Note: Descriptions are shown in the official language in which they were submitted.
CA 02192813 2004-10-22
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DESCRIPTION
METHODS OF INDUCING GENE EXPRESSION
BY IONIZING RADIATION
BACKGROUND OF. THE INVENTTON
The United. States gbvernment may own rights in the
present invention pursuant to Government'Grants.
1. Field of the Invention
The present invention relates to methods of
regulating gene transcription and polypeptide expression
by ionizing radiation.
2. Description of the Related Art
The effects of ionizing radiation on living
cells generally result in DNA damage and cell death; the
effects being proportional to the dose rate. Ionizing
radiation has been postulated to induce multiple
biological effects by direct interaction with DNA or
through the formation of free radical species leading to
DNA damage (Hall, 1988). These effects include gene
. mutations, malignant transformation, and cell killing.
Although ionizing radiation has been demonstrated to
induce expression of certain DNA repair genes in some
prokaryotic and lower eukaryotic cells, little is known
about the effects of ionizing radiation on the regulation
of mammalian gene expression (Borek, 1985). Several
studies have described changes in the pattern of protein
synthesis observed after irradiation of mammalian cells.
For example, ionizing radiation treatment of human
malignant melanoma cells is associated with induction of
several unidentified proteins (Boothman, et al., 1989).
Synthesis of cyclin and co-regulated polypeptides is
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suppressed by ionizing radiation in rat REF52 cells but
not in oncogene-transformed REF52 cell lines (Lambert and
Borek, 1988). Other studies have demonstrated that
certain growth factors or cytokinea may be involved in x-
ray-induced DNA damage. In this regard, platelet-derived
growth factor is released from endothelial cells after
irradiation (Witte, et al., 1989).
Initiation of mRNA synthesis is a critical
1D control point in the regulation of cellular processes and
depends on binding of certain tranacriptional regulatory
factors to specific DNA sequences. However, little is
known about the regulation of transcriptional control by
ionizing radiation exposure in eukaryotic cells. The
effects of ionizing radiation on posttranscriptional
regulation of mammalian gene expression are also unknown.
Many diseases, conditions, and metabolic
deficiencies would benefit from destruction, alteration,
2D or inactivation of affected cells, or by replacement of a
missing or abnormal gene product. In certain situations,
the affected cells are focused in a recognizable tissue.
Current methods of therapy which attempt to seek and
destroy those tissues, or to deliver necessary gene
products to them, have serious limitations. For some
diseases, e.g., cancer, ionizing radiation is useful as a
therapy. Methods to enhance the effects of radiation,
thereby reducing the necessary dose, would greatly
benefit cancer patients. In a general sense, ionizing
radiation is specifically delivered to cells either by
external sources, such as a Co-60 gamma source or X-
irradiation through a linear accelerator, or through
internal means, for example radioactively tagged
monoclonal antibodies. A major problem with external
irradiation is that many cancers are not localized to a
limited area, but are spread throughout the body as
distant metastases. Furthermore, monoclonal antibodies
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useful in internally irradiating cancerous tissue must be
directed specifically to a particular tumor type. An -
important goal of the present invention was to develop
alternative methods to specifically target ionizing
radiation doses to tissues containing genes under the
control of enhancers-promoters that are inducible by the -
radiation.
SUMMARY OF THE INVENTION
The present invention seeks to overcome these
and other drawbacks inherent in the prior art by
providing methods of specifically irradiating tissues
that contain radiation,reaponsive enhancers-promoters
operatively linked to structural genes encoding
polypeptides having the ability to inhibit the growth of
a cell, and in particular, a tumor cell_ In particular,
the invention overcomes the limitations by using the
radioisotope Technetium (Tc99m), which may be chemically
modified to preferentially accumulate in particular
tissue types. Tranafection of cells with radiation
responsive enhancers-promoters operatively linked to a
structural gene followed by treatment with Tc99m results
in a two to twenty-fold increase in gene activation. The
activation of promoter using this method is gradual and
consistent compared to that obtained with X-rays,
resulting in promoter activation over a longer duration,
and in principle, at multiple metastatic sites.
In embodiments of the present invention, a
radiation responsive enhancer-promoter comprises a CArG
domain of an Egr-1 promoter, a TNF-a promoter or a c-Tun
promoter. In one preferred embodiment, an encoding
region encodes a single polypeptide. A preferred
polypeptide encoded by such an encoding region has the
ability to inhibit the growth of a cell and, particularly
a tumor cell.
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An exemplary and preferred polypeptide is a
cytokine, a-dominant negative, a tumor suppressing
factor, an angiogenesis inhibitor or a monocyte
chemoattractant. More particularly, such a preferred
polypeptide is TNF-a, interleukin-4, JE, ricin, PF4
Pseudomonas toxin, p53, the retinoblastoma gene product
or the Wilms' tumor gene product.
Another preferred polypeptide encoded by such
an encoding region has radioprotective activity toward
normal tissue. An exemplary and preferred such
polypeptide having radioprotective activity is
interleukin-1; TNF; a tissue growth factor such as a
hematopoietic growth factor, a hepatocyte growth factor,
a kidney growth factor, an endothelial growth factor or a
vascular smooth muscle growth factor; interlEUkin-6; a
free radical scavenger or a tissue growth factor
receptor.
Another preferred polypeptide encoded by such
an encoding region has radioprotective activity toward
normal tissue. An exemplary and preferred such
polypeptide having radioprotective activity is
interleukin-1; TNF; a tissue growth factor such as a
hematopoietic growth factor, a hepatocyte growth factor,
a kidney growth factor, an endothelial growth factor or a
vascular smooth muscle growth factor; interleukin-6; a
free radical- scavenger or a tissue growth factor
receptor.
Preferably, 1) a hematopoietic growth factor is
interleukin-3 or a colony stimulating factor (CSF) such
as GM-CSF, G-CSF and M-CSF; 2) an endothelial growth
factor is basic fibroblast growth factor (bFGF); 3) a
vascular smooth muscle growth factor is platelet derived
growth factor (PDGF); and 4) a free radical scavenger is
manganese superoxide dismutase (MnSOD).
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Yet another preferred polypeptide encoded by
such an encoding region has anticoagulant, thrombolytic
or thrombotic activity as exemplified by plasminogen
activator, a streptokinase or a plasminogen activator
inhibitor.
A further preferred polypeptide encoded by such
an encoding region has the ability to catalyze the
conversion of a pro-drug to a drug. Exemplary and
preferred such polypeptides are herpes simplex virus
thymidine kinase and a cytosine deaminase.
A further preferred polypeptide encoded by such
an encoding region is a surface antigen that is a gene
product of a major histocompatibility complex. Exemplary
and preferred such polypeptides are H2 proteins and HLA
protein.
In another aspect, an encoding region of a DNA
molecule of the present invention encodes the whole or a
portion of more than one polypeptide. Preferably, those
polypeptides are transcription factors. In accordance
with such an embodiment, an encoding region comprises:
(a) a first encoding sequence that encodes a
DNA binding domain of a first transcription factor;
(b) a second encoding sequence that encodes an
activation or repression domain of a second transcription
factor;
(c) a third encoding sequence that encodes a
nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
frame to each other in any order with the proviso that
the third encoding sequence need be present only if the
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first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region that is
operatively linked to any of the first, second or third
encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences.
In a preferred embodiment, a first encoding
sequence encodes a DNA binding domain of transcription
factor GAL4, a second encoding sequence encodes the VP-16
activation domain, the NF-KB activation domain, the
repression domain of the Wilms' tumor suppressor gene WT1
or the repression domain of Egr-1_
In yet another aspect, a DNA molecule of the
present invention comprises a binding region that is
capable of binding a DNA binding domain of a
transcription factor, which binding region is operatively
linked to a minimal promoter that is operatively linked
to an encoding region that encodes a polypeptide, which
encoding region is operatively linked to a transcription-
terminating region.
Preferably, the transcription factor is GAL4
and the polypeptide is the same as set forth above.
The present invention also contemplates a
pharmaceutical composition comprising a DNA molecule of
the present invention and a physiologically acceptable
carrier.
In another aspect, the present invention
contemplates a cell transformed or transfected with a DNA
molecule of .this invention or a transgenic cell derived
from such a transformed or transfected cell. Preferably,
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a transformed or transgenic cell of the present invention
is a leukocyte such as a tumor infiltrating lymphocyte or
a T cell or a tumor cell.
In another aspect, the present invention
contemplates a process of regulating the expression of a
polypeptide comprising the steps of:-
(a) operatively linking a radiation responsive
enhancer-promoter to an encoding region that encodes the
polypeptide, which encoding region is operatively linked
to a transcription-terminating region to form a DNA
molecule; and
(b) exposing the DNA molecule to an effective
expression-inducing dose of ionizing radiation.
In an alternate embodiment, more than one DNA
molecule is prepared. Preferably, those DNA molecules
comprise:
(1) a first DNA molecule comprising a radiation
responsive enhancer-promoter operatively linked to an
encoding region that comprises:
(a) a first encoding sequence that encodes
a DNA binding domain of a first transcription factor;
(b) a second encoding sequence that
encodes an activation or repression domain of a second
transcription factor;
(c) a third encoding sequence that encodes
a nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
frame to each other in any order with the proviso that
the third encoding sequence need be present only if the _
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_a_
first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region
that is operatively linked to any of the first, second or
third encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences; and
IO (2) a second DNA molecule comprising a binding
region that is capable of binding the DNA binding domain
of the first transcription factor, which binding region
is operatively linked to a minimal promoter that is
operatively linked to an encoding region that encodes a
polypeptide, which encoding region is operatively linked
to a transcription-terminating region.
A radiation responsive enhancer-promoter, a
transcription factor, a binding domain of a transcription
factor and an activation or repression domain of a
transcription factor are preferably those set forth
above. A polypeptide encoded by an encoding region is
also preferably the same as set forth above.
Where regulating is inhibiting, an encoding
region preferably comprises:
(a) a first encoding sequence that encodes a
DNA binding domain of positively acting transcription
factor for a gene encoding the polypeptide;
(b) a second encoding sequence that encodes a
repression domain of a transcription factor;
(c) a third encoding sequence that encodes a
nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
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frame to each other in any order with the proviso that
the third encoding sequence need be present only if the
first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region that is
operatively linked to any of the first, second or third
encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences.
Preferably the second encoding sequence encodes
the repression domain of the Wilms' tumor suppressor gene
WT1 or the repression domain of Egr-1.
In yet another aspect, the present invention
contemplates a process of inhibiting growth of a tumor
comprising the steps of:
(a) delivering to the tumor a therapeutically
effective amount of a DNA molecule comprising a radiation
responsive enhancer-promoter operatively linked to an
encoding region that encodes a polypeptide having the
ability to inhibit the growth of a tumor cell, which
encoding region is operatively linked to a transcription-
terminating region; and
(b) exposing the tumor to an effective
expression-inducing dose of ionizing radiation.
