p53 Gene Therapy inaRatModel ofHepatocellular Carcinoma: Intra … · surgery, radiation therapy, radioimmunotherapy, andlivertrans-plantation. Alternatively, modified intra-arterial

Vol. 4, 1649-1659, July 1998 Clinical Cancer Research 1649

p53 Gene Therapy in a Rat Model of Hepatocellular Carcinoma:

Intra-Arterial Delivery of a Recombinant Adenovirus

Scott C. Anderson, Duane E. Johnson,Matthew P. Harris,’ Heidrun Engler,Wendy Hancock, Whei-Mei Huang, Ken N. Wills,

Richard J. Gregory,2 Suganto Sutjipto,Shu Fen Wen, Sam Lofgren, H. Michael Shepard,and Daniel C. Maneval3Canji Inc., San Diego, California 92121

ABSTRACTp53 tumor suppressor gene therapy has been proposed

for cancers characterized by inactivation of p53 function,

and successful therapy will require efficient strategies for

gene delivery. To maximize transgene expression in tumors,

a clinical strategy has been proposed to treat neoplasms in

the liver via hepatic artery administration of a recombinant

adenovirus encoding wild-type p53 (rAd-p53). We have de-

veloped a syngeneic rat model using a p53mut hepatocellubar

carcinoma cell line (McA-RH7777) that results in multifocal

liver tumor nodules to provide experimental support for this

strategjr. Treatment of McA-RH7777 cells with rAd-p53 in

vitro resulted in efficient transgene expression, growth suppres-

sion, and apoptosis. Intrahepatic artery dosing with rAd-p53 or

an adenovirus encoding �-galactosidase (rAd-f3gal) increased

transgene expression in tumor tissue and decreased systemic

exposure when compared with i.v. dosing. Daily hepatic artery

dosing of rAd-p53 suppressed tumor growth when compared

with untreated rats or animals treated with rAd-(3gaI. These

data demonstrate the potential for arterial gene delivery to

tumors using recombinant adenoviruses, and support contin-

ued investigation of rAd-p53 gene therapy for liver malignan-

cies.

INTRODUCTIONHCC4 is perhaps the most common form of lethal cancer,

resulting in greater than 1 million deaths/year worldwide (1, 2).

Received 12/2/97; revised 4/16/98; accepted 4/29/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I Present address: Program in Cellular and Molecular Biology, Depart-ment of Anatomy, University of Wisconsin, Madison, WI 53715.2 Present address: Genzyme Corp., One Mountain Road, Framingham,

MA 01701.3 To whom requests for reprints should be addressed, at Canji Inc., 3030Science Park Road, San Diego, CA 92121. Phone: (619) 597-0177; Fax:

(619) 623-2032; E-mail: [email protected] The abbreviations used are: HCC, hepatocellular carcinoma; rAd-p53,recombinant adenoviruses encoding wild-type p53; rAd-�3gal, rAd en-coding �3-galactosidase; RT-PCR, reverse transcription-PCR; CMV, cy-

Although its incidence in the United States and Europe is

modest, the disease is endemic in regions of South Africa and

Southeast Asia. Conventional chemotherapy is essentially inef-

fective in this disease and current treatment of HCC includes

surgery, radiation therapy, radioimmunotherapy, and liver trans-

plantation. Alternatively, modified intra-arterial chemotherapy

has been investigated to capitalize on the unique physiology of

tumor blood flow (2). In contrast to normal hepatocytes that

receive blood primarily from the portal vein, the hepatic artery

supplies nearly all (80-100%) of the blood to malignancies in

the liver (3). Clinical trials have compared iv. and IHA admin-

istration of conventional chemotherapeutics, and improved re-

sponse rates in both primary and secondary liver tumors have

been reported with arterial delivery (4-8). The overall clinical

and economic benefit of intra-arterial chemotherapy is under

discussion, and effective therapies for hepatocellular carcinoma

remain elusive (9).

The p53 tumor suppressor is a 393 amino acid nuclear

transcription factor that plays a central role in cell cycle regu-

lation and apoptosis (10). Inactivation of p53 function has been

reported in approximately 50% of all human cancers, including

primary and secondary malignancies of the liver ( 1 1 , 1 2). Re-

introduction of wild-type p53 into p53-altered cells can suppress

tumors (13), lead to apoptosis (14), increase the sensitivity of

tumor cells to chemotherapeutic agents (15), and inhibit angio-

genesis (16). Recombinant adenoviruses encoding wild-type

p53 (rAd-p53) can suppress growth of a spectrum of human

cancer cell lines (17), and direct injection of tumors in nude

mice with rAd-p53 can inhibit growth of tumors derived from

malignancies of the lung, head and neck, colon, breast, brain,

and prostate (17-22).

As an alternative to direct intratumoral administration, we

have considered rAd-p53 delivery via the primary tumor blood

supply (i.e., hepatic artery). The unique circulation of liver

tumors provides an attractive system to test this concept. Be-

cause iv. administered recombinant adenovirus has a natural

tropism for the liver (23), regional delivery via the hepatic artery

should also limit systemic exposure of rAd-p53. Increased local

exposure coupled with decreased systemic exposure provides

advantages for evaluating gene therapy in this disease (24). To

evaluate the potential for gene delivery to liver malignancies, we

have developed a syngeneic rat model of HCC. IHA adminis-

tration of recombinant adenoviruses resulted in transgene cx-

pression in the tumor tissue and p53-specific effects on tumor

growth. Data from this model support continued investigation of

rAd-p53 gene therapy for hepatocellular carcinoma.

tomegabovirus; MOI, multiplicity of infection; GCV, gancicbovir; FBS,

fetal bovine serum; IHA, intrahepatic artery.

Research. on March 21, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1650 p53 Gene Therapy of HCC

MATERIALS AND METHODS

Tumor Cell Lines. The McA-RH7777 rat hepatoma cell

line was originally derived from carcinogenicity studies using

female Buffalo rats that were maintained on a diet including N-2

fluorenylphthalamic acid (25). McA-RH7777 cells were ob-

tamed from the American Type Culture Collection and propa-

gated in Ham’s F12IDME supplemented with 2 mtvi L-gluta-

mine, 20% horse serum, and 5% FBS at 37#{176}C,7% CO2. The

HLE human hepatocellular carcinoma cell line containing a

mutation in p53 at codon 249 was obtained from Dr. Takahashi

Morisaki (Kyushu University, Japan). These cell lines were

propagated in DME supplemented with 1% L-glutamine, and

10% FBS at 37#{176}C,7% CO2. A549 (human non-small cell lung

cancer), SW480 (human colon carcinoma), and 293 (human

embryonic kidney) cells were obtained from Amercan Type

Culture Collection.

Sequence Analysis of the McA-RH7777 Cells. Total

RNA was isolated from McA-RH7777 cells using a TRI Rca-

gent kit (Molecular Research Center, Inc., Cincinnati, OH). The

primers CCCCTGAAGACTGGATAA and AAATGGCAA-

GAAAGGAGG were used to amplify the entire rat p53 cDNA

by RT-PCR. Sequence analysis of the 1.3 Kb PCR product was

carried out using an ABI 373 automated sequencer (Applied

Biosystems, Foster City, CA) and the additional sequencing

primers: AGTFCCAGGTFCCTGTGCT, GTCATCTFCCGTC-

CCTFCTC, AGAAGA11�CCCACTGGAGTC, and TCCAC-

TACAAGTACATGTGC. Sequence analysis identified a bp

change at amino acid 280 of the rat p53 cDNA from CGT

(Arginine) to CCI (Proline). This mutation corresponds to

amino acid 282 of human p53 at Exon 8 (26, 27).

