http://cancerres.aacrjournals.org/content/55/8/1660.full.pdf

[CANCER RESEARCH 55, 1660-1663,
April 15, 1995]
Advances in Brief
Use of the Stress-inducible grp78/BiP Promoter in Targeting High Level Gene
Expression in Fibrosarcoma in Vivo1
Gadi Gazit, Susan E. Kane, Peter Nichols, and Amy S. Lee2
Departments of Biochemistry and Molecular Biology [G. G., A. S. L.} and Pathology [P. N.], and the Norris Cancer Center [G. G., P. N., A. S. L], University of Southern
California School of Medicine, Los Angeles, California 90033-0800, and Department of Cell and Tumor Biology, City of Hope National Medical Center, Duarte, California
91010-3000 [S. E. K.I
Abstract
Current advances in human gene therapy open up new frontiers for
molecular therapies of cancer. However, one major limitation in cancer
gene therapy is the lack of a general tumor-specific promoter which allows
stringent and high level expression of the therapeutic reagent in malig
nantly transformed but not normal tissues. Hallmark features of solid
tumors such as glucose deprivation, chronic anoxia, and acidic pH induce
the glucose-regulated proteins, in particular, GRP78/BÃŒP,a A/,. 78,000
endoplasmic reticulum-localized
protein with chaperone and calciumbinding properties. We report here that a truncated rat grp78 promoter
with most of the distal basal elements removed can be utilized as a potent
internal promoter in a retroviral vector to drive high level expression of a
reporter gene in a murine fibrosarcoma model system. The stress-inducible grp78 promoter offers a novel approach for gene delivery systems
targeting transcription in tumorigenic cells.
Introduction
Glucose deprivation, chronic anoxia, and acidic pH known to
persist in solid tumors with poor vascularization induce GRPs3 (1-3).
For example, GRP78, also known as BiP, is induced about 10-fold
under these stress conditions, primarily through transcriptional control
(1). As molecular chaperones and calcium-binding proteins localized
in the endoplasmic reticulum, elevated levels of GRPs protect cells
against physiological adverse conditions (4, 5). Increased GRP78
expression is detected in virus-, chemical-, and radiation-transformed
cells (6). In B/C10ME tumor cells, resistance to cell-mediated cytotoxicity is mediated by GRP induction (7). During growth of a murine
tumor from radiation-induced fibrosarcoma, GRPs are increased, cor
relating with size of the tumor (8). Thus, GRPs are major cellular
proteins specifically induced in transformed cells and in tumor envi
ronments to confer a protective role in cell survival.
For successful administration of gene therapy in cancer, the ability
to achieve high level gene expression in a tumor-specific manner will
gene expression in retroviral vectors (12). Cellular promoters from the
phosphoglycerate kinase, ß-actin,and histone genes (13) have also
been used as internal promoters with varying degrees of success in a
number of cell lines and tissues. While these viral and cellular
promoters are effective in driving high level expression, nonetheless,
their effect is constitutive and cannot offer tissue- or tumor-specific
selectivity.
Recently, tissue-specific cellular promoters have been identified
which allowed targeted gene expression in vivo. For example, a
2.5-kilobase fragment of the tyrosinase promoter stringently restricts
the expression of the reporter gene in melanomas, although normal
melanocytes infected with the retroviral vector will also express the
reporter gene (14). In a transgenic mouse model, a 7.6-kilobase
fragment of the 5'-flanking sequence DNA of the mouse fetal a-fetoprotein gene, which can be abnormally reactivated in hepatocellular
carcinoma, has been exploited to direct expression of a foreign gene
in liver cancers (15). Unique properties of the stress-inducible grp78
promoter suggest that it may offer special advantages for high level
expression in malignantly transformed cells and a variety of solid
tumors. grp78 is a single copy gene in mammalian cells, and its
promoter, when fused to a reporter gene, is highly inducible by
glucose-deprivation in vitro (16). While this gene is expressed at a low
basal level in most tissues, the promoter elements controlling basal
level expression are located upstream with the stress-response ele
ments residing within 155 base pairs proximal to the TATA element
(17). We report here that a truncated grp78 promoter with most of the
distal basal elements removed can be utilized as a potent internal
promoter in a retroviral gene delivery system to drive high level
expression of a marker gene in a murine fibrosarcoma B/CIOME
system.
