paclitaxel induces programmed cell death in...

Vol. 2, 847-854, May 1996 Clinical Cancer Research 847

Paclitaxel Induces Programmed Cell Death in MDAMB-468 Human

Breast Cancer Cells1

Diane E. McCloskey, Scott H. Kaufmann,

Laura J. Prestigiacomo, and Nancy E. Davidson2

The Johns Hopkins Oncology Center, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland 21231 [D. E. M., L. J. P.,N. E. DI, and Division of Oncology Research, Mayo Clinic,

Rochester, Minnesota 55905 [S. H. K.]

ABSTRACTThe ability of paditaxel, one of the most active chemo-

therapeutic agents against breast cancer, to induce pro-

grammed cell death in hormone-independent MDA-MB-468

human breast cancer cells was assessed. Treatment of MDA-MB-468 cells led to growth inhibition, high-molecular-weightand oligonucleosomal DNA fragmentation, and apoptosis-asso-

ciated morphological changes after either 3- or 24-h exposure

to paditaxel concentrations �1O ni�i. Additionally, cleavageproducts of poly(ADP.ribose) polymerase and lamin B1, twoproteins that are cleaved early in the execution phase of pro-grammed cell death, were detected. Quantitative studies mdi-

cated that exposure to paditaxel for 24 h resulted in more DNAfragmentation than did 3-h exposure. Rapid induction of theearly-response gene c-jun but not c-myc was associated with

paditaxel treatment. The ability of paditaxel to induce high-molecular-weight DNA fragmentation and apoptosis-associ-

ated morphological changes in three other breast cancer celllines was also established. These data suggest that paditaxel, an

agent known to stabilize microtubules and prevent cell divisionbut not to act directly on DNA, induces programmed cell deathin breast cancer cells.

INTRODUCTION

Breast cancer is the most common cancer in American

women. New approaches to systemic therapy are needed, as

age-adjusted mortality has not changed greatly for 50 years (1).

Exploitation of pathways of programmed cell death (2-4), a

mechanism of cellular suicide, is one potential approach. We

have previously used human breast cancer cells to show that

hormone-dependent MCF-7 cells growing in vivo undergo pro-

grammed cell death upon estrogen withdrawal (5). Furthermore,

even hormone-independent breast cancer cells retain the ability

to undergo programmed cell death in response to diverse agents,

including fluoropyrimidines (6).

Received 8/3 1/95; revised 1/25/96; accepted 1/31/96.I This work was supported by NIH Grants 5T32CA0907 1 , RO1 -

CA57545, UO1CA66084, and CA69008 and the Susan 0. Komen

Foundation. S. H. K. is a Leukemia Society of America Scholar.2 To whom requests for reprints should be addressed, at the JohnsHopkins Oncology Center, Johns Hopkins University School of Medi-cine, 422 North Bond Street, Baltimore, MD 21231. Phone: (410)

955-8489; Fax: (410) 955-0840.

The critical event(s) necessary for activation of pro-

grammed cell death pathways have not yet been character-

ized, and specific targeting of a chemotherapeutic agent to

these pathways is not yet possible. Increased understanding

of the mechanisms by which existing chemotherapeutic

agents act may lead to the development of new drugs or more

effective use of the currently available ones. Paclitaxel

(Taxo!; Bristol-Myers/Squibb, Wallingford, CT) is one of the

most active new agents currently used for the clinical treat-

ment of breast cancer (7-10). It is known for its unique

ability to stabilize microtubules, thus preventing chromo-

some segregation and subsequent cellular division. The goal

of these studies was to determine whether paclitaxel, an agent

that does not cause direct DNA damage, induces programmed

cell death in human breast cancer cells.

MATERIALS AND METHODS

Cell Line and Culture Conditions. MDA-MB-468 cells

were purchased from American Type Culture Collection (Rock-

ville, MD) and maintained in improved minimal essential me-

dium supplemented with 5% fetal bovine serum (Biofluids,

Rockville, MD) and 2 mtvt glutamine. MCF-7, MDA-MB-23 1,

and Hs578t cells (obtained from Dr. Marc Lippman, Vincent T.

