Bryostatin 1 Affects P-Glycoprotein Phosphorylation but not...

Vol. 1. 1581-1587. December 1995 Clinical Cancer Research 1581

Bryostatin 1 Affects P-Glycoprotein Phosphorylation but not

Function in Multidrug-resistant Human Breast Cancer Cells

Stefania Scala, Bruce Dickstein, Joanna Regis,

Zoltan Szallasi, Peter M Blumberg,

and Susan E. Bates’

Medicine Branch, Clinical Oncology Program. Division of Cancer

Treatment IS. S.. B. D., J. R., S. E. B.l, and Laboratory of Cellular

Carcinogenesis and Tumor Proiiiotion, Division of Cancer Etiology

lZ. S.. P. M. B.l, National Cancer Institute. NIH. Bethesda, Maryland

20892

ABSTRACT

The function of P-glycoprotein (Pgp), which confers

multidrug resistance by active efflux of drug, is thought tobe dependent on phosphorylation. Previous studies have

suggested that protein kinase C (PKC) plays an important

role in Pgp phosphorylation. We report here the effects of

bryostatin 1, a unique PKC activator and inhibitor, on Pgp

function in a multidrug-resistant MCF-7 human breast can-

cer subline which overexpresses PKC-a. Bryostatin 1 (100

nrsi) decreased Pgp phosphorylation after 24 h of treatment.

In contrast, it did not affect Pgp function as demonstratedby the accumulation of [3H]vinblastine and rhodamine 123.

We compared the effect of bryostatin 1 treatment on PKC-awith that of the phorbol ester 12-O-tetradecanoylphorbol-

13-acetate (200 nsi). 12-O-tetradecanoylphorbol-13-acetate

caused translocation of PKC-a from the cytosol to the cell

membrane after a 10-mm treatment and its down-regulationafter 24 h of treatment. Likewise, bryostatin 1 (100 nsl)

caused translocation, but only after longer treatment ( 1 h),

and it caused down-regulation of PKC-a at 24 h of treat-ment. Thus, while the MCF-7TH cells overexpress thePKC-a isoform, and its down-regulation by bryostatin 1 isassociated with decreased Pgp phosphorylation, these alter-ations do not modulate drug transport. We conclude that,

while bryostatin 1 may be useful clinically because of itsability to inhibit PKC, it is not able to reverse Pgp-mediated

multidrug resistance.

INTRODUCTION

The identification of clinically suitable agents able to oven-

come drug resistance would have great impact for cancer treat-

ment. In tumor cell lines. the best characterized mechanism of

drug resistance is ‘ ‘multidrug resistance’ ‘ mediated by overex-

pression of an energy-dependent membrane-bound drug effiux

pump known as Pgp2. Pgp undergoes covalent modification by

Received 2/6/95: revised 8/15/95: accepted 8/17/95.

I To whom requests for reprints should be addressed. at NIH. Building10/Room 12N226, 9000 Rockville Pike, Bethesda. MD 20892. Phone:

(301) 402-0984; Fax: (301) 402-0172.

2 The abbreviations used are: Pgp. P-glycoprotein: PK. protein kinase:TPA, I 2-O-tetnadecanoylphonbol- I 3-acetate.

rapid cycles of phosphorylation and dephosphorylation in which

several different protein kinases have been implicated ( 1-6).

The earliest reports suggested that phosphorylation played a role

in the activity of the efflux pump ( 1. 7-9). Increased transport

results from increased Pgp phosphorylation, whereas impaired

function occurs with inhibition of phosphorylation (2. 3).