Preferably, a radiation responsive enhancer-
promoter comprises a CArG domain of an Egr-1 promoter, a
TNF-a promoter or a c-Jun promoter and a polypeptide is a
cytokine, a dominant negative, a tumor suppressing factor -
or an angiogenesis inhibitor.
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Delivering is preferably introducing the DNA
molecule into the tumor. Where the tumor is in a
subject, delivering is administering the DNA molecule
into the circulatory system of the subject. In a
preferred embodiment, administering comprises the steps
of
(a) providing a vehicle that contains the DNA
molecule; and
(b) administering the vehicle to the subject.
A vehicle is preferably a cell transformed or
transfected withthe DNA molecule. An exemplary and
preferred transformed or transfected cell is a leukocyte
such as a tumor infiltrating lymphocyte or a T cell or a
tumor cell from the tumor being treated. Alternatively,
the vehicle is a virus or an antibody that immunoreacts
with an antigen of the tumor.
In a preferred embodiment, exposing comprises
the steps of: -
a) providing a radiolabeled antibody that
immunoreacts with an antigen of the tumor; and
b) delivering an effective expression inducing
amount of the radiolabeled antibody to the tumor.
Alternatively, a process of inhibiting growth
of a tumor comprises the steps of:
(a) delivering to the tumor a therapeutically
effective amount of
3 5
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(1) a first DNA molecule comprising a
radiation responsive enhancer-promoter operatively linked
to an encoding region that comprises:
(i) a first encoding sequence that
encodes a DNA binding domain of a first transcription
factor;
(ii) a second encoding sequence that
encodes an activation or repression domain of a second
transcription factor;
(iii) a third encoding sequence that
encodea~a nuclear localization signal, whereby the first,
second and third encoding sequences are operatively
linked in frame to each other in any order with the
proviso that the third encoding sequence need be present
only if the first or second encoding sequence does not
encode a nuclear localization signal; and
(iv) a transcription-terminating
region that is operatively linked to any of the first,
second or third encoding sequences such that the
transcription-terminating region is located 3' to all of
the first, second and third encoding sequences; and
(2) a second DNA molecule comprising a
binding region that is capable of binding the DNA binding
domain of the first transcription factor, which binding
region is operatively linked to a minimal promoter that
is operatively linked to an encoding region that encodes
a polypeptide that has the ability to inhibit the growth
of a tumor cell, which encoding region is operatively
linked to a transcription-terminating region; and
(b) exposing the cell to an effective
expression-inducing dose of ionizing radiation.
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Preferably, a radiation responsive enhancer-
promoter and a polypeptide are the same as set forth
above. Delivering is preferably the same as set forth
above.
In a preferred embodiment, cells are exposed to
ionizing radiation by delivery of radionuclides that
specifically target, or that can be conjugated or
otherwise modified to specifically target, tumor cells.
Preferred radionuclides include, but are not limited to
Technetium-99, Sodium Iodide-123, Sodium Iodide-125,
Sodium Iodide-131, Potassium Chloride-42, Gold-198, or
Sodium Phosphate-32, with Technetium-99 being
specifically preferred. Intravenous dosages of
radionuclidea may range from 0.005 mCi to 100 mCi. In
certain embodiments, the dose will preferably be in the
range of 0.4 to 5 mCi. Radionuclides, or conjugates
thereof, are preferably supplied in a sterile, pyrogen-
free solution suitable for intravenous administration.
Alternatively, radionuclides may be administered orally
or by topical application.
Radionuclides suitable for the invention may be
gamma or beta emitters. In preferred embodiments, the
radionuclide is a gamma emitter.
Specific targeting of a radionuclide to a
particular tissue or cell type may be accomplished
through the use of derivatives of the radionuclide that
have the effect of causing it to localize in specific
tissues. As an example, over 80% of the activity of
Technetium-99 sodium phytate will localize in the liver
and spleen within 30 minutes following intravenous
administration, since it is cleared rapidly from the
blood by the reticuloendothelial system (Remington's
Pharmaceutical Sciences). For purposes of this
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invention, preferred intravenous dosages of this
radionuclide range from 1 to 4 mCi.
As a further example, Technetium-99m Sodium
Methylene Diphosphonate is known to concentrate in areas
of altered osteogenesis. As applied to the invention,
preferred intravenous dosage is up to 4 mCi.
In other specific targeting embodiments, the
radionuclides may be administered directly to solid
tumors. For example, Cobalt-60 rods ensheathed in
stainless steel may be embedded in the solid tumor that
has been previously transfected with the radiation
responsive enhancer-promoter. Alternatively, small,
stainless steel ensheathed seeds of Iridium-192 embedded
in a nylon ribbon may be employed as a therapeutic
applicator of ionizing radiation.
It is further envisioned that specific
targeting to solid tumors will be accomplished by the
administration by direct injection or infusion of
radionuclides or a radionuclide conjugate composition to
the site of the tumor. This method is particularly
suitable for all solid tumors, the general location of
which can be readily determined by a variety of means
known to physicians. In such embodiments, the
'targeting' aspect of the invention comes from the
generally specific administration of the radionuclides
rather than any inherent physical or chemical properties
thereof.
Studying the effects of such protocols on tumor
cells in vitro is an effective means by which to assess
the effectiveness of these new treatment methods. Model
systems to assess the killing of transformed (cancerous)
cells are known to be predictive of success in human
treatment regimens, partly as the cell types are
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essentially the same and all malignant cells simply
proliferate, having little interaction with other
systems. This is different to the problems found using
other more interactive biological systems, such as, for
example, when studying components of the immune system in
isolation.
However, other models designed to allow
optimizationof these methods will naturally be employed
prior to translating to a clinical environment. In
particular, -one may assess the effects in various animal
model systems of cancer, including those in which human
cancer cells-are localized within an animal.
~tIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present
specification and are included to further demonstrate
certain aspects of the present invention. The invention
may be better understood by reference to one or more of
these drawings in combination with the detailed
description of specific embodiments presented herein.
FIG. 1. Egr-1 enhancer-promoter cloned into the XhoI
and SacI restriction endonuclease sites of the luciferase
reporter vector.
FIG. 2. Stimulation of Erg-1-LUC by 131I and Tc99m in
human pancreatic cell line AsPC-1.
FIG. 3. The effect of 14.3 MBq of Tc99m on stimulation
of Erg-1-LUC in the human pancreatic cancer cell line
AePC-1_
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to compositions
and methods for regulating transcription of an encoding -
DNA sequence and expression of a polypeptide encoded by
that sequence. A composition of the present invention
comprises one or more synthetic DNA molecules comprising
an enhancer-promoter region that is responsive to
ionizing radiation and an encoding region that encodes at
least one polypeptide. In this invention, control is
exerted over transcription of an encoding DNA sequence by
an enhancer-promoter region responsive to ionizing
radiation. The enhancer-promoter region is used as a
switch to selectively affect expression of a polypeptide
encoded by that sequence. The regulation of specific
polypeptide expression in a distinct target cell or
tissue provides opportunities for therapeutic
destruction, alteration, or inactivation of that cell or
tissue.
1. Radiation responsive enhancer-promoter
A promoter is a region of a DNA molecule
typically within about 100 nucleotide pairs in front of
(upstream of) the point at which transcription begins
(i.e., a transcription start site). That region
typically contains several types of DNA sequence elements
that are located in similar relative positions in
different genes. As used herein, the term "promoter~~
includes what is referred to in the art as an upstream
promoter region, a promoter region or a promoter of a
generalized eukaryotic RNA Polymerase II transcription
unit. Exemplary and preferred promoters are the TATA
box, the CART box and GC-rich sequence elements.
Another type of discrete transcription
regulatory sequence element is an enhancer. An enhancer
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provides specificity of time, location and expression
level for a particular encoding region (e.g., gene). A
major function of an enhancer is to increase the level of
transcription of an encoding region in a cell that
contains one or more transcription factors that bind to
that enhancer. Unlike a promoter, an enhancer can
function when located at variable distances from
transcription start sites so long as a promoter is
present.
As used herein, the phrase '~enhancer-promoter"
means a composite unit that contains both enhancer and
promoter elements. As used herein, a "radiation
responsive enhancer-promoter" indicates an enhancer-
i5 promoter whose transcription controlling function is
affected by ionizing radiation. Typically, upon exposure
to an effective dose of ionizing radiation, a radiation
responsive enhancer-promoter of the present invention
stimulates or increases the rate of transcription of an
encoding region controlled by that enhancer-promoter. An
exemplary and preferred enhancer-promoter for use in a
DNA molecule of the present invention is a CArG domain of
an Egr-1 promoter, a promoter for tumor necrosis factor-
alpha (TNF-a)gene or a c-Jun promoter.
a. CArG Domain of Ear-1 Promoter
Exposure of mammalian cells to ionizing
radiation is associated with induction of Egr-1 gene
expression. The Egr-1 gene (also known as zif/268, TIS-
8, NFGI-A and Krox-24; Sukhatme, et al. 1985; Christy, et
al., 1988; Milbrandt, 1987; Lemaire, et al., 1988; Lim,
et al., 1987; Geasler, 1990) encodes a 533-amino acid
- residue nuclear phosphoprotein with a Cyst-His2 zinc
finger domain that is partially homologous to the
corresponding domain in the wilms' tumor-susceptibility
gene (Gessler, 1990). The Egr-1 protein binds to the DNA
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sequence CGCCCCCGC in a zinc-dependent manner and -
functions as a regulator of gene transcription (Chriaty,
et al., 1989; Cao, et al., 1990; Lau, et al., 1987).
Both mitogenic and differentiation signals have been
shown to induce the rapid and transient expression of
Egr-1 in a variety of cell types. Exposure of human HL-
525 cells to x-rays was associated with increases in Egr-
1 mRNA levels. Those increases were maximal at 3 hours
and transient. Nuclear run-on assays demonstrated that
this effect was related at least in part to activation of
Egr-1 gene transcription.
Sequences responsive to ionizing radiation-
induced signals were determined by deletion analysis of
the Egr-1 promoter. X-ray inducibility of the Egr-1 gene
was conferred by a region containing six serum response
or CC(A/T)6GG (CArG) domains or domains.
A region encompassing the three distal or
upstream CArG elements was functional in the x-ray
response as sequential deletion of those three CArG
domains progressively decreased the response. A single
CArG domain, however, was found to be sufficient to
confer X-ray inducibility. Those results indicate that
ionizing radiation induces Egr-1 transcription through
one or more CArG domains.
In order to identify cis elements responsible
for x-ray-induced Egr-1 transcription, the Egr-1 promoter
region extending from position -957 upstream to the
transcription start site to position +248 was ligated to
a chloramphenicol acetyl transferase (CAT) reporter gene
to form plasmid pEgr-1 P1.2. The Egr-1 promoter region
contains several putative cis elements including six CArG
domains (Christy, et al., 1989; Qureshi, et al., 1991).