Lombardi Cancer Center, Washington, DC) were maintained in

DMEM supplemented with 5% fetal bovine serum and 2 mr�i

glutamine. Cultures were incubated at 37#{176}Cin a humidified 5%

CO2 atmosphere and passaged every S days. Mycoplasma test-

ing was routinely negative.

Paclitaxel. Paclitaxel was a gift from Bristol-Myers/

Squibb. For all experiments, a concentrated paclitaxel solu-

tion (10 mM in DMSO, stored at 4#{176})was diluted in medium

to the desired concentration. In each experiment, control cells

were exposed to a DMSO concentration equivalent to the

highest DMSO concentration present in the paclitaxel-treated

cells.

Growth Inhibition Assay. Exponentially growing cells

were plated in triplicate at 3-5 X l0� cells/cm2 in 24-well

plates. After attachment, medium was changed, and cells were

incubated in the presence or absence of at least eight paclitaxel

concentrations. If drug exposure time was less than 120 h, cells

were washed three times with medium (�30-fold drug dilution

with each wash) to ensure that the residual extracellular drug

concentration was below that which would have effects over the

total time of the experiment. After 120 h, the cells were de-

tached by trypsinization and counted using a Coulter counter.

Initial experiments demonstrated that cell numbers determined

using a Coulter counter were equivalent to numbers of trypan

blue-excluding cells (viable cells) determined by cell counting

using a hemocytometer. IC50 values were determined from plots

of the percentage of the control cell number versus the logarithm

of the drug concentration. All experiments were carried out at

Research. on September 10, 2018. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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848 Paclitaxel-induced Cell Death

3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase;EGF, epidermal growth factor.

least twice, and values reported are means ± SDs of individual

determinations from all experiments.

Assessment of Morphology. Exponentially growing

MDA-MB-468 cells were incubated in the presence or absence of

100 mvt pacitaxel for 3 or 24 h. Cells were then washed and

incubated in drug-free medium for the additional periods of time

before fixation in methanol. Fixed cells were stained with 0.1

mg/mi Hoechst dye no. 33342 (Sigma Chemical Co., St. Louis,

MO) and visualized by fluorescence microscopy using a Zeiss

Axioskop microscope (Zeiss, Hanover, MD) with filter set 48179/

02.

DNA Fragmentation Assays. Exponentially growing

cells were plated at 3-5 X i0’� cells/cm2. After attachment, the

medium was changed, and cells were incubated in the presence

or absence of paclitaxel for the desired exposure time. If the

drug exposure time was shorter than the time to cell harvest,

cells were washed and incubated in drug-free medium for the

remainder of the total incubation time. At harvest, medium and

trypsinized cells were combined and cells pelleted by centrifu-

gation. For analysis of oligonucleosomal DNA fragmentation,

DNA was isolated as previously described (6). Equivalent

amounts of DNA (15-20 p.g) were loaded into wells of a 1.6%

agarose gel and electrophoresed in Tris-borate EDTA. For ana]-

ysis of high-molecular-weight DNA fragmentation, the cell pe!-

let was resuspended in 1% low-melting-point agarose and cast

in a plug mold. After digestion with proteinase K and RNase A,

plugs were loaded into wells of a 0.8% agarose gel and elec-

trophoresed 12-14 h at 6 V/cm in 0.5X Tris-borate EDTA (1 X

Tris-borate EDTA is 89 m�i Tris, 89 mrvi boric acid, and 2 mn

EDTA) using program 6 of a PPI-200 field inversion timer (M.

J. Research, Inc., Watertown, MA) to provide a linear time ramp

from 0.3 to 30 s in the forward direction and a forward:reverse

ratio of 3.

Quantitation of DNA Fragmentation. Experiments

were carried out as stated above for analysis of high-molec-

ular-weight fragmentation, with the following modifications.

