Cytotoxic Activity of Epidermal Growth Factor-Genistein ......vital PTK shows considerable promise...

Vol. 4, 901-912, April 1998 Clinical Cancer Research 901

Cytotoxic Activity of Epidermal Growth Factor-Genistein against

Breast Cancer Cells’

Fatih M. Uckun,2 Rama Krishna Narla, Xiao Jun,

Tamer Zeren, Taracad Venkatachalam,

Kevin G. Waddick, Alexander Rostostev, and

Dorothea E. Myers

Departments of Oncology [F. M. U., R. K. N.], Chemistry [T. V.],Biochemistry [A. R.], Immunobiobogy [X. J.l, and Biotherapy[D. E. M., X. J., K. G. W.] and Drug Discovery Program [F. M. U.,R. K. N., T. V., A. R., D. E. M.], Wayne Hughes Institute, St. Paul,Minnesota 55103, and Department of Therapeutic Radiology,University of Minnesota, Minneapolis, Minnesota 55455 [T. Z.,K. G. W.]

ABSTRACT

The receptor (R) for epidermal growth factor (EGF) isexpressed at high levels on human breast cancer cells andassociates with ErbB2, ErbB3, and Src proto-oncogene fam-

ily protein tyrosine kinases (PTKs) to form membrane-

associated PTK complexes with pivotal signaling functions.

Recombinant human EGF was conjugated to the soybean-

derived PTK inhibitor genistein (Gen) to construct an EGF-

R-directed cytotoxic agent with PTK inhibitory activity. The

EGF-Gen conjugate was capable of binding to and enteringEGF-R-positive MDA-MB-231 and BT-20 breast cancercells (but not EGF-R-negative NALM-6 or HL-60 leukemiacells) via its EGF moiety, and it effectively competed withunconjugated EGF for target EGF-R molecules in ligand

binding assays. EGF-Gen inhibited the EGF-R tyrosine Id-nase in breast cancer cells at nanomolar concentrations,

whereas the IC50 for unconjugated Gen was > 10 riM. No-

tabby, EGF-Gen triggered a rapid apoptotic cell death inMDA-MB-231 as well as BT-20 breast cancer cells at nano-molar concentrations. The EGF-Gen-induced apoptosis wasEGF-R-specific because cells treated with the control gran-ulocyte-colony stimulating factor-Gen conjugate did not be-come apoptotic. Apoptosis was dependent both on the PTK

inhibitory function of Gen and the targeting function ofEGF, because cells treated with unconjugated Gen plus

Received 9/17/97; revised 1/14/98; accepted 1/16/98.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.1 Supported in part by a research grant from Parker Hughes Trust, GrantU0l-CA-72l57 from the National Cancer Institute, and Contract No.DAMDI7-96-C-6064 from the United States Army Medical Researchand Material Command. This work is part of a doctoral thesis by T. Z.to fulfill the requirements of the University of Minnesota GraduateSchool.2 To whom requests for reprints should be addressed, at the WayneHughes Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55 1 13.Phone: (612) 697-9228; Fax: (612) 697-1042; E-mail: [email protected].

unconjugated EGF did not undergo apoptosis. The IC5�s of

EGF-Gen versus unconjugated Gen against MDA-MB-231and BT-20 cells in clonogenic assays were 30 ± 3 n�i versus

120 ± 18 �LM (P < 0.001) and 30 ± 10 flM versus 112 ± 17

�LM (P < 0.001), respectively. Thus, the EGF-Gen conjugateis a > 100-fold more potent inhibitor of EGF-R tyrosineIdnase activity in intact breast cancer cells than unconju-gated Gen and a > 100-fold more potent cytotoxic agentagainst EGF-R’ human breast cancer cells than unconju-

gated Gen. Taken together, these results indicate that theEGF-R-associated PTK complexes have vital antiapoptoticfunctions in human breast cancer cells and may therefore beused as therapeutic targets.

INTRODUCTION

Breast cancer is the most common tumor in women, rep-

resenting 32% of all new cancer cases and causing 18% of the

cancer-related deaths among women in the United States ( 1).

Although the majority of patients with metastatic breast cancer

will experience an initial response, survival is only modestly

improved with contemporary chemotherapy programs (2-4).

Consequently, the development of new potent anti-breast cancer

drugs has emerged as an exceptional focal point for translational

research in treatment of breast cancer (5).

Human EGF3 is a 53-amino acid, single-chain polypeptide

(Mr 62 16) that exerts biological effects by binding to a specific

Mr 170,000 cell membrane EGF-R (EGF-R/ErbB-l ; Refs. 6-9).

The human EGF-R consists of an extracellular domain with a

high cysteine content and N-linked glycosylation, a single trans-

membrane domain, and a cytoplasmic domain with PTK activ-

ity. Binding of EGF to EGF-R/ErbB-l results in receptor dimer-

ization with itself or other members of the Erb-B (subtype I)

transmembrane PTK family (e.g., Erb-B2 and Erb-B3), resulting

in activation with autophosphorylation of the PTK domain (10,

1 1). Recent studies demonstrated that the EGF-R is physically

and functionally associated with the Src proto-oncogene family

PTK, including p60s� (10-12). This association is believed to

be an integral part of the signaling events in breast cancer cells

mediated by the EGF-R and contributes to proliferation and

survival of breast cancer cells (1 2-14). Many types of cancer

cells display enhanced EGF-R expression on their cell surface

membranes (8). Enhanced expression of the EGF-R on cancer

cells has been associated with excessive proliferation and me-

tastasis (9). Examples include breast cancer, prostate cancer,

lung cancer, head and neck cancer, bladder cancer, melanoma,

and brain tumors (8). In breast cancer, expression of the EGF-R

3 The abbreviations used are: EGF, epidermal growth factor; rhEGF,recombinant human EGF; EGF-R, EGF receptor; PTK, protein tyrosinekinase; Gen, genistein; HPLC, high-performance liquid chromatogra-phy; TFA, trifluoroacetic acid; G-CSF, granulocyte colony-stimulatingfactor; P1, propidium iodide.

on May 4, 2021. © 1998 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

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902 Cytotoxic Activity of EGF-Genistein

is a significant and independent indicator for recurrence and

poor relapse-free survival (15-17).

