arrest transiently or proceed throughmitosis without arresting (27, 28). The damage causes theimproper segregation of chromosomes resulting in aberrantmitosis, multiple micronuclei, and an eventual necrosis-likedeath. Although checkpoint signaling may be involved inthe drug response, it is not required for mitotic catastropheto occur. In some cases, cells are observed to undergoapoptosis following mitotic catastrophe (18, 19). However,in mitotic catastrophe, apoptosis is not required for celldeath to occur (see below).In general, breast cancers are resistant to the therapeutic

induction of apoptosis; this is likely due to the status ofgene products, such as Bcl-2, p21, and p53 (29). Therefore,therapies that promote other types of death, such asmitotic catastrophe, may be preferential for use in treatingbreast cancer. Three human breast cancer cell lines ofincreasing degree of cancer progression were used forthis study: MCF-10A, MCF-7, and MDA-mb-231, repre-senting the nontumorigenic, tumorigenic/nonmetastatic,and metastatic stages of disease, respectively. As suggested

by the gene expression phenotypes of these cells (Table 1;see Materials and Methods), we hypothesized that thetumorigenic lines would be at least partially deficient indocetaxel-induced apoptosis but that the normal MCF-10A cells would exhibit apoptosis. However, at thedosages and time points examined in this study, mitoticcatastrophe was the predominant mechanism of celldeath observed in all three cell lines, with a lessercontribution by other necrotic modes of death andpractically no contribution by apoptosis. In MCF-10Aand MDA-mb-231 cells, mitotic catastrophe was nearlythe only mode of death observed. A mixture of mitoticcatastrophe and other nonapoptotic modes of death wasobserved for MCF-7 cells, with mitotic catastrophe stillbeing the predominant mechanism. Mitotic catastropheincreased with increasing cancer progression, althoughthis may simply be a function of the fraction ofproliferating cells at confluence. The absence of apoptoticmorphologies was confirmed by transmission electronmicroscopy (TEM), which is the ‘‘gold standard’’ fordetection of apoptosis (30).

Materials andMethodsMaterialsDocetaxel was acquired from spent clinical stocks

originally purchased from Aventis Pharmaceuticals (Par-sippany, NJ). The commercial sources of reagents used inthis study are as follows: epidermal growth factor (EGF;Upstate Biotechnology, Lake Placid, NY), fetal bovineserum (Omega Scientific, Inc., Tarzana, CA), trypsin(Invitrogen/Life Technologies, San Diego, CA), AnnexinV-FITC apoptosis detection kit (PharMingen, San Diego,CA), and the Diff-Quik system for cytology staining (DadeBehring, Inc., Newark, DE). All other media, chemicals, andreagents were purchased from Sigma (St. Louis, MO)unless otherwise stated.

StatisticsFor mode of cell death determination by cytology, at

least 500 cells were scored per treatment group and theresults were presented as a percentage. In all other cases,the results from at least three individual experiments(n z 3) were averaged to determine mean and SE. Whereappropriate, significance was determined by Student’st test. 95% Confidence intervals are reported for IC50

values.

Cell LinesMCF-10A cells were obtained from the Michigan Cancer

Foundation (now Karmanos Cancer Institute, Detroit, MI),MCF-7 cells were obtained from the Arizona Cancer Centercell culture shared service (Tucson, AZ), and MDA-mb-231cells were obtained from the American Type CultureCollection (Rockville, MD). Cells were passaged weeklyin DMEM/F-12 supplemented with 10% fetal bovine serumand 5 mg/mL insulin. MCF-10A medium also contained20 ng/mL EGF (31). For all assays involving therapeuticresponse, cells were grown to confluence plus 1 day; themedium was then replaced with medium containing 10 or

Docetaxel-Induced Mitotic Catastrophe in Breast Cancer1496




100 nmol/L docetaxel or docetaxel carrier as a control andallowed to grow for the indicated duration. Table 1 lists thegenetic and functional status of pathways known to beinvolved in proliferation and therapeutic response for thesecell lines.MCF-10A cells were originally isolated from fibrocystic

breast disease, were spontaneously immortalized, and donot form tumors when xenografted into severe combinedimmunodeficient mice (32). MCF-10A cells have intact cellcycle checkpoint controls (33–38), are aneuploid but havestable genomes comparable with human mammary epithe-lial cells (36), and have normal proliferation controls,including contact inhibition (31, 39–43) and normalapoptotic machinery (37, 38, 44, 45).MCF-7 cells were isolated from a pleural effusion of stage

IV invasive ductal carcinoma (46), form tumors whenxenografted into severe combined immunodeficient micebut are not metastatic (47), are aneuploid with high chro-mosomal instability, have a mismatch repair defect (48) anda single-strand break repair defect (49), and are partially

defective for the G1 and mitotic spindle checkpoints (36, 50).MCF-7 cells are proficient for apoptosis but not by acaspase-3-mediated pathway (49, 51) and overexpress Bcl-2.Enhanced proliferation is suggested by EGF-independentgrowth (31), elevated Her-2/Neu/c-ErbB-2 expression (52),amplified N-ras (53), and rapid phosphorylation of theRb gene product (43).MDA-mb-231 cells were isolated from stage IV invasive

ductal carcinoma (54), form tumors, and are metastaticwhen xenografted into severe combined immunodeficientmice (55). These cells are aneuploid and have anintermediate level of chromosomal instability (36), withpartial defects in mismatch repair (48) and single-strandbreak repair (49). Although at least partially proficient forall cell cycle checkpoints (34, 36, 56), MDA-mb-231 cellsexpress mutated p53 (49) and low p21 expression withsubdued induction (35). MDA-mb-231 cells are proficientfor apoptosis (51, 57) but resistant (58) due to elevatedexpression of the apoptosis-inhibiting Bcl-xL (49). Regula-tion of proliferation is perturbed by the following

Table 1. Status of key pathways and gene products of cell lines used in this study

Mechanism MCF-10A MCF-7 MDA-mb-231 References

Apoptosis Proficient. NormalBcl-2, p53, andp21 expressionand regulation.