Exponentially growing cells were labeled with Emethyl-

‘4C]thymidine (0.05 mCi/ml medium, Amersham Life Sci-

ence, Arlington Heights, IL) for 24 h before plating for DNA

fragmentation assays. Throughout the cell harvest and proc-

essing of the agarose plugs, aliquots were removed and

counted in the presence of fluor (Bio-Safe II, Research Prod-

ucts International, Mount Prospect, IL) to determine the

percentage of total radioactivity in the medium and the

washes. Gels were photographed with UV illumination and

then dried onto DE8 1 paper on a vacuum drier. Fragmenta-

tion, as indicated by migration of DNA out of the wells, was

quantitated by integration of signals using a phosphorimager

(Molecular Dynamics, Sunnyvale, CA), and that percentage

was added to the percentage of fragmentation determined

from the medium and washes to assess the total percentage of

DNA fragmentation.

Assessment of Apoptosis-related Proteolytic Cleavage.

Cleavage of nuclear proteins was examined as described previ-

ously (1 1 , 12, and references therein). In brief, samples contain-

ing 50 jig of protein from paclitaxel-treated cells were subjected

to SDS-PAGE, transferred to nitrocellulose, and probed with the

following antibodies: C-2-lO, a murine monoclonal antibody

against PARP3 (12); a chicken antiserum raised against mam-

malian !amin � (1 1); and a chicken antiserum raised against the

abundant nucleolar protein B23 (1 1, 12).

RNA Isolation and Northern Blot Analysis. After in-

cubation with paclitaxel or vehicle control, adherent and non-

adherent cells were combined, and total cellular RNA was

isolated using the urea-lithium chloride method of Auffray and

Rougeon (13). Northern blot analysis was performed as de-

scribed previously (6, 1 1). Quantitation of signals on autoradio-

graphs was carried out using a phosphorimager, and results were

normalized by expressing the units obtained for a specific Iran-

script relative to the units obtained for the l8s ribosome iran-

script.

RESULTS

Inhibition of MDA-MB-468 Cell Growth by Paclitaxel.

The effect of paclitaxe! on MDA-MB-468 human breast cancer

cells was examined. Initial experiments examined cell number

120 h after the start of 3-, 24-, or 120-h paclitaxel exposure (Fig.

1). These exposure times were chosen to model clinically used

treatment times of 3, 24, and �96 h. Cells exposed to paclitaxel

for only 3 h demonstrated concentration-dependent growth in-

hibition at � 10 nM paclitaxel and an IC50 of 17 nrvi. Increasing

the exposure time to 24 h resulted in increased sensitivity of

MDA-MB-468 cells to paclitaxel, with concentration-dependent

growth inhibition occurring at � 1 flM paclitaxel and an IC50 of

2.6 nM. It is interesting to note that further increasing the

paclitaxel exposure time had little effect, as the concentration

dependence and the IC50 (1.8 nM) resulting from 120-h pacli-

taxel exposure were similar to those resulting from 24-h expo-

sure. Ongoing clinical studies are addressing the optimal dura-

tion of paclitaxel treatment in breast cancer with particular

interest in durations of 3 and 24 h. Pharmacokinetic studies

during Phase I and II clinical trials have demonstrated peak

plasma levels of 2.5-4.3 p.M and 0.2-0.9 p.M paclitaxel during

3- and 24-h infusions, respectively, and a steady-state plasma

level of 0.05-0.09 p.M paclitaxel during 96-h infusions (7, 9,

14). Therefore, 3- or 24-h exposure times and clinically achiev-

able paclitaxel concentrations (0. 1 rust to 1 JiM) were used for all

subsequent studies.

Induction of Programmed Cell Death by Paclitaxel.