PKC (2. 10-13). PKA (4. 13). and other potentially more

specific kinases (3. 5. 6) have been linked to Pgp phosphoryla-

tion. Chambers et a/. ( 12) reported that PKC phosphorylates Pgp

at multiple sites in the linker region between the two halves of

the molecule. Characterization of specific residues for PKC and

PKA phosphorylation has been reported in both munine and

human Pgp (11, 13). Several studies have confirmed that acti-

vation of PKC by the phorbol ester TPA results in an increase in

Pgp phosphorylation associated with increased drug efflux, de-

creased drug accumulation, and a more resistant phenotype

( 1-3. 7. 14. 15). Conversely, PKC down-regulation by long-

term TPA treatment or inhibition by staunosponne on calphostin

C impairs Pgp function, resulting in decreased drug efflux and

increased accumulation (7, 14, 16). Increased expression or

activity of the PKC isoforms has been reported in multidrug-

resistant cell lines (2, 7, 17), with a differential increase in the

expression of the PKC-a isoform being the most frequent ob-

servation ( 17-19). This finding has been reported for a multi-

drug-resistant subline of MCF-7 human breast cancer cells (19).

Bryostatin 1 . which is structurally distinct from the phorbol

esters, can activate PKC in vitro but in t’is’o it can paradoxically

antagonize other activators of PKC such as the tumor-promoting

phonbol esters (20. 2 1 ). Bryostatin 1 is the best studied member

of a family of more than I 3 compounds that have a macrocyclic

lactone structure with varying side chains (22. 23). As the most

abundant of the bryostatins isolated from the marine bryozoan

Bugu/a neritina, bryostatin 1 is the first member of this class of

compounds to be tested in humans. These macrocyclic lactones

possess potent antineoplastic properties in human and murine

tumor cell lines (22. 24-26) and in tim activity in murine B16

melanoma. The range of in vitro activities includes cytotoxicity

and terminal differentiation of malignant cells (27, 28). More-

over, in contrast to the phorbol esters, bryostatin I lacks tumor-

promoting activity. These properties suggested that bryostatin I

could be useful as an antineoplastie agent. The first Phase I

clinical trial with bryostatin 1 was recently completed and

demonstrated that concentrations of drug as high as 25 jig/mI

could be safely achieved in humans (29).

Since PKC modulation has been shown to impair Pgp

function, and bryostatin 1 has been shown to be well tolerated

clinically. we studied the effect of bryostatin I on a multidrug-

resistant MCF-7 human breast cancer subbine which overex-

presses PKC-a. We sought an agent suitable for clinical studies

which could decrease Pgp phosphorylation and thereby impair

its function. The results of these studies are described here.

Research. on June 1, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

MCF-7

‘Parent TH I Rat

S P S PBrain

� - PKCa

I� PKC’y

100�

75.

50’

25’

0� �- C

- C

1582 Bryostatin I in Breast Cancer Cells

...

. PKC�I

I PKC#{128}

.. PKC�

Fig. I Expression of PKC isoforms in MCF-7 (Parent) and MCF-7TH

(TI-f) cells. Cells were harvested and fractionated into a cytosol (S) and

a membrane (P) fraction. Following electrophoresis and transfer to

nitrocellulose, immunoblotting was performed with antibodies specific

for the isoenzymes detected. Identical results were obtained in a second

series of independent experiments.

MATERIALS AND METHODS

Materials. Bryostatin 1 was provided by the Develop-

mental Therapeutics Program (Division of Cancer Treatment,

National Cancer Institute); TPA was obtained from Sigma;

monoelonal anti-PKC-a antibody was obtained from United

Biomedical, Inc.; and polycbonal anti-PKC-�3, -#{128},-�, -“y, and �

antibodies were obtained from GIBCO Life Technobogies-BRL.

Cell Lines. MCF-7TH multidrug-resistant cells were Se-

leeted from the parental MCF-7 human breast cancer cells by

intermittent exposure to doxorubicin and were maintained in

200 ng/ml drug. The cells were cultured in DMEM, 10% FCS.

penicillin, streptomycin, and glutamine at 37#{176}Cin 5% CO2.