Proficient via caspase-6,no caspase-3 expression," Bcl-2 expression, andnormal p53 and p21expression and regulation.

Proficient but resistant," Bcl-xL expression,a p53 mutation with" expression, andlower than normalp21 expression withsubdued induction.

37, 38, 44, 45, 49,51, 57, 58, 65–67

Cell cyclecheckpoint

G1, S, and mitoticspindle checkpointproficient. Normalp53 and p21expression andregulation.

Transient G1 and mitoticspindle checkpoints.S and G2 checkpointproficient. Normal p53and p21 expressionand regulation.

G1, S, G2, and mitoticspindle checkpointproficient. p53 mutationwith " expression andlower than normal p21expression withsubdued induction.

33–38, 49, 50,56, 67, 68

Genomestability

Low/normalchromosomalinstability.

High chromosomal instability.High single-nucleotideinstability (mismatchrepair defect). High single-strand break repair defect.

Moderate chromosomalinstability. Slight increasein single-nucleotideinstability. Moderatesingle-strand breakrepair defect.

36, 48, 49

Contactinhibition

Normal down-regulationof mitogen-activatedprotein kinase. Normalanchorage-dependentgrowth.

Cell density does not decreasemitogen-activated proteinkinase activity.

Cell density does notdecrease mitogen-activated proteinkinase activity.

39

Proliferation Estrogen receptor positive.Normal anchorage-dependent growth.EGF-dependentgrowth. NormalHer-2/Neu/c-ErbB-2expression. NormalRas expression. Normalphosphorylation of Rb.

" Estrogen receptor expression,estrogen-dependent growth.EGF-independent growth." Her-2/Neu/c-ErbB-2expression. Amplified N-ras .Rapid phosphorylation of Rb.

Estrogen receptor negative,estrogen independent/unresponsive.EGF-independent growthbut responsive formigration. Normal/lowHer-2/Neu/c-ErbB-2expression. ActivatedKi-ras mutation. Deregulateddephosphorylation of Rb.

31, 40–43,52, 53, 59

Molecular Cancer Therapeutics 1497




observations: MDA-mb-231 cells are both estrogen andEGF independent for growth (53, 59), have an activatedKi-ras mutation (53), and deregulated dephosphorylationof Rb (43).

Cell Proliferation and Cell CyclePopulation doubling times were determined by plating

cells at 1 � 104, 3 � 104, and 1 � 105 cells/mL in 24-wellplates and detaching (0.25% trypsin) and counting threewells per day on a hemacytometer for 3 subsequent daysand then every other day until the culture reached steadystate. The slope of the growth curves during log phase of atleast three individual experiments (n z 3) was used todetermine population doubling time. Pretreatment andpost-treatment cell cycle status was determined by stainingthe DNA of 1 � 106 cells/mL with propidium iodide(PI; modified Krishan buffer) and performing fluorescence-activated cell sorting (FACS) following treatment periods of0, 8, 16, 24, and 48 hours. FACS analysis was done by theArizona Cancer Center Flow Cytometry Shared ServiceLaboratory using a FACScan instrument (Becton Dickinson,Franklin Lakes, NJ). The percentage of DNA at G0-G1,S, and G2-M phases of the cell cycle was determined usingthe modeling program ModFit LT 2.0 (Verity SoftwareHouse, Inc., Topsham, ME).

Nucleoprotein Content AssayPost-treatment nucleoprotein content was determined by

crystal violet staining (60). A decrease in nucleoprotein isinterpreted as resulting from drug-induced cell detachmentor decreased proliferation. Cells were grown in 96-wellplates, 10,000 per well, and each row of wells was treatedwith increasing concentration of drug. Following 24 or 48hours of treatment, the medium was aspirated from eachwell and washed with medium so that only viable adherentcells remain. Cells were either allowed to grow for anadditional 24 hours before fixation or fixed immediately in0.025% glutaraldehyde for 30 minutes. The nucleoprotein offixed cells was stained by 0.1% crystal violet for 60 minutesand then destained by multiple washes with doubledistilled water. After air drying overnight, the remainingcrystal violet was solubilized in 0.1 mL of a 100� dilution ofglacial acetic acid. Absorbance was measured at 590 nmwavelength in a microplate autoreader model EL311 (Bio-Tek Instruments, Winooski, VT). The measured absorbanceis proportional to the protein content of the living-adhered

cells remaining in the well following treatment. Inhibitoryconcentration values were determined using GraphPadPrism version 3.01 (GraphPad Software, Inc., San Diego,CA) to generate a nonlinear regression line fit.

CellViabilityAssayCell viability was determined by scoring trypan blue

uptake 48 hours after treatment with 10 nmol/L docetaxelor docetaxel carrier as a control. One hundred cells pertreatment group were scored and results are reported as apercentage.

FACSApoptosis AssayAnnexin V binding assays were done by incubating cells

in 10 nmol/L docetaxel or docetaxel carrier for 0, 2, 4, 8,16, 24, and 48-hours followed by retrieval of detachedcells suspended in the buffer and dispersal of adheredcells using cell dispersal buffer (HBSS prepared with K+

or Na+ salts substituted for Mg2+ or Ca2+ salts). AnnexinV-FITC staining was done in conjunction with PI todistinguish early from late apoptosis and living from deadcells. Annexin V stains cells with accessible phosphati-dylserine. During early apoptosis, phosphatidylserine istranslocated to the outer membrane surface. However,cells that are dying and have permeabilized membranes asin early necrosis will have Annexin V–accessible internalphosphatidylserine. PI stains cells with permeable mem-branes, thus distinguishing cells with permeabilizedmembranes from those with healthy membranes. Follow-ing FACS, fluorescence of PI was plotted over AnnexinV-FITC fluorescence. Healthy, nonapoptotic cells have lowFITC fluorescence and low PI fluorescence. Early apopto-tic cells have high FITC fluorescence (bound phosphati-dylserine) but low PI fluorescence (intact membranes).Late apoptotic or early necrotic cells have high FITCfluorescence and high PI fluorescence. Late necrotic ordead cells have low FITC fluorescence but high PIfluorescence.