Characteristic morphological changes are associated with pro-

grammed cell death. These morphological changes include chro-

matin aggregation, nuclear condensation, and cellular fragmen-

tation into apoptotic bodies (3, 4). MDA-MB-468 cells exposed

to 100 flM paclitaxel for 3 h were assessed for these changes at

times ranging from 6 to 48 h after initial drug exposure (Fig. 2).

Chromatin aggregation and nuclear condensation were observed

in a small number of the treated cells by 6 h (not shown), and the

number of cells exhibiting these changes increased at 12 and 24

h (Fig. 2, B and C). Fragmentation of the cell into membrane-

bound apoptotic bodies was also detected at 24 h and was

markedly increased by 48 h (Fig. 2D). Cells exposed to pacli-

taxel for 24 h showed the same progression of morphological



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Clinical Cancer Research 849

Fig. 1 Time and concentration

dependence of growth inhibition

of MDA-MB-468 cells by pacli-

taxel. Exponentially growing

cells were plated, incubated in

the presence or absence of pacli-

taxel, harvested, and counted as

described in “Materials and

Methods.” The duration of drugexposure was 3 (A), 24 (U)’ or120 h (0), and the total experi-mental time was 120 h. Values

reported are the averages of atleast six individual data points

determined in at least two sepa-

rate experiments; bars. SD.

changes, but the time course was accelerated, with a larger

number of apoptotic bodies present by 24 h (not shown). Un-

treated control cells fixed at each time point showed no evidence

of these apoptosis-associated morphological changes, and mi-

totic figures were observed through the 48-h time point (Fig.

2A).

These morphological changes are frequently accompanied

by fragmentation of genomic DNA into oligonucleosomal frag-

ments ( 1 5). Paclitaxel exposure for 3 h resulted in time- and

concentration-dependent oligonucleosomal DNA fragmentation

characteristic of programmed cell death (Fig. 3A). Fragmenta-

tion induced by 3-h exposure to paclitaxel concentrations �lOO

nM was detected at 24 h (Fig. 3A, Lanes 4 and 5), whereas that

resulting from 10 ns� paclitaxel was not detectable until 48 h

after initial drug exposure (Fig. 3A, Lane 7). No oligonucleo-

somal DNA fragmentation was detected in DNA isolated from

untreated controls at either 24 or 48 h (Fig. 3A, Lanes 2 and 6).

In additional experiments, paclitaxel exposure for 24 h at con-

centrations � 10 ns� induced oligonucleosomal DNA fragmen-

tation that was detectable at �24 h.

In some cell types undergoing programmed cell death,

formation of oligonucleosomal DNA fragments appears to be

preceded by two distinct events: (a) cleavage of DNA to do-

main-sized (�50 kb) fragments (16); and (b) selective cleavage

of several nuclear proteins by proteinases related to interleukin-

l-� converting enzyme (12, 17). As one or both of these steps

might play a role in commitment to programmed cell death (16,

17), the timing of both of these processes in paclitaxel-induced

programmed cell death was examined.

Paclitaxel treatment of MDA-MB-468 cells resulted in a

time- and concentration-dependent pattern of high-molecular-

weight DNA fragmentation. Exposure to 100 flM paclitaxel for 3

or 24 h resulted in DNA fragments of -50-150 kb detectable at

24, 48, and 72 h, but not at 6 h after initial drug exposure (Fig.

3B). Assay at 24 h indicated that paclitaxel concentrations �l0

nM were sufficient to induce DNA fragmentation even if the

duration of drug exposure was only 3 h (not shown).

Examination of programmed cell death-related cleavage of

nuclear proteins revealed a similar picture. Cleavage of the Mr

1 16,000 repair-related protein, PARP, to a Mr 85,000 fragment

(12, 17) was detectable at �24 h in cells exposed to 100 nM

paclitaxel for 3 or 24 h (Fig. 3C, Lanes 3-5 and 7-9, respec-

tively), whereas no cleavage of PARP was detectable at 6 h (Fig.