MCF-7TH cells overexpress Pgp and are cross-resistant to

doxorubicin (250-fold), vinblastine (480-fold), paclitaxel (73-

fold), and actinomycin D (220-fold), relative to MCF-7 cells. A

multidrug-resistant human colon carcinoma subline, SW620

Ad300, was utilized as a control in certain experiments. These

0

.0

.00

.0

SC

Bryostatin-l, nM

Fig. 2 Effect of bryostatin 1 on cell growth inhibition in MCF-7 and

MCF-7TH cells. Cells were plated in 96-well plates. and 24 h later weretreated with bryostatin 1 in the presence or absence of verapamil (10

pg/mb) for 4 days. Each point represents the average of two independent

experiments. �, MCF-7: El. MCF-7 + verapamil: #{149},MCF-7TH: 0,MCF-7TH + verapamil.

cells overexpress mndr-I and are maintained in 300 ng/ml doxo-

rubicin (30). The multidrug-resistant sublines were cultured in

media free of drug for I week prior to each experiment.

Drug Accumulation Assay. The accumulation of yin-

blastine, daunornycin. and colehicine were assayed as previ-

ously described (31). Briefly, cells were plated in 6-well dishes

(350,000/well), and after 24 h were treated with TPA and

bryostatin 1 for the indicated periods of time. Cells were then

rinsed once and incubated for 10 mm with assay medium before

starting the incubation. Cells were incubated for 45 mm with 2

x l0� dpm/well. which is equivalent to 14 nsi [3Hlvinblastmne.

Rhodamine I 23 accumulation was also assayed as previously

described ( I 6). Briefly. after treatment for the indicated time

period, cells were trypsinized and resuspended at 1 X 106/ml in

phenol red-free improved MEM + 10% FBS containing 0.5

jig/mb rhodamine. Following a 30-mm incubation at 37#{176}Cin 5%

CO,. cells were washed in ice-cold improved MEM and ana-

byzed on a FACSort flow cytometer (Becton Dickinson)

equipped with a 488-nm laser. The green fluorescence of rho-

damine 123 was detected with a 530-nm band-pass filter.

PKC Isoform Immunoblotting. Pretreated and un-

treated cells were harvested in PKC buffer [2 mM EDTA. 0.5

mM EGTA, 5 mM DTT, 25 msi Tris-CI (pH 7.4), and 50 jig/mI

aprotinin (32)1. After sonication. the disrupted cells were cen-

trifuged at 1,000 X g to remove nuclei; the samples were then

centrifuged at 100.000 X g for 30 mm. The supernatant. con-

taming the cytosol, was removed, and the membrane pellet was

resuspended in PKC bufThr. After protein quantitation (Bio-

Rad), equal amounts (25 jig) of cytosolic and membrane protein

were subjected to SDS-PAGE on a 7.5% SDS-polyaerylamide



C/)

C

>

IC,)

A

B

00 BlOt)

0 0.15

Time(h)Rhodamine accumulationMean channel number. FL1

Time Q 2.ti 2�4�h ZZb

TPA (200 nM)

Bryostatin-1 (100 nM)

543 531 574 566

543 550 546 543

Clinical Cancer Research 1583

Fig. 3 Effect of TPA and bryostatin 1 (BRY) on [3Hlvinblastine accu-

mulation in MCF-7TH cells. Cells were treated with TPA (200 nsi) or

bryostatin 1 (100 n�i) for the indicated time prior to the accumulation

assay (31). Vinblastine accumulation in MCF7-TH is 3.5% of the

MCF-7 parental cell line (data not shown). 13H]Vinblastine accumula-tion (ordinate) is shown as the percentage of the value found in the

untreated MCF-7TH control. Bars, average ± SE of three independent

sets of experiments. These results are representative of another five

experiments. Inset, verapamil (Vu. 10 jig/nil) and staurosporinc (Se. 2(X)

nM) effects on l3H]vinblastine accumulation in MCF-7TH cells. C.

control.

gel. The fractionated proteins were then transferred to a nitro-

cellulose membrane in Towbin transfer buffer. The nitroceblu-

lose was blocked for I h with 3% BSA in TBST [20 hiM Tris-Cl

(pH 7.4, pH 7.5). 0.15 si NaCI, and 0.1% Tween 20). Blots were

then incubated for I h in TBST with the appropriate PKC

isoform-specific antibody. After three washes in TBST. the

blots were then incubated with horseradish peroxidase-labeled

secondary antibody in TBST for 60 mm. After three more

washes, the immunolabeled protein was detected by chernilu-

mineseence (enhanced chernilurninescence; Amersharn).