Recombinant Adenoviruses. An El-deleted replication-

deficient recombinant adenovirus encoding wild-type human

p53 (rAd-p53) under the control of the human CMV immediate

early promoter was used. El-deleted control adenoviruses were

constructed either containing the bacterial lacZ gene (rAd-�3gal)

under the control of the human CMV gene promoter (18) or

without a specific gene in the expression cassette (rAd-control;

28). Adenovirus production was carried out using the 293 hu-

man embryonic kidney cell line that provides the El-deleted

function in trans. CsC1-purified recombinant human adenovi-

ruses were stored in a PBS solution containing 2-3% sucrose,

and 2 m� MgCl2 (VPBS). Infectious titers (iu/ml) in adenovirus

stocks or serum samples were determined in a limiting dilution

bioassay using the 293 cell line (29), and testing for replication

competent adenovirus was performed.

Detection of Transgene Expression in Vitro. �3-galac-

tosidase expression was assayed by plating cells in 24-well

plates (Costar Corp., Cambridge, MA) at 1 X i05 cells/well.

After overnight incubation, infections with rAd-�3gal were per-

formed using a MOI of 0, 1, 3, 10, 30, and 100 in a volume of

1 ml. After 24 h, cells were fixed with 3.7% formaldehyde/PBS,

washed, and stained overnight with 1 mg/ml X-gal solution

containing 10% DMF, 1.2 mn MgCl2, 3 mM K3FeCN, and 3 mi�i

K4FeCN in PBS (17).

Detection of cellular p53 protein was accomplished by

Western blot in untreated cells or after treatment with rAd-p53.

Cells were lysed in buffer containing 50 mM Tris-HC1, pH 7.5,

250 m�i NaCl, 0.1% NP-40, 50 mist NaF, 5 misi EDTA, 10 jig/mI

Lii

0 Lii 0� � C�) C�)�. -

��OO

�mAb24O �, �PAb18OI

Fig. 1 McA-RH7777 cells were treated with rAd-p53 overnight at a

MOI of 3 or 30. Cells were harvested and lysed, and samples ofsupernatant were loaded onto a precast 10% tris-glycine gels. Afterelectrophoresis, proteins were transferred and blotted with an antibody

that detects both human and rodent p53 (mAb 240) or an antibody thatdetects human p53 (PAb 1801). Endogenous p53 was detected by mAb240, and adenovirus-mediated expression of p53 was detected by bothantibodies. Untreated McA-RH7777 cell lysates and cell lysates from

the SW480 human colon carcinoma cell line that expresses mutant p53

were used as controls.

aprotinin, 10 p.g/ml leupeptin, and 1 mr�i PMSF. After centrif-

ugation, samples of supernatant were normalized for total pro-

tein content and loaded onto precast 10% Tris-glycine gels

(Novex, San Diego, CA). After electrophoresis, proteins were

transferred onto 0.2 p.m nitrocellulose membranes and blocked

with a 0.5% casein solution for 1-2 h. The membrane was then

blotted for 1 h with PAb 1801 antibody that detects human p53

(Novacastra, San Diego, CA) or mAb 240 antibody that is

pantropic and detects both human and rodent p53 (Oncogene

Science, Uniondale, NY). Blots were then incubated with the

appropriate secondary antibody for 1 h. p53 protein was de-

tected by enhanced chemiluminescence (ECL kit; Amersham,

Arlington Heights, IL) using Kodak XAR-5 film.

[3H]Thymidine Incorporation in Vitro. Cells were

plated in 96-well micro-titer plates (Costar) and allowed to

incubate overnight. Serial dilutions of recombinant adenovirus

were made in DME:Fl2/15% FBS/l% glutamine and cells were

infected in triplicate at half-log increments of MOI ranging from

0.3 to 100 (200 p.1 total volume). Untreated controls were not

exposed to virus. Approximately 24 h after infection, 100 p.1

from each well was replaced with fresh media, and cells were

returned to the incubator. Five hours before harvest, 1 p.Ci of

[3H]thymidine (Amersham) was added to each well. Cells were

harvested onto glass-fiber filters (Filtermate; Packard Instru-

ments, Meriden, CT) 72 h after adenovirus infection, and total

incorporated radioactivity was detected using liquid scintillation

(TopCount; Packard Instruments). Incorporated [3H]thymidine

(cpm/well) at each MOI was expressed as a percentage of

untreated control cells (17). The MOI resulting in 50% inhibi-

tion of [3H]thymidine incorporation (ED50) was estimated from

a curve fitted to the data.



A

;); �0 1 3 10

B

D

0 1 3 10

HLE(Human, HCC)

%

Media

Control

0.1 1 10 100

Multiplicity of Infection

S

C

00

a

4_______�____ rAd-control

A-_____� rAd-p53

lou 10’

L10’

TUNEL Labling

‘ - ‘ � ‘-‘‘‘‘‘

Clinical Cancer Research 1651

Fig. 2 Rat (McA-RH7777; A)

and human (HLE, p53 mutant;B) hepatocellular carcinomacell lines were treated with

rAd-�3ga1, and transgene cx-pression was evaluated using an

X-gal stain. Dose-dependenttransgene expression was de-

tected for both cell lines. Pro-

liferation of HCC cell linesMcA-RH7777 (C) and HLE(D) was measured by [3H]thy-

rnidine incorporation 72 h after

adenovirus infection and cx-pressed as a percentage of un-treated controls (mean ± SD).The solid symbols representcells treated with rAd-p53;open symbols represent cellstreated with rAd-control. Esti-mates of ED50 were determinedfrom logistic equations fitted tothese data.

. �‘

“,.‘�‘�: “�

C McARH7777(Rat, 14CC)

Table 1 Cell cycle analysis after treatment of McA-RH7777 cells

Rat hepatoma cells (McA-RH7777) were treated with recombinantadenovirus at a MO! of 30. Cells were harvested 42 hours later togetherwith untreated cells and stained with propidium iodide. The percentage

of cells in each phase of the cell cycle was determined by fluorescence-activated cell-sorting analysis.

rAd-control rAd-p53

Cell cycle Untreated (MOI = 30) (MOI = 30)

G1 (%) 58 22 23S(%) 14 17 12

G2 (%) 25 58 42

subG1(%) 2 1 21

Detection of Apoptosis in Vitro. McA-RH7777 cells

were plated in a volume of 5 ml in T25 flasks (1 X 106

cells/flask), infected with rAd-p53 or rAd-control at MOl 30,

and incubated for 42 h. Cells were harvested, fixed with 1%

formalin and postfixed with 70% ethanol. After equilibration

buffer was added to each sample for 1-5 mm, TdT enzyme/

reaction buffer solution was added, and the cells were incubated

for 30 mm at 37#{176}C.The reaction was stopped by applying

stop/wash buffer according to the manufacturer’s instructions

(ApopTag; Oncor, Gaithersburg, MD). After the wash, anti-

digoxigenin fluorescein solution was applied and incubated for

30 mm at room temperature, and cells were additionally washed

in 0.1% Triton X-l00. Cells were then counterstained with

propidium iodide and analyzed by flow cytometry (Becton

Dickinson, San Jose, CA). A minimum of 10,000 events were

counted at a 515-565 nm wavelength for propidium iodide

staining and TUNEL (TdT-mediated dUTP-x Nick End Label-

ing).