3C, Lanes 2 and 6). When cells were separated into adherent

(not yet apoptotic) and nonadherent (apoptotic) cell populations

as described previously (6, 1 1 ), PARP cleavage was detected

exclusively in the nonadherent population (Fig. 3C, Lanes 11

and 14). No PARP cleavage was detectable in vehicle-treated

control cells through 72 h (Fig. 3C, Lanes 16-19). Examination

of lamin , an abundant nuclear envelope polypeptide that is

cleaved to a Mr �45,000 fragment during the course of pro-

grammed cell death ( 1 7 and references therein), revealed that

cleavage of lamin B1 paralleled cleavage of PARP (Fig. 3C). In

contrast, the abundant nucleolar protein B23 was not cleaved at

any of the time points tested (Fig. 3C), an observation that is

consistent with previous claims that the proteolytic process is

selective for certain nuclear proteins (12, 17).

Effects of Paclitaxel on Expression of Early-ResponseGenes. In view of previous observations that different effec-

tors of programmed cell death can have different effects on the

expression of early-response genes (6, 1 1 ), the effects of pacli-

taxel on c-jun and c-mvc expression in MDA-MB-468 cells

were determined. A 2-fold increase in c-jun mRNA expression

was observed 15 mm after exposure to 100 nnvt paclitaxel.

Expression of c-jun peaked at 2.4-fold induction at 30 mm and

returned to baseline levels at 1 h (Fig. 3D). Expression of c-mvc

did not change significantly during the 48-h period (Fig. 3D).

Quantitation of Programmed Cell Death Induced by

Paclitaxel. The results described above provide clear evidence

that 3- or 24-h paclitaxel exposure activates programmed cell



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Control 48 h

Paclitaxel 24 h

B.Paclitaxel 1 2 h

Paclitaxel 48 h� #{149}� �

Fig. 2 Fluorescent micrographs

of MDA-MB-468 cells incubated

in the presence or absence of 100

nM paclitaxel. MDA-MB-468

cells were exposed to 100 nM pa-

clitaxel (B-D) or vehicle control

(A) for 3 h and then incubated in

drug-free medium until fixationand staining with Hoechst’s dye

no. 33342 at 12 (B), 24 (C), or 48

(A and D) h.

death pathway(s) in MDA-MB-468 cells. To assess whether the

extent of programmed cell death resulting from 3-h versus 24-h

paclitaxel exposures was comparable, DNA fragmentation and

cell number were quantitated (Fig. 4). At all times >24 h, DNA

fragmentation of treated cells was greater than that of untreated

cells (Fig. 4A). Moreover, the amount of DNA fragmentation

resulting from a 24-h exposure to 100 nM paclitaxel was greater

than that resulting from a 3-h exposure (Fig. 4A). When the

effects of 24-h versus 3-h paclitaxel were evaluated by compar-

ing DNA fragmentation 72 h after the initiation of treatments

that involved similar concentration X time parameters (“area

under the curve”), greater induction of programmed cell death

by the 24-h treatment remained evident. For example, treatment

with 10 nM paclitaxel for 24 h (240 nM . h) provided lower

exposure than 100 nM paclitaxel for 3 h (300 nM . h), yet the

DNA fragmentation with the 24-h treatment was 1 .5-fold higher

(63.5 ± 8.6% versus 42.9 ± 3.1%; Fig. 4B). Similarly, treat-

ment with 100 nM paclitaxel for 24 h (2400 nn�t � h) involved

lower exposure than 1 p.M for 3 h (3000 nM . h), yet the DNA

fragmentation 72 h after the initiation of the 24-h treatment was

greater than that 72 h after the initiation of the 3-h treatment

(76.6 ± 6.9 versus 53.9 ± 9.9%; Fig. 4B). Indeed, 24-h expo-

sure to 10 nM paclitaxel produced more DNA fragmentation

than 3-h exposure to 1 p.M (63.5 ± 8.6% versus 53.9 ± 9.9%),

although the area under the curve with the 24-h treatment (240

flM � h) was only 8% of the exposure in the 3-h treatment (3000

nM . h). These comparisons indicate that lower concentrations of

paclitaxel are required to achieve equal or greater DNA frag-

mentation when the longer exposure time of 24 h is used.