Cell Death by Cytology andTEMFollowing 48-hour treatment of cells with 10 or 100

nmol/L docetaxel or docetaxel carrier, cells were detachedfrom the cell culture plate using 0.25% trypsin, resus-pended in 20% bovine serum albumin, cytospinned ontoslides, fixed, and stained using the Diff-Quik system. Usinga �100 objective lens, at least 500 cells per treatment groupwere scored as being normal nonmitotic, normal mitotic,

Figure 1. G2 + M checkpointarrest detected by DNA-FACS andindicated by the increase in G2-M/G0-G1 ratio over time following treat-ment with 10 nmol/L docetaxel (.)or drug-carrier only (o). Plots areshown for MCF-10A cells (left ),MCF-7 cells (center ), and MDA-mb-231 cells (right ). RepresentativeFACS histograms, plotted as countsover DNA content (top ), are posi-tioned above respective time points.





aberrant mitotic, having multiple micronuclei, having avacuolated nucleus, being dark and shrunken, or beingapoptotic. Images of cytostained cells were generated usinga SPOT model 1.5.0 CCD camera (Diagnostic Instruments,Inc., Sterling Heights, MI) mounted on a Nikon EclipseE600 microscope (Nikon, Melville, NY). To verify cytologicresults, TEM images of cells were acquired following48-hour treatment with 10 and 100 nmol/L docetaxel anddocetaxel carrier. Following treatment, cells were caused todetach using 0.25% trypsin, fixed in 3% glutaraldehyde inphosphate buffer, postfixed in 1% osmium tetroxide, andembedded in Spurr low viscosity resin. Sections (1 Amthick) were prepared and stained with toluidine blue O.Ultrathin sections were prepared and stained with uranylacetate and lead citrate. TEM was done by the ArizonaResearch Laboratories Biotechnology Imaging Facilityusing a Philips CM12 system.

ResultsCell CycleRelative proliferation rates of MCF-10A, MCF-7, and

MDA-mb-231 cells during exponential growth (doublingtime), percent G0 + G1 DNA at confluence (steady-stategrowth), and G2 + M checkpoint arrest following treatmentwith 10 nmol/L docetaxel were determined. In thesegrowth conditions, no significant difference between theexponential growth rates of the three cell lines wasobserved, with doubling times of 1.52 F 0.23 for MCF-10A cells, 1.60 F 0.21 for MCF-7 cells, and 1.67 F 0.23 forMDA-mb-231 cells. However, at confluence, a higherfraction of cells remained proliferating (non-G0/G1) in thetumorigenic and metastatic MDA-mb-231 cells (43.9 F2.5%) and tumorigenic MCF-7 cells (27.8 F 0.5%) compared

with the relatively normal MCF-10A cells (18.2 F 2.5%).As an indicator of checkpoint arrest, the (G2 + M) / (G0

+ G1) ratios were determined over a period of 48 hoursfollowing treatment with 10 nmol/L docetaxel (Fig. 1).MCF-10A cells had a small increase peaking at 8 hoursand returned to pretreatment levels by 24 hours. Cellcycle arrest in MCF-7 and MDA-mb-231 cells steadilyincreased to 24 hours and remained elevated but leveledoff with a lesser degree of increase at 48 hours.

Nucleoprotein ContentAs a measure of cell detachment or decreased prolifer-

ation following treatment, nucleoprotein content of ad-hered cells was quantified by crystal violet staining (60) atincreasing doses of docetaxel and at different time pointsfollowing treatment. To assess clonogenic death (senes-cence), crystal violet staining was also assayed after48 hours of drug followed by 24 hours in normal medium.From these experiments, IC50 values were determined forall three cell lines and are shown in Table 2. As shown inTable 2, the two cancer lines, MCF-7 and MDA-mb-231,were similarly sensitive to drug at 24 and 48 hours butwere less sensitive than the normal MCF-10A cell line.Only MCF-7 cells had a further decrease in crystal violetstaining following 24 hours of recovery in normalmedium, and drug sensitivity under these conditionswas similar to that of MCF-10A cells. From these data,10 and 100 nmol/L docetaxel were chosen for subsequentexperiments, as they bracket the IC50 response in all celllines under all conditions. The responses at these doseswere determined and are presented in Table 3. As shownin Table 3, virtually all of the MCF-10A and MCF-7 cellsare destined to die by 48 + 24 hours at the higher dose of100 nmol/L, whereas nearly one third of the MDA-mb-231cells survive. Similarly, even the lower dose of 10 nmol/L

Table 2. IC50 docetaxel at 24- and 48-hour chronic treatment, and at 48-hour treatment followed by 24-hour incubation in drug-freemedium

Cell line IC50, 24 hours(95% confidence interval)

IC50, 48 hours(95% confidence interval)

IC50, 48 + 24 hours(95% confidence interval)

MCF-10A 3.25e�8 (1.10e�8 to 9.66e�8) 2.24e�8 (1.02e�8 to 4.93e�8) 1.97e�8 (1.49e�8 to 2.63e�8)MCF-7 3.87e�7 (8.69e�8 to 1.72e�6) 8.34e�8 (1.03e�8 to 6.73e�7) 1.47e�8 (2.53e�9 to 8.52e�8)MDA-mb-231 9.28e�8 (1.63e�9 to 5.28e�6) 5.12e�8 (3.25e�8 to 8.07e�8) 5.00e�8 (3.43e�8 to 7.29e�8)