Targeted delivery of Gen, a naturally occurring PTK in-

hibitory isoflavone (5,7,4 ‘ -trihydroxyisoflavone) from fermen-

tation broth of Pseudomonas spp. , to CD 19 receptor-associated

vital PTK shows considerable promise for more effective treat-

ment of human leukemias and lymphomas (18, 19). We postu-

bated that the EGF-R-associated PTK may be of similar impor-

tance for the survival of breast cancer cells and, therefore, may

serve as a suitable target for biotherapy of breast cancer using

EGF-Gen conjugates. In this study, rhEGF was conjugated to

Gen to construct an EGF-R-directed cytotoxic agent with PTK

inhibitory activity. Here, we report the effects of the EGF-Gen

conjugate on human breast cancer cells.

MATERIALS AND METHODSPreparation of the EGF-Gen. rhEGF was produced in

Escherichia coli harboring a genetically engineered plasmid that

contains a synthetic gene for human EGF fused at the NH2

terminus to a hexapeptide leader sequence for optimal protein

expression and folding. The rhEGF fusion protein precipitated

in the form of inclusion bodies, and the mature protein was

recovered by trypsin-cleavage, followed by purification using

ion exchange chromatography and HPLC. rhEGF was 99% pure

by reverse-phase HPLC and SDS-PAGE with an isoebectric

point of 4.6 ± 0.2. The endotoxin level was 0. 172 EU/mg. The

recently published photochemical conjugation method using the

hetero-bifunctional photoreactive cross-linking agent, sulfosuc-

cinimidyl 6-(4’azido-2’-nitrophenybamino)hexanoate (Sulfo-

SANPAH; Pierce Chemical Co., Rockford, IL; Ref. 18) has

been used in the synthesis of the EGF-Gen conjugate. Sulfo-

SANPAH-modified rhEGF was mixed with a 10:1 molar ratio

of Gen (LC Laboratories, Woburn, MA) (50 msi solution in

DMSO) and then irradiated with gentle mixing for 10 mm with

UV bight at wavelengths 254-366 nm with a multiband UV

light-emitter (model UVGL-l5 Mineralight; UVP, San Gabriel,

CA). Photolytic generation of a reactive singlet nitrene on the

other terminus of EGF-SANPAH in the presence of a 10-fold

molar excess of Gen resulted in the attachment of Gen via its

available C7-hydroxyl group to lysine 28 or lysine 48 residues

of EGF. Excess Gen in the reaction mixture was removed by

passage through a PD-lO column, and Mr 12,000 EGFEGF

homoconjugates, with or without conjugated Gen, as well as

higher molecular weight reaction products were removed by

size-exclusion HPLC. Reverse-phase HPLC using a Hewlett-

Packard 1 100 series HPLC instrument was used for separation

of EGF-Gen from EGF-SANPAH. After the final purification,

analytical HPLC was performed using a Spherisorb ODS-2

reverse-phase column (250 X 4-mm; Hewlett-Packard). Prior to

the HPLC runs, a Beckman DU 7400 spectrophotometer was

used to generate a UV spectrum for each of the samples to

ascertain the Xmax for EGF-Gen, EGF-SANPAH, and unmod-

ified EGF. Each HPLC chromatogram was subsequently run at

wavelengths of 214, 265, and 480 nm using the multiple wave-

length detector option supplied with the instrument to ensure

optimal detection of the individual peaks in the chromatogram.

Analysis was achieved using a gradient flow consisting of

0-100% eluent in a time interval of0-30 mm. Five-�i.L samples

applied to the above column were run using the following

gradient program: 0-5 mm, 0-20% eluent; 5-20 mm, 20-100%

eluent; 25-30 mm, 100% eluent; and 30-35 mm, 100-0%

eluent. The eluent was a mixture of 80% acetonitrile (CH3CN),

20% H,O, and 0.1% TFA.

Electrospray ionization mass spectrometry (20, 2 1 ) was

performed using a PE SCIEX API triple quadruple mass spec-

trometer (Norwalk, CT) to determine the stoichiometry of Gen

and EGF in EGF-Gen. ‘25I-Gen was also used to confirm the

stoichiometry of Gen and EGF in EGF-Gen and to verify the

removal of free genistein and genistein-labeled EGF-EGF ho-

moconjugates by the described purification procedure. Gen (in

65% ethanol, 35% PBS, pH 7.5; LC Laboratories, Woburn, MA)

was radioiodinated at room temperature in Reacti-Vials contain-

ing lodo-beads (Pierce Chemical Co., Rockford, IL) and 1251

(Na, carrier-free, 17.4 Ci/mg; NEN, Boston, MA) as per man-

ufacturer’s instructions (18, 19). The purity of EGF-’25I-Gen

was assessed by SDS-PAGE (20% separating gels, nonreducing

conditions) and autoradiography using intensifying screens and

Kodak XAR-5 film. EGF-’25I-Gen was also used for in vitro

ligand binding assays (18, 21) and EGF-Gen internalization

studies (18).

Breast Cancer Cells. MDA-MB-23 1 (ATCC HTB-26)

is an EGF-R-positive breast cancer cell line initiated from

anaplastic carcinoma cells of a 5 1-year-old patient. BT-20

(ATCC HTB-l9) is another EGF-R-positive breast cancer cell

line isolated from the primary breast tumor of a 74-year-old

patient with grade II mammary adenocarcinoma. MDA-MB-231

and BT-20 breast cancer cell lines were maintained in RPM!

1640 supplemented with 10% fetal bovine serum. For subcul-

turing, medium was removed from the flasks containing a con-

fluent layer of cells, and fresh 0.25% trypsin was added for 1-2

mm. Trypsin was removed, and cultures were incubated for

5-10 mm at 37#{176}Cuntil cells detached. Fresh medium was then

added, and cells were aspirated and dispensed into new flasks.

Binding of EGF-’�I-Gen to Breast Cancer Cells. Li-gand binding assays using EGF-’25I-Gen (2.0 X 108 cpml

pmol), ‘251-Gen (3.8 X 108 cpm/p.mol), and ‘251-EGF (2.2 X

1012 cpm/p.mob; Amersham) were performed using standard

procedures, as described previously ( 18, 22). The cell lines in

ligand binding assays included the EGF-R-positive breast can-

cer cell lines, MDA-MB-23 1 and BT-20, as well as the EGF-

R-negative human leukemia cell lines, NALM-6 (pre-B leuke-

mia) and HL-60 (promyelocytic leukemia).