The enhanced effectiveness of the prolonged paclitaxel

exposure was also evident when cell numbers were exam-

med. For example, 10 flM paclitaxel for 24 h (240 nnvi - h) and

100 nM for 3 h (300 nM . h) resulted in 74% and 59%

decreases in cell number, respectively. Likewise, 100 nM

paclitaxel for 24 h (2400 nM . h) and 1 p.M paclitaxel for 3 h

(3000 nM . h) resulted in 83% and 70% decreases in cell

number, respectively. Although the area under the curve was

smaller in each case, the cell decrease was more profound



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with the 24-h treatment. Indeed, decreases in cell number lions. After the 3-h exposure to 100 n�i paclitaxel, an increase

were similar after treatment with 10 nM paclitaxel for 24 h in cell number over the number at the time of initial drug

(74%) and 1 p.M paclitaxel for 3 h (70%), although the area exposure was observed at times >24 h, and cell number at 96

under the curve of the 24 h treatment was less than 10% of h was 2.4 times greater than the original number (Fig. 4C).

that obtained with the 3-h treatment (240 nM . h versus 3000 Even at the highest paclitaxel concentration tested (1 p.M), the

flM � h). There are also additional indications that the longer 3-h exposure resulted in a net increase in cell number over

exposure time is more toxic even at lower drug concentra- that at the time of initial drug exposure (Fig. 4D). In contrast,


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Fig. 3 Paclitaxel induction of programmed cell death in MDA-MB-468 cells. A, induction of oligonucleosomal-sized DNA fragmentation by

paclitaxel. Cells were exposed to paclitaxel (P) or vehicle (U) for 3 h and then incubated in drug-free medium until 24 or 48 h after initial drugexposure. Isolated DNA was analyzed by gel electrophoresis. Lane 1, l23-bp ladder marker; Lane 2. vehicle control, 24 h; Lanes 3-5, 10 nsi, 100flM, and 1 p.M paclitaxel, respectively, 24 h; Lane 6, vehicle control, 48 h; Lanes 7 and 8, 10 nM and 1 p.M paclitaxel, respectively, 48 h. B, paclitaxel

induction of high-molecular-weight DNA fragmentation. MDA-MB-468 cells were incubated in paclitaxel or vehicle for up to 24 h and incubated indrug-free medium for any additional period before harvest for field inversion gel electrophoresis. Lane 1, X Hindlll molecular weight marker; Lane

2, vehicle control, 6 h; Lane 3, 100 nM paclitaxel, 6 h; Lane 4, vehicle control, 24 h; Lanes 5-9, 0.1 nM, 1 nM, 10 nM, 100 nsi, and 1 p.M paclitaxel,

respectively, 24 h; Lane 10, vehicle control, 48 h; Lane 11, 100 nM paclitaxel, 48 h; Lane 12, untreated control, 72 h; Lane 13, 100 nM paclitaxel,

72 h. C, paclitaxel induction of proteolytic cleavage. MDA-MB-468 cells were exposed to 100 nM paclitaxel for 3 or 24 h. Cleavage of PARP, laminB1, and B23 was assessed at 3, 6, 24, 48, and 72 h after initial drug exposure. Lanes 1-5, 24-h paclitaxel exposure, harvest at 3, 6, 24, 48, and 72h, respectively; Lanes 6-9, 3-h paclitaxel exposure, harvest at 6, 24, 48, and 72 h, respectively; Lanes 10-12, 24-h paclitaxel exposure with immediate

harvest for total cell population, nonadherent cells, and adherent cells, respectively; Lanes 13-15, 24-h paclitaxel exposure with harvest at 48 h for

total cell population, nonadherent cells, and adherent cells, respectively; Lanes 16-19, vehicle controls, 6, 24, 48, and 72 h, respectively. D, Northernblot analysis of RNA isolated from MDA-MB-468 cells exposed to paclitaxel for 3 h. U, vehicle-treated control cells; P, paclitaxel-treated cells;

subscripts, time of harvest (h).