Materials and Methods
be extremely useful since it could alleviate the need for precise
targeting of the gene delivery system (9). Current efforts in designing
promoters in gene delivery systems have focused mainly on using
strong viral and cellular promoters to drive the expression of the
foreign gene. Commonly used viral promoters include Moloney mu
rine leukemia virus and HaMSV enhancer/promoter, both located in
the retrovirus LTR (10, 11). A potent viral promoter derived from
cytomegalovirus is used widely as an internal promoter to enhance
Received 2/9/95; accepted 3/2/95.
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.
' Supported in part by NIH Grants CA27607 (A. S. L.) and CA59308 (S. E. K.). G. G.
is a recipient of the NIH predoctoral fellowship T32 CA09569.
2 To whom requests for reprints should be addressed, at Norris Comprehensive Cancer
Retroviral Construction. To construct pHaMAGRP.neo, PCR amplifica
tion was used to obtain a fragment of the grp78 promoter spanning nucleotides
-599 to +33 (18), with a Mlul site at its 5' end and a Sail site at its 3' end. The
PCR fragment was cloned into the Mlul-Sall
sites of pHaMA,
which is
described elsewhere (19) and which contains the MDR\ gene as a selectable
marker. A cDNA encoding the bacterial neoR gene flanked by Sail and Xhol
sites was then inserted into the Sail site downstream of the grp78 promoter. For
pHaMASV.neo, a Mlul-Sall fragment carrying the SV40 early promoter/
enhancer (20) was removed from vector pSKl.MDR (21) and cloned into the
same sites of pHaMa. The neo cDNA was inserted as above.
Retroviral Production and Titering on NIH3T3 Cells. NIH3T3 cells
were maintained in DMEM containing 10% bovine serum (Irvine Scientific),
5 mM glutamine, 50 units of penicillin/ml, and 50 ^tg of streptomycin/ml.
GP+E86 and GP+envAml2 cells (kindly provided by A. Bank) were main
tained in the same medium but with 10% fetal bovine serum. Plasmids
pHaMAGRP.neo and pHaMASV.neo were transfected into GP+E86 ecotropic
packaging cells (22) by the CaPO4-DNA coprecipitation method. Cells were
Center, University of Southern California School of Medicine, P.O. Box 33800, 1441
Eastlake Avenue, Los Angeles, CA 90033-0800.
3 The abbreviations used are: GRP, glucose-regulated proteins; LTR, long terminal
selected in the presence of 20 ng/ml colchicine (Sigma Chemical Co., St.
Louis, MO) as described previously (21), and drug-resistant cells were pooled
repeat; HaMSV, Harvey murine sarcoma virus.
and grown to 80% confluence. Fresh medium lacking colchicine was added to
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IN VIVO FIBROSARCOMA
GENE TARGETING
the cells, and viruses were collected after 20-24 h. Ecotropic viruses were used
BY GRP78 PROMOTER
Results and Discussion
to transduce the GP+envAml2 amphotropic packaging cell line (23), trans
duced cells were selected in 20 ng/ml colchicine, and viruses were collected as
above. Amphotropic virus supernatants were titered on NIH3T3 cells and
determined to be 3.6 X IO4 colony-forming units/ml for pHaMAGRP.neo and
5.6 X IO4 colony-forming units/ml for pHaMASV.neo.
Retroviral Transduction. B/C10ME cells were grown in high glucose
DMEM containing 4.5 mg/ml glucose supplemented with 10% PCS and 2 mM
glutamine. Cells (5 X 10") were plated on 60-mm dishes. On day 1, the
medium was changed with the addition of 8 fxg/ml polybrene. The cells were
infected with the virus at a multiplicity of infection of 1 for 48 h. On day 3, the
cells were trypsinized and replated at 1:4 in growth media containing 60 ng/ml
colchicine for the selection of MDRl gene expression. On day 12, the surviv
ing cells were pooled and expanded.