Phosphorylation Studies. Untreated cells or cells pre-

treated for 24 h with bryostatin 1 were labeled as previously

described (33. 34) with 250 jiCi I32Plorthophosphonic acid

(specific activity, 6000 Ci/rnrnol) in 4 ml phosphate-free

DMEM without serum for 1 h. After lysis of cells in RIPA

buffer (1% Triton X-l00/0.l% NaDodSO.4/I% sodium deoxy-

colate/0.15 M NaC1/20 m�vi Tris-CI, pH 7.2) with 20 jig/mb

aprotinin, samples were centrifuged to remove insoluble mate-

rial. lrnrnunoprecipitation was carried out with a polyclonal

antibody raised against recombinant protein fragments (35): the

product was analyzed on a 7.5% SDS-polyacrylamide gel.

Cytotoxicity Assay. Cells were seeded in 96-well plates

( 1000-2000/well); 24 h later different doses of bryostatin I

were added. The cells were incubated for 4 days in drug prior to

fixation with 10% TCA and staining with 0.4% sulforhodamine

B in 1% acetic acid (36). The bound dye was solubilized in

100-200 jil 10 mM unbuffered Tnis solution. and the absorbance

at 540 nrn was determined in a microplate reader (Bio-Rad).

Fig. 4 Effect of bryostatin I on Pgp function. A. l3Hlvinhlastine

(VBL), l3Hldaunomycin (DAUNO). and l3Hlcolchicine (COLCHI) ac-

cumulation in MCF-7TH cells. Cells were plated in 6-well dishes and

the day after treated with 100 n�i of bryostatin 1 for 24 h hefcre the

incubation with labeled drugs. B. rhodamine 123 accumulation in

MCF7-TH cells treated with bryostatin I. Cells were plated in 100-mm

Petni dishes, and 24 h later were treated with bryostatin 1 (1(X) nM) at the

indicated time points. Results are presented in channel numbers which

reflect the fluorescence intensity. Similar results were obtained in two

separate experiments.

RESULTS

We first studied the pattern of expression of PKC isoforms

in MCF-7TH multidrug-resistant breast cancer cells. As shown

in Fig. 1 , MCF-7TH cells overexpress PKC-ct when compared

with parental MCF-7 cells. This blot, which compares equal

amounts of protein in the cytosobic. or supernatant fraction, and

in the membrane. or particulate, fraction, demonstrates that

PKC-ot is overexpressed in both fractions. The isoforms #{128}and �

are expressed at comparable levels in both cell lines. while �, �,

and -y are not reliably detected in either cell line. The overex-

pression of PKC-a in the multidrug-resistant cells suggested

that inhibition could affect Pgp phosphorylation and function.

To exclude the possibility that bryostatin I is a substrate for

Pgp. cytotoxicity assays were performed in parental and resis-

tant cells, with and without verapamil. Doses of bryostatin I of

less than or equal to 100 nM did not result in growth inhibition

over the 4-day incubation in the cytotoxicity assay. The 50%

inhibitory concentration for MCF-7TH was 20 jisi and for

MCF-7 was 2 jiM, suggesting that MCF-7TH is 10-fold resistant

to bryostatin I as shown in Fig. 2. However, the addition of

venapamil (10 jig/mI) did not significantly increase the sensi-

tivity to bryostatin I (Fig. 2). The effect of verapamil on

bryostatin 1 cytotoxicity was also tested in multidrug-resistant

SW620 Ad300 human colon carcinoma cells. again demonstrat-

ing no sensitization by the Pgp antagonist (data not shown).