B. 100

900 24 h Paclitaxel0 3 h Paclitaxel

80

70

60

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30

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Time (h)

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Fig. 4 Quantitation of DNA fragmentation and cell numbers after exposure of MDA-MB-468 cells to paclitaxel. A, time dependence of DNA

fragmentation resulting from 3- or 24-h exposure to 100 nM paclitaxel. B, concentration dependence of DNA fragmentation assayed 72 h after initialexposure of cells to paclitaxel for 3 or 24 h. At 72 h, there was 23% DNA fragmentation in vehicle-treated control cells. DNA fragmentation resulting

from paclitaxel was quantitated as described in “Materials and Methods.” Values reported in A and B are the means of three or more individualdeterminations from at least two separate experiments; bars, SD. C, time dependence of cell number after 3- or 24-h exposure to 100 nM paclitaxel.

D, concentration dependence of cell number assayed 72 h after initial exposure of cells to paclita.xel for 3 or 24 h. Cell number at the time of initial

drug exposure was 2.5 X l0’� cells/cm2. Values reported are from a representative experiment and represent means of at least three individualdeterminations for C and averages of two individual determinations for D; bars, SD.

0 24 h Paclitaxel0 3 h Paclitaxel

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Time(h)

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after 24-h paclitaxel (100 nM) exposure, the cell numbers at

each assay time remained at or below the original number

(Fig. 4C), and a net decrease in cell number was evident after

24-h exposure to concentrations � 100 nM (Fig. 4D).

Effects of Paditaxel in Other Breast Cancer Cell Lines.

To determine whether paclitaxel induces programmed cell death

in other human breast cancer cell lines, MDA-MB-23l, HS578t,

and MCF-7 breast cancer cells were exposed to 100 nM pacli-

taxel for 24 h and monitored over a 72-h period. Morphological

changes and high-molecular-weight DNA fragmentation were

observed in each of these cell lines (data not shown), indicating

induction of programmed cell death by paclitaxel. Quantitation

of the amount of DNA fragmentation indicated that 24-h pacli-

taxel exposure resulted in rapid and extensive DNA fragmenta-

tion in H5578t cells, in as much as there was 53% DNA

fragmentation by 24 h and 96% at 72 h after initial drug

treatment (Fig. SA). Although the effects were not as rapid in the

other cell lines, at 72 h, DNA fragmentation was 40% and 21%,

respectively, for MDA-MB-23 1 and MCF-7 cells, levels signif-

icantly greater than those observed in the untreated control cells

(Fig. SA). Examination of cell numbers present at each harvest

time indicated that 24-h exposure to 100 nnvi paclitaxel prevented

an increase in total cell number of MDA-MB-23l and MCF-7

cell populations over the 72 h observed, and resulted in a



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Fig. 5 Quantitation of DNA fragmentation and cell numbers after exposure of MDA-MB-23l, HS578t, and MCF-7 cells to paclitaxel. A, timedependence of DNA fragmentation resulting from 24-h exposure to 100 flM paclitaxel. B, time dependence of cell number after 24-h exposure to 100nM paclita.xel. Values reported in A and B are the average of two individual determinations.

decrease in total cell number of the HSS78t cell population by

48 h (Fig. SB).

DISCUSSION

Paclitaxel has substantial clinical activity against a variety

of malignancies, including breast cancer. The present study

shows that exposure to clinically attainable paclitaxel concen-

trations (7, 9, 14) for 24 h induces programmed cell death in all

four breast cancer cell lines tested. In addition, exposure to

clinically attainable paclitaxel concentrations (7, 9, 14) for 3 h is

sufficient to activate programmed cell death pathway(s) in

MDA-MB-468 cells. Even this brief exposure resulted in char-

acteristic morphological changes, DNA fragmentation to both

high-molecular-weight and oligonucleosomal-sized fragments,

and proteolytic cleavage of PARP and lamin , although the

changes were not evident until some hours later.