RNA Isolation and Northern Blot Analysis. Transduced or nontransduced B/C10ME cells were plated on 15-cm dishes and grown to 80%
confluency. For glucose starvation treatment, the cells were changed to glu
cose-free media with 10% dialyzed PCS and 2 mM glutamine and incubated for
30 h prior to RNA extraction. Conditions for total cytoplasmic RNA isolation,
gel electrophoresis, and blot hybridization with the grp78 probe have been
described (24). The neo probe, containing 1 kilobase of the coding region of
the neomycin resistance gene, was generated by digesting the pNEO3 plasmid
(16) with Bglll and Smal. The 1-kilobase band was isolated from a low melting
point agarose gel and hexamer labeled (25) to a specific activity of 6.5 X IO7
cpm/jxg. The grp78 probe was generated by hexamer labeling the p3C5
plasmid. The specific activity of the grp probe was 5.2 X IO8 cpm/ng. The
autoradiogram was quantitated by an LKB ultroscan XL densitometer.
Tumor Generation. Approximately 3 x IO7 transduced B/C10ME cells
grown in culture were injected s.c. into a BALB/c mouse and allowed to form
tumors. After 3 weeks, tumors were dissected and immediately placed in 4%
paraformaldehyde for 24 h at 4°C.The tumors were then transferred to a 0.5-M
sucrose solution (in PBS) at 4°C.After 24 h, the tumors were frozen and cut
at 7-fim thickness in a cryostat, and sections were mounted on slides coated
previously with a 2% 3-aminopropyl-triethoxysilane
solution in acetone and
stored at -70°C.
In Situ Hybridization.
Slide sections were fixed in 4% paraformaldehyde
(in PBS, pH 7) for 30 min at room temperature. Sections were then sequen
tially rinsed 3 times in PBS for 10 min. Afterward, slides were rinsed in
RNase-free water for 1 min. Next, sections were placed in 0.1 M triethanolamine for l min (pH 8). To 200 ml of 0.1 M triethanolamine, 0.5 ml of acetic
anhydride was added and slides were rinsed in this solution for 10 min. Slides
were then rinsed in RNase-free water for 1 min, dehydrated with a series of
ethanol concentrations (30, 50, 70, 85, 95, and 100%), and air dried. The in
vi/ro-transcribed probes were generated as follows: a 3-kilobase grp78 hamster
cDNA EcoRV/Sall fragment was subcloned into the corresponding sites of the
pBS plasmid flanked by T7 and T3 promoters. Prior to the in vitro transcription
reaction, the gip subclone was digested with Pvull to generate a 0.95-kilobase
fragment which was transcribed as a probe. The grp antisense and sense probes
were generated by using T7 and T3 polymerases, respectively, in the presence
of [35S]UTP to yield the RNA probes. The grp antisense and sense probes are
both 950 base pairs long. To generate the neo probe, pNEO3 was digested with
Pstl to generate a 0.92-kilobase fragment of the neomycin resistance genecoding sequence. This 0.92-kilobase fragment was subcloned into the Pst] site
of pBS plasmid adjacent to a T7 and T3 promoter. T3 and T7 polymerases
were used to yield the antisense and sense RNA probes, respectively. All the
probes had a specific activity of about 1.0 X IO6 cpm/ng of RNA. Two to three
Retroviral vectors enabling us to compare directly grp78 as an
internal promoter to the well characterized SV40 viral promoter were
prepared. As shown in Fig. 1, this set of retroviral vectors contained
the human multidrug resistance gene MDRl gene driven by the
HaMSV LTR. MDRl is a dominant, selectable, and amplifiable
marker that allows selection of the transduced cells by colchicine (21).