Treatment of pEgr-1 P1.2 tranafected cells with ionizing
radiation was associated with a 4.1-fold increase in CAT
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activity as compared to transfected but unirradiated
cells. In contrast, similar studies performed with
plasmid p~Egr-1 P1.2 (similar to pEgr-1 P1.2 except that
nucleotides from position -55~ to -5o are deleted)
demonstrated little if any inducibility by x-rays. Thus,
x-ray inducibility of Egr-I is likely mediated by
sequences present between -550 and -50 of the Egr-1
promoter. -
Irradiation of cells transfected with plasmid
pE425, which plasmid contains an about 491 base pair
region of the Egr-1 promoter with six CArG domains
operatively linked to a CAT gene, was associated with a
3.6-fold induction of CAT activity compared to that in
non-irradiated cells transfected with this construct.
Studies have been performed with fragments of
the Egr-1 promoter linked to elements of a herpes simplex
virus-thymidine kinase (HSV-TK) gene and the CAT gene.
There was no detectable x-ray inducibility of CAT
activity in cells transfected with plasmid pTK35CAT
(containing a thymidine kinase promoter but no Egr-1
promoter regions). In contrast, cells transfected with
plasmid pE425/250TK (containing the four distal CArG
domains and using HSV-TK promoter), CAT activity was
inducible by x-rays. The region of the Egr-I promoter
extending from -395 to -250, which region excludes the
first CArG element, was also functional in conferring
x-ray inducibility to the heterologous promoter.
Although those findings provided further
support for the involvement of CArG domains in x-ray
induced Egr-1 transcription, other sequences between
these domains could also serve as functional cis
elements. Transfection of cells with plasmid pSREITK
(containing the first CArG domain having seven base pairs
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of the 5' and 3' flanking sequences) induced
transcription of pTK35CAT.
Thus, x-ray inducibility of the Egr-1 gene is
conferred by a region of the Egr-1 promoter that contains
CArG domains. The six CArG domains of the Egr-1 promoter -
are located within a region of the Egr-1 promoter located
about 960 nucleotide bases upstream from the
transcription initiation site of the Egr-1 gene
(reference). A single CArG domain is sufficient to
confer radiation inducibility. Preferably, a radiation
responsive enhancer-promoter comprises at least one of
the three most distal (i.e. upstream) CArG domains. A
detailed description of the radiation inducibility of the
Egr-1 gene by CArG domains upstream to the transcription
initiation site can be found in Example 4 hereinafter.
Studies with the c-foa promoter have
demonstrated that the CArG domain or serum response
element is functional in inducing transcription of this
gene in response to serum and other signals (Triesman,
1990). The CArG element is required for c-foa induction
by both PKC-mediated signaling pathways and by growth
factor-induced signals independent of PKC (Fisch, et al.,
1987; Gilman, 1988; Buscher, et al., 1988; Sheng, et al.,
1988; Stumpo, et al., 1988; Graham, et al., 1991). The
kinetics of induction, as well as repression, of c-fos
expression are similar to those of Egr-1 in other models
(Sukhatme, et al., 1988; Guis, et al., 1990). Indeed,
x-ray-induced changes in c-fos transcripts are similar to
those obtained for Egr-1 in HL-525 cells and TPA-induced
c-fos expression, like that for Egr-1, is attenuated in
these cells. Studies with the c-fos promoter have
demonstrated that the CArG domain functions as a binding
site for the serum response factor (SRF) (Treiaman, 1986;
Prywes, et al., 1988). SRF binds, but with varying
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affinity, to the different CArG elements in the Egr-1
promoter (Christy, et al., 1989).
Previous studies have demonstrated that binding
of SRF to CArG in the c-fos promoter is not detectably
altered by serum and other conditions (Treisman, 1986;
Prywes, et al., 1986; Sheng, et al., 1988). Nuclear
proteins from quiescent and serum-stimulated 3T3 cells
have also shown little if any difference in binding to
the first CArG element of the Egr-1 promoter (Gius, et
al., 1990). These findings suggest that ionizing
radiation, like serum, induces a posttranscriptional
modification of SRF. Other studies have demonstrated
that phosphorylation of SRF is required for activation or
transcription (Prywes, et al., 1988). The kinases
responsible for this effect, however, remain unclear.
Alternatively, ionizing radiation may result in
the modification of other proteins that interact with the
SRF or CArG domain. Both SAP-1 and p62TCF (ternary
complex factor) recognize SRF-DNA complexes (Dalton, et
al., 1992; Shaw, et al., 1989), while p62DBF (direct
binding factor) binds directly to the SRE (Ryan, et al.,
1989; Walsh, 1989). Other studies have demonstrated that
SRE-ZBP undergoes posttranslational modification and
binds to this element (Attar, et al., 1992). One or more
of these proteins may therefore be involved in x-ray-
induced Egr-I transcription.
b. c-Jun promoter
Exposure of cells to x-rays is associated with
activation of the c-Jun/c-fos gene families, which encode
transcription factors (Hallahan, et al., 1991; Sherman,
et al., 1990).
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The c-Jun gene encodes the major form of the
40-44 kD AP-1 transcription factor (Mitchell, et al.,
1989). The Jun/AP-1 complex binds to the heptomeric DNA
consensus sequence TGAG/~TCA (Mitchell, et al., 1989).
The DNA binding domain of c-Jun is shared by a family of
transcription factors, including Jun-B, Jun-D and c-fos.
Moreover, the affinity of c-Jun binding to DNA is related
to the formation of homodimers or heterodimers with
products of the fos gene family (ZOrial, et al., 1989;
Nakabeppa, et al., 1988; Halazonetis, et al., 1988).
Phorbol ester activation of c-Jun transcription
in diverse cell types has implicated the involvement of a
protein kinase C (PKC)-dependent mechanism (Brenner, et
al., 1989; Angel, et al., 1988b; Hallahan, et al.,
1991a). A similar pathway likely plays a role, at least
in part, in the induction of c-Jun expression by ionizing
radiation. Prolonged treatment with phorbol esters to
down-regulate PKC is associated with decreases in the
effects of x-rays on c-Jun transcription (Hallahan, et
al., 1991a). Furthermore, non-specific inhibitors of
PKC, such as the isoquinolinesulfonamide derivative, H7,
block x-ray-induced c-Jun gene product expression
(Hallahan, et al., 1991a).
The effects of ionizing radiation on c-Jun gene __
product expression were studied in an HL-60 cell variant,
designated HL-525, which variant is deficient in
PKC-mediated signal transduction (Homma, et al., 1986).
That variant is resistant to both phorbol ester-induced
differentiation and x-ray-induced TNF gene product
expression (Hallahan, et al., 1991b; Homma, et al., 1986)
and resistant to the induction of c-Jun gene product
expression by phorbol esters.
Treatment of those cells withionizing
radiation was associated with a superinduction of c-Jun
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mRNA levels compared to phorbol ester-responsive HL-60
cells. Transcription of c-Jun was low in untreated
HL-525 cells. However, exposure of those cells to
ionizing radiation resulted in c-Jun mRNA levels which
were substantially higher at 3, 6 and 8 hours after x-ray
exposure than in non-irradiated cells. Expression of the
Jun-B and Jun-D gene products was also transiently
increased following x-irradiation of the HL-525 cells.
The kinetics of those increases in foa gene product
expression were similar to that obtained for members of
the Jun genefamily.
The activation of Jun likely results in
increased transcription of the AP-1 binding site
following ionizing radiation exposure. The plasmid
p3xTRE-CAT (containing three AP-1 sites upstream of the
minimal tk promoter from plasmid pBLCAT2) was tranafected
into RIT-3 cells. Irradiation of p3xTRE-CAT
transfectants resulted in a 3-fold increase in CAT
expression.
Where RIT-3 cells tranafected-with a DNA
molecule (c-Jun-CAT) comprising a 1840-base pair (-1.1 kb
to +740 bp) segment of the c-Jun promoter placed upstream
of the CAT gene were exposed to ionizing radiation, CAT
expression increased about 3-fold relative to
tranafected, non-irradiated cells. Transfection of those
cells with a plasmid having a deletion of the AP-1 site
located at +150-by (-132/+170 D AP-1CAT) resulted in a
loss of x-ray-mediated induction of CAT expression.
Thus, activated AP-1 likely participates in the
transcription of c-Jun and the AP-1 DNA sequence is
likely sufficient and necessary to confer x-ray-mediated
c-Jun gene induction. A detailed description of x-ray
induced transcription of DNA molecules containing a c-jun
promoter can be found hereinafter in Examples 2, 3, 5 and
6.
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c. TNF-a promoter
Tumor necrosis factor a (TNF-a) is a
polypeptide mediator of the cellular immune response with
pleiotropic activity. TNF-a acts directly on vascular
endothelium to increase the adhesion of leukocytes during
the inflammatory process (Bevelacqua, et al., 1989).
This in vivo response to TNF-a was suggested to be
responsible for hemorrhagic necrosis and regression of
transplantable mouse and human tumors (Carswell, 1975).
TNF-a also has a direct effect on human cancer cell lines
in vitro, resulting in cell death and growth inhibition
(Sugarman, et al., 1985; Old, 1985). The cytotoxic
effect of TNF-a correlates with free-radical formation,
DNA fragmentation, and microtubule destruction (Matthews,
et al., 1988; Rubin, et al., 1988; Scanlon, et al., 1989;
Yamauchi, et al., 1989; Matthews, et al., 1987; Neale, et
al., 1988). Cell lines that are resistant to oxidative
damage by TNF-a also have Elevated free-radical buffering
capacity (Zimmerman, et al., 1989; Wong, et al., 1988).
In addition, TNF-a causes hydroxyl radical
production in cells sensitive to killing by TNF-a
(Matthews, et al., 1987). Cell lines sensitive to the
oxidative damage produced by TNF-a have diminished
radical-buffering capacity after TNF-a is added
(Yamauchi, et al., 1989). Lower levels of hydroxyl -
radicals have been measured in cells resistant to TNF-a
cytotoxicity when compared with cells sensitive to TNF-a
killing (Matthews, et al., 1987).
TNF-a is increased after treatment with x-rays
in certain human sarcoma cells (e. g., STSAR-13 and STSAR-
48). TNF-a mRNA levels were substantially elevated 3 and
6 hours after irradiation of STSAR-13 and STSAR-48
cells. TNF-a mRNA levels in cell line STSAR-13 increased
by >2.5-fold as measured by densitometry 3 hours after
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exposure to 500 cGy and then declined to baseline levels
by 6 hours. TNF-a transcripts increased at 6 hours after
irradiation in cell line STSAR-48, thus indicating some
heterogeneity between cell lines in terms of the kinetics
of TNF-a gene expression. In contrast, irradiation had
no detectable effect on 7S RNA levels or expression of
the polymerase (3 gene.