Rat Model of Hepatocellular Carcinoma. Experimen-

tal protocols conformed with the Guidefor the Care and Use of

Fig. 3 McA-RH7777 cells were plated and infected with rAd-p53 or

rAd-control at MOI 30. Forty-two hours after treatment, cells wereharvested, and fragmentation of DNA was detected using a commer-cially available kit (ApopTag; Oncor). Flow cytomeffic analysis dem-

onstrated a significant increase in TUNEL staining after treatment with

rAd-p53 as compared with rAd-control.

Laboratory Animals published by the NIH (NIH publication No.

85-23, revised 1985). Young adult female Buffalo rats (175-

200 g) were obtained from Harlan Sprague Dawley (Indianap-

ohs, IN) and allowed ad libitum access to food (Pico rodent

chow; PMI feeds, St. Louis, MO) and tap water. To establish

liver tumors, rats were anesthetized with isoflurane (IsoFlo;

Abbott Laboratories, North Chicago, IL) administered by a




7 Days 17 Days 32 Days Fig. 4 After anesthesia and

aseptic surgery, rats received a

single injection of McA-

RH7777 cells (1 X 106 cells/

rat) intrasplenically, and thespleen was ligated and removed

1 mm after the injection. Rats

were permitted to recover, and

serial sacrifices were performed

on days 7, 17, and 32 after cell

injection. Representative photo-

graphs of tumor-bearing livers

are shown.

vaporizer. Animals were prepared for surgery and a 1-cm mci-

sion was made along the left side approximately 5 mm distal to

the rib cage. The spleen was retracted and exteriorized. McA-

RH7777 cells (1 X 106 in 1 ml sterile HBSS) were injected

directly into the spleen over a 10-15-s interval. Pressure was

applied to the injection site for 1 mm after injection, and the

spleen was removed after ligation of blood vessels (30). The

abdominal musculature was sutured and the skin was closed

using wound clips (Clay Adams, Parsippany, NJ). After animals

recovered on a thermal barrier, they were returned to their cages

for routine observation.

Hepatic Artery Catheterization. At scheduled times af-

ter tumor cell inoculation, animals were anesthetized with

isoflurane, prepared for surgery, and a mid-line ventral incision

was made. The liver was then retracted and connective tissues

were cleared to visualize the hepatic and gastroduodenal arteries

(3 1 , 32). A polyethylene catheter (modified PE-50 tubing, Clay

Adams) was placed in the gastroduodenal artery, forwarded to

the bifurcation of the common hepatic artery and secured. For

single administration studies, recombinant adenovirus was de-

livered in a 1 ml volume via infusion pump (0.33 mI/mn.).

Following dosing, the catheter was removed, the gastroduodenal

artery was ligated, and the animals were sutured as described

previously. For studies involving multiple administration, cath-

eters were filled with a heparin saline solution and partially

exteriorized to a s.c. pouch to facilitate subsequent dosing.

Detection of Transgene Expression in Vivo. Two to

three weeks after intrasplenic injection multifocal tumor lesions

were established, and Buffalo rats received a single infusion of

rAd-�3gal either via the hepatic artery or via tail vein. Forty-eight

hours after dosing, animals were euthanized. Liver samples

were embedded in OCT compound (Miles, Inc., Elkhart, IN),

frozen, and sectioned on a cryostat. Frozen sections (8-12 jim)

were fixed (2% (v/v) phosphate buffered formalin, 2% (v/v)

glutaraldehyde, 2 mM MgCl2, in PBS) at room temperature for

45 mm. Sections were then placed in a rinse solution (2 msi

MgC12, 0.1% (w/v) sodium deoxycholate, 0.2% (v/v) NP4O, in

PBS) and incubated overnight at 4#{176}C(33). Sections were stained

in an X-gal solution for 6 - 8 h at room temperature and counter

stained with H&E for morphological examination. Positive

staining in both tumor and normal tissue was quantitated using

Image Pro Plus software (Media Cybernetics, Silver Spring,

MA).

Detection of p53 Expression in Vivo. Two weeks after

intrasplenic injection (2 X 106 cells in 1 ml) multifocal tumor

lesions were established, and Buffalo rats received a single

infusion of either rAd-�3gal or rAd-p53 via the hepatic artery.

Forty-eight hours after dosing, animals were euthanized and

liver samples were frozen in liquid nitrogen. Tissues were

quickly thawed, rinsed in RNase free water, minced, and 1 ml of

Tri-Reagent (Molecular Research Center) was added to 100 mg

of tissue for homogenization. Chloroform (200 p.1) was then

added to homogenates and pelleted. Pellets were washed with

isopropanol, 75% ethanol, air dried, resuspended in water, and

incubated at 55#{176}C for 5 rain. Samples were then incubated with

DNase for 15 mm, and EDTA (10 mist) was added and incubated

for an additional 15 mm at 65#{176}C.Absorbance was determined

(260/280 nm), and less than 1 jig of total RNA was used for

amplification. Primers were designed to amplify recombinant

p53 mRNA expression. Primers used were a 5 ‘ from the CMV

region (CCACTGCTFACTGGC’VFATCGAAA) and a 3’ from

the p53 region (AAGCGTGTCACCGTCGTGGAAA-GC’IT).

RT-PCR was carried out by preparing a 50 p.1 sample containing

0.33 p.M of each primer, 1 X of Gene Amp PCR buffer II

(Perkin-Elmer Biosystems, Foster City, CA), 1.5 miii MgC12,

0.5 mM dNTPs, 5 units of 0.5 msi AMV-RT (BM), 2.5 units of

a 0.5 mM Ampli Tag DNA Polymerase (Perkin-Elmer, Foster

City, CA), 16 units RNase inhibitor (Perkin-Elmer), and RNase

free water. Reverse transcription was at 65#{176}C10 mm, 50#{176}C8

mm, and 95#{176}C5 mm, and PCR amplification was for 32 cycles

at 94#{176}30 5, 56#{176}C30 s, 72#{176}C1 mm, and a 10 mm 72#{176}Cfinal

extension period in a Perkin-Elmer 9600 thermal cycler (Foster

City, CA). PCR products were loaded into a 10% TBE poly-

acrylamide precast gel (Novex) and visualized with ethidium

bromide staining.

Serum Pharmacokinetics of rAd-p53. Five young adult

female S/A Simonsen Albino rats (Simonsen Laboratories, Gil-

roy, CA) were anesthetized with isoflurane, and catheters were

secured in the carotid artery for serial blood sampling. An

additional catheter was placed either in the femoral vein (n = 3)

or gastroduodenal artery (n = 2) for i.v. or IHA injection,

respectively. A single 3-mn infusion of rAd-p53 (2.4 X iO� iu)

was administered in 1 ml to each animal. Blood samples (0.5

ml) were collected before administration and at scheduled times

during the 120 mm after infusion. Anesthesia was maintained

throughout the experiment. Blood samples were centrifuged,

and serum was frozen. Serum concentrations (iu/ml) of recom-

binant adenovirus were determined using a limiting dilution

bioassay (29).



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Fig. 5 Animals received an injection of McA-RH7777 cells, and tumors were permitted to form for 2-3 weeks. Rats then received a single IHA oriv. infusion of rAd-�3gal at doses of either 4 X l0� iu (IHA) or 1.5 X l0’#{176}iu (iv.). Two days after infusion, livers were harvested, stained for �3-gal

activity, and counterstained with H&E for morphology. Representative histological sections are shown at low (top) and high (bottom) magnification.