100

e� 80

� � 60lace

� .� 40

� 20

C

Control 10” lh 24h �

Is pH� pl� � p’ �

TPA � -_ __ � - - PKC a

Bryo � � - � � - - PKC a

TPA .� -PKC#{128}

Bryo . - -PKCe

TPA ‘�ZW� � __� - � - � - PKC �

Bryo -� - � � - � - - - PKC �

1584 Bryostatin 1 in Breast Cancer Cells

MCF-7TH Cells

I BryostatinI I

Control 10 pM 100 nM TPA

�#{248}..

�llUnUControl � pM 100 nM � TPA

Bryostatin

Fig. 5 Effect of bryostatin I on Pgp phosphorylation in MCF-7TH

cells. Cells were treated for 24 h with 10 �M or 100 nsi of bryostatin 1

or 200 nM TPA prior to metabolic labeling with 250 p.Ci [32P1P for 90mm. Next, immunoprecipitation was carried out with a polyclonal

antibody against recombinant protein fragments of Pgp. Similar results

were obtained in three independent experiments.

To study the effect of bryostatin I on Pgp function, drug

accumulation studies were performed in MCF-7TH cells, as

shown in Fig. 3. Previous studies described an increase in

vinblastine accumulation to 300% of control in MCF-7TH cells

after treatment with calphostin C, a PKC inhibitor ( 16). As

shown in Fig. 3, treatment of MCF-7TH cells with either 200 nst

TPA or 100 nM bryostatin 1 decreased [3Hjvinblastine aecurnu-

lation 10-30% after 15 mm. Treatment with TPA for 72 h

increased vinblastine accumulation to 160% ofeontrol, which is

suggestive of PKC inhibition. However, 100 nrvt bryostatin 1 did

not significantly affect [3Hlvinblastine accumulation after a 24-

or 72-h treatment. Likewise, no effect of 10 �M bryostatin I on

vinblastine accumulation could be observed (data not shown).

For comparison, vinblastine accumulation in the presence of

verapamil and after staurosporine treatment is shown (Fig. 3,

inset). Verapamil increased vinblastine accumulation to 700%

of control, while staurosporine, a nonspecific PKC inhibitor,

increased vinblastine accumulation to 350% of control. Thus,

neither TPA nor bryostatin I increased vinblastine accumulation

as expected. Accumulation experiments with [3Hldaunomyein,

[3Hjeolehieine, [3H]vinblastine, and rhodamine 123 also

showed no appreciable effect of treatment with either bryostatin

1 or TPA (Fig. 4). No significant effect of bryostatin 1 was

observed in vinblastine accumulation in MCF-7 parental cells

(data not shown).

We asked whether Pgp dephosphorylation and PKC-a

down-regulation were occurring under the conditions used in the

vinblastine accumulation assay. Cells were treated with 10 �M

and 100 nM bryostatin I for 24 h prior to labeling with [32P]P1

at 37#{176}Cfor 90 mm in the presence of bryostatin 1. Immunopre-

cipitation of the labeled Pgp is shown in Fig. 5, with densito-

metric quantitation in the lower panel. There is a significant

reduction in Pgp phosphorylation in cells treated with 100 nrvt

bryostatin 1 for 24 h, while a slight reduction is also detected

after a 10 �M treatment. Pgp phosphorylation is also reduced by

Fig. 6 Effect of TPA and bryostatin I (Brvo) on PKC isoforms inMCF-7TH cells. Cells were treated with TPA ( 100 nM) and bnyostatin-l

(100 nM) for 10 mm, 1 h, and 24 h. They were then harvested and

fractionated into a cytosol (S) and a membrane (P) fraction. Equal

amounts of protein (25 jig) were loaded in each lane for the cytosolic

(S) and the membrane (P) fraction. Following electrophoresis and trans-

fen to nitnocellulose, immunoblotting was performed with antibodies

against the PKC-a, -#{128}, and -� isoforms. Results shown are representative

of three different experiments, all of which gave similar results.