Immunocytochemistry. Immunocytochemistiy was used

to: (a) examine the surface expression of EGF-R on breast cancer

cells; (b) evaluate the uptake of EGF-Gen by breast cancer cells;

and (c) examine the morphological features of EGF-Gen-treated

cancer cells. In uptake studies, the culture medium was replaced

with fresh medium containing 10 p.g/ml EGF or EGF-Gen, and

cells were incubated at 37#{176}Cfor 5, 10, 15, 30, and 60 mm, and 24 h.

For EGF-R expression studies, cells were plated on poby-L-lysine-

coated, glass-bottomed, 35-mm Petri dishes and maintained for

48 h. At the end of the incubation, cells were washed with PBS and

fixed in 2% paraformaldehyde. The cells were permeabilized, and

nonspecific binding sites were blocked with 2.5% BSA in PBS

containing 0.1% Triton X-lOO for 30 mm. To detect the EGF-R/

EGF-Gen complexes, cells were incubated with a mixture of a

monocbonal antibody (1:10 dilution in PBS containing BSA and



mAU mAU

250 . Retentiontime: 18.840

--- Retention time: 18.874

- Retentiontime: 18.807

EGF

IFig. 1 A, HPLC chromatogram of EGF-Gen. Asample of the EGF-Gen conjugate was analyzedon a Spherisorb ODS, 250 X 4-mm reverse-phasecolumn using a 0. 1% TFA-H20/0. 1% TFA-80%acetonitribe-20% H,O gradient as described in“Materials and Methods.” The retention time ofEGF-Gen was 18.84 mm. Solid line, absorbance at265 nm; dotted line. absorbance at 480 nm. B, UVspectrum of the EGF-Gen. The EGF-Gen peakobtained from the HPLC run shown in B wasfurther analyzed by the diodide array multiplewavelength detector.

Genistein

00C(U

.00U)

.0

0

>

(U0

cc

SANPAH

1875 19.00

Time (mm) Wavelength (nm)

Clinical Cancer Research 903

EC

0

‘�.

EC

U,(0CsJ

00C0

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00

Triton X-lOO) directed to the extracelbular domain of the human

EGF-R (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and a

polyclonal rabbit anti-Gen antibody (1:500 dilution) for 1 h at room

temperature. After rinsing with PBS, cells were incubated for 1 h

with a mixture of a goat anti-mouse IgG antibody conjugated to

FITC (Amersham Corp., Arlington Heights, IL) at a dilution of

1:40 in PBS and donkey anti-rabbit IgG conjugated to Texas Red

(Amersham Corp.). Cells were washed in PBS and counterstained

with toto-3 (Molecular Probes, Inc., Eugene, OR) for 10 mm at a

dilution of 1:1000. Cells were washed again with PBS, and the

coverslips were mounted with Vectashield (Vector Labs, Burl-

ingame, CA) and viewed with a confocal microscope (Bio-Rad

MRC 1024) mounted in a Nikon Labhophot upright microscope.

Digital images were saved on a Jaz disc and processed with Adobe

Photoshop software (Adobe Systems, Mountain View, CA).

in Vitro Treatment of Cells with EGF-Genistein. To

determine the cytotoxic activity of EGF-Gen against breast

cancer cells, cells in a-MEM supplemented with 10% (v/v) FCS

were treated with various concentrations of EGF-Gen for 24 h at

37#{176}C,washed twice in a-MEM, and then used in either apop-

tosis assays or clonogenic assays, as described hereafter. Con-

trols included: (a) cells treated with G-CSF-Gen (an irrelevant

cytokine-Gen conjugate that does not react with EGF-R); (b)

cells treated with unconjugated EGF plus unconjugated Gen; (c)

cells treated with unconjugated Gen or unconjugated EGF; and

(‘0 cells treated with PBS, pH 7.4. In some experiments, excessG-CSF or EGF was added to the EGF-Gen-containing treatment

medium to show that the cytotoxicity of EGF-Gen can be

selectively blocked by excess EGF but not G-CSF.

Immune-Complex Kinase Assays and Anti-Phosphoty-rosine Immunoblotting. Twenty-four h after treatment with

EGF-Gen, cells were stimulated with 20 ng/ml EGF for 5 mm

and lysed in 1% NP4O buffer, and cell bysates were immuno-

precipitated with an anti-EGF-R antibody reactive with the

sequence ‘ �AspS&� of the human EGF-R (Upstate Biotech-

nobogy, Inc.). EGF-R immune complexes were examined for

tyrosine phosphorylation by Western blot analysis, as described

previously (23). All anti-phosphotyrosine Western blots were

subjected to densitometric scanning using the automated AM-

BIS system (Automated Microbiology System, Inc., San Diego,

CA), and for each time point, a percentage inhibition value was

determined by comparing the density ratios of the tyrosine-

phosphorylated EGF-R protein bands with those of the baseline

sample and using the formula:

% inhibition

(Density of tyrosine phosphorylated

EGF-R band)te� sample

=100- . . XlOO(Denslty of tyrosine-phosphorybated

EGF-R band)basel,fl. control sample

The IC,0s were determined using an Inpbot program (Graphpad

Software, Inc., San Diego, CA). The Src immune complexes

were then subjected to immune complex kinase assays, as de-

scribed (18, 19, 23).

Apoptosis Assays. Loose packing of membrane phos-

pholipid head groups and cell shrinkage precede DNA fragmen-




Table 1 Specific binding of EGF-’25I-Gen to Breast Cancer Cells

The binding of EGF-Gen, unconjugated Gen, and unconjugated EGF to EGF-R� breast cancer cells and EGF-R leukemia cells was examined

in ligand binding assays. as described in “Materials and Methods.” Each cpm determination was performed in duplicate.