The increase in TNF-a mRNA was accompanied by
an increased expression of TNF-a protein, which increase
was accompanied by secretion of TNF-a protein into the
medium in which those cells were grown. Levels of TNF-a
in the medium of human tumor cell lines and fibroblaste
were quantified before and after exposure to ionizing
radiation. Five of 13 human bone and soft tissue sarcoma
cell lines (STSAR-5, -13, -33, -43, and -48) released
TNF-a into the medium after irradiation, whereas TNF-a
levels were not elevated in supernatant from normal human
fibroblast cell lines (GM-1522 and NFIF-235) and four
human epithelial tumor cell lines (HN-SCC-68, SCC-61,
SCC-25, and SQ-20B) after exposure to radiation. Tumor
cell line STSAR-13 produced undetectable amounts of TNF-a
before x-irradiation and 0.35 unita/ml after x-ray
exposure. Cell lines STSAR-5 and -33 responded to x-
irradiation with increases in TNF-a concentrations of >5-
to 10-fold. Cell lines STSAR-43 and -48 demonstrated
increases in TNF-a of 1..5- to 3-fold. TNF-a protein in
the medium was first elevated at 20 hr after x-ray
treatment, reached maximal levels at 3 days, and remained
elevated beyond 5 days. Furthermore, supernatant from
irradiated, but not control STSAR-33 cells, was cytotoxic
to TNF-a-sensitive cell line SQ-20B. A detailed
description of x-ray induced transcription of DNA
molecules containing the TNF-a promoter can be found
hereinafter in Example 1.
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2. Encoding Region
A radiation responsive enhancer-promoter is
operatively linked to an encoding region that encodes at
least one polypeptide. As used herein, the phrase
"operatively linked" means that an enhancer-promoter is
connected to an encoding region in such a way that the
transcription of that encoding region is controlled and
regulated by that enhancer-promoter. Means for
operatively linking an enhancer-promoter to an encoding
region are well known in the art. As is also well known
in the art, the precise orientation and location relative
to an encoding region whose transcription is controlled,
is dependent inter alia upon the specific nature of the
enhancer-promoter. Thus, a TATA box minimal promoter is
typically located from about 25 to about 30 base pairs
upstream of a transcription initiation site and an
upstream promoter element is typically located from about
100 to about 200 base pairs upstream of a transcription
initiation site. In contrast, an enhancer can be located
downstream from the initiation site and can be at a
considerable distance from that site.
a. Sin~cle polvneptide
In one embodiment, an encoding region of a DNA -
molecule of the present invention encodes a single
polypeptide. As used herein, the term "polypeptide"
means a polymer of amino acids connected by amide
linkages, wherein the number of amino acid residues can
range from about 5 to about one million. Preferably, a
polypeptide has from about 10 to about 1000 amino acid
residues and, even more preferably from about 20 to about
500 amino residues. Thus, as used herein, a polypeptide
includes what fs often referred to in the art as an
oligopeptide (5-10 amino acid residues), a polypeptide
(11-100 amino acid residues) and a protein (>100 amino
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acid residues). A polypeptide encoded by an encoding
region can undergo post-translational modification to
form conjugates with carbohydrates, lipids, nucleic acids
and the like to form glycopolypeptides (e. g.,
glycoproteins), lipopolypeptides (e.g. lipoproteins) and
other like conjugates.
Any polypeptide can be encoded by an encoding
region of a DNA molecule of the present invention. An
encoding region can comprise introns and exons so long as
the encoding region comprises at least one open reading
frame for transcription, translation and expression of
that polypeptide. Thus, an encoding region can comprise
a gene, a split gene or a cDNA molecule. In the event
that the encoding region comprises a split gene (contains
one or more introna), a cell transformed or transfected
with a DNA molecule containing that split gene must have
means for removing those introns and splicing together
the exons in the RNA transcript from that DNA molecule if
expression of that gene product is desired.
In a preferred embodiment, a polypeptide
encoded by an encoding region of a DNA molecule of the
present invention interferes with the structural or
functional integrity of a cell exposed to that
polypeptide. Such a polypeptide has the ability to
inhibit the growth of a cell and, particularly a tumor
cell. A polypeptide is preferably a cytokine, a dominant
negative, a tumor suppressing factor, an angiogenesis
inhibitor, or a monocyte chemoattractant.
Dominant negatives to cellular enzymes such as
Raf-1 kinase are cytotoxic to human tumor cells (Qureshi,
et al., 1991). Dominant negatives to oncogenes such as
N-myc may also be effective in the treatment of cancer.
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Expression of-tumor suppreasor genes such as
p53, the retinoblastoma (Rb) susceptibility gene, Wilms'
tumor gene can be controlled by radiation. Transfection
of p53 deficient tumor cells with a p53 expression vector
abrogates cell growth (Johnson, et al., 1991).
Tumor growth is angiogenesis-dependent and
angiogenesis is directly or indirectly induced by the
tumor. Induction of angiogenesis is an important step in
carcinogeneais and in metastatic development.
Angiogeneais is induced during the transition from
hyperplasia to neoplasia. Since angiogenesis is
necessary for tumor growth, any natural or synthetic
antiangiogenic compound may have an antineoplastic
potential. Inhibition of-tumor angiogenesis through
controlled expression of an anti-angiogenesis gene could
play an important role in cancer treatment. Inhibitors
of capillary endothelial cell proliferation and/or
angiogenesis are a cartilage-derived inhibitor and
platelet factor 4 (PF4) (reviewed in Neta, et al., 1991;
Zucker, et al., 1991).
The mouse fibroblast gene is induced by PDGF.
The fibroblast gene product, JE or monocyte
chemoattractant protein-1 (MCP-1) is a member of a family
of cytokine-like glycoproteins whose expression is
induced by a mitogenic signal in monocytes, macrophages
and T cells. JE has been identified, characterized and
recombinantly produced from both mouse and human
fibroblasts (Rollins et al., 1989). The mouse and human
fibroblast gene products are designated mJE and hJE,
respectively.
MCP-1 or JE is a monocyte-specific
chemoattractant in vitro that is structurally related to
a family of proinflammatory cytokines such as macrophage
inflammatory proteins.
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Exemplary and preferred polypeptidea are tumor
necrosis factor (TNF), interleukin-4, SE, PF4 ricin, a
bacterial toxin such as Pseudomonas toxin; p53, the
retinoblastoma gene product or the Wilms' tumor gene
product.
Ire another preferred embodiment a polypeptide
encoded by an encoding region has radioprotective
activity toward normal cells (i.e., the polypeptide
protects a normal cell or tissue from a deleterious
effect of radiation). Exemplary and preferred
polypeptides having radioprotective activity are
interleukin-1; tumor necrosis factor; a tissue growth
factor such as a hematopoietic growth factor, a
hepatocyte growth factor, a kidney growth factor, an
endothelial growth factor or a vascular smooth muscle
growth factor; interleukin-6, a free radical scavenger or
a tissue growth factor receptor.
Preferably, 1) a hematopoietic growth factor is
a colony stimulating factor such as GM-CSF, G-CSF, M-CSF
or interleukin-3; 2) an endothelial growth factor is
basic fibroblast growth factor; 3) a vascular smooth
muscle growth factor is platelet derived growth factor
(PDGF); and 4) a free radical scavenger is manganese
auperoxide dismutase (MnSOD).
The radioprotective effect of administered IL-1
and IL-6 have been demonstrated (Nets, et al., 1991;
Neta, et al., 1992). The added benefit of
radioprotection of hematopoietic cells was demonstrated
by exogenous TNF added prior to irradiation which has
been demonstrated to protect the hematopoietic system in
animals (Nets, et al., 1991).
Studies by Neta et al have demonstrated that
IL-1 inducea-Beveral hematopoietic growth factors
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(GM-CSF, G-CSF, M-CSF, IL 3, and IL 6) which clearly
contribute to the accelerated growth and differentiation
of hematopoietic progenitor cells (Nets, et al, 1991).
Uckun et al have examined the radioprotective effects of
pre-total body irradiation (TBI) conditioning with
recombinant granulocyte colony-stimulating factor (rG-
CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF)
in a large series of lethally irradiated mice (Uckun, et
al, 1989). Administration of rG-CSF or rGM-CSF before
TBI protects a significant fraction of mice from the
lethal effects of LD 100/30 TBI (Waddick, et al., 1991).
At equivalent doses, rG-CSF displayed a more potent
radioprotective activity than rGM-CSF. The survival rate
after lethal TBI was also significantly higher in mice
receiving optimally radioprotective doses of rG-CSF as
compared with mice receiving optimally radioprotective
doses of rGM-CSF. Pretreatment with rG-CSF followed by
rGM-CSF was slightly more effective than rG-CSF alone in
aupralethally irradiated mice but not in lethally
irradiated mice. Nets et al. have also shown that
administration of suboptimal, nonradioprotective doses of
IL-1 alpha also synergize with GM-CSF or G-CSF to confer
optimal radioprotection (Nets, et al., 1988), suggesting
that such an interaction may be necessary for
radioprotection of hemopoietic progenitor cells.
TNF may induce radioprotection through the
production of manganese superoxide dismutase (MnSOD),
which has been shown to be associated with radiation
resistance in the T-cell line HUT-78 (WOng, et al.,
1991). C-met is the receptor for hepatocyte growth
factor and is activated during kidney and liver
regeneration. These genes can be used to prevent
radiation injury to these organs.
Arteriovenous malformations (AVMs) in the
cerebrum have been treated with radiosurgery. This
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technology involves the direction of high dose
irradiation to the AVM. The intima of AVMs thickens
through endothelial proliferation and the
microvasculature is obliterated (Steiner, 1984).
Endothelial and smooth muscle proliferation have been
shown to be associated with the production of bFGF and
PDGF. Clinical results may be improved by the addition
of bFGF and PDGF.
In yet another preferred embodiment, the
polypeptide encoded by the encoding region has
anticoagulant, thrombolytic or thrombotic activity as
exemplified by plasminogen activator, a streptokinase or
a plasminogen activator inhibitor.
The value of coronary artery re-perfusion
resulting from pharmacologically induced fibrinolysis in
patients with evolving myocardial infarction has been
rigorously evaluated (reviewed in Tiefenbrunn, 1992;
Becker, et al_, 1991). Improved left ventricular
function and even more impressive improvements in
survival rates have been demonstrated consistently~in
controlled studies. Benefit is related to the
restoration of myocardial blood flow. Maximal benefit is
achieved with early and sustained restoration of coronary
artery patency. Patients must be assessed carefully
prior to initiating treatment, especially for potential
bleeding hazards,and appropriate follow-up evaluation and
concomitant therapy needs to be planned. However, given
the overwhelming body of data now available regarding its
benefits and relative safety, thrombolysis should be
considered as conventional therapy for patients with
acute evolving myocardial infarction.
Animal studies of stroke have been encouraging
with regard to arterial recanalization and safety
(reviewed in Brott, 1991; Levine, et al., 1992).