24-h treatment with 200 nM TPA. Since both Pgp dephospho-

rylation and PKC-cs down-regulation result from bryostatin 1

treatment. we sought an explanation for the failure to see an

effect on drug accumulation and examined the possibility that

increased mdr-1 expression results from bryostatin I treatment.

Induction of mdr-1 expression could confound the effect of

bryostatin 1 . Northern blot analysis was performed following

bryostatin I treatment of MCF-7TH cells; ,i,dr-I expression was

not regulated (data not shown). Thus. up-regulation of Pgp

expression cannot explain the failure of bryostatin 1 to modulate

Pgp activity.

The effect of bryostatin 1 on the PKC isoforms in MCF-

7TH was compared to the previously reported results with TPA

in human multidrug-resistant breast cancer cells (7, 19). As

shown in Fig. 6, in the MCF-7TH cells PKC-a was translocated

by TPA after a 10-mm treatment and was still in the active form

after a 1-h treatment as demonstrated by a shift in signal from

the supernatant to the particulate fraction, representing transbo-

cation from the cytosol to the cell membrane. After 24 h. the

level of PKC-a was reduced in both cytosolie and membrane

fractions, consistent with down-regulation of this isoform. Bryo-

statin 1 treatment with I 00 rust showed a weaker effect compared

to TPA in PKC-a translocation. A partial shift was detected

after a 10-mm treatment, and the shift from the cytosol to the

membrane was still not complete at 1 h of treatment. Subse-

quently, PKC-ct was completely down-regulated, as shown at



MCF-7

I � � 100 � C .01

Is P S P S P�S

MCF-7TH .�

100 I

PSPSP’c�

..

S

PKC#{128}


Fig. 7 Effect of bryostatin 1 onPKC isofonms in MCF-7 parental and

multidrug-resistant MCF-7TH. Cells

were treated for 24 h with 10 �M

0 “ (0.01 nM) and 100 flM bryostatin 1.,,� . . l’\Li 13 The cells were harvested, and immu-

noblotting was performed with anti-

bodies against the indicated isoforms.

Identical results were obtained in a

second set of independent experi-

ments. C. control (untreated) cells.

� � � PKC�

the 24-h time point. Modulation of the other two detectable

isoforms of PKC, #{128}and �, was also evaluated in MCF-7TH cells.

As shown in Fig. 6, PKC-#{128} appeared to be partially down-

regulated by both agents at 24 h, whereas no significant effect

on PKC-� was observed (the slight differences observed in Fig.

6 are experimental variations). These results demonstrate that

PKC-a in MCF-7TH responded to both TPA and bryostatin I,

but a more pronounced effect was detected in TPA-treated cells

compared to bryostatin 1-treated cells. Since differential results

have been observed with bower doses of bryostatin 1 in other

model systems (20. 37, 38), we next examined the effect of 10

�M bryostatin 1 in both MCF-7 and MCF-7TH cells. Bryostatin

I at 10 �M failed to down-regulate PKC-a levels after 24-h

treatment in contrast to 100 nM bryostatin 1 (Fig. 7). Vinblastine

accumulation performed with 10 �M bryostatin 1 also demon-

strated no significant difference in accumulation after 15 mm, 1

h, or 24 h of treatment (data not shown).

DISCUSSION

The present study reports the effect of bryostatin I . a PKC

activator and inhibitor, on drug transport and Pgp phosphoryla-

tion in multidrug-resistant human breast cancer cells. Bryostatin

I was unable to increase rhodamine or vinbbastine accumulation

in MCF-7TH cells, although it did reduce Pgp phosphorylation.

PKC-a, which is overexpressed in MCF-7TH cells relative to

the parental MCF-7 cells, was effectively down-regulated by

bryostatin 1 . Partial down-regulation of PKC-#{128} was also ob-

served, with no major effect on PKC-�. Thus. although the PKC

isoforms responded to bryostatin I as expected, no effect on Pgp

function could be measured.

The lack of an effect on Pgp function was unexpected,

since a role for PKC-a in stimulating Pgp activity has been

suggested in previous reports. Increased PKC-a expression has

been described in other MCF-7 doxorubiein-nesistant cells, a KB

cell line, and multidrug-resistant murine cell lines including

P388, murine fibrosarcoma cells, and UV-2237 (19, 39-42).