Table 3. Percent death following treatment with 10 and 100 nmol/L docetaxel at 24- and 48-hour chronic treatment and at 48-hourtreatment followed by 24-hour incubation in drug-free medium

Cell line Dose (nmol/L), docetaxel % Death, 24 hours (SE) % Death, 48 hours (SE) % Death, 48 + 24 hours (SE)

MCF-10A 10 30.7 F 6.9 34.4 F 5.2 30.2 F 2.1100 68.9 F 6.9 78.9 F 5.4 90.0 F 2.1

MCF-7 10 4.5 F 7.7 11.2 F 13.0 28.0 F 5.7100 25.4 F 7.7 53.2 F 15.0 99.6 F 5.6

MDA-mb-231 10 13.4 F 25.6 21.8 F 2.9 12.9 F 2.7100 51.5 F 31.6 62.5 F 3.1 68.4 F 2.8





kills significant fractions of the MCF-10A and MCF-7 cells(30% and 28%, respectively) and only a small number(13%) of the MDA-mb-231 cells. Similar to the data ofTable 2, the MCF-7 cells were slowest to respond at thelow dose, and the MCF-10A and MDA-mb-231 cells didnot have a further decrease in cell number following therecovery period.

CellViabilityTo determine whether cell killing occurred in response to

docetaxel, trypan blue uptake was scored 48 hours aftertreatment with the lowest dosage of docetaxel used(10 nmol/L) or with docetaxel carrier as a control. MCF-10A cells had 18% lower viability, MCF-7 cells had 17%lower viability, and MDA-mb-231 cells had 10% lowerviability in treated versus untreated cells.

Apoptosis Assay by FACSTo determine the proportion of cell death due to

apoptosis, Annexin V binding was determined at 0, 2, 4,8, 16, 24, and 48 hours following treatment with 10 nmol/Ldocetaxel in tandem with untreated controls (61). For allthree cell lines and all time points, post-treatment increasesin Annexin V binding in the absence of PI stainingcompared with controls were minimal and not significant,with a maximal increase of 4.68% (P = 0.21) at 8 hours forMCF-10A cells, 1.05% (P = 0.35) at 16 hours for MCF-7 cells,and 0.97% (P = 0.39) at 8 hours for MDA-mb-231 cells (datanot shown). A significant increase in Annexin V + PIcostaining was observed for MCF-10A cells at 16 hoursafter treatment. Costaining increased by 17.8% (P = 0.003).Because this increase in costaining was not preceded withincreased staining by Annexin V alone at earlier timepoints, it is attributed to loss of membrane integrity dueto a necrotic type of cell death.

Cell Death Assays by Cytology andTEMFigure 2 shows both cytology and TEM images from

untreated MCF-7 cells and those treated with 10 nmol/Ldocetaxel. Normal nonmitotic nuclei (Fig. 2A and E) arejuxtaposed with multiple micronucleated cells (Fig. 2B

Figure 2. Mitotic catastrophe in MCF-7 cells following 48-h treatmentwith 10 nmol/L docetaxel. Shown are Diff-Quik-stained cells (A–D), TEMimages (E–H), normal untreated nuclei (A and E), treated cells withmultiple micronuclei (B and F), untreated cells undergoing normal mitosis(C and G), and treated cells undergoing aberrant mitosis (D and H).

Figure 3. Other forms of death in cells treated with docetaxel. Shownare Diff-Quik-stained cells (A–C), TEM images (D–F), MCF-7 cells withlarge vacuolated nuclei (A and D), small nonapoptotic MCF-7 cells withdark-staining nuclei and cytoplasm (B and E), an MDA-mb-231 apoptoticcell (C), and a MCF-10A apoptotic cell (F). In addition, the MCF-7 cells in(D and E) are being engulfed via phagocytosis by viable cells in the culture.





and F). Normal mitotic cells (Fig. 2C and G) are jux-taposed with cells undergoing aberrant mitosis (Fig. 2Dand H). Figure 3 illustrates other nonmitotic modes ofdeath observed in these samples. Cells with largevacuolated nuclei were observed (Fig. 3A and D), withthe cell in Fig. 3D being phagocytosed by another cell.Nonapoptotic cells with condensed nuclei and cytoplasmwere also observed (Fig. 3B and E) along with apoptoticcells (Fig. 3C and F). By TEM only, cells with condensedmitochondria were observed (Fig. 4). The mitochondriaof these cells have a ‘‘state III’’ conformation (ref. 62;e.g., energized mitochondria), which is hypothesized torepresent a compensating effect in cells struggling tomaintain adequate energy metabolism. Recent studiesalso indicate that condensed mitochondria can beobserved following a drop in mitochondrial membranepotential (63).

From these preparations, a minimum of 500 cells percondition were scored for morphologies and the results arepresented in Table 4. For all three cell lines at all doses,apoptosis accounted for <0.5% of all cells scored. Mitoticcatastrophe (aberrant mitoses + multinucleated cells) wasthe major cause of death for all cell lines and treatments.Other nonapoptotic forms of death increased with dosefor MCF-10A and MCF-7 cells but did not increase forMDA-mb-231 cells. MDA-mb-231 cells had the highestnumber of dead cells at all doses, with mitotic catastrophebeing the only relevant cell death response.