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Arterial recanalization has been demonstrated in patients
with ischemic stroke following the administration of any
one of several thrombolytic drugs. Placebo-controlled
trials have not been completed, and so clinical benefit
has not been established. Even though the development of
brain hemorrhage has been an infrequent complication, the
very high morbidity and mortality have been worrisome.
Ironically, thrombolytic therapy holds promise for
treatment of subarachnoid hemorrhage and perhaps also for
spontaneous intracerebral hemorrhage. Human studies have
been limited, but complications have been modest, and
clinical outcomes have been encouraging.
In still yet another preferred embodiment, a
polypeptide encoded by an encoding region has the ability
to catalyze the conversion of a pro-drug to a drug or to
sensitize a cell to a therapeutic agent. By way of
example, cells manipulated to contain a herpes simplex
virus (HSV) gene for thymidine kinase (tk) and to express
HSV-tk become sensitive to the action of the antiviral
agent ganciclovir (GCV) (Culver et al., 1992). By way of
further example, cells manipulated to contain a gene for
bacterial cytosine deaminase and to express that enzyme
can catalyze the conversion of inactive, non-toxic 5'-
fluorocytosine to the active cytotoxin 5-fluorouracil
(Culver et al., 1992).
Thus, a preferred polypeptide that has the
ability to catalyze the conversion of a pro-drug to a
drug or to sensitize a cell to a therapeutic agent is
herpes simplex virus thymidine kinase or a cytosine
deaminase.
A further preferred polypeptide encoded by an
.35 encoding region is a surface antigen that is a gene
product of a major histocompatibility complex (MHC). Ae
is well known in the art, MFiC represents a set of linked
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genetic loci involved in regulating the immune response.
MHC gene products occur on cell surfaces where they act
as antigenic-markers for distinguishing self from non-
self. Typically, MHC-gene products are classified as
being of a class I or Class II depending upon their
function. MHCa from different animals have been given
different and corresponding designations. By way of
example, human MHC gene products are designated by the
prefix HL; mouse MHC gene products are designated by the
prefix H-2; rat MHC gene products are designated by the
prefix RT1 and chimpanzee MHC gene products are
designated by the prefix ChLA.
Exemplary and preferred human MHC gene products
are class I antigens HLA-A, HLA-B and HLA-D and class II
antigens HLA-Dr and HLA-Dc.
b. More than one polvoeptide
In another aspect, an encoding region of a DNA
molecule of the present invention encodes the whole or a
portion of more than one polypeptide. Preferably, those
polypeptides are transcription factors.
A transcription factor is a regulatory protein
that binds to a specific DNA sequence (e. g., promoters
and enhancers) and regulates transcription of an encoding
DNA region. Typically, a transcription factor comprises
a binding domain that binds to DNA (a DNA binding domain)
and a regulatory domain that controls transcription.
Where a regulatory domain activates transcription, that
regulatory domain is designated an activation domain.
Where that regulatory domain inhibits transcription, that
regulatory domain is designated a repression domain.
In accordance with such an embodiment, an
encoding region comprises:
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(a) a first encoding sequence that encodes a
DNA binding domain of a first transcription factor;
(b) a second encoding sequence that encodes an
activation or repression domain of a second transcription
factor;
(c) a third encoding sequence that encodes a
nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
frame to each other in any order with the proviso that
the third encoding sequence need be present only if the
first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region that is
operatively linked to any of the first, second or third
encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences.
As used herein, the phrase "operatively linked
in frame" means that encoding sequences are connected to
one another such that an open reading frame is maintained
between those sequences. Means for linking DNA encoding
sequences in frame are well known in the art.
DNA binding domains of transcription factors
are well known in the art. Exemplary transcription
factors known to contain a DNA binding domain are the
GAL4, c-fos, c-Jun, lacl, trpR, CAP, TFIID, CTF, Spl,
fiSTF and NF-rcB proteins. Preferably, a DNA binding
domain is derived from the GAL4 protein.
The GAL4 protein is a transcription factor of
yeast comprising 881 amino acid residues. The yeast
protein GAL4 activates transcription of genes reguired
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for catabolism of galactose and melibiose. GAL4
comprises numerous discrete domains including a DNA
binding domain (Marmorstein et al., 1992).
The DNA sequences recognized by GAL4 are 17
base pairs (bp) in length, and each site binds a dimer of
the protein. Four such sites, similar but not identical
in sequence, are found in the upstream activating
sequence (UASG) that mediates GAL4 activation of the GAL1
and GAL10 genes, for example (Marmorstein et al., 1992).
Functions have been assigned to various parts
of the 881-amino-acid GAL4 protein, including DNA binding
(residues 1-65) and dimerization (residues 65-94). In
addition, three acidic activating regions have been
identified (residues 94-106; 148-196; 768-881) as has a
region near the carboxyl terminus that binds the
inhibitory protein GAL80 (Marmorstein et al., 1992).
The DNA-binding region of GAL4 has eix cysteine
residues, conserved among a set of homologous proteins,
that coordinate two Zn2+ ions in a bimetal-thiolate
cluster. Residues 10-40, which form the metal binding
domain, are a compact globular unit. Residues 1-9 and
residues C-terminal to 41 are disordered (Marmorstein et
al., 1992).
The protein fragment binds to its DNA site as a
symmetrical dimer. Each subunit folds into three
distinct modules: a compact, metal-binding domain
(residues 8-40), an extended linker (residues 41-49), and
an a-helical dimerization element (residues 50-64). The
metal-binding domain contacts three DNA base pairs in the
major groove, and is therefore referred to as a
recognition module (Marmorstein et al., 1992).
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The recognition module is held together by two
metal ions, tetrahedrally coordinated by the six
cysteines. The recognition module of GAL4 defines a
class in the group of DNA-binding domains that have Zn2+
as a structural element.
Residues 50-64 form an amphipathic a-helix.
The complete GAL4 molecule contains additional residues
between 65 and 100 that contribute to dimer interactions
l0 and maintain the protein as a dimer even when it is not
bound to DNA. The amino-acid sequence of GAL4 is
consistent with a coiled-coil that may continue for one
heptad repeat beyond the C terminus of GAL4 (1-65).
Moreover, residues 79-99 include three potentially a-
helical heptad sequences. The intervening segment
(residues 72-78) contains a proline. The full
dimerization element of GAL4 could therefore share some
structural features with the 'helix-loop-helix'
transcription factors.
At least eleven other fungal DNA-binding
proteins are known to contain repeated CXaCX6C sequences
like those found in the GAL4 recognition module: LAC9,
PPR1, QA-1F, QUTA1, ARGRII, HAP1, MAL63, LEU3, PUTS, and
AMDR. In most of them, the 'loop' between the third and
fourth cysteines is six residues long, but it is one
residue shorter in MAL63, PDR1, PUT3 and AMDR, and
several residues longer in LEU3. GAL4 residues 15-20,
which are in closest proximity to DNA in the complex, are
highly conserved in these homologues. Arg 15 and Lys 20,
which form phosphate salt links that anchor the first
helix of the recognition module to DNA, are conserved in
all but AMDR, which has His and Arg at these positions,
respectively. Residue 19 is usually hydrophobic, and
residue 17 is basic, except in QA-1F and LEU3. Lys 18,
which makes base-specific contacts in the GAL4 complex,
is conserved in all but three cases. In two of the
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exceptions (QA-1F and MAL63) it is Arg; in the other
(PUT3), it is His. These conaervationa suggest that the
recognition modules of these GAL4 homologues all approach
DNA in a similar way (Marmoratein et al., 1992).
There are symmetrically disposed CCG sequences
in known sites for LAC9, PPR1, LEU3 and PUT3.
Characteristic heptad sequences suggest that several of
the homologue (LAC9, QA-1F, QUTA1, PPR1) contain coiled-
l0 coil dimerization elements similar to the one in GAL4.
In others such as ARGRII, HAP1, and LEU3, no obvious
heptad sequences occur in the 60 residues immediately C-
terminal to the recognition modules. In LEU3, the
heptads lie one residue closer to the recognition module
than in GAL4; in HAP1, they appear to be displaced toward
the C terminus by seven residues. Some heterogeneity of
dimerization structures and of linker lengths is implied
by these observations (Marmorstein et al., 1992).
The closest relatives of GAL4 are LAC9, which
carries out the same function in K. Iactica, and PPR1,
which regulates pyrimidine biosynthesis in S. cereviaiae.
GAL4 and LAC9 bind to the-same DNA sites; PPR1 recognizes
sites with the CCG triplet separated by six, rather than
11, base pairs. GAL4 and LAC9 have similar amino-acid
sequences in their linker and dimerization segments; the
linker and dimerization elements of PPRl bear no sequence
similarity to those of GAL4, aside from the rough
characteristics of their heptad regions (Marmorstein et
al., 1992).
In a preferred embodiment, therefore, a first
encoding sequence of a DNA molecule of the present
invention encodes a DNA binding domain of GAL4.
Preferably, that binding domain comprises amino acid
residue sequences 1 to about 147 of GAL4, which numerical
designations refer to amino acid residue sequences
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numbered consecutively beginning at the amino terminus.
Thus, a first encoding sequence comprises about 444
nucleotide base pairs of the GAL4 gene, which base pairs
encode amino acid residue sequences 1 to 147 of GAL4.
In another preferred embodiment, a first
encoding sequence of a DNA molecule of the present
invention encodes a DNA binding domain of GAL4 that
comprises amino acid residue sequences 1 to about 65 of
GAL4, which numerical designations refer to amino acid
residue sequences numbered consecutively beginning at the _
amino terminus. Thus, a first encoding sequence
comprises about 198 nucleotide base pairs of the GAL4
gene-, which base pairs encode amino acid residues 1 to 65
of GAL4.
Transcription factors having activation or
repression domains are well known in the art. Exemplary
transcription factors having activation domains are GAL4,
c-Jun, viral protein VP-16, and nuclear factor NF-rcB.
As set forth above, GAL4, a protein of 881
amino acid residues, activates transcription of factors
involved in carbohydrate metabolism of yeast. There are
likely two or three acidic activation domains in the GAZ,4
protein. Those activation domains comprise (1) amino
acid residues 94 to 106, (2) amino acid residues 148 to
196, and (3) amino acid residues 768 to 881, where amino
acid residues are numbered consecutively beginning at the
amino terminus (Marmoratein et al., 1992).
In one embodiment, a second encoding sequence
encodes an activation domain of GAL4.- Such an encoding
sequence comprises nucleotide base sequences of about,
69, 147 and 342 base pairs, respectively that encode the
activation domains aet forth above.
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C-Jtzn is a major form of the 40 to 44 kD AP-1
transcription factor. Several regulatory and DNA binding
domains exist within the Jun protein. Close to the DNA
binding domain is a region designated as A2, which is
required to activate transcription (Lewin, 1991). Al, an
additional transcriptional activation domain is found
near the N terminus adjacent to a region termed Delta (D)
which is proposed to bind a cellular protein that
inhibits the transcriptional activating properties of Jun
(Baichwal, 1990 and Baichwal, 1991. Jun transcriptional
activity can be conferred through either or both
activation domains A1 and A2.