Bbobe et al. ( 19) reported a 10-fold increase in total PKC

activity in multidrug-resistant MCF-7 cells with increased phos-

phorylation following TPA treatment. Ahmad and Glazer (43)

reported that transfection of MCF-7Adr cells with an expression

vector containing the eDNA for PKC-a in the antisense orien-

tation inhibited PKC-a, which then sensitized the cells to ehe-

motherapeutic agents. These studies documented an overexpres-

sion of PKC-rt in multidrug-resistant cells and raised the

possibility that ehemosensitivity could be modified through

PKC-a inhibition.

The lack of an effect on drug accumulation in the MCF-

7TH subline was also unexpected in view ofobservations show-

ing decreased Pgp function and increased drug accumulation

following treatment of MCF-7TH cells with both ealphostin C

and staurosporine. Similarly, bryostatin 1 had no effect on drug

accumulation in a multidrug-resistant subline of SW620 human

colon cancer cells (data not shown).

Bryostatin I and TPA treatment effects on the various PKC

isoforms were comparable and similar to findings in previously

published reports (21, 44-46). Both induced an early translo-



1586 Bryostatin 1 in Breast Cancer Cells

3 z. Szallasi et a!. , submitted for publication.

cation of PKC-a from the cytosol to the membrane in MCF-

7TH cells, although bryostatin 1 activation of PKC-a was less

pronounced than that of TPA and both resulted in down-regu-

lation by 24 h. Identical results were observed in a multidrug-

resistant human colon carcinoma subline (data not shown)

which does not overexpress PKC-a compared to parental cells

as do the multidrug-resistant MCF-7TH cells reported here.

TPA and bryostatin 1 induced a partial down-regulation of

PKC-#{128}, and had no obvious effect on PKC-�. This was also

consistent with previous reports showing partial down-regula-

tion of PKC-#{128}in response to TPA or bryostatin 1 treatment of

NIH3T3 cells, mouse keratinocytes, and SH-SY5Y neuroblas-

toma cells (21, 46),� while neither transloeation nor down-

regulation of PKC-� was observed in NIH3T3 mouse fibroblast

cells, platelets, and murine epidermis (21, 47-49).

The present results contrast with previous observations

showing that decreased Pgp phosphorylation is associated with

decreased drug transport (2, 3, 16, 33). Decreased Pgp phos-

phorylation and impaired vinblastine transport were seen after

sodium butyrate treatment of SW620 human colon cancer cells,

although colehicine transport was preserved (33). Similar find-

ings were observed in SW62OAd300 cells treated with calphos-

tin C, a PKC inhibitor which impaired vinblastine while inereas-

ing verapamil transport (1 6). We now report a decrease in Pgp

phosphorylation following bryostatin 1 treatment accompanied

by unaffected vinblastine, rhodamine, and daunomycin trans-

port. These discrepancies may be explained by a differential

effect of these agents on distinct sites of phosphorylation by

several kinases which may include but are not limited to the

PKC family. These data suggest that kinases other than PKC

may be more important in regulating Pgp. Taken together, these

data suggest that Pgp phosphorylation modulates drug binding

and affinity and resulting transport, rather than having a direct

effect on Pgp function.

Our results show that bryostatin 1 down-regulates PKC-a

and reduces Pgp phosphorylation in MCF-7TH multidrug-resis-

tant cells overexpressing PKC-a, without an effect on drug

transport. Although bryostatin 1 appears to be clinically well

tolerated and may have a role as an antineoplastie agent, based

on these observations, its use clinically to modulate Pgp-rnedi-

ated multidrug resistance cannot be recommended.

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1995;1:1581-1587. Clin Cancer Res S Scala, B Dickstein, J Regis, et al. function in multidrug-resistant human breast cancer cells.Bryostatin 1 affects P-glycoprotein phosphorylation but not

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