DiscussionThe type of cell death resulting from a given therapy isdetermined by the mechanism of action of the drug, dosingregimen of the therapy, and genetic background of the cellsbeing treated. Nonapoptotic cell death (i.e., mitoticcatastrophe) is the major response to docetaxel in threehuman breast lines at the dosages and timing used in thisstudy. This action of taxanes may be partly responsible fortheir efficacy in treating breast cancers, which may beapoptosis deficient. Absolutely no apoptotic response wasobserved for MCF-7 cells, which overexpress the apoptosis-inhibiting Bcl-2 and do not express caspase-3, a majorcomponent of the effector or executionary phase of themajority of apoptotic signaling pathways (ref. 49; Table 1).In contrast, MCF-10A and MDA-mb-231 cells had slight(<0.5%) but measurable numbers of apoptotic figures.FACS assays for apoptosis support the cytologic results,with minimal and insignificant increases in Annexin Vbinding following treatment of all three cell lines. Inaddition, the cytology and Annexin V results weresupported by TEM images of treated cells (64). TEM isconsidered to be the gold standard for detection ofapoptosis (30). For TEM, multiple preparations were madefor each cell line and treatment condition. Each preparationwas scanned thoroughly and only two examples ofapoptosis were observed in all of the preparations. Incontrast, other mechanisms of death were ubiquitouslyobserved in TEM images.As scored by cytology, mitotic catastrophe is the

predominant mode of death for all docetaxel dosingregimens and all human breast cancer cell lines used inthis study. In general, the percentage of cell death by thismechanism was higher in cells of increasing cancerprogression. At the highest dose, the normal MCF-10A cellline had the lowest measure of cell death (15%), withmitotic catastrophe being the major component (12%); thetumorigenic MCF-7 cells had a considerably higherpercentage of death (56%), with f46% attributed to mitoticcatastrophe; and the tumorigenic and metastatic MDA-mb-231 cells had the highest percentage of death (80%), withnearly all cell death attributed to mitotic catastrophe (78%).The TEM images support the cytologic results, becausecells undergoing aberrant mitoses and having multiplemicronuclei were common observations following doce-taxel treatment of all three cell lines.

Figure 4. Condensed mitochondria in treated MDA-mb-231 cells. Boxin A indicates expanded region (B).





Long-term assays show that the percentage of cellsundergoing mitotic catastrophe is proportional to thedecrease in clonogenic survival and that these morpholo-gies ultimately result in cell death (11, 13, 22). Withoutperforming clonogenic assays, viability (trypan blue incor-poration) measurements showed a loss of membraneintegrity and ultimate cell death 48 hours after treatmentwith the lowest dosage used. Percentages of cell deathmeasured by this assay were comparable with percentagesscored by both biomass (nucleoprotein) assay and cytology.This increase in death by mitotic catastrophe may be

caused by ineffective checkpoint arrest due to a partialdefect in the mitotic spindle checkpoint or the higher ratesof proliferation in confluent cells of increasing metastaticpotential (Table 1). At least 18% of MCF-10A cells areproliferative (i.e., in non-G0/G1) at steady-state growth(confluence), whereas 28% of the MCF-7 cells and 44% ofMDA-mb-231 cells are proliferating at steady state. Cellsactively proceeding through the cell cycle would be moresusceptible to mitotic block and subsequent mitotic death.This could also explain the apparently discrepant resultsbetween the dose-responses that show the MDA-mb-231cells to be least sensitive (Tables 2 and 3) and the cytologicscoring (Table 4) that shows the MDA-mb-231 cells to bemost sensitive. Because the MDA-mb-231 culture isreplenishing itself more rapidly, fewer cells would be lost(as determined by crystal violet staining), although thereis a higher proportion of cell death (as determined bycytology). Furthermore, the number of cells scored as being‘‘normal’’ following treatment may have been ultimatelydestined for death, causing a disconnect between cytologyand survival.Modes of cell death other than mitotic catastrophe and

apoptosis were detected by cytology and TEM. Other formsof death scored by cytology were small, dark nonapoptoticcells and cells with highly vacuolated nuclei at varyingdegrees of disintegration. These types of death are possiblydue to disruption of the cellular infrastructure in termsof uptake, secretion, transport, and energy metabolism due

to the mechanism of action of the drug. An inhibitionof energy metabolism is implicated by the observation ofstate III mitochondria (Fig. 4), which may represent theenergized state of mitochondria (62). It is also possible thatthe increase in condensed mitochondria may represent aloss of mitochondrial membrane potential (63), althoughthis loss in membrane potential does not translate intoapoptosis. In contrast to mitotic catastrophe, which requirescells to progress through mitosis, all cells regardless of theircell cycle status would be susceptible to these nonapoptoticforms of death. Hence, the nonmetastatic MCF-10A andMCF-7 cells are more competent for contact inhibition thanthe metastatic MDA-mb-231 cells, thus are more likely tobe quiescent (G0), and have a higher proportion of celldeath by these other means (Table 4).In conclusion, mitotic catastrophe is the primary mode

of death observed in response to the treatment regimenwith docetaxel in the human breast cancer cell linesincluded in this study, with additional death attributed toother nonapoptotic mechanisms. The proportional mixtureof cell death responses was cell line specific and could bedue to differential regulation of proliferative controls,contact inhibition, or cell cycle checkpoints (Table 1). BothMCF-7 and MDA-mb-231 cells have compromised contactinhibition and altered proliferative signaling [i.e., ampli-fied ras (MCF-7) and activated Ki-ras (MDA-mb-231)].Both have partial defects in the mitotic spindle check-point, the checkpoint activated by microtubule-hyper-polymerizing drugs, such as docetaxel. MDA-mb-231cells have mutated p53, which is implicated in checkpointmaintenance. In addition, both MCF-7 and MDA-mb-231cell lines are partially resistant to apoptosis due toelevations in the apoptosis-inhibiting products Bcl-2(MCF-7) and Bcl-xL (MDA-mb-231) and caspase-3 defectsin MCF-7 cells. Therefore, it is not surprising thattreatment of these two cell lines with docetaxel resultedin death primarily by mitotic catastrophe with nosignificant increase in apoptosis. In addition, the mecha-nism of action of docetaxel, the binding to microtubules,