Increased Jun binding to AP--1 sequences
following irradiation suggest that Jun protein is
modified following irradiatiori. Taken together with the
recent findings that protein kinase C (PKC) is activated
following irradiation of cells and that PKC depletion
suppress c-Jun induction by irradiation (Hallahan, 1992),
it is likely that irradiation activates Jun through MAP-K
modification of the A1 domain (Binetruy, 1991 and
Pulverer, 1991).
The ability of a Jun activation domain to
stimulate transcription was demonstrated in studies of
cells transformed or tranafected with DNA molecules
comprising such domains. HeLa and RIT-3 cells were
transfected with two plasmids. Plasmid pSG-JunS-253
contained the SV40 promoter (not transcriptionally
responsive to radiation) upstream of an encoding region
that encoded a chimeric protein (GAL4-Jun? comprising a
sequence for d, Al, and AZ (Baichwal, 1990) and the DNA
binding domain of the yeast GAL4 gene (the DNA binding
domain of Jun was replaced with the DNA binding domain of
the GAL4 gene, Baichwal, 1990). A second plasmid, GSBCAT
was constructed to contain the DNA sequence that binds
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Gal4 protein placed 5' of the Elb TATA box and upstream
of the CAT reporter gene (Baichwal, 1990).
Transcriptional activation of the activation
domain of Jun by irradiation of transfected cells
stimulated transcription and expression of the chimeric
Gal-Jun protein, which protein bound to the Gal4 binding
sequence and initiated transcription and expression of
CAT. Irradiation of RIT-3 cells tranafected with GSBCAT
alone demonstrated no increase in CAT activity. Similar-
results were obtained in HeLa cells which contain the Jun
inhibitor.
However, Hep G2 cells (which do not contain the
Jun inhibitor; Baichwal, 1990) tranafected with
pSG-Jun5-235 and GSBCAT demonstrated no x-ray-induced
activation of the Gal4-Jun chimeric-protein. These data
suggest that the Gal-Jun chimeric protein is activated
following irradiation resulting in DNA binding to
accelerate transcription of CAT.
Because X-ray induced c-Jun gene expression is
attenuated when PKC is depleted or inhibited, the PKC
inhibitor H7 was added to RIT-3 cells transfected with
pSG-JunS-235 and GSBCAT. H7 treatment abrogated the
x-ray induced increase in CAT activity suggesting that
irradiation induced PKC activation is required for gene
expression (Hallahan, 1991a; Hallahan, 1991b). These
data suggest that dissociation from the Jun inhibitor may
be one mechanism of regulating radiation-mediated
transcription.
In yet another aspect, a DNA molecule of the
present invention comprises a binding region that is
capable of binding a DNA binding domain of a
transcription factor, which binding region is operatively
linked to a minimal promoter that is operatively linked
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to an encoding region that encodes a polypeptide, which
encoding region ie operatively linked to a transcription-
terminating region.
Preferably, a binding region is capable of
binding the DNA binding domain of the first transcription
factor set forth above. By way of example, where the
binding domain is a Gal4 binding domain, a binding region
of a DNA molecule binds that Gal4 binding domain. A
binding region is operatively linked to a minimal
promoter (e.g., a TATA box) that is operatively linked to
an encoding region that encodes a polypeptide. An
exemplary preferred DNA molecule comprising a binding
region, minimal promoter and encoding region is plasmid
pGSBCAT. Plasmid pGSBCAT comprises a binding region that
binds the DNA binding domain of Gal4 (amino acid residues
1-147) operatively linked to an Elb TATA box that is
operatively linked to CAT gene (See Example w
hereinafter).
In a preferred embodiment, an activation domain
is an activation domain of viral protein VP-16 or nuclear
factor NK-fB.
Viral protein VP-16 is a 65 kD polypeptide of
about 490 amino acid residues that is expreased~during
the immediate early phase of herpes simplex viral
infection and activates transcription and subsequent
expression o~ infected cell proteins (ICP) such as ICP4
(Trienzenberg et al., 1988).
The activation domain of VP-16 comprises an
amino acid residue sequence of about 78 amino acid
residues located at the carboxy-terminus of VP16 (amino
acid residues 413 to 490 as numbered from the amino-
terminus). The activation domain of VP16 is further
likely centered in a 61 amino acid residue sequence
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located from about residue 429 to about residue 456
(Trienzenberg et al., 1988).
Thus, in a preferred embodiment, a second
encoding sequence encodes amino acid residue sequences
from about residue number 413 to about residue number 490
of VP16 and, more preferably from about residue number
429 to about residue number 456 of VP16.
Nuclear factor NF-xB is a transcription factor.
The activation domain of NF-xB comprises amino acid
residue sequences from about residue position 414 to
about residue position 515, numbered from the amino-
terminus. Thus, a second encoding sequence preferably
comprises nucleotide base pairs that encode amino acid
residues from about residue position 414 to about residue
position 515 of NF-xB (Ballard, 1992).
c. Nuclear localization signal
At least one of the encoding sequences contains
a nuclear localization signal. Such a signal permits the
encoded transcription factor to enter the nucleus and
interact with DNA in the nucleus. Preferably, such a
nuclear localization signal is contained in the first or
second encoding sequence. Where a nuclear localization
signal is not present in a first or second encoding
sequence such a signal is contained in a third encoding
sequence.
Nuclear localization signals are well known in
the art. An exemplary and preferred such signal is
derived from Simian Virus 40 (SV40) large T antigen. In
a preferred embodiment, a SV40 nuclear localization
signal comprises an amino acid residue sequence of from
about 7 to about 15 amino acid residues around a lysine
(Lys) residue at position 128 of SV40 large T antigen
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(Kalderon et al. 1984). In a more preferred embodiment a
nuclear localization signal comprises the amino acid
residue sequence of SV40 extending from about residue
position 126 to about residue position 132.
d. Transcription-terminating region
RNA polymerase transcribes an encoding DNA
sequence through a site where polyadenylation occurs.
Typically, DNA sequences located a few hundred base pairs
downstream of the polyadenylation site serve to terminate
transcription. Those DNA sequences are referred to
herein as transcription-termination regions. Those
regions are required for efficient polyadenylation of
transcribed messenger RNA (mRNA).
Transcription-terminating regions are well
known in the art. A preferred transcription-terminating
region used in a DNA molecule of the present invention
comprises nucleotides 1533 to about 2157 of the human
growth hormone (Seeburg, 1982).
3. Preparation of a DNA Molecule
A DNA molecule of the present invention is
prepared in accordance with standard techniques well
known to a skilled worker in the art. First, DNA
fragments containing the various regions of a desired DNA
molecule are prepared or isolated. Those regions are
then ligated to form a DNA molecule of this invention.
Means for synthesizing, isolating and ligating DNA
fragments are-well known in the art.
DNA sequences of up to about 200 base pairs can
be prepared using well known solid phase synthetic
techniques. Thus, by way of example, where a radiation
responsive enhancer-promoter is a CArG domain of an Egr-I
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promoter, one or more of that domain can be synthetically
prepared.
Where a desired DNA sequence is of about 200 or
more nucleotides, that sequence is typically obtained
from tissues, cells or commercially available constructs
(e. g. vectors or plasmida) known to contain that desired
sequence.
By way of example, a cDNA clone of Egr-1 has
been isolated and sequenced from mouse liver (TSa7.-
Morris, 1988). A cDNA of c-Jun has been isolated and
sequenced from rat (Hatori, 1988 and Unlap, 1992).
Sources known to contain encoding DNA regions that encode
specific polypeptides are well known in the art. Table 1
below summarizes sources known to contain encoding DNA
sequences for exemplary polypeptides.
Table 1
Polvueptide Source of Encoding DNA
CETUS
TNF
ricin CETUS
p53 Dr. Vogelstein,
Johns Hopkins
University
MnSOD Genentech
Pseudomonas Dr. Steve Lory, Univ.
exotoxin of Washington
Where a DNA fragment is obtained from a cell or
other organism, total DNA is extracted from that organism
or.cell and fragmented using restriction enzymes. The
choice of what restriction enzyme or enzymes to use is
dependent upon the desired DNA sequence being obtained.
Particular DNA sequences of interest are then isolated,
identified and purified using standard techniques well
known in the art. If needed, an encoding DNA sequence
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can be amplified prior to isolation. A preferred means
of amplifying a DNA sequence of interest is the
polymerase chain reaction.
A wide variety and number of DNA molecules
comprising a radiation responsive enhancer-promoter and
an encoding region have been prepared (See Examples 1-6,
hereinafter). Table 2, below, summarizes the composition
of exemplary such DNA molecules.
Table 22
Plasmid Designation Enhancer-Promoter Encoding Region
pE425-TNF CArG domain of Egr-1TNF
pE425-CAT CArG domain of Egi CAT
1
pE425-p53 CArG domain of Egi p53
1
pE425-raf 301-1 CArG domain of Egr raf 301-1
1
pE425-MnSOD CArG domain of Egr-1MnSOD
pE425-Ga14NP16 CArG domain of Egr-1Ga14NP16
c-Jun-CAT c-Jan promoter CAT
2o AP-1CAT AP-1 CAT
a. pE425-TNF
Plasmid pE425-TNF comprises nucleotide bases
from nucleotide position -425 to nucleotide position +65
(relative to the transcription start site) of the Egr-i
gene operatively linked to an encoding region that
encodes TNF-a. pE425-TNF was constructed from plasmids ,
3D pE-TNF, which-contains TNF cDNA, and plasmid pE425-CAT,
which contains the Egr-1 segment, a transcription- -
terminating region and a polyadenylation segment from
CAT. ,
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pE-TNF was digested with the restriction enzyme
Pst I to yield a 1.1 kilobase (kb} fragment containing
TNF cDNA. pE425-CAT was digested with the restriction
enzyme Hind III to yield a 3.3 kb fragment containing the
CAT gene and a 3.2 kb segment containing the Egr-1
fragment and the polyadenylation signal from CAT. The
1:1 kb fragment from pE-TNF and the 3.2 kb fragment from
pE425-CAT were blunt ended at the 3' overhang with T4 DNA
polymerase and at the 5' overhang with Klenow using
standard procedures well known in the art.
The resulting pE425 and TNF cDNA were blunt-end
ligated using T4 DNA lipase and T4 RNA lipase using
standard procedures well known in the art to form pE425-
TNF. Digestion of pE425-TNF with BamHl and Hind II
yielded 1.4 kb and 4.5 kb segments of the sense construct
and a 0.9 kb segment of the antisense construct
indicating the sense orientation of the plasmid.
Plasmid pE425-CAT was prepared from an about
491 base pair fragment of the Egr-3 promoter, which
fragment is located from nucleotide base -425 to
nucleotide base +65 relative to the transcriptional start
site and plasmid pCATm (Gius et al. 1990).