The neomycin resistance gene (neo) was used as a reporter gene
driven by either the grp78 or the SV40 promoter. In the pHa
MAGRP.neo retroviral vector, the rat grp78 promoter fragment con
tained 632 base pairs upstream of the transcription initiation site.
While it contained some residual basal elements, the stress-inducible
regulatory elements located in two critical regions spanning -155 to
-135 and -95 to -85 were fully retained (26, 27). The pHaMASV.neo
vector was identical to the GRP vector except that it contained 340
base pairs of the immediate early SV40 promoter.
The B/C10ME cells were transduced with the retroviral vectors
carrying either the grp78 or the SV40 promoter driving neo. These
cells were chosen as recipients since they could form fibrosarcomas
when injected s.c. into BALB/c mice. Further, the induction of GRP78
by various stress conditions is well documented in these cells (7). The
transduced cells were selected by their MD/?l-mediated resistance to
colchicine, the expression of which was driven by the HaMSV LTR.
The two populations of transduced cells expressed equal levels of
colchicine resistance. Further, this provided an unbiased selection
since the level of colchicine resistance was not determined by the
relative strengths of the internal grp78 or SV40 promoters being tested
in these experiments. The transduction efficiency was high as esti
mated by the high fraction (~80%) of transduced cells which survived
the colchicine selection. The resistant cells were pooled and ex
panded, and integration of the provirus was confirmed by PCR (data
not shown). The two populations of transduced cells showed equiv
alent copy number of the integrated neo gene.
To measure the relative levels of neo expressed from the grp78 and
SV40 promoter, total cytoplasmic RNA was extracted from B/C10ME
cells and from the same cells transduced with vHaMAGRP.neo or
vHaMASV.neo virus. Prior to RNA extraction, the subconfluent cells
were cultured for 30 h either in normal medium containing 4.5 mg/ml
glucose or in glucose-free medium, neo and endogenous grp78 mRNA
were measured by Northern analysis (Fig. 2A). Equal amounts of
RNA were loaded on each lane, and based on the strong induction of
the endogenous grp78 RNA in the glucose starved samples, it was
evident that the transduced cells were being stressed. Our results
indicated that the level of neo mRNA driven by the internal grp78
promoter was induced 8-fold under glucose starvation conditions (Fig.
2B). In contrast, the level of neo mRNA driven by the SV40 promoter
remained at the same low level under both culture conditions. The
level of the longer neo-containing transcript driven by the HaMSV
LTR in both the grp78 and the SV40 constructs was also low and not
pHaMAGRP.neo
ng of RNA probe were used per slide for hybridization. The probes and slides
were incubated at 50°Cfor 3 h in a moist chamber in a hybridization solution
containing 50% formamide, 4X SSC, 5X Denhardt's, 1% SDS, 10% dextran
LTRMDR
sulfate, and 250 /xg/ml tRNA. Slides were then soaked in 20 mM DTT/4X SSC
for 10 min and placed in RNase digestion buffer for 30 min at 37°C(5 M
NaCl-1 M Tris, pH 8-0.5 M EDTA-10 mg/ml RNase A). The slides were then
washed overnight in 2x SSC/0.02 M ß-mercaptoethanol. The following day,
slides were washed for 1 h in 0.1 X SSC at 60°C.For high stringency wash, the
slides were further incubated at 70°Cfor 1 h in 50% formamide, 0.5 M NaCl,
5% sodium phosphate, 0.2 M ß-mercaptoethanol, and 1% SDS. All sides were
dehydrated with a series of ethanol concentrations
dried, and exposed to film.
(30, 70, 95, and 100%), air
pHaMASV.neo
LTRMDR
¡.TO
Fig. 1. Schematic
drawing of the retroviral vectors. In both vectors, the human MDR
gene was driven by the Harvey murine sarcoma virus LTR. The pHaMAGRP.neo
contains
632 base pairs of the rat grp78 promoter driving the expression
of the neomycin resistance
gene (neo). The pHaMASV.neo
contains 340 base pairs of the SV40 promoter.