Table 4. Results of scoring cell death morphologies of Diff-Quik-stained cytospins (nz500 cells)

Cell line Dose (nmol/L),docetaxel*

Normal Normalmitotic

Nonapoptotic deathc Apoptosis

Aberrantmitoses

Multinucleate Other nonapoptoticcell death

Total

MCF-10A 0 98.8 1.2 0.0 0.0 0.0 0.0 0.010 76.0 0.0 5.7 17.9 0.2 23.8 0.1100 85.0 0.0 2.2 9.0 3.4 14.6 0.4

MCF-7 0 97.7 1.0 1.1 0.0 0.2 1.3 0.010 81.6 0.5 6.1 6.1 5.6 17.8 0.0100 43.5 0.7 10.7 35.5 9.6 55.8 0.0

MDA-mb-231 0 94.0 2.3 0.9 0.7 2.0 3.6 0.010 61.4 0.6 9.3 25.6 2.9 37.8 0.1100 19.3 0.4 11.3 66.4 2.5 80.2 0.2

* 0 nmol/L dose contained diluted docetaxel carrier.cOther nonapoptotic cell death includes vacuolated nuclei and nonapoptotic darkly stained nuclei.





would favor a type of cell death associated with aberrantassembly and separation of chromosomes during mitosis.However, the normal MCF-10A cells, which are proficientfor proliferative controls, contact inhibition, checkpointcontrols, and apoptosis, were also negative for a signifi-cant increase in apoptosis. In contrast, MCF-10A cells hadlower levels of mitotic catastrophe possibly due to aproficient G1 checkpoint and fewer numbers of prolifer-ative cells due to normal regulation of proliferation andcontact inhibition. This study underscores the importanceof evaluating the specific mechanism of cell death inbreast cancer cells (i.e., apoptosis, necrosis, and mitoticcatastrophe) because chemotherapeutic agents that targetthe mitotic spindle may be most effective at killing theseapoptosis-resistant cells.

References

1. Ringel I, Horwitz SB. Studies with RP 56976 (Taxotere): a semisyn-thetic analogue of Taxol. J Natl Cancer Inst 1991;83:288–91.

2. Crown J, O’Leary M. The taxanes: an update. Lancet 2000;355:1176–8.

3. Diaz JF, Andreu JM. Assembly of purified GDP-tubulin into micro-tubules induced by Taxol and Taxotere: reversibility, ligand stoichiometry,and competition. Biochemistry 1993;32:2747–55.

4. Lavelle F, Bissery MC, Combeau C, Riou JF, Vrignaud P, Andre S.Preclinical evaluation of docetaxel (Taxotere). Semin Oncol 1995;22:3–16.

5. Saunders DE, Lawrence WD, Christensen C, Wappler NL, Ruan HM,Deppe G. Paclitaxel-induced apoptosis in MCF-7 breast-cancer cells. Int JCancer 1997;70:214–20.

6. Haldar S, Basu A, Croce CM. Bcl2 is the guardian of microtubuleintegrity. Cancer Res 1997;57:229–33.

7. Lin HL, Liu TY, Chau GY, Lui WY, Chi CW. Comparison of2-methoxyestradiol-induced, docetaxel-induced, and paclitaxel-inducedapoptosis in hepatoma cells and its correlation with reactive oxygenspecies. Cancer 2000;89:983–94.

8. Wang LG, Liu XM, Kreis W, Budman DR. The effect of antimicrotubuleagents on signal transduction pathways of apoptosis: a review. CancerChemother Pharmacol 1999;44:355–61.

9. Schimming R, Mason KA, Hunter N, Weil M, Kishi K, Milas L. Lack ofcorrelation between mitotic arrest or apoptosis and antitumor effect ofdocetaxel. Cancer Chemother Pharmacol 1999;43:165–72.

10. Chang BD, Broude EV, Dokmanovic M, et al. A senescence-likephenotype distinguishes tumor cells that undergo terminal proliferationarrest after exposure to anticancer agents. Cancer Res 1999;59:3761–7.

11. Roninson IB, Broude EV, Chang BD. If not apoptosis, then what?—treatment-induced senescence and mitotic catastrophe in tumor cells.Drug Resist Updat 2001;4:303–13.

12. Galons JP, Morse DL, Jennings DR, Gillies RJ. Diffusion MRI andresponse to anti-cancer therapies. Israel J Chem 2003;43:91–101.

13. Lock RB, Stribinskiene L. Dual modes of death induced by etoposidein human epithelial tumor cells allow Bcl-2 to inhibit apoptosis withoutaffecting clonogenic survival. Cancer Res 1996;56:4006–12.

14. Wouters BG, Giaccia AJ, Denko NC, Brown JM. Loss of p21(Waf1/Cip1) sensitizes tumors to radiation by an apoptosis-independentmechanism. Cancer Res 1997;57:4703–6.

15. Rein DT, Schondorf T, Breidenbach M, et al. Lack of correlationbetween P53 expression, BCL-2 expression, apoptosis and ex vivochemosensitivity in advanced human breast cancer. Anticancer Res 2000;20:5069–72.

16. Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity toanticancer agents. Cancer Res 1999;59:1391–9.

17. Cohen-Jonathan E, Bernhard EJ, McKenna WG. How does radiationkill cells? Curr Opin Chem Biol 1999;3:77–83.

18. Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L.Mitotic block induced in HeLa cells by low concentrations of paclitaxel

(Taxol) results in abnormal mitotic exit and apoptotic cell death. CancerRes 1996;56:816–25.