The 491 base pair fragment of Egr-1 was
obtained from plasmid p2.4, which contained a 2.4 kb
fragment of the 5' flanking sequence of the Egr-1 gene
(Tsai-Morris, 1988). Briefly, Balb/c 3T3 liver DNA was
used to construct a Fix genomic library using a well
known partial fill-in cloning procedure. About 100,000
unamplified clones in E.coli strain JC7623 (rec B, rec C,
sbc B; Winas et al., 1985) were screened with a 32P-
labeled Egr-1 plasmid OC 3.1 (Sukhatme et al., 1988) that
contained a full length 3.1 kb cDNA insert. Membranes
(GeneScreenPlus, New England Nuclear) were hybridized for
about 16 hours at about 65°C in 1 percent SDS, 10 percent
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dextran sulfate and 1 M NaCl. The filters were washed to
a final stringency of 65°C in D.2 x SSC. Autoradiographs
were prepared by exposing the filters for about 18 hours
at -70°C with an intensifying screen. A single clone,
designated mgEgr-1.1 was obtained, which clone hybridized
to the extreme 5' 120 by EcoRI-ApaI fragment from plasmid
OC 3.1.
A 2-.4 kb PvuII-PvuII fragment and a 6.6 kb
XbaI-XbaI fragment derived from mgEgr-1.1 were subcloned
into the SmaI and XbaI sites of pUCl3 and pUCl8 (Promega
Corp. Madison, WI), respectively, to form plasmids p2.4
and p6.6 respectively.
An about I2D6 base pair fragment (nucleotide
base position -957 to nucleotide base position +248
relative to the transcription start site) was obtained
from plasmid-p2.4 to form plasmid pEgr-1 P1.2. A
deletion mutant was constructed from pEgr-1 P1.2 using
oligomers and polymerase chain reaction to form the 491
base pair fragment extending from nucleotide base
position -425 to nucleotide base position +65.
Plasmid pCAT3m was obtained from Dr. Laimonis
A. Laimins, Howard Hughes Medical Institute Research
Laboratories, University of Chicago, Chicago, IL).
Plasmid pE-TNF was prepared in accordance with the
procedure of Hlong(WOng, 1985).
b. pE425-p53
Plasmid pE425-p53 comprises an about 491 base
pair fragment of the Egr-1 promoteroperatively linked to
an encoding region for the tumor suppressing factor p53.
pE425-p53 was constructed from a plasmid (pC53SN3;
Diner, 1990) that contains p53 cDNA, and plasmid pE425-
CAT, which contains the Egr-1 segment and a
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transcription-terminating region, the polyadenylation
segment from CAT. Plasmid pE425-CAT was prepared as
described above.
c. nE425-raf 301-1
Plasmid pE425-raf 301-1 comprises an about 491
base pair fragment of the Egr-1 promoter operatively
linked to an encoding region for a serine/threonine-
specific protein kinase product of an oncogene from a
3611 murine sarcoma cell. pE425-raf 301-1 was
constructed from plasmids pMN301-1, which contains the
raf dominant negative (Kolch, 1991), and pE425-CAT, which
contains the Egr-1 segment and a transcription-
terminating region, the polyadenylation segment from CAT.
d. pE425-Mn50D
Plasmid pE425-MnSOD comprises an about 491 base
pair fragment of the Egr-I promoter operatively linked to
an encoding region for the free-radical scavenger
manganese superox:i.de dismutase (MnSOD). pE425-MnSOD was
constructed from a plasmid nMnSOD #0664 (Genentech)
(Wong, 1989) which contains MnSOD cDNA and pE425-CAT,
which contains the Egr-I segment and a transcription-
terminating region, the polyadenylation segment from CAT.
a . G5TNFTNF
Plasmid G5-TNF comprises the DNA binding domain
of the yeast GAL4 gene and the Eib minimal promoter TATA
box operatively linked to an encoding region that encodes
TNF-a. pG5-TNF was constructed from plasmid GSBCAT and
plasmid pE-TNF.
Plasmid GSBCAT, which contains the DNA sequence
which binds Gal4 protein placed 5' of the E1b TATA box
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upstream of the CAT reporter gene (Baichwal, 1990). The
GSBCAT plasmid was digested with the EcoRl restriction
enzyme. The large fragment was isolated and blunt ended
at the 3' overhang using T4 DNA polymerase and at the 5'
overhang with_Klenow. This digestion removes the minimal
promoter but retains the poly-A end.
TNF cDNA was removed from the pE4 plasmid using
the Pst I restriction enzyme and the 1.1 kb fragment
containing TNF cDNA was isolated and blunt ended at the
3' overhand using T4 DNA polymerase and at the 5'
overhang withKlenow.
The resulting G5B- and TNF cDNA were blunt-end
ligated usingthe T4DNA ligase and T4 RNA ligase. The
resulting G5-TNF plasmid underwent restriction enzyme
mapping and DNA sequencing to assure the sense
orientation of TNF. Plasmid GSBCAT was prepared by the
method of Baichwal (Baichwal, et al., 1990). Plasmid pE-
TNF was prepared as set forth above.
f. c-Jun CAT
Plasmid c-Jun-CAT comprises an about 11D0 base
pair fragment of the c-Jun promoter operatively linked to
an encoding region for CAT. Plasmid c-Jun-CAT was
constructed from plasmid h-jun-CAT in accordance with the
procedure of Angel (Angel, 1988).
g. pE25-Pseudomonas exotoxin-
A plasmid comprising-a CArG domain of- an Egr-1
promoter and an encoding region that encodes Pseudomonas
exotoxin was prepared from plasmid PE425 and plasmid
.35 pMS150A (Lory, 1988), which contains the Pseudomonas
exotoxin encoding region.
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h. Other constructs
Other DNA molecules of the present invention
are made using techniques similar to those set forth
above. Specific examples of the preparation of other DNA
molecules can be found in Examples 1-6 hereinafter.
B. Pharmaceutical Composition
In another aspect, the present invention
contemplates a pharmaceutical composition comprising a
therapeutically effective amount of at least one DNA
molecule of the present invention and a physiologically
acceptable carrier.
A therapeutically effective amount of a DNA
molecule that is combined with a carrier to produce a
single dosage form varies depending upon the host treated
and the particular mode of administration.
As is well known in the art, a specific dose
level for any particular patient depends upon a variety
of factors including the activity of the specific
compound employed, the age, body weight, general health,
sex, diet, time of administration, route of
administration, rate of excretion, drug combination, and
the severity of the particular disease undergoing
therapy.
A composition of the present invention is
typically administered orally or parenterally in dosage
unit formulations containing standard, well known
nontoxic physiologically acceptable carriers, adjuvants,
and vehicles as desired. The term parenteral as used
herein includes subcutaneous injections, intravenous,
intramuscular, intraarterial injection, or infusion
techniques.
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Injectable preparations, for example, sterile
injectable aqueous or oleaginous suspensions are
formulated according to the known art using suitable
dispersing or wetting agents and suspending agents. The
sterile injectable preparation can also be a sterile
injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example,
as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile,
fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil
can be employed including synthetic mono- or di-
glyceridea. In addition, fatty acids such as oleic acid
find iiae in the preparation of injectables.
A DNA molecule of the present invention can
also be complexed with a poly(L-Lysine)(PLL)-protein
conjugate such~as a transferrin-PLL conjugate or an
asialoorosomucoid-PLL conjugate.
Liquid dosage forms for oral administration
include pharmaceutically acceptable emulsions, syrups,
solutions, suspensions, and elixirs containing inert
diluenta commonly used in the art, such ae water. Such
compositions can also comprise adjuvants, such as wetting
agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
C. Transformed or Tranafected and Transcrenic
Cells _
In another aspect, the present invention
provides a cell transformed or transfected with one or
more DNA molecules of the present invention as well as
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transgenic cells derived from those transformed or
transfected cells. Means of transforming or transfecting
cells with exogenous DNA molecules are well known in the
art.
A DNA molecule is introduced into a cell using
standard transformation or transfection techniques well
known in the art such as calcium-phosphate- or DEAE-
dextran-mediated transfection, protoblast fusion,
electroporation, liposomea and direct microinjection
(Sambraok, Fritsch and Maniatis, 1989).
The most widely used method is transfection
mediated by either calcium phosphate or DEAE-dextran.
Although the mechanism remains obscure, it is believed
that the transfected DNA enters the cytoplasm of the cell
by endocytoais and is transferred to the nucleus.
Depending on the cell type, up to 20% of a population of
cultured cells can be transfected at any one time.
Because of its high efficiency, transfection mediated by
calcium phosphate or DEAE-dextran is the method of choice
for experiments that require transient expression of the
foreign DNA in large numbers of cells. Calcium
phosphate-mediated transfection is also used to establish
cell lines that carry integrated copies of the foreign
DNA, which are usually arranged in head-to-tail tandem
arrays.
In the protoplast fusion method, protoplasts
3D derived from bacteria carrying high numbers of copies of
a plasmid of interest are mixed directly with cultured
mammalian cells. After fusion of the cell membranes
(usually with polyethylene glycol), the contents of the
bacteria are delivered into the cytoplasm of the
mammalian cells and the plasmid DNA is transferred to the
nucleus. Protoplast fusion is not as efficient as
transfection for many of the cell lines that are commonly
WO 95131559 PCT/US95/05959
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used for transient expression assays, but it is useful
for cell lines in which endocytosis of DNA occurs
inefficiently. Protoplast fusion frequently yields
multiple copies of the plasmid DNA tandomly integrated
into the host chromosome.
The application of brief, high-voltage electric
pulses to a variety of mammalian and plant cells leads to
the formation of nanometer-sized pores in the plasma
membrane. DNA is taken directly into the cell cytoplasm
either through these pores or as a consequence of the
redistribution of membrane components that accompanies
closure of the pores. Electroporation can be extremely
efficient and can be used both for transient expression
of clones genes and for establishment of cell lines that
carry integrated copies of the gene of interest.
Electroporation, in contrast to calcium phosphate-
mediated transfection and protoplast fusion, frequently
gives rise to cell lines that carry one, or at most a
few, integrated copies of the foreign DNA.
Ligosome transformation involves encapsulation
of DNA and RNA within liposomes, followed by fusion of
the liposomes with the cell membrane. In addition, DNA
that is coated with a synthetic cationic lipid can be
introduced into cells by fusion.
Direct microinjection of a DNA molecule into
nuclei has the advantage of not exposing DNA to cellular
compartments such as low-pFI endosomes. Microinjection is
therefore used primarily as a method to establish lines
of cells that carry integrated copies of the DNA of
interest.
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D. Process of Revulatina Expression
In another aspect, the present invention
contemplates a process of regulating the expression of a
polypeptide. Polypeptide expression is regulated by
stimulating or inhibiting transcription of an encoding
region that encodes that polypeptide. In accordance with
one embodiment, a process of regulating polypeptide
expression comprises the steps of:
(a) operatively linking a radiation responsive
enhancer-promoter to an encoding region that encodes that
polypeptide, which encoding region is operatively linked
to a transcription-terminating region to form a DNA
molecule; and
(b) exposing the DNA molecule to an effective
expression-inducing dose of ionizing radiation.