Paclitaxel effects in MDA-MB-468 cells differ from those

that result from activation of the cell death program by fluoro-

pyrimidines or EGF (6, 1 1). Quantitation of DNA fragmentation

indicated that 24-h paclitaxel exposure (100 nM) results in

-2-fold greater DNA fragmentation at 48 h than does contin-

uous exposure to either EGF (10 ng/ml) or 5-fluoro-2’-de-

oxyuridine (100 p.M), although similar amounts of fragmenta-

tion are observed at 72 and 96 h. Exposure to paclitaxel or EGF,

but not to 5-fluoro-2’-deoxyuridine, led to rapid induction of the

early response gene c-jun. However, the peak level of induction

after paclitaxel was 2.4-fold compared with 16-fold induction of

c-jun after EGF. Neither paclitaxel nor 5-fluoro-2’-deoxyuridine

induced c-myc expression, whereas EGF resulted in an 1 1 -fold

increase of c-myc expression. The variety of cellular responses

to different effectors of programmed cell death within the same

cell type suggests that these responses are secondary to the

as-yet-unidentified critical event(s) that actually trigger activa-

tion of the cell death pathway.

Paclitaxel effects in the breast cancer cells during activa-

tion of programmed cell death also differ from those reported in

other cell types. In HL-60 leukemia cells, paclitaxel-induced

programmed cell death is accompanied by a decrease in c-myc

expression without a change in c-jun expression at �24 h. The

possibility that a transient induction could have occurred at

earlier times was not examined (18).

Finally, it is currently unclear whether the variety of din-

ical protocols that include infusion times of 3, 24, or 96 h,

currently used for paclitaxel, are equally efficacious (7, 8, 19).

It has been reported that 4- and 12-h paclitaxel exposures are

required to induce programmed cell death in HL-60 cells (18)

and 0V2008 ovarian carcinoma cells (20), respectively. Our

studies provide clear evidence that paclitaxel exposure for either

3 or 24 h can increase the rate of cell death in MDA-MB-468

breast cancer cells (Figs. 2 and 3), but the two treatments differ

in their ability to alter the overall growth of the MDA-MB-468

cell population. After 3-h paclitaxel exposure, the overall cell

number continued to increase despite evidence of programmed

cell death occurring in some cells (Fig. 4, C and D). This

indicates that a portion of the cell population was still actively

proliferating, and the rate of cell proliferation still exceeded the

rate of cell death. In contrast, after exposure to paclitaxel for 24

h, the rate of cell death exceeded the rate of cell proliferation,

because the population cell number remained at or below the

original number at all times, and no net increase in cell number

occurred (Fig. 4, C and D). These results demonstrate quantita-

tively that 24-h paclitaxel exposure was more effective than 3-h

exposure at inducing programmed cell death and stopping cell

proliferation, even when treatments with similar areas under the




curve were compared. These in vitro studies cannot mimic the

pharmacokinetic or pharmacodynamic situation encountered

clinically. However, these results do support those clinical stud-

ies that suggest that longer paclitaxel infusion times are of

greater benefit, and suggest an explanation at the cellular level

for this observed outcome. Additionally, if the same situation

exists in vivo, these results suggest that at least part of the lack

of response seen clinically to 3-h paclitaxel infusions may not

result from inherent resistance of the cells to this agent, but from

a lack of adequate time for a large portion of the cell population

to initiate a response.

ACKNOWLEDGMENTS

We thank Drs. John Isaacs and Lars Cisek for helpful discussions.

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1996;2:847-854. Clin Cancer Res D E McCloskey, S H Kaufmann, L J Prestigiacomo, et al. human breast cancer cells.Paclitaxel induces programmed cell death in MDA-MB-468

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