19. Blajeski AL, Kottke TJ, Kaufmann SH. A multistep model forpaclitaxel-induced apoptosis in human breast cancer cell lines. Exp CellRes 2001;270:277–88.

20. Torres K, Horwitz SB. Mechanisms of Taxol-induced cell death areconcentration dependent. Cancer Res 1998;58:3620–6.

21. Yeung TK, Germond C, Chen XM, Wang ZX. The mode of action ofTaxol: apoptosis at low concentration and necrosis at high concentration.Biochem Biophys Res Commun 1999;263:398–404.

22. Blagosklonny MV, Robey R, Sheikh MS, Fojo T. Paclitaxel-inducedFasL-independent apoptosis and slow (non-apoptotic) cell death. CancerBiol Ther 2002;1:113–7.

23. Suzuki A, Kawabata T, Kato M. Necessity of interleukin-1h convert-ing enzyme cascade in Taxotere-initiated death signaling. Eur J Pharmacol1998;343:87–92.

24. Zeng S, Chen YZ, Fu L, Johnson KR, Fan W. In vitro evaluation ofschedule-dependent interactions between docetaxel and doxorubicinagainst human breast and ovarian cancer cells. Clin Cancer Res 2000;6:3766–73.

25. Kolfschoten GM, Hulscher TM, Duyndam MCA, Pinedo HM, Boven E.Variation in the kinetics of caspase-3 activation, Bcl-2 phosphorylation andapoptotic morphology in unselected human ovarian cancer cell lines as aresponse to docetaxel. Biochem Pharmacol 2002;63:733–43.

26. Wang Q, Wieder R. All-trans retinoic acid potentiates Taxotere-induced cell death mediated by Jun N-terminal kinase in breast cancercells. Oncogene 2004;23:426–33.

27. Lanni JS, Jacks T. Characterization of the p53-dependent postmitoticcheckpoint following spindle disruption. Mol Cell Biol 1998;18:1055–64.

28. Vogel C, Kienitz A, Hofmann I, Muller R, Bastians H. Crosstalk of themitotic spindle assembly checkpoint with p53 to prevent polyploidy.Oncogene 2004;23:6845–53.

29. Gewirtz DA. Growth arrest and cell death in the breast tumor cell inresponse to ionizing radiation and chemotherapeutic agents which induceDNA damage. Breast Cancer Res Treat 2000;62:223–35.

30. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biologicalphenomenon with wide-ranging implications in tissue kinetics. Br J Cancer1972;26:239–57.

31. Blagosklonny MV, Bishop PC, Robey R, Fojo T, Bates SE. Loss of cellcycle control allows selective microtubule-active drug-induced Bcl-2phosphorylation and cytotoxicity in autonomous cancer cells. CancerRes 2000;60:3425–8.

32. Soule HD, Maloney TM, Wolman SR, et al. Isolation and character-ization of a spontaneously immortalized human breast epithelial-cell line,MCF-10. Cancer Res 1990;50:6075–86.

33. Benaud CM, Dickson RB. Adhesion-regulated G1 cell cycle arrest inepithelial cells requires the downregulation of c-Myc. Oncogene 2001;20:4554–67.

34. Eastman A, Kohn EA, Brown MK, et al. A novel indolocarbazole, ICP-1,abrogates DNA damage-induced cell cycle arrest and enhances cytotox-icity: similarities and differences to the cell cycle checkpoint abrogatorUCN-01. Mol Cancer Ther 2002;1:1067–78.

35. Upadhyay S, Neburi M, Chinni SR, Alhasan S, Miller F, Sarkar FH.Differential sensitivity of normal and malignant breast epithelial cells togenistein is partly mediated by p21(WAF11). Clin Cancer Res 2001;7:1782–9.

36. Yoon DS, Wersto RP, Zhou WB, et al. Variable levels of chromosomalinstability and mitotic spindle checkpoint defects in breast cancer. Am JPathol 2002;161:391–7.

37. Thomas TJ, Faaland CA, Adhikarakunnathu S, Watkins LF, Thomas T.Induction of p21 (CIP1/WAF1/SID1) by estradiol in a breast epithelial cellline transfected with the recombinant estrogen receptor gene: a possiblemechanism for a negative regulatory role of estradiol. Breast Cancer ResTreat 1998;47:181–93.

38. Majumder B, Wahle KWJ, Moir S, et al. Conjugated linoleic acids(CLAs) regulate the expression of key apoptotic genes in human breastcancer cells. FASEB J 2002;16:U85–105.

39. Zhang LF, Bewick M, Lafrenie RM. Role of Raf-1 and FAK in celldensity-dependent regulation of integrin-dependent activation of MAPkinase. Carcinogenesis 2002;23:1251–8.

40. Kawai H, Li HC, Chun P, Avraham S, Avraham HK. Direct interactionbetween BRCA1 and the estrogen receptor regulates vascular endothelial





growth factor (VEGF) transcription and secretion in breast cancer cells.Oncogene 2002;21:7730–9.

41. Ignatoski KMW, Lapointe AJ, Radany EH, Ethier SP. erbB-2 over-expression in human mammary epithelial cells confers growth factorindependence. Endocrinology 1999;140:3615–22.

42. Basolo F, Elliott J, Tait L, et al. Transformation of human breastepithelial-cells by c-Ha-ras oncogene. Mol Carcinog 1991;4:25–35.

43. Botos J, Barhoumi R, Burghardt R, Kochevar DT. Rb localization andphosphorylation kinetics correlate with the cellular phenotype of culturedbreast adenocarcinoma cells. In Vitro Cell Dev Biol Anim2002;38:235–41.

44. Starcevic SL, Elferink C, Novak RF. Progressive resistance toapoptosis in a cell lineage model of human proliferative breast disease.J Natl Cancer Inst 2001;93:776–82.