I 251-Gen binding to breast cancer cells

Cell line EG-coldF (cpm)

+cobd Specific InhibitableEGF (cpm) binding binding pm 01/106 cells

Moleculesper cell

MDA-MB-231 4531 1983 2548 56% 7.5 4.5 X 106

BT-20 7511 2663 4848 65% 9.5 5.7x 106

NALM-6 2708 3091 0 None None NoneHL-60 788 1346 0 None None None

‘251-Gen binding to breast cancer cells

MDA-MB-23l 852 860 0 None None NoneBT-20 2439 2540 0 None None NoneNALM-6 1098 NDU ND ND ND NDHL-60 814 ND ND ND ND ND

‘25I-EGF binding to breast cancer cells

-coldEGF

(cpm)

+coldEGF

(cpm)

Specific +coldbinding Inhibitable EGF-Gen Inhibition by(cpm) binding (cpm) EGF-Gen

+coldGM-CSF

(cpm)Inhibition by

GM-CSF

MDA-MB-23b 15,102 1,100 14,002 93% 1,398 91% 15,440 0%BT-20 17,351 ND ND ND 1,624 91% 16,486 5%

a ND, not done.

tation in apoptotic cells, thereby providing MC540 binding as an

early marker for apoptosis (24). Plasma membrane permeability

to P1 (Sigma) develops at a later stage of apoptosis (24). MC540

binding and P1 permeability were simultaneously measured in

breast cancer cells 24 h after exposure to EGF-Gen (either

without any cytokine preincubation or after preincubation with

excess unconjugated EGF or G-CSF), unconjugated Gen, un-

conjugated EGF + unconjugated Gen, or G-CSF-Gen, as de-

scribed (24). Stock solutions of MC540 and P1, each at 1 mg/ml,

were passed through a 0.22 p.m filter and stored at 4#{176}Cin the

dark. Shortly before analysis, suspensions containing 1 X 106

cells were suspended in S jig/mb MCS4O and 10 pig/mb P1 and

kept in the dark at 4#{176}C.Whole cells were analyzed with a

FACStar Plus flow cytometer (Becton Dickinson, San Jose,

CA). All analyses were done using 488-nm excitation from an

argon laser. MC540 and P1 emissions were split with a 600-nm

short pass dichroic mirror; a 575-nm band pass filter was placed

in front of one photomultiplier tube to measure MC540 emis-

sion, and a 635-nm band pass filter was used for P1 emission. To

detect the DNA fragmentation in apoptotic cells, cells were

harvested 24 h after treatment with EGF-Gen, and DNA was

prepared from Triton-X-l00 detergent lysates for analysis of

fragmentation, as described (24). In brief, cells were lysed in

hypotonic 10 msi Tris-Cb (pH 7.4), 1 m�i EDTA, 0.2% Triton-

X-lOO, and subsequently centrifuged at 11,000 X g. This pro-

tocol allows the recovery of fragmented DNA in the superna-

tant. To detect apoptosis-associated DNA fragmentation,

supernatants were ebectrophoresed on a 1 .2% agarose gel, and

the DNA fragments were visualized by UV light after staining

with ethidium bromide.

Clonogenic Assays. After treatment with EGF-Gen, G-CSF-Gen, unconjugated EGF, unconjugated Gen, or PBS, cells

were resuspended in clonogenic medium consisting of a-MEM

supplemented with 0.9% methylcellulose, 30% fetal bovine

serum, and 50 Ji.M 2-mercaptoethanol. Cells were plated in

duplicate Petri dishes at 100,000 cells/ml/dish and cultured in a

humidified 5% CO2 incubator for 7 days. Cancer cell colonies

were enumerated on a grid using an inverted phase microscope

of high optical resolution. Results were expressed as percentage

inhibition of clonogenic cells at a particular concentration of the

test agent using the formula:

1 - mean no. of colonies (test)% inhibition = x 100

Mean no. of colonies (control)

Furthermore, the dose-survival curves were constructed using

the percentage of control survival [mean no. of colonies (test)/

mean no. of colonies (control) X 100] results for each drug

concentration as the data points, and the IC50s were calculated.

The IC50s were determined using an Prism Version II Inplot

program (Graphpad Software, Inc.). The mean IC50s for EGF-

Gen and Gen were compared using Student’s t tests.

RESULTS

Composition of EGF-Gen Conjugate. EGF-Gen wasconsistently found to contain, in four independent conjugations,

one molecule of Gen per each EGF molecule, as determined by

the specific activity of EGF-Gen prepared with ‘25I-Gen. The

electrospray ionization mass spectrum of EGF-Gen also showed

a single Mr 7287 EGF-Gen species containing one EGF mole-

cule, three SANPAH molecules, and one Gen molecule. Fig. lA

depicts the analytical HPLC chromatogram of purified EGF-

Gen, which eluted as a single peak at 18.84 mm. The UV

spectral scan of this HPLC peak revealed: (a) a peak at a

wavelength of 220 nm (due to peptide bonds) and a shoulder at

280 nm (due to aromatic amino acid residues) representing



0mm

A A’

B

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Fig. 2 Binding and internalization ofEGF-Gen in BT-20 cells. Cells were incu-bated with EGF-Gen (10 p.g/ml) for theindicated times (0 mm, A and A’; 5 mm, B

and B’; 30 mm, C and C’; 24 h, D and D’).

Cells were then processed for immunocy-tochemistry using a monocbonal anti-EGF-R antibody and FITC conjugated goatanti-mouse IgG for EGF-R (green fluores-

cence, lefipanet). Gen was detected using apobycbonal anti-Gen antibody and TexasRed conjugated anti-rabbit IgG (red fluo-rescence, right panel), as described in “Ma-terials and Methods.” Blue fluorescence,

the nuclei stained with toto-3. In A and A’,

BT-20 cells showed a high bevel of EGF-Rexpression; no red fluorescent staining wasobserved in untreated cells incubated withthe anti-Gen antibody. In B and B’, after 5mm exposure, EGF-Gen was bound to thecell surface EGF-R (arrowheads), and theinternalization of the EGF-R was detectedby cytoplasmic green fluorescent staining,whereas the internalization of EGF-Genmolecules was evident from the red fluo-rescent staining. In C and C’, by 30 mm,most of the EGF-R/EGF-Gen complexeswere internalized and deposited in the pe-rinuclear region (arrows). In D and D’,

after 24 h exposure, the cells lost theiradherent features and showed morphobogi-cal changes consistent with apoptosis (open

arrow).