Regions of transgene expression are indicated by the blue color.

p53 Gene Therapy. For experiments testing the efficacy

of rAd-p53 in vivo, six female Buffalo rats received intrasplenic

injections of approximately 1 X 106 McA-RH7777 cells. Seven

days later, rats were grouped in pairs to receive either rAd-p53

or rAd-�3gal (3 x l0� iu/infusion). Surgery was performed to

catheterize the gastroduodenal artery (described above), and

each rat received a 1 ml infusion (0.33 ml/min) of rAd-p53

while its matched pair received rAd-j3gal. Catheters were par-

tially exteriorized to a s.c. pouch, and daily infusions were

administered for 5 consecutive days. At day 3 1 , animals were

euthanized and livers were harvested, evaluated for tumor mass,

and photographed. In a separate experiment six female Buffalo

rats received injections of McA-RH7777 as described above.

Seven days post cell injection, paired rats received either rAd-

p53 or rAd-�3gal treatment (4 X l0� iu/infusion) via the hepatic

artery catheter for 4 consecutive days. At day 3 1 , animals were

euthanized, livers were weighed, tumor nodules were counted,

and livers were photographed.

RESULTS

Cell Line Characterization. The McA-RH7777 cell line

is one of a series of previously characterized Morris hepatoma

cells lines derived from female Buffalo rats after exposure to

N-2-fluorenylphthalanic acid (25). A Western blot of cell lysates

from untreated McA-RH7777 cells probed with an antibody that

detects both human and rodent p53 (mAb 240) demonstrated

high levels of endogenous protein (Fig. 1, Lane 2), typically

associated with the accumulation of mutated p53. No endoge-

nous protein was detected with the PAb-1801 antibody that

detects human p53. Sequence analysis confirmed a COT to CCT

mutation at codon 280, corresponding to an arginine to proline



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#1 0 (2.5x1 � iu)Fig. 6 Tumor bearing rats received single 1-mm infusions of rAd-f3gal (I X l0�-2.5 X 10)0 iuldose) via the hepatic artery catheter. Two days afterinfusion, livers were harvested, stained for 3-gal activity, and counterstained with H&E for morphology. Representative histological sections are

shown for six different animals (indicated by ID#). Regions of transgene expression are indicated by the blue color. Expression was evident in tumors,

but the extent of expression varied both within and among animals.

amino acid substitution in this conserved region of the protein

(data not shown).

Adenovirus-mediated Transgene Expression in Vitro.

Treatment of McA-RH7777 cells with rAd-p53 resulted in an

increase of p53 detected by mAb 240. Western blot analysis

using an antibody (PAb 1801) that detects human but not rodent

p53 further demonstrated the presence of transgene-mediated

expression following treatment of cells with rAd-p53 (Fig. 1).

Because differential levels of transgene expression have been

reported for human tumor cell lines, and human adenoviruses

may not efficiently transduce rodent cells, a recombinant ade-

sion in rat (McA-RH7777) and human (HLE) cells. Expression

of 3-gal was dose-dependent in cell lines from both species (Fig.

2A and B). Transgene expression was detected in >90% of the

McA-RH7777 cells at MOI 3 (3 X l0� iu/ml), indicating a level

of transduction comparable to human hepatocellular carcinoma

cell lines (17). These data demonstrate efficient adenovirus-

mediated gene transfer and expression in McA-RH7777 cells at

relatively low adenovirus concentrations.

rAd-p53 Growth Inhibition and Apoptosis in Vitro.

Adenovirus mediated transfer of wild-type p53 has been shown

to inhibit the proliferation of a spectrum of human tumor cell

lines, and the degree of growth inhibition depends on the extent




Table 2 Quantification of reporter gene expression in liver tumors

Female Buffalo rats received single intrasplenic injections of McA-RH7777 cells, and tumors were permitted to establish. Single iv. or IHA

infusions of rAd-�3gal were administered at various doses in multipleexperiments. Two days after rAd-�3gal administration, livers were har-vested, sectioned, and stained for reporter gene expression. Multiplehigh-power fields containing tumor and normal tissue (n 2-10/

animal) were assessed for X-gal staining. Images were quantitated forpositive staining with Image Pro Plus software. Data were collected as

a percentage of tumor or nontumor tissue within each field of view andtabulated as an average of multiple images for each animal. IHAadministration resulted in increased transgene expression in tumor and

decreased expression in normal tissue.

Animal IDNo.

Route ofadministration

Dose(iu)

% Positive

in Tumor± SD

% Positive

in Normal± SD

730 iv. 7.4 X l0� 0.6 ± 0.3 19.4 ± 8.4

732 iv. 7.4 X l0� 0.2 ± 0.3 13.5 ± 1.5

734 iv. 7.4 X l0� 0.2 ± 0.1 20.6 ± 11.6738 iv. 1.5 X 10’#{176} 1.6 ± 1.8 61.9 ± 9.4

749 iv. 1.5 x lO’#{176} 0.3 ± 0.4 43.7 ± 13.0

47 IHA 1 x 10’ 2.4 ± 1.2 0.3 ± 0.2

55 IHA 4.0 X i09 63.1 ± 1.6 14.6 ± 0.1

48 IHA 4.0 X l0� 16.2 ± 16.8 4.4 ± 3.9

505 IHA 4.3 x l0� 8.7 ± 5.0 27.4 ± 25.3

508 IHA 4.3 x l0� 13.5 ± 12.0 10.2 ± 5.5300 IHA 5.5 x l0� 5.2 ± 3.6 0.9 ± 0.6

294 IHA 5.5 x 10’ 1 .5 ± I .5 0.4 ± 0.2

296 IHA 5.5 x 10’ 4.2 ± 5.7 0.3 ± 0.2

295 IHA 5.5 x I0� 15.0 ± 12.9 0.8 ± 0.8

418 IHA 1.0 x l0’#{176} 15.1 ± 19.9 3.9 ± 3.8

103 IHA 1.0 X 1010 27.5 ± 16.9 4.8 ± 5.0

10 IHA 2.5 x l0’� 6.2 ± 4.8 3.6 ± 2.9

of transgene expression (17, 34, 35). A 3-day [3H]thymidine

incorporation assay was used to evaluate the effects of rAd-p53.

Treatment of HLE or McA-RH7777 cells with rAd-p53 inhib-

ited [3H]thymidine incorporation in a p53-specific dose-depen-

dent manner (Fig. 2C and D). Interestingly, the dose required to

inhibit 50% of McA-RH7777 (rat) cell proliferation was approx-

imately 10-fold greater than that required to inhibit HLE (hu-

man) cell growth. The reduced effect on McA-RH7777 cell

proliferation cannot be attributed to differential transgene cx-

pression (Fig. 2A and B), suggesting a less potent effect of the

human p53 protein in the rat tumor cell line.

Reintroduction of wild-type p53 can trigger apoptosis in

p53-altered cells. Flow cytometry was used to detect changes

associated with programmed cell death. Untreated McA-

RH7777 cells, or cells treated with rAd-p53 or rAd-control were

fixed and stained for total DNA content using propidium iodide.

Flow cytometric analysis demonstrated a significant increase in

the hypo-diploid fraction after rAd-p53 treatment (21%) versus

rAd-control (1%; Table 1). The p53-specific apoptosis was

further confirmed using a terminal deoxynucleotidyl transferase

reaction (ApopTag; Oncor). Flow cytometric detection of the

labeled DNA fragments was notable after rAd-p53 treatment

(Fig. 3). Together with the inhibition of cell proliferation, these

data confirm the p53-specific effects of rAd-p53 on the McA-

RH7777 cells in vitro.

In Vivo Tumor Formation. To test the effects of p53

gene therapy in vivo, an orthotopic model of HCC was devel-

oped with the p53mUl McA-RH7777 cell line. McA-RH7777

tumor cells were introduced to female Buffalo rats by intras-

plenic injection, and rats subsequently developed aggressive

multifocal tumors localized to the liver. Microscopic examina-

tion of liver sections collected 7 days after cell injection dem-

onstrated the presence of tumor, and macroscopic tumors were

typically visible by 14 days. Consistent with other rat models of

liver malignancy, tumor burden increased with time resulting in

death 4-6 weeks after cell injection (Fig. 4).