45. Kim HH, Park CS. Lipotropes regulate Bcl-2 gene expression in thehuman breast cancer cell line, MCF-7. In Vitro Cell Dev Biol Anim 2002;38:205–7.

46. Soule HD, Vazquez J, Long A, Albert S, Brennan M. Human cell linefrom a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst1973;51:1409–16.

47. Kurebayashi J, McLeskey SW, Johnson MD, Lippman ME, DicksonRB, Kern FG. Quantitative demonstration of spontaneous metastasis byMCF-7 human breast cancer cells cotransfected with fibroblast growthfactor 4 and LacZ. Cancer Res 1993;53:2178–87.

48. Watanabe N, Okochi E, Mochizuki M, Sugimura T, Ushijima T. Thepresence of single nucleotide instability in human breast cancer cell lines.Cancer Res 2001;61:7739–42.

49. Nieves-Neira W, Pommier Y. Apoptotic response to camptothecin and7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell linesof the NCI anticancer drug screen: multifactorial relationships withtopoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspasepathways. Int J Cancer 1999;82:396–404.

50. Nagasawa H, Keng P, Maki C, Yu YJ, Little JB. Absence of aradiation-induced first-cycle G(1)-S arrest in p53(+) human tumor cellssynchronized by mitotic selection. Cancer Res 1998;58:2036–41.

51. Im EO, Choi YH, Paik KJ, et al. Novel bile acid derivatives induceapoptosis via a p53-independent pathway in human breast carcinomacells. Cancer Lett 2001;163:83–93.

52. Rait AS, Pirollo KF, Rait V, Krygier JE, Xiang LM, Chang EH. Inhibitoryeffects of the combination of HER-2 antisense oligonucleotide andchemotherapeutic agents used for the treatment of human breast cancer.Cancer Gene Ther 2001;8:728–39.

53. Sutherhland RL, Watts CKW, Lee CSL, Musgrove EA. Breast cancer.In: Masters JRW, Palsson B, editors. Human cell culture. Vol. II. Cancercell lines. Part 2. Boston: Kluwer Academic Publishers; 1999. p. 79–106.

54. Cailleau R, Young R, Olive M, Reeves WJ. Breast tumor-cell lines frompleural effusions. J Natl Cancer Inst 1974;53:661–74.

55. Russell RL, Geisinger KR, Mehta RR, White WL, Shelton B, Kute TE.

nm23—relationship to the metastatic potential of breast carcinoma celllines, primary human xenografts, and lymph node negative breastcarcinoma patients. Cancer 1997;79:1158–65.

56. Pervin S, Singh R, Chaudhuri G. Nitric oxide-induced cytostasisand cell cycle arrest of a human breast cancer cell line (MDA-MB-231): potential role of cyclin D1. Proc Natl Acad Sci U S A 2001;98:3583–8.

57. Kim KI, Jung YK, Noh DY, et al. Functional screening of genessuppressing TRAIL-induced apoptosis: distinct inhibitory activities ofBcl-X-L and Bcl-2. Br J Cancer 2003;88:910–7.

58. Hunakova L, Chorvath M, Duraj J, et al. Radiation-induced apoptosisand cell cycle alterations in human carcinoma cell lines with differentradiosensitivities. Neoplasma 2000;47:25–31.

59. Price JT, Tiganis T, Agarwal A, Djakiew D, Thompson EW. Epidermalgrowth factor promotes MDA-MB-231 breast cancer cell migrationthrough a phosphatidylinositol 3V-kinase and phospholipase C-dependentmechanism. Cancer Res 1999;59:5475–8.

60. Gillies RJ, Didier N, Denton M. Determination of cell number inmonolayer cultures. Anal Biochem 1986;159:109–13.

61. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novelassay for apoptosis. Flow cytometric detection of phosphatidylserineexpression on early apoptotic cells using fluorescein labelled Annexin V.J Immunol Methods 1995;184:39–51.

62. Hackenbrock CR. Ultrastructural bases for metabolically linkedmechanical activity in mitochondria. I. Reversible ultrastructural changeswith change in metabolic steady state in isolated liver mitochondria. J CellBiol 1966;30:269–97.

63. Gottlieb E, Armour SM, Harris MH, Thompson CB. Mitochondrialmembrane potential regulates matrix configuration and cytochrome crelease during apoptosis. Cell Death Differ 2003;10:709–17.

64. Otsuki Y, Li Z, Shibata MA. Apoptotic detection methods– frommorphology to gene. Prog Histochem Cytochem 2003;38:275–339.

65. Simoes-Wust AP, Olie RA, Gautschi O, et al. bcl-xL antisensetreatment induces apoptosis in breast carcinoma cells. Int J Cancer2000;87:582–90.

66. Sarker M, Ruiz-Ruiz C, Robledo G, Lopez-Rivas A. Stimulation of themitogen-activated protein kinase pathway antagonizes TRAIL-inducedapoptosis downstream of BID cleavage in human breast cancer MCF-7cells. Oncogene 2002;21:4323–7.

67. Jeffy BD, Chen EJ, Gudas JM, Romagnolo DF. Disruption of cellcycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1and p53 in MCF-7 cells arrested in S and G2. Neoplasia 2000;2:460–70.

68. Shao RG, Cao CX, Shimizu T, OConnor PM, Kohn KW, PommierY. Abrogation of an S-phase checkpoint and potentiation of campto-thecin cytotoxicity by 7-hydroxystaurosporine (UCN-O1) in human can-cer cell lines, possibly influenced by p53 function. Cancer Res 1997;57:4029–35.





2005;4:1495-1504. Mol Cancer Ther David L. Morse, Heather Gray, Claire M. Payne, et al. human breast cancer cellsDocetaxel induces cell death through mitotic catastrophe in

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