3Qmln



EGF

.‘��‘#‘__. - . �

A

EGF-GEN

A’

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a �. . . � �

B

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#{149}p.�


Fig. 3 Binding and internalizationof EGF-Gen in MDA-MB-23 I cells.Cells were incubated with either un-conjugated EGF (10 p.s/mI; A andA’) or EGF-Gen (10 �ig/ml; B and B’)

for 15 mm and processed for the de-tection of EGF-R (green fluores-

cence, left panel) and EGF-Gen (red

fluorescence, right panel) by im-munocytochemistry. EGF-Gen wasvery similar to EGF with respect toits ability to induce internalization of

the surface EGF-R molecules. Nota-bly, the intracellular staining patternsfor EGF-R and EGF-Gen were verysimilar.

EGF; (b) a peak at 267 nm representing Gen; and (c) a peak at

480 nm corresponding to the nitrobenzene-substituted structure

in the SANPAH moiety (Fig. 1B). The EGF-Gen conjugate was

highly stable in mouse, monkey, and human plasma with no

detectable decrease in concentration, as examined by quantita-

tive autoradiography of EGF-’251-Gen, as well as quantitative

anti-Gen Western blot analysis of nonradioactive EGF-Gen,

even after 3 days of continuous incubation at 37#{176}C(data not

shown).

Binding of EGF-Genistein to EGF-R-positive BreastCancer Cells. We examined the in vitro binding of radioio-

dinated EGF-Gen (EGF-’25I-Gen: final concentration, 260 nr�t

= 1700 ng/ml) to EGF-R on these breast cancer cells in the

presence and absence of a 100-fold molar excess of nonradio-

active EGF using standard ligand binding assays (18, 19, 22).

EGF- ‘25I-Gen was able to bind to MDA-MB-23 1 and BT-20

human breast cancer cells, and this binding was blocked by

excess nonradioactive EGF (% EGF-inhibitable binding = 56%

for MDA-MB-23 1 and 65% for BT-20; 4.5 X 106 EGF-Gen

molecules/cell for MDA-MB-23 1 cells and 5.7 X 106 EGF-Gen

molecules/cell for BT-20 cells; Table 1), but not by excess

nonradioactive GM-CSF, which was used as a control ligand

(data not shown). 25I-Gen did not bind to EGF-R-negative

HL-60 or NALM-6 leukemia cell lines. EGF-Gen was as effec-

tive as unconjugated EGF in blocking the binding of ‘ 251-EGF

to breast cancer cells, whereas granubocyte-macrophage-CSF

did not block the binding of ‘251-EGF (Table 1). Thus, EGF-Gen

was able to bind to EGF-R-positive breast cancer cells via its

EGF moiety. However, because: (a) 35-44% of the EGF-Gen

binding to breast cancer cells was not inhibitable by excess

unconjugated EGF; (b) EGF-Gen binding not inhibitable by

excess EGF was also observed with EGF-R-negative leukemia

cell lines NALM-6 and HL-60; and (c) unconjugated Gen

showed binding to all cell lines, which was not inhibitable by

EGF, the Gen moiety as well as nonspecific surface adherence

may also contribute to the observed binding of EGF-Gen to

breast cancer cells.

We next examined the kinetics of uptake and cytotoxicity

of unlabeled EGF-Gen in BT-20 (Fig. 2) and MDA-MB-23 1

(Fig. 3) human breast cancer cells using immunocytochemistry

and confocal laser microscopy for tracing the internalized

EGF-R and EGF-Gen molecules as well as evaluating the mor-

phological changes in treated cells. EGF-Gen was very similar

to unconjugated EGF with respect to its ability to bind to and

induce internalization of EGF-R molecules. Within S mm after

exposure to EGF-Gen, the EGF-RIEGF-Gen complexes begin

being internalized, as determined by colocalization of EGF-R

(detected by anti-EGF-R antibody, green fluorescence) and

EGF-Gen (detected by anti-Gen antibody, red fluorescence) in

the cytoplasm of treated cells (Figs. 2 and 3). By 15-30 mm, the



EGF-R�


EGF-R/EGF-Gen complexes were detected in the perinuclear

region of the cells. The examination of the morphological fea-

tures of EGF-Gen-treated (but not EGF-treated) cells after 24 h

of exposure showed distinct changes consistent with apoptosis

including marked shrinkage, nuclear fragmentation, and forma-

tion of apoptotic bodies (Fig. 2).

Biological Activity of EGF-Gen. EGF-Gen treatment

resulted in decreased tyrosine phosphorylation of the EGF-R in

a dose-dependent fashion (Fig. 4A). Whereas EGF-Gen exhib-

ited marked PTK-inhibitory activity in MDA-MB-231 cells at

concentrations as low as 0. 1 p.M in the treatment medium,

unconjugated Gen did not significantly affect the EGF-R tyro-

sine phosphorylation, even at a 10 p.M concentration (Fig. 4A).

The inhibitory effect of EGF-Gen was blocked by preincubation

of cells with excess EGF but not by excess G-CSF, a control

cytokine that does not react with EGF-R (Fig. 4B). We next used

immune complex kinase assays to assess the effects of EGF-Gen

on the enzymatic activities of EGF-R-associated Src PTK in

MDA-MB-23l cells. As shown in Fig. 4C, EGF-Gen treatment

inhibited the Src kinase. Unlike EGF-Gen, a mixture of uncon-

jugated Gen and EGF or G-CSF-Gen did not inhibit the Src

kinase activity in MDA-MB-23l cells. Thus, EGF-Gen is a

potent inhibitor of both the EGF-R tyrosine kinase as well as

other PTKs that are associated with the EGF-R.

Targeting Gen to vital PTKs in leukemia cells results in

apoptotic cell death (18, 19). Furthermore, the examination of

the morphological features of EGF-Gen-treated BT-20 and

MDA-MB-23l cells by immunocytochemistry suggested that

these cells might be undergoing apoptosis. Therefore, we de-

cided to formally study whether EGF-Gen could trigger apop-

tosis in breast cancer cells. To this end, we first used a quanti-

tative flow cytometric apoptosis detection assay. MC540

binding and P1 permeability of MDA-MB-23 1 breast cancer

cells were simultaneously measured before and after treatment

with 1 p.g/ml EGF-Gen (0.1 taM), 10 jig/mb EGF (1 �M), plus 10

�j.g/ml unconjugated Gen (37 p.M), or 1 p.g/ml G-CSF-Gen.