Detection of Transgene Expression in Tumor and Nor-

mal Tissue. To evaluate in vivo gene transfer and expression

in normal and tumor tissue, rAd-�3gal was injected i.v. or intra-

arterially (hepatic) to tumor-bearing rats at doses ranging from

I X l0� to 2.5 x l0�0 iu/dose. When the adenovirus was

administered by tail vein (n - 5), hepatocytes stained positive

for the transgene product. In contrast, low levels of transgene

expression were detected in the tumor tissue (Fig. 5). After

intra-arterial delivery of rAd-�3gal (n 12), transgene expres-

sion was clearly evident in the tumor tissue, with greater than

50% of tumor cells expressing �3-ga1 in some sections. Repre-

sentative sections from six animals are shown in Fig. 6. When

compared with i.v. dosing, IHA delivery resulted in increased

tumor to nontumor ratios (Table 2), consistent with dye-injec-

tion studies that demonstrated preferential arterial vasculariza-

tion of tumor nodules (data not shown). X-gal positive stained

tissue was not detected in untreated rats or rats treated with

rAd-p53.

To confirm p53 transgene expression in vivo, tumor bear-

ing rats were treated via the hepatic artery with rAd-�3ga1 or

rAd-p53 (approximately i0� iu/dose). Two days after injection,

liver samples containing both normal and tumor tissue were

homogenized and evaluated by RT-PCR using primers specific

for exogenous p53. p53 mRNA was detected in samples from

rats treated with rAd-p53 but not rAd-�3gal (Fig. 7). The PCR

reaction was also performed with these samples in the absence

of reverse-transcriptase to confirm that measured expression

was not a result of DNA contamination (data not shown).

Serum Pharmacokinetics of rAd-p53. To compare sys-

temic exposure after single iv. or lEA administration, rAd-p53

(2.4 X l0� iu) was administered to nontumor bearing female

rats. Serial blood samples were collected from a catheter placed

in the carotid artery, and serum samples were analyzed for

recombinant adenovirus concentration. Infectious adenovirus

concentrations (iu/ml) were detected in all serum samples ana-

lyzed from I mm to 30 mm after i.v. and IHA administration.

No infectious rAd-p53 levels were detected in the samples

collected before dosing or 2 h after administration. Peak serum

concentrations of rAd-p53 measured in rats dosed iv. were

10-100-fold greater than those measured after IHA administra-

tion (Fig. 8). Data from this study indicate that the first-pass

effect after IHA administration can limit the systemic exposure

of rAd-p53 in rats. However, detection of infectious rAd-p53 in

the circulation after IHA administration demonstrates that the

hepatic extraction of recombinant adenovirus was not complete.

In Vivo Effects of rAd-p53. To test efficacy in vivo,

multiple injections of rAd-p53 or rAd-�3gal were administered to

rats via IHA administration beginning 7 days after tumor cell

inoculation. Because of interexperiment variations in tumor

formation, direct comparisons were made between animals that

received intrasplenic injections of McA-RH7777 cells on the



108

1 �

106

1 �

0 20 40 60limo (mm)

�r . . I


.�

D� 0.�

,�

� 0� 0

Ii .�! #{149}j � C�) Cd) C�) ‘i �i .� � �, LI) tt) It) Lb D� �

� 0 � �, 0. 0. �, � � E�

� .��*-p53. ‘ S

. ‘� S ‘Fig. 7 Animals received an injection of McA-RH7777 cells, and tumors were allowed to grow for 2 weeks. The gastroduodenal artery was thencatheterized, and rats received single infusions (IHA) of rAd-p53 or rAd-�3gal (approximately l0� iuldose). Two days after test article administration,livers were harvested and evaluated for mRNA by RT-PCR. Primers specific for recombinant p53 were used. mRNA was extracted from A549 (humanNSCLC) cells treated with rAd-p53 as a positive control. p53 mRNA was detected in samples that were treated with rAd-p53 and absent in samplestreated with rAd-�3gal. No contaminating DNA was detected when homogenates were evaluated in the absence of reverse transcriptase (data notshown).

-J

E3

C

0

C

UC0

C.)

E3

Cl)

80 100

Fig. 8 Serum samples were analyzed for infectious adenovirus con-

centration (iulmb) after a single iv. or IHA administration of rAd-p53(2.4 X l0� iuldose) to nontumor bearing rats. Data shown representsamples collected from three animals after iv. injection (closed sym-bobs) and two animals after IHA injection (open symbols). Pooledpharmacokinetic analysis of area-under the curve suggests first-pass

extraction of rAd-p53 exceeds 90%. No infectious virus concentrationswere detected in samples drawn I 20 mm after injection from anyanimal. Limit of detection was approximately l0� iu/ml.

same date. In one experiment, rats received five daily doses of

rAd-p53 or rAd-�3gal (3 X iO� iu/injection). Livers were har-

vested and photographed 31 days after cell inoculation. Un-

treated animals were used as a positive control for tumor

growth. In a second experiment, paired animals were treated

with four daily injections (4 X l0� iu/injection) of rAd-p53 or

rAd-�3ga1 beginning 7 days after intrasplenic injection of cells.

Rats treated with recombinant adenoviruses were harvested 31

days after tumor cell inoculation together with untreated con-

trots. Tumors were present in the livers of all animals, but

growth was visibly reduced in rats that received rAd-p53 (Fig.

9, A and B). In the second experiment tumor-bearing livers were

weighed wet, and visible tumor nodules on the liver surface

were counted to quantify tumor growth (Table 3). Consistent

with qualitative observations, the liver weights and number of

tumors were reduced in animals that received rAd-p53. In con-

trast to IHA administration, no reduction in tumor burden was

detected after i.v. injection of rAd-p53 or rAd-�3gal (n =

4/group) when compared with untreated controls (data not

shown).

DISCUSSION

Strategies for p53 tumor suppressor gene therapy have

been proposed that use retroviral (36), adenoviral (18), and

lipid-based delivery (37) systems. Clinical trials are currently

underway to evaluate the biological efficacy of intratumoral

administration of an adenovirus encoding wild-type p53 to

patients with lung or head and neck cancers (38). As an alter-

native to intralesional injection, we have proposed adenoviral

delivery of the p53 gene via the vascular supply to the tumor.

Using a syngeneic model of HCC, we have demonstrated trans-

gene expression in tumor tissue and antitumor effects of rAd-

p53 after IHA administration. Dosing via the arterial rather than

venous circulation increased delivery of recombinant adenovi-

rus to the tumor and reduced exposure to the systemic circula-

tion. The data presented provide preclinical support for adeno-

virus-mediated p53 gene therapy of hepatocellular carcinoma.