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IN VIVO FIBROSARCOMA
Fig. 2. RNA blot analysis of neo and grp78
mRNA levels in B/C10ME cells. A, B/C10ME cells
were stably transduced with vHaMAGRP.neo
(GRP.neo) or vHaMASV.neo
(SV.neo). The cells
were grown in either normal medium (+) or glucosestarved (GS) for 30 h prior to total cytoplasmic RNA
extraction. Each lane contained 15 /J.g of RNA. The
RNA blots were hybridized with either neo or grp78
probes. The autoradiograms are shown. The posi
tions of the transcript driven by the viral LTR [neo
(LTR)] and the internal promoter [neo (int.)] are
indicated. Both transcripts contain neo sequences,
although only the internal one is used for translation
of the neo gene. Shown below are the endogenous
grp78 mRNA levels in the corresponding RNA
samples. B, quantitation of the neo (int.} mRNA
levels in the B/C10ME-transduced cells (GRP.neo
and SV.neo). The neo (int.) band intensities in A
were scanned by laser densitometry. The level of
neo (int.) in the GRP.neo cells grown under normal
culture conditions {+) was set as 1. This value was
used for the normalization of the other neo (int.)
levels in the transduced cells grown either in glu
cose-containing (+) or glucose-free media under
glucose starvation conditions (GS).
GENE TARGETING
BY GRP7H PROMOTER
B
10
0_
Å’
O
+ GS
D.
OC
o
+
«neo (LTR)
«neo (int.)
ce
E
o
<D
C
«grp78
+
GS
+
GS
Fig. 3. In situ hybridizations of tumor sections. A,
Tumors derived from B/C10ME cells stably trans
duced with vHaMASV.neo (Sections 1 and 2) or
vHaMAGRP.neo (Sections 3 through 5) after s.c.
injection in BALB/c mice were sectioned and hy
bridized to [35S]UTP-labeled probes to detect the
B
endogenous grp78 mRNA (7K) level or background
(bkg) hybridization as indicated on top. Sections 1,
2, and 3 were subjected to a high stringency wash, ß,
sections of tumors derived from vHaMAGRP.neotransduced cells were hybridized with either
[35S]UTP-labeled neo (grp/neo) probe or sense
probe to detect background (bkg) as indicated on
lop. Two sample slides are shown. C, sections of
tumors derived from vHaMASV.neo-transduced
cells were hybridized with the probes as indicated on
top. Two sample slides are shown.
SV/neo
bkg
SV/neo
bkg
inducible by glucose deprivation (Fig. 24). Quantitation of the neo HaMSV LTR and the SV40 promoter, and (b) while both HaMSV and
SV40 promoters were nonresponsive to glucose-stressed conditions,
mRNA driven by the internal promoters showed that the grp78 pro
moter had a 2-fold lower basal activity than the SV40 promoter (Fig. the internal grp78 promoter was able to confer high inducibility to the
2ß).
The low basal activity of the truncated grp78 promoter could be reporter gene.