Whereas < 10% of MDA-MB-23 1 or BT-20 cells showed ap-

optotic changes after EGF plus unconjugated Gen treatment or

G-CSF-Gen treatment, a significant portion of cells underwent

apoptosis within 24 h after EGF-Gen treatment (95.1% = 57.9%

MC540� early stage apoptosis plus 37.2% MC54047P14 ad-

vanced stage apoptosis at 24 h; Fig. 5). Excess EGF (10 p.g/ml),

but not excess G-CSF (10 p.g/ml), could prevent EGF-Gen-

induced apoptosis. Thus, EGF-Gen causes apoptosis in an EGF-

R-specific fashion, and this activity requires both its EGF-R

binding growth factor moiety as well as its PTK inhibitory Gen

moiety.

As shown in Fig. 6, DNA from Triton-X-100 lysates of

EGF-Gen-treated MDA-MB-231 or BT-20 breast cancer cells

showed a ladder-like and dose-dependent fragmentation pattern,

consistent with apoptosis. The EGF-Gen-induced DNA frag-

mentation was EGF-R-specific because DNA from cells treated

with the control cytokine-Gen conjugate G-CSF-Gen showed no

fragmentation. DNA fragmentation was dependent both on the

PTK inhibitory function of Gen and the targeting function of

EGF because cells treated with unconjugated Gen plus uncon-

jugated EGF did not show apoptotic DNA fragmentation

(Fig. 6).

We compared the ability of equimolar concentrations of

EGF-Gen� (KM)

A. c� ‘io 1 O.1’�

�

B. cP #��<�4W

EGF..R� � �

‘:� c�

�c? #{231}?�,

(‘ ��?‘A c,’�’ �-4’%.1. cY4YC.i ((�‘

Src � �

Fig. 4 Inhibitory activity of EGF-Gen on EGF-R-associated PTKs ofhuman breast cancer cells. CON, control. In A, after a 24-h incubation

with EGF-Gen (0.1, 1.0, or 10.0 p.M) or unconjugated Gen (10 p.M),

MDA-MB-23l cells were lysed in 1% NP4O buffer, and cell bysateswere immunoprecipitated with an anti-EGF-R antibody reactive with thesequence � -Asp3� of the human EGF-R. The EGF-R immunecomplexes were then subjected to APT immunoblotting. In B, after a24-h incubation with 10.0 p.g/mb (1.3 p.M) EGF-Gen, MDA-MB-23lcells were lysed in 1% NP4O buffer, cell lysates were immunoprecipi-tated with an anti-EGF-R antibody, and the EGF-R immune complexeswere subjected to APT immunobbotting as in A. Controls includeduntreated cells as well as cells pretreated with 10-fold molar excess ofunconjugated EGF (13 p.M) or unconjugated G-CSF (13 p.M) prior toEGF-Gen incubation. In C, Src immune complex kinase assays in thepresence of [-y-32P]ATP (50 p.Ci/p.mol) were performed on the lysatesof MDA-MB-23 I cells treated with 1 j.g/ml EGF-Gen (0. 1 p.M), 1

p.g/ml G-CSF-Gen, or 1 �g/mb EGF (0. 1 p.M) + 1 p.g/ml Gen (3.7 p.M).

Controls included untreated cells.



PBS EGF+Gen

2.4%

I.

1.5%

48

G-CSF-Gen

64

48

EGF-Gen

32

1.5% 37.2%

EGF-EGF-Gen

48

64

G-CSF-EGF-Gen

32

1.6%

48

32

16

I

. .. .flI..

�

. ... �

529%16 32 48 64


>�

Cl)

a)C

a)0Ca)0U)a)0

U-

0)

0-J

44.0%

Log (MC 540 Fluorescence Intensity)

Fig. 5 EGF-Gen induces apoptosis in human breast cancer cells. Fluorescence-activated cell sorting correlated two-parameter displays of MDA-MB-23 1 cells stained with MCS4O and P1 24 h after treatment with PBS, 10 jig/mb EGF + 10 p.g/ml Gen (37 p.M), 1 ��.g/ml G-CSF-Gen, I �xg/ml

EGF-Gen (0.1 fiM), 10 ��.g/ml EGF + I p.g/ml EGF-Gen, or 10 p.g/mb G-CSF + I jig/mb EGF-Gen. The percentages indicate the fraction of cells

at an early stage of apoptosis, as measured by single MC540 fluorescence, and the fraction of cells at an advanced stage of apoptosis, as measured

by dual MCS4OIPI fluorescence.



‘V �

Vt

a. . �

� �

Clinical Canccr Research 909

23,130 -

9,416 -

6,557 -

4,361 -

2,322 -

2,027 -

1,353 -

1,078 -

872 -

603 -

310 -

M 0#{176}

MDA-MB-231 BT-20

Fig. 6 Internucleosomal DNA fragmentation in EGF-Gen-treated breast cancer cells. Cells were harvested 24 h after treatment with PBS (CON.

control), EGF-Gen, G-CSF-Gen, or unconjugated EGF + unconjugated Gen, and DNA was prepared for analysis of fragmentation. DNA was then

separated by electrophoresis through a 1% agarose gel. and the DNA bands were visualized by UV light after staining with ethidium bromide. Lane

M, molecular size markers in bp.

EGF-Gen and unconjugated Gen to induce apoptosis in dose-

response studies using the MDA-MB-23 1 breast cancer cell line.

Whereas EGF-Gen caused apoptosis in 98.7% of treated breast

cancer cells at concentrations as low as 0. 1 tiM, Gen was

significantly less active and caused apoptosis in only 15.5% of

the breast cancer cells, even at a 100 p.M concentration (Fig. 7).