The direct comparison of livers from paired rats treated

with rAd-�3gal indicates that the effects of rAd-p53 treatment

were not due to an immune response or to nonspecific cyto-

pathic effects of the adenoviral delivery. Although we observed

some antitumor effects of rAd-�3gal, the in vivo results are

consistent with the in vitro data and demonstrate a p53-specific

effect of rAd-p53 on the growth of established tumors. Impor-

tantly, complete tumor suppression was not observed with the

dosing regimen described. Limited delivery of the recombinant

adenovirus to all tumor cells provides a likely explanation for

tumor growth after rAd-p53 treatment. The variation in gene

transfer and expression detected in tumors after a single injec-



ACN53

O� j’S.. ‘,SS�’ a’3:s�#{149},,-S�S�#{149}#{149}’A.5u(-”s �C � #{149}�5555, �‘,., #{149},‘#{149}‘#{149}#{149}

‘,&. � �9,s �


A

B

DAY 31

UNTREATED

ACBGL

Fig. 9 A, animals received an injection of McA-RH7777 cells, and

tumors were allowed to grow for 7 days. The gastroduodenal artery wasthen catheterized, and paired rats received five daily 1-mb infusions (3 X

l0� iuldose) of rAd-p53(ACN53) or rAd-�3ga1(ACBGL). Thirty-one

days after cell injection, livers were harvested and photographed. B,animals received an injection of McA-RH7777 cells, and tumors were

allowed to grow for 7 days. The gastroduodenal artery was then cath-eterized, and paired rats received four daily 1-mb infusions (IHA) ofrAd-p53 or rAd-�3gab (4 X tO� iu/dose). Thirty-one days after cell

injection, livers were harvested, weighed, photographed, and visible

tumor nodules were counted. Tumor nodule counts and liver weights aresummarized in Table 3.

Table 3 Effects of multiple rAd-p53 treatment on tumor growth

Female Buffalo rats (n = 6) received single intrasplenic injectionsof McA-RH7777 cells, and tumors were permitted to grow for 7 days.

Intra-arterial (hepatic) infusions of rAd-p53 or rAd-�3gab were adminis-

tered daily over the following 4 days. Thirty-one days after cell inocu-

bation, livers were harvested, weighed, and tumor burden was evaluated

by counting tumor nodules.

Treatment No. tumors Liver weight (g)

Untreated >150 22.90

Untreated >150 24.43

rAd-�3gal 92 27.45

rAd-�3gab 111 28.02

rAd-p53 63 13.58

rAd-p53 46 16.01

tion of rAd-�3gal supports this contention. Furthermore, collat-

eral circulation has been reported for liver neoplasms (39, 40),

suggesting that the injected arterial blood vessel may not vas-

cularize each tumor nodule in this animal model. Alternatively,

the incomplete tumor suppression in vivo may be attributed to

the reduced function of wild-type human p53 in rodent tumor

cells. In vitro, rAd-p53 was approximately 10-fold less potent in

McA-RH7777 cells than in human hepatocellular carcinoma

cells comparably transduced by recombinant adenovirus. Re-

sults with this model may, therefore, underestimate the effects

of rAd-p53 expected in human malignancies.

i.v. delivery of rAd-p53 did not significantly suppress

tumor growth in this model. The lack of efficacy following i.v.

dosing corresponded to limited transgene expression detected

via this route of administration (Fig. 5). These data are consist-

ent with the limited transgene expression and efficacy of rAd-

p53 after portal vein injection to transgenic mice bearing hep-

atocellular carcinoma (41). When delivered i.v., transgene

expression from recombinant adenoviruses is localized primar-

ily to the liver, presumably because the fenestrated nature of the

microvasculature facilitates direct interaction between the cx-

travasated adenoviral particles and normal parenchymal cells

(23). As primary or secondary malignancies grow in the liver,

the nature of the developing tumor vasculature will depend on

various factors, including location in the liver, tissue of origin,

age, and size of tumor (39). Primary hepatocellular carcinoma in

particular may be expected to retain the hypervascular nature of

normal parental tissue, where as tumor microcirculation may

differ and adenovirus delivery may be less efficient in metastatic

tumors of nonliver origin. Additional experimental data are

required to determine whether adenovirus-mediated gene trans-

fer will be efficient after arterial delivery to secondary malig-

nancies in the liver, or to other localized tumors with a defined

arterial blood supply (e.g., glioblastoma, soft-tissue sarcomas).

Physiological and pharmacological methods may also in-

crease adenovirus targeting to liver malignancies, including

obstruction of the hepatic blood supply after administration (42),

chemoembolization (43), and use of vasoactive agents to en-

hance tumor delivery (44). Such aggressive strategies have been

evaluated for more conventional therapeutics and may warrant

investigation with rAd-p53 in hepatocellular carcinoma because

of the serious nature of the disease.

In vivo transfer of the herpes simplex virus-thymidine




kinase gene that confers sensitivity to the prodrug GCV has

been proposed for the treatment of hepatocellular carcinoma

(28) and colorectal cancer (45, 46). One theoretical advantage of

this strategy is the potential for a bystander effect that can

inhibit the growth of neighboring nontransduced tumor cells

(47). Adenovirus-mediated delivery of the herpes simplex virus-

thymidine kinase gene (rAd-TK) with GCV may, therefore, be

efficacious after transduction of only a limited number of cells

in the tumor, and tissue-specific promoters may add selectivity

to this antitumor approach. Adenovirus transgene expression

after intra-arterial delivery supports not only the potential for

p53 gene therapy, but also the potential for recombinant adeno-

virus delivery of enzymes for directed prodrug therapy of HCC.

Indeed, a pilot study with this Buffalo rat model demonstrated a

reduction in tumor growth after rAd-TK/GCV treatment (data

not shown). Antitumor effects have also been reported in a

carcinogen-induced model of hepatocellular carcinoma after

portal vein administration of rAd-TKJGCV (48). However,

these investigators observed deaths in 47% of rats treated with

rAd-TK/GCV, and other investigators have recently demon-

strated severe liver toxicity associated with the rAd-TK/GCV

combination in mice (49). In contrast, rAd-p53 toxicities have

not been reported in a regenerating liver model (50). Because

p53 overexpression is tolerated in normal cells, an improved

therapeutic index may be expected with tumor suppressor gene

therapy. Further experimentation is warranted to compare the

effects of these alternative gene therapy strategies for liver

malignancies.

A CTL response to recombinant adenoviruses has been

reported to limit the duration of transgene expression in vivo and

cause an inflammatory response to transduced cells (5 1). This

cellular response may limit the use of adenoviral vectors for

diseases requiring prolonged transgene expression. However, in

vitro data demonstrate that only short-term expression of p53

(on the order of days) is sufficient to trigger apoptosis in

p53-altered cells (2 1 ). If delivery of rAd-p53 to tumor cells can

be optimized by arterial administration, the Cli response may

not compromise the antitumor effects of recombinant p53 cx-

pression. In addition, the inflammatory response to positively

transduced tumors after rAd-p53 treatment may result in anti-

tumor effects independent of the transgene.

Development of neutralizing antibodies after iv. adminis-

tration of recombinant adenovirus has been reported, and this

antibody response can be expected to limit transgene expression

after subsequent cycles of treatment (52). The extent of antibody

neutralization will likely depend on dose and route of delivery.

High local concentrations after arterial infusion of rAd-p53 may

enable transgene expression even in the presence of serum

antibodies. Transient immunosuppression with available phar-

maceutical agents has been shown to facilitate gene transfer and

expression after multiple administration (53-55). The ability to

overcome a neutralizing humoral response may be of critical

importance for successful rAd-p53 gene therapy.

ACKNOWLEDGMENTS

We thank Drs. John Park, Robert Warren, Alan Venook, and Jordan

Gutterman for advice; Dr. Elaine Sigurdson and Erika Sutanto-Ward for

valuable discussions concerning the animal model; and Drs. Monica Ze-

peda and Robert Bookstein for critical review of the manuscript.

REFERENCES

I . Ravoet, C., Bleiberg, H., and Gerard, B. Non-surgical treatment of

hepatocellubar carcinoma. J. Surg. Oncol., 3 (Suppl.): 104-ill, 1993.