To examine whether the enhanced neo expression in glucoseattributed to the deletion of much of the upstream enhancing sequence
from the native promoter (18). Low basal level under normal growth starved cells in vitro could be reproduced in a tumor microenvironconditions is important because an optimal promoter for tumor-spe
ment in vivo where glucose supply might be limiting, B/C10ME cells
cific expression would require that the promoter be suppressed in stably transduced with vHaMAGRP.neo and vHaMASV.neo were
normal cells. Thus, the truncated grp78 promoter exhibited the desir
injected s.c. into BALB/c mice. After 3 weeks, progressively growing
able properties that (a) its basal level was lower than that of the tumors (size, ~20 mm) were harvested and sectioned. In situ hybrid1662
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IN VIVO FIBROSARCOMA
izations were performed
with [35S]UTP-labeled
"antisense"
GENE TARGETING
RNA
BY GRP78 PROMOTER
References
probes able to detect endogenous grp78 and neo mRNA. To monitor
for background hybridizations, equivalent sections were hybridized
with the corresponding "sense" probes labeled to equal specific ac
tivities. Previously, with the use of RNA blots, it has been shown that
grp78 mRNA levels are elevated in radiation-induced fibrosarcoma
tumors, correlating with the size of the tumors (8). Here, using in situ
hybridization, we observed strong hybridization of the grp78 probe to
the tumor sections, in contrast to the uniform low background ob
tained with the control probe (Fig. 3A, compare sections 4 and 5). The
level of grp78 was much lower in the muscle sections surrounding the
tumor (data not shown). Upon a higher stringency wash, we detected
pockets of higher intensity grp78 mRNA levels in localized, center
regions of the tumor (Fig. 3A, Sections 1-3). Similar results were
observed with the neo probe in tumors derived from pHaMAGRP.neo-transduced cells, neo expression was much enhanced at
or near the center of the fibrosarcoma, and the overall neo level was
higher than that of the background control (Fig. 3B), suggesting that
the internal grp78 promoter in the retroviral vector was effective in
conferring similar inducibility to the reporter gene. The neo mRNA
levels in cells transduced with the SV40 internal promoter were
uniformly low. Their levels were comparable to, or at best, minimally
higher than that of the background control (Fig. 3C), showing low
SV40 promoter activity in these tumor environments. Microscopic
examination
revealed that the fibrosarcomas
derived from
vHaMAGRP.neo and vHaMASV.neo tumors were similar in appear
ance (data not shown). Within the central portion of the tumors were
areas of necrosis associated with polymorphonuclear leukocytes.
Thus, the enhanced neo expression in these regions in the
vHaMAGRP.neo tumors strongly suggests that the cells were expe
riencing a stress response resulting in the specific activation of the
grp78 promoter but not of the SV40 promoter.
These combined results demonstrate that a 600-base pair subfrag
ment of the grp78 promoter, as an internal promoter within a HaMSV/
MDR\ retroviral vector, is able to confer high level expression of a
reporter gene in a murine fibrosarcoma in vivo. Unlike other viral or
cellular promoters which express constitutively in normal and malig
nant cells, the stress-inducible grp78 promoter offers a novel approach
for targeted gene therapy for tumors in vivo. The truncated grp78
promoter described here may be useful to express a wide range of
therapeutic reagents in retroviral vectors or other gene delivery sys
tems in a variety of malignantly transformed cells and tumors which
exhibit properties of increased glucose utilization and anaerobic glycolysis. Possible applications could include the expression of tumor
suppressor genes, foreign MHC genes, or cytokines (28, 29) to induce
cell death or inflammation at the site of a progressively growing
tumor, leading to systemic immunity against metastasized tumor cells.
Since most anticancer agents are extremely toxic when expressed at
high levels in normal cells, the discovery of a candidate promoter,
such as that of grp78, with stringently enhanced expression in a tumor
environment, could advance cancer gene therapy. By coupling the
grp78 promoter to a delivery system which can effectively target
tumor cells, the cumbersome and expensive requirement for near
absolute targeted tumor delivery will be alleviated.
Acknowledgments
We thank Dr. GüntherDennert for helpful discussions and assistance in s.c.
injection. Colin Jamora and Mariane Metz provided excellent technical assist
ance. We are grateful to Xiaomei Cui and Dr. Charles Schuler for tumor
sectioning. We thank Drs. W. French Anderson and GüntherDennert for
critical review of the manuscript.
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Use of the Stress-inducible grp78/BiP Promoter in Targeting
High Level Gene Expression in Fibrosarcoma in Vivo
Gadi Gazit, Susan E. Kane, Peter Nichols, et al.
Cancer Res 1995;55:1660-1663.
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