We next tested the anticancer activity of EGF-Gen against

MDA-MB-23 1 and BT-20 breast cancer cell lines using in vitro

clonogenic assays. The EGF-R-negative leukemia cell line

NALM-6 was used as a negative control, and the EGF-R-

positive prostate cancer cell line PC-3 was used as a positive

control. As shown in Table 2, 24-h treatment with 10 p.g/ml

EGF-Gen killed >99% of cbonogenic MDA-MB-23 1 and

BT-20 cells as well as >99% of PC-3 cells, under conditions

that did not affect the cbonogenic growth of EGF-R-negative

NALM-6 leukemia cells. The lack of toxicity to NALM-6 cells

was not caused by a cellular resistance to Gen. because B43-

Gen. an anti-CD19 immunoconjugate (18), killed >99% of

NALM-6 cells. Unlike EGF-Gen, neither EGF ( 10 �ig/ml: un-

modified or Sulfo-SANPAH-modified) nor Gen ( 10 �ig/ml)

were able to inhibit the clonogenic growth of EGF-R-positive

cancer cell lines. Similarly, G-CSF-Gen (10 �i.g/ml) did not

affect the cbonogenic growth of these breast and prostate cancer

cell lines (Table 2). To more accurately compare the cytotoxic

activities of EGF-Gen and unconjugated Gen, we performed

detailed dose-response studies using in vitro cbonogenic assays.

As shown in Fig. 8, EGF-Gen inhibited in each of three inde-

pendent experiments the cbonogenic growth of MDA-MB-23 1



ControlTAF= 0.8%

0.1%

Gen, 1pMTAF= 6.3%

0.8%

Gen, 10pMTAF= 10.7%

1 :

1.2%

.;;.a

(I)C4)C

4)0C4)0(I)4)’,

0

U-

0)03

_1

Gen, 100pMTAF= 15.5%

2.0%

20.0%

� 78 7%

61.0%I.42.2%

Log (MC 540 Fluorescence Intensity)

Fig. 7 EGF-Gen induces apoptosis in human breast cancer cells. FACS correlated two-parameter displays of MDA-MB-231 cells stained withMC540 and P1 24 h after treatment with PBS, EGF-Gen (0.1, 1.0. and 10 p.M), or unconjugated Gen (1.0, 10. and 100 p.M). The percentages indicate

the fraction of cells at an early stage of apoptosis, as measured by single MC540 fluorescence, and the fraction of cells at an advanced stage of

apoptosis. as measured by dual MC540/PI fluorescence. For each treatment. the total apoptotic fraction (TAF, percentage of MC540 single fluorescentcells + percentage of MC54OIPI double fluorescent cells) is also provided.

Table 2 Cytotoxic activity of EGF-Gen against cbonogenic breast cancer cells

Cancer cells were treated with the indicated agents for 24 h at 37#{176}C,washed twice. and then plated in duplicate Petri dishes at l0� cells/mI. The

clonogenic medium was a-MEM supplemented with 0.9% methylceblubose. 30% fetal bovine serum. and 50 p.M 2-mercaptoethanol. Colonies were

enumerated on day 7 using an inverted phase microscope of high optical resolution.

Cell line

MDA-MB-23l (breast cancer)

BT-20 (breast cancer)

PC-3 (prostate cancer)

NALM-6 [Pre-B ALL” EGFR�(-)]


0.7%

ControlTAF=2.2% 0.8%

�fic��

�“ � 1.4%

‘a..

/#{231}��o� 5.5%

�. .

EGF-Gen, O.lpM

TAF= 98.7%

Treatment

PBSEGF, 10 p.g/mlGen. 10 p.g/mlEGF-Gen, 10 pig/mb

GCSF-Gen, 10 �ig/ml

PBSEGF, 10 p.g/mlGen, 10 p.g/mlEGF-Gen, 10 p.g/ml

GCSF-Gen, 10 �i.g/ml

PBSEGF, I 0 p.g/mlGen, 10 �ig/ml

EGF-Gen, 10 p.g/mlGCSF-Gen, 10 p.g/mlPBSGen. 10 p.g/mlEGF-Gen, 10 �g/ml

B43-Gen, 10 �ig/ml

.c�

EGF-Gen, 1pM

TAF= 97.6%

36.6%

.

Mean no. of coloniesper I X 10� cells

394 (368,420)

524 (512,536)

275 (268.282)

0 (0,0)

395 (390,400)

155 (143,167)

161 (152,170)

I 17 (113,121)0 (0,0)

154 (150.158)

298 (287,309)

355 (307,403)

256 (253,259)

0 (0.0)309 (298,320)

214 (209,219)

175 (142,188)

210 (187,233)

0 (0,0)

‘Y�;:!13.5%

EGF-Gen, 10pM

TAF= 98.9%

56.7%

% inhibition of

cbonogenic cells

0.0

30.2

>99.40.4

0.0

24.5

>99.4

0.6

0.0

14.1

>99.7

0.0

I 8.2

0.0

>99.5

‘, ALL, acute lymphocytic leukemia.

as well as BT-20 cells at nanomolar concentrations with mean

IC511s of 30 ± 3 nM (range, 21-42 nM) and 30 ± 10 nr�i (range,

17-64 nM), respectively (-196 ng/ml), whereas unconjugated

Gen elicited substantially less inhibitory activity with >1000-

fold higher mean IC50s [I 20 ± 18 p.�i; range, 99-154 �i.M) for

MDA-MB-231 cells (-32 �i.g/ml) and 1 12 ± 17 �iM (range,

80-139 �i.M) for BT-20 cells (-30 p.g/ml). The Ps for the

Student’s t test comparisons of the IC50s for EGF-Gen versus

Gen were <0.001 for both cell lines. The IC50s derived from the

composite MDA-MB-3 1 cbonogenic cell survival curves were



20±1 8�tM)

�

125

100

75

50

25

0

-25

125

100

75

50

25�

0�

0

C0

C.)

0

(U>

Cl)

000Ca,a)0C0

0

0.1 � 1’o i#{224}o iooo

4�.5t�/.Gert(�IC5O=1 1 2±1 7�sM)

�

1000


-25 .