A DNA molecule used with such a method is a DNA
molecule of the present invention as set forth above.
As used herein, the phrase "effective
expression-inducing dose of ionizing radiation" means
that dose of ionizing radiation needed to stimulate or
turn on a radiation responsive enhancer-promoter of the
present invention. The amount of ionizing radiation
needed in a given cell depends inter alia upon the nature
of that cell. Typically, an effective expression-
inducing dose is less than a dose of ionizing radiation
that causes cell damage or death directly. Means for
determining an effective expression inducing amount are
well known in the art.
In a preferred embodiment an effective
expression inducing amount is from about 2 to about 2D
Gray (Gy) administered at a rate of from about 0.5 to
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about 2 Gy/minute. Even more preferably, an effective
expression inducing amount of ionizing radiation is from
about 5 to about 15 Gy.
As used herein, ~~ionizing radiation~~ means
radiation comprising particles or photons that have
sufficient energy or can produce sufficient energy via
nuclear interactions to produce ionization (gain or loss
of electrons). An exemplary and preferred ionizing
radiation is an x-radiation. Means for delivering x-
radiation to a target tissue or cell are well known in
the art.
Cells containing a DNA molecule of the present
invention encoding a particular polypeptide express that
polypeptide when exposed to ionizing radiation.
By way of example, treatment of cells
transfected with plasmid pEgr-IP1.2 with ionizing
radiation was associated with a 4.1-fold increase in CAT
activity ae compared to tranafected but unirradiated
cells. Plasmid pEgr-1 P1.2 comprises a radiation
responsive enhancer-promoter (the Egr-1 promoter region
extending from position -957 upstream to the
transcription start site to position +248) operatively
linked to the CAT reporter gene. Indeed, irradiation of
pE425-CAT transfected cells was associated with a 3.6-
fold induction of CAT activity compared to that in non-
irradiated cells tranafected with this construct.
A aeries of deleted Egr-I promoter constructs
was next used to further define the x-ray responsive
elements in pE425-CAT_ Sequential deletion of the three
distal CArGs progressively decreased CAT activity.
Plasmid pE395-CAT (first CArG deleted) conferred x-ray
inducibility to a lesser extent than pE425-CAT. Deletion
of the first and second CArG domains (pE359-CAT) resulted
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in further decreases in CAT activity, while deletion of
the first three CArG domains (pE342-CAT)) was associated
with minimal increases in CAT activity.
Other studies were performed with fragments of
the Egr-1 promoter linked to HSV-TK and the CAT gene.
There was no detectable inducibility of pTK35CAT (not
containing any CArG domains of an Egr-1 promoter) by
x-rays. In contrast, cells transfected with plasmid
pE425/250TK (containing the four distal Egr-1 CArG
domains) were responsive to x-ray treatment. The region
of the Egr-1 promoter extending from nucleotide positions
-395 to -250, which region does not include the first
CArG domain, was also functional in conferring x-ray
inducibility to the heterologous promoter, but to a
lesser extent than pE425/250TK. X-ray inducibility of
CAT expression was also observed in cells transfected
with a plasmid comprising only one CArG domain.
By way of further example, TNF-a protein
expression was induced by ionizing radiation in cells
tranafected with plasmid pE425-TNF. SQ-20B, RIT-3 and -
HL-525 cells were transfected with plasmid pE425-TNF by
DEAF precipitation. Tranafected cells were exposed to 10
Gy of x-radiation at a rate of-1 Gy/minute. TNF-a
expression was increased about 2-fold, 5-fold and 4-fold,
respectively in SQ-20B, RIT-3 and HL-525 cells when
compared to transfected, non-irradiated cells.
By way of still further example, CAT expression
was induced by ionizing radiation in RIT-3 cells
transfected with plasmid c-Jun-CAT, which plasmid
comprises a 1100 base pair segment of the c-Jun promoter
operatively linked to a CAT gene. Cells were co-
transfected with an SV40 promoter-(3 galactosidase
expression vector to control for tranafection efficiency.
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Transfectants were irradiated (10 Gy, 1 Gy/min,
GE Maxitron) 40 hours after transfection. CAT was
extracted 6 hours after irradiation. CAT activity
increased about 3-fold following irradiation of RIT-3
cells tranefected with pc-Jun-CAT. (3 gal expression was
not affected by radiation. Ionizing radiation did not
increase CAT,expresaion in cells transfected with a
plasmid comprising the minimal Jun promoter (nucleotide
base position-18 to nucleotide base position +170
relative to the transcription start site) operatively
linked to CAT.
The data set forth above show that ionizing
radiation can be used as a trigger to regulate
transcription of an encoding region in a DNA molecule of
the present invention and expression of a polypeptide
encoded by that region.
In an alternate embodiment, polypeptide
expression is regulated by the use of two DNA molecules.
One of those DNA molecules comprises a radiation
responsive enhancer-promoter operatively linked to an
encoding region that comprises:
(a) a first encoding sequence that encodes a
DNA binding domain of a first transcription factor;
(b) a second encoding sequence that encodes an
activation or repression domain of a second transcription
factor;
(c) a third encoding sequence that encodes a
nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
frame to each other in any order with the proviso that
the third encoding sequence need be present only if the
WO 95131559 PCTIUS95105959
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first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region that is
operatively linked to any of the first, second or third
encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences.
A second DNA molecule comprises a binding
region that is capable of binding the DNA binding domain
of the first transcription factor, which binding region
is operatively linked to a minimal promoter that is
operatively linked to an encoding region that encodes a
polypeptide, which encoding region is operatively linked
to a transcription-terminating region.
A radiation responsive enhancer-promoter, a
transcription factor, a binding domain of a transcription
factor and an activation or repressor domain of a
transcription factor are preferably those set forth -
above. A polypeptide encoded by an encoding region is
also preferably the same as set forth above.
Cells transfected with such DNA molecules show
ionizing radiation-inducible polypeptide expression.
By way of example, two plasmids were
transfected into HeLa and RIT-3 cells using a calcium
precipitation method. The first plasmid (pSG424)
contained the SV40 promoter (not transcriptionally
responsive to radiation) upstream of the coding sequence,
for b, A1, and A2 regions of the activation domain of the
Jun protein, wherein the DNA binding domain of Jun was
replaced with the DNA binding domain of GAL4. A second
plasmid, GSBCAT contained the DNA sequence which binds
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Gal4 protein linked to a minimal TK promoter upstream of
the CAT reporter gene.
Transfected cells were irradiated with 10 Gy of
x-rays. CAT activity increased in the irradiated,
tranafected HeLa and RIT-3 cells as compared to
transfected, non-irradiated cells.
By way of further example, irradiation induced
an increase in TNF-a expression in RIT-3- cells
transfected with plasmids pE425-Gal4/VP-16 and pG5-TNF.
Plasmid pE425-Gal4/VP-16 comprises an about 491 base pair
fragment of the Egr-1 promoter containing 6 CArG domains,
which fragmentis operatively linked to an encoding
I5 region comprising a first encoding sequence encoding DNA
binding domain-of Gal4 operatively linked in frame to a
second encoding sequence encoding the activation domain
of viral protein VP-16. Plasmid GS-TNF comprises a DNA
segment that binds the Gal4 binding domain operatively
linked to minimal promoter operatively linked to an
encoding region that encodes TNF-a. RIT-3 cells were co-
tranafected with the pE425-Gal/VP16 and G5-TNF plasmids
using lipofectin. Transfected cells were irradiated
36 hours following transfection and TNF was assayed
10 hours following irradiation. The concentration of
intracellular-TNF increased about 9-fold as compared to
cells tranafected with G5-TNF alone.
Where regulating is inhibiting, an encoding
region preferably comprises:
(a) a first encoding sequence that encodes a
DNA binding domain of a first transcription factor;
(b) a second encoding sequence that encodes an
activation or repression domain of-a second transcription
factor;
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(c) a third encoding sequence that encodes a
nuclear localization signal, whereby the first, second
and third encoding sequences are operatively linked in
frame to each other in any order with the proviso that
the third encoding sequence need be present only if the
first or second encoding sequence does not encode a
nuclear localization signal; and
(d) a transcription-terminating region that is
operatively linked to any of the first, second or third
encoding sequences such that the transcription-
terminating region is located 3' to all of the first,
second and third encoding sequences.
Preferably the second encoding sequence encodes
the repression domain of the Wilms' tumor suppressor gene
WT1 or the repression domain of Egr-1. A radiation
responsive enhancer-promoter and a first transcription
factor are the same as set forth above.
E. Process of Inhibitina Tumor Growth
In yet another aspect, the present invention
contemplates a process of inhibiting growth of a tumor
comprising the steps of:
(a) delivering to the tumor a therapeutically
effective amount of a DNA molecule comprising a radiation
responsive enhancer-promoter operatively linked to an
encoding region that encodes a polypeptide having the
ability to inhibit tumor cell growth, which encoding
region is operatively linked to a tranacription-
terminating region; and
(b) exposing the tumor to an effective
expression-inducing dose of ionizing radiation.
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Preferably, a radiation responsive enhancer-
promoter comprises a CArG domain of an Egr-I promoter, a
TNF-a promoter or a c-Jun promoter and a polypeptide
having the ability to inhibit tumor cell growth is a
cytokine, a dominant negative, a tumor suppressing factor
or an angiogeneaie inhibitor. Exemplary and preferred
polypeptides are TNF-a, interleukin-4, ricin, Pseudomonas
toxin, p53, the retinoblastoma gene product or the Wilma'
tumor gene product.
TNF-is cytotoxic to tumor cells. An
interaction between TNF and radiation was found in 12
human epithelial tumor cell lines analyzed for
cytotoxicity to TNF and synergistic killing by combining
the two agents (Hallahan, et al., 1990; Hallahan, et al.,
1989). TNF was found to have cytotoxic effects at
concentrations of 10 to 1000 units/ml in ten of twelve
tumor cell lines studied (Hallahan, et al., 1990;
Hallahan, et al., 1989). Furthermore, synergistic or
additive killing by TNF and x-rays was observed in seven
of those ten cell lines.
When cells from a murine renal cell tumor were
engineered to secrete large doses of interleukin-4 (IL-4)
locally, they were rejected in a predominantly T cell-
independent manner (Golumbek, et al., 1985). However,
animals that rejected the IL-4 transfected tumors
developed T cell-dependent systemic'immunity to the
parental tumor. This systemic immunity was tumor-
specific and primarily mediated by CD8+ T cells.
Established parental tumors could be cured by the
systemic immune response generated by injection of the
genetically engineered tumors. These results provide a
rationale for the use of lymphokine gene-transfected
tumor cells that are activated by