2. Venook, A. P. Treatments of hepatoceblubar carcinoma: too manyoptions? J. Clin. Oncol., 12: 1323-1334, 1994.

3. Breedis, C., and Young, G. The blood supply of neoplasms in the

liver. Am. J. Pathol., 30: 969-985, 1954.

4. Ravikumar, T. S., Pizzorno, G., Bodden, W., Marsh, J., Strair, R.,

Pollack, J., Hendler, R., Hanna, J., and D’Andrea, E. Percutaneous

hepatic vein isolation and high-dose hepatic arterial infusion chemo-

therapy for unresectable liver tumors. J. Clin. Oncob., 12: 2723-2736,

1994.

5. Atiq, 0. T., Kemeny, N., Niedzwiecki, D., and Botet, J. Treatment ofunresectabbe primary liver cancer with intrahepatic fluorodeoxyuridineand mitomycin C through an implantabbe pump. Cancer (Phila.), 69:

920-924, 1992.

6. Yodono, H., Sasaki, T., Tarusawa, K., Midorikawa, H., Saito, Y., and

Takekawa, S. D. Arterial infusion chemotherapy for advanced hepato-

cellular carcinoma using EPF and EAR therapies. Cancer Chemother.

Pharmacob., 31: S89-S92, 1992.

7. Doci, R., Bignami, P., Bozzetti, F., Giuliano, B., Audisio, R., Co-

lombo, M., and Gennari, L. Intrahepatic chemotherapy for unresectabbe

hepatocellubar carcinoma. Cancer (Phila.), 61: 1983-1987, 1988.

8. Stagg, R. J., Lewis, B. J., Friedman, M. A., Ignoffo, R. J., and Hohn,D. C. Hepatic arterial chemotherapy for colorectal cancer metastatic tothe liver. Ann. Intern. Med., 100: 736-743, 1984.

9. Meta-Analysis Group In Cancer. Reappraisal of hepatic arterial in-fusion in the treatment of nonresectable liver metastases from coborectal

cancer. J. Natl. Cancer Inst., 88: 252-258, 1996.

10. Lane, D. P., Midgley, C. A., Hupp, T. R., Lu, X., Vojtesek, B., and

Picksley, S. M. On the regulation of the p53 tumour suppressor, and its

role in the cellular response to DNA damage. Philos. Trans. R. Soc.

Lond. B. Biol. Sci., 347: 83-87, 1995.

11. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53

mutations in human cancers. Science (Washington DC), 253: 49-53,

1991.

12. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C.

Mutations in the p53 tumor suppressor gene: clues to cancer etiology

and molecular pathogenesis. Cancer Res., 54: 4855-4878, 1994.

13. Chen, P-L., Chen, Y., Bookstein, R., and Lee, W-H. Genetic mech-

anisms of tumor suppression by the human p53 gene. Science (Wash-

ington DC), 250: 1576-1580, 1990.

14. Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B., and Costa,

J. Induction of apoptosis by wild-type p53 in a human colon tumor-

derived cell line. Proc. Natl. Acad. Sci. USA, 89: 4495-4499, 1992.

15. Fujiwara, T., Grimm, E. A., Mukhopadhyay, T., Zhang, W-W.,Owen-Schaub, L. B., and Roth, J. A. Induction of chemosensitivity in

human lung cancer cells in vivo by adenovirus-mediated transfer of

wild-type p53 gene. Cancer Res., 54: 2287-2291 , 1994.

16. Dameron, K. M., Volpert, 0. V., Tainsky, M. A., and Bouck, N.

Control of angiogenesis in fibroblasts by p53 regulation of throm-

bospondin. Science (Washington DC), 265: 1582-1584, 1994.

17. Harris, M. P., Sutjipto, S., Wills, K. N., Hancock, W., Cornell, D.,

Johnson, D. E., Gregory, R. J., Shepard, H. M., and Maneval, D. C.Adenovirus-mediated p53 gene transfer inhibits growth of human tumor

cells expressing mutant p53 protein. Cancer Gene Ther., 3: 121-130,

1996.

18. Wills, K. N., Manevab, D. C., Menzel, P., Harris, M. P., Sutjipto, S.,

Vaillancourt, M-T., Huang, W-M., Johnson, D. E., Anderson, S. C.,

Wen, S-F., Bookstein, R., Shepard, H. M., and Gregory, R. J. Devel-opment and characterization of recombinant adenoviruses encoding p53

for gene therapy of cancer. Hum. Gene Ther., 5: 1079-1088, 1994.

19. Clayman, G. L., El-Naggar, A. K., Roth, J. A., Zhang, W-W.,

Goepfert, H., Taylor, D. L., and Liu, T-J. In vivo molecular therapy withp53 adenovirus for microscopic residual head and neck squamous car-

cinoma. Cancer Res., 55: 1-6, 1995.




20. Nielsen, L. L., Del, J., Maxwell, E., Armstrong, L., Manevab, D.,

and Catino, J. Efficacy of p53 adenovirus-mediated gene therapy against

human breast cancer xenografts. Cancer Gene Ther., 4: 129-138, 1997.

21. Kock, H., Harris, M. P., Anderson, S. C., Machemer, T., Hancock,W., Sutjipto, S., Wills, K. N., Gregory, R. J., Shepard, H. M., Westphal,M., and Maneval, D. C. Adenovirus-mediated p53 gene transfer sup-presses growth of human glioblastoma cells in vitro and in vivo. Int. J.Cancer, 67: 808-815, 1996.

22. Eastham, J. A., Hall, S. J., Sehgal, I., Wang, J., Timme, T. L., Yang,

0., Connell-Crowley, L., Elbedge, S. J., Zhang, W-W., Harper, J. W.,and Thompson, T. C. In vivo gene therapy with p53 or p21 adenovirus

for prostate cancer. Cancer Res., 55: 5151-5155, 1995.

23. Li, Q., Kay, M. A., Finegold, M., Stratford-Perricaudet, L. D., andWoo, S. L. C. Assessment of recombinant adenoviral vectors for hepatic

gene therapy. Hum. Gene Ther., 4: 403-409, 1993.

24. Bookstein, R., Demers, W., Gregory, R., Maneval, D., Park, J., andWills, K. p53 Gene therapy in vivo of hepatocellular, and liver meta-

static colorectal cancer. Semin. Oncob., 23: 66-77, 1996.

25. Morris, H. P., and Meranze, D. R. Induction and some character-istics of “minimum deviation” and transplantable rat hepatomas. Recent

Results in Cancer Res., 44: 103-1 14, 1974.

26. Zakut-Houri, R., Bienz-Tadmor, B., Givol, D., and Oren, M. Hu-man p53 cellular tumor antigen: cDNA sequence and expression in COScells. EMBO., 4: 1251-1255, 1985.

27. Soussi, T. Nucleotide sequence of a cDNA encoding the rat p53

nuclear oncoprotein. Nucleic Acids Res., 16: 1 1384, 1988.

28. Wills, K. N., Huang, W-M., Harris, M. P., Machemer, T., Maneval,

D. C., and Gregory, R. J. Gene therapy for hepatocellubar carcinoma:chemosensitivity conferred by adenovirus-mediated transfer of the

HSV-1 thymidine kinase gene. Cancer Gene Ther., 2: 191-197, 1995.

29. Huyghe, B. G., Liu, X., Sutjipto, S., Sugarman, B. J., Horn, M. T.,

Shepard, H. M., Scandelba, C. J., and Shabram, P. Purification of a type5 recombinant adenovirus encoding human p53 by column chromatog-

raphy. Human Gene Ther., 6: 1403-1416, 1995.

30. Lafreniere, R., and Rosenberg, S. A. A novel approach to the

generation and identification of experimental hepatic metastases in amurine model. J. Nat!. Cancer Inst., 76: 309-322, 1986.

3!. Sutanto-Ward, E., Sigurdson, E. R., Tremiterra, S., Lincer, R.,

Chapman, D., and Niedzwiecki, D. Adjuvant chemotherapy for cob-rectal hepatic metastases: role of route of administration and timing.

Surg. Oncol., 1: 87-95, 1992.

32. Than, T. T., Sitzmann, J. V., Li, S-S., Lin, P-W., and Grochow,L. B. A method for hepatic arterial perfusion studies in the rat. J. Surg.Res., 47: 251-254, 1989.

33. Bass, C., Cabrera, G., Elgavish, A., Robert, B., Siegal, G. P.,

Anderson, S. C., Maneval, D. C., and Curiel, D. T. Recombinant

adenovirus-mediated gene transfer to genitourinary epithebium in vitro

and in vivo. Cancer Gene Ther., 2: 97-104, 1995.

34. Roemer, K., and Friedmann, T. Mechanisms of action of the p53tumor suppressor and prospects for cancer gene therapy by reconstitu-tion of p53 function. Ann. NY Acad. Sci., 716: 265-282, 1994.

35. Katayose, D., Gudas, J., Nguyen, H., Srivastava, S., Cowan, K. H.,and Seth, P. Cytotoxic effects of adenovirus-mediated wild-type p53

protein expression in normal and tumor mammary epithebial cells. Clin.Cancer Res., 1: 889-897, 1995.

36. Roth, J. A., Nguyen, D., Lawrence, D. D., Kemp, B. L., Carrasco,

C. H., Ferson, D. Z., Hong, W. K., Komaki, R., Lee, J. J., Nesbitt, J. C.,Pisters, K. M. W., Putnam, J. B., Schea, R., Shin, D. M., Walsh, G. L.,

Dobormente, M. M., Han, C-I., Martin, F. D., Yen, N., Xu, K., Stephens,L. C., McDonne., T. J., Mukhopadhyay, T., and Cai, D. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung

cancer. Nat. Med., 2: 985-991, 1996.

37. Lesoon-Wood, L. A., Kim, W. H., Kleinman, H. K., Weintraub,B. D., and Mixson, A. J. Systemic gene therapy with p53 reduces growthand metastases of a malignant human breast cancer in nude mice. Hum.Gene Ther., 6: 395-405, 1995.

38. Roth, J. A. Modification of tumor suppressor gene expression and

induction of apoptosis in non-small cell lung cancer (NSCLC) with an

adenovirus vector expressing wildtype p53 and cicpbatin. Hum. Gene

Ther., 7: 1013-1030, 1996.

39. Ackerman, N. B. The blood supply of experimental liver metasta-

ses. IV. Changes in vascularity with increasing tumor growth. Surgery

(St. Louis), 75: 589-596, 1974.

40. Lien, W. M., and Ackerman, N. B. The blood supply of experimen-

tab liver metastases II. A microcircubatory study of the normal and tumor

vessels of the liver with the use of perfused silicone rubber. Surgery (St.

Louis), 68: 334-340, 1970.

41. Bao, J., Zhang, W., and Kuno, M. T. Adenovirab delivery ofrecombinant DNA into transgenic mice bearing hepatocebbular carcino-

mas. Hum. Gene Ther., 7: 355-365, 1996.

42. Roos, G., Christensson, P-I., Hag, I. A. E., Jakobsson, B., Teder, H.,

and Stenram, U. Degradable starch microspheres in cytostatic treatment

of a liver carcinoma; experimental studies in rats with 5-fluorouracib,

tauromustine, carmustine, doxorubibin and RSU- 1069. Anticancer Res.,

13: 635-642, 1993.

43. Civalleri, D., Scopinaro, G., Balbetto, N., Claudiani, F., DeCian, F.,Camerini, G., DePaobi, M., and Bonabumi, U. Changes in vascularity ofliver tumors after hepatic arterial embolization with degradable starch

microspheres. Br. J. Surg., 76: 699-703, 1989.

44. Takats, P. 0. D., Kerr, D. J., Poole, C. J., Warren, H. W., andMcArdle, C. S. Hepatic arterial chemotherapy for metastatic coborectabcarcinoma. Br. J. Cancer, 69: 372-378, 1994.

45. Caruso, M., Panis, Y., Gagandeep, S., Houssin, D., Sabzmann, J-L.,

and Klatzmann, D. Regression of established macroscopic liver metas-tases after in situ transduction of a suicide gene. Proc. Natl. Acad. Sci.

USA, 90: 7024-7028, 1993.

46. Chen, S-H., LiChen, X. H., Wang, Y., Kosai, K-I., Finegold, M. J.,

Rich, S. S., and Woo, S. L. C. Combination gene therapy for liver

metastasis of colon carcinoma in vivo. Proc. Natl. Acad. Sci. USA, 92:

2577-2581, 1995.

47. Culver, K. W., Ram, Z., Walbbridge, S., Ishii, H., Obdfiebd, E. H.,

and Blaese, R. M. In vivo gene transfer with retrovirab vector-producer

cells for treatment of experimental brain tumors. Science (Washington

DC), 256: 1550-1552, 1992.

48. Qian, C., Idoate, M., Bilbao, R., Sangro, B., Bruna, 0., Vazquez, J.,and Prieto, J. Gene transfer and therapy with adenovirab vector in rats

with diethylnitrosamine-induced hepatocelbubar carcinoma. Hum. Gene

Ther., 8: 349-358, 1997.

49. Brand, K., Arnold, W., Bartels, T., Lieber, A., Kay, M. A., Strauss,M., and Dorken, B. Liver-associated toxicity of the HSV-tk/GCV ap-proach and adenoviral vectors. Cancer Gene Ther., 4: 9-16, 1997.

50. Drazan, K. E., Shen, X. D., Csete, M. E., Zhang, W. W., Roth, J. A.,Busuttil, R. W., and Shaked, A. In vivo adenovirab-mediated human p53

tumor suppressor gene transfer and expression in rat liver after resec-

tion. Surgery (St. Louis), 116: 197-204, 1994.

5!. Yang, Y. P., and Wilson, J. M. Clearance of adenovirus-infectedhepatocytes by MHC class I-restricted CD4(+) CTLs in vivo. J. Immu-nol., 155: 2564-2570, 1995.

52. Trapnebl, B. C. Adenovirab vectors for gene transfer. Adv. Drug

Delivery Syst., 12: 185-199, 1993.

53. Engebhardt, J. F., Ye, X., Doranz, B., and Wilson, J. M. Ablation ofE2A in recombinant adenoviruses improves transgene persistence and

decreases inflammatory response in mouse liver. Proc. Natb. Acad. Sci.

USA, 91: 6196-6200, 1994.

54. Jooss, K., Yang, Y., and Wilson, J. M. Cycbophosphamide dimin-

ishes inflammation and prolongs transgene expression following dcliv-

cry of adenoviral vectors to mouse liver and lung. Hum. Gene Ther., 7:

1555-1566, 1996.

55. Vibquin, J., Guerette, B., Kinoshita, I., Roy, B., Goulet, M., Gravel,

C., Roy, R., and Tremblay, J. FKSO6 immunosuppression to control theimmune reactions triggered by first-generation adenovirus-mediated

gene transfer. Hum. Gene Ther., 6: 1391-1401, 1995.



1998;4:1649-1659. Clin Cancer Res S C Anderson, D E Johnson, M P Harris, et al. intra-arterial delivery of a recombinant adenovirus.p53 gene therapy in a rat model of hepatocellular carcinoma:

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