0.1 1 10 100

Drug Concentration (riM)

Fig. 8 EGF-Gen is more cytotoxic against clonogenic MDA-MB-23land BT-20 human breast cancer cells than unconjugated Gen. MDA-MB-23l (A) and BT-20 (B) cells were treated with 0.1, 0.3, 1.0, 3.0, 10,30, or 100 p.M EGF-Gen or equimolar concentrations of unconjugatedGen for 24 h. Subsequently, cells were assayed for cbonogenic growth in

vitro, as described in “Materials and Methods.” The colony numbers forMDA-MB-231 cells ranged from 385 colonies/l05 cells to 510 colonies/i0� cells (mean ± SE; 450 ± 36 cobonies/l05 cells). The colonynumbers for BT-20 cells ranged from 193 cobonies/105 cells to 276colonies/HI’ cells (mean ± SE; 224 ± 26 colonies/l05 cells). Composite

clonogenic cell survival curves were generated using the dose-response

data from three independent experiments, each performed in duplicate.Each data point on the composite survival curve represents the meanpercentage of control clonogenic cell survival at a given drug concen-tration. Bars, SE. The individual IC54�s for EGF-Gen from the threeexperiments ranged from 21 to 42 nM for MDA-MB-23l cells (mean ±

SE; 30 ± 3 nM) and 17 nM to 64 nM for BT-20 cells (mean ± SE; 30 ±

10 nM). The corresponding IC50s for Gen ranged from 99 to 154 p.M forMDA-MB-23l cells (mean ± SE; 120 ± 18 p.M) and from 80 to 139 �.Mfor BT-20 cells (mean ± SE; 1 12 ± 17 ELM). The EGF-Gen IC,0sderived from the composite MDA-MB-31 and BT-20 clonogenic cellsurvival curves were 39 and 20 nM, respectively. By comparison, theIC,0s of the composite cbonogenic survival curves for unconjugated Genwere 160 and 147 �i.M, respectively.

39 nM for EGF-Gen and 160 p.M for unconjugated Gen. The

IC50s derived from the composite BT-20 clonogenic cell sur-

vival curves were 20 rest for EGF-Gen and 147 p�M for uncon-

jugated Gen (Fig. 8).

DISCUSSION

In this report, we presented experimental evidence that

the EGF-Gen conjugate inactivates the EGF-R tyrosine ki-

nase as well as ErbB2, ErbB3, and Src proto-oncogene family

PTKs in breast cancer cells triggering apoptosis and clono-

genic cell death. These results indicate that the EGF-R-

associated PTK complexes have vital antiapoptotic functions

in human breast cancer cells and may, therefore, be used as

therapeutic targets.

EGF-Gen is a cytotoxic drug, and its effects on breast

cancer cells are irreversible. We favor the hypothesis that the

observed ability of EGF-Gen to cause apoptosis in breast cancer

cells is at least in part due to inhibition of EGF-R-associated Src

family PTKs rather than inhibition of the EGF-R family PTKs

because several studies have demonstrated previously that

EGF-R tyrosine kinase activity is not essential for the survival

of cancer cells (25-29). For example, Fry et a!. (25) reported

that the PTK inhibitor PD153035 inhibited EGF-R tyrosine

kinase with an IC50 of 29 �M but failed to kill EGF-R positive

cells. Tanaka et al. (26) reported that BE-23372M, which in-

hibits the EGF-R with an IC50 of 30 nM, results only in 50%

inhibition of target cell growth. RG-13022, a potent inhibitor of

EGF-R tyrosine kinase, also elicited only a transient cytostatic

effect on breast cancer cells, even at a 10 �i.M concentration, and

its inhibitory effects were completely abolished after its removal

from the culture medium (27). Others similarly reported that

inhibition of EGF-R tyrosine kinase with specific inhibitors

elicits only transient cytostatic but not cytotoxic effects on

cancer cells (28, 29). However, a more recent study using

CP-358,774, a potent quinazoline derivative PTK inhibitor, in-

dicates that apoptotic cell death can be induced by inhibition of

EGF-R tyrosine kinase (30).

In apoptosis assays, 10 �j.�ml (37 �i.M) Gen was not active

against MDA-MB-231 and BT-20 breast cancer cells, whereas 1

�ig/ml (0.137 p.M) EGF-Gen, which contains 270-fold less Gen,

was active. In clonogenic assays, the IC50s for EGF-Gen against

MDA-MB-231 and BT-20 breast cancer cells were >1000-fold

lower than those of unconjugated Gen (30 nr�i versus 112-120

�J.M). Thus, the conjugation of Gen to the targeting EGF mole-

cule substantially enhances its cytotoxic activity against human

breast cancer cells. This may in part be due to the delivery of

more Gen molecules to cancer cells, thereby increasing the

intracellular Gen concentration, by this targeted biotherapy ap-

proach. We further postulate that the binding of EGF-Gen to the

EGF-R brings Gen in direct contact with EGF-R tyrosine kinase

as well as Src family PTK associated with the EGF-R. The

inhibitor is held in close proximity to the EGF-R and associated

VTK because of its covalent attachment to EGF. Localization of

the Gen molecule in close proximity to the ATP-binding do-

mains of the EGF-R-associated PTK may increase the effective

binding constant by both reducing entropy and providing addi-

tional linker binding contacts and lead to sustained inhibition of

the PTK. Decreasing the effective off-rate of Gen by conjugat-

ing it to EGF may also promote covalent modification of the

EGF-R-associated PTK, reminiscent of the oxidative inactiva-

tion of CD19-associated Src family PTK by B43-Gen, an anti-

CD19 antibody-Gen immunoconjugate (18).

EGF-R overexpression is found in many types of cancer

besides breast cancer (31, 32). Thus, EGF-Gen could potentially

be useful in several different types of cancer. The EGF-R on

cancer cells represents a potential target for other forms of

biotherapy as well (27, 33, 34). Whether EGF-Gen will prove




superior to such anti-EGF-R antibodies or EGF-R-directed re-

combinant toxins (33, 34) should be examined in appropriate

preclinical and clinical settings.

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1998;4:901-912. Clin Cancer Res F M Uckun, R K Narla, X Jun, et al. breast cancer cells.Cytotoxic activity of epidermal growth factor-genistein against

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Cytotoxic Activity of Epidermal Growth Factor-Genistein ......vital PTK shows considerable promise...

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Transcript of Cytotoxic Activity of Epidermal Growth Factor-Genistein ......vital PTK shows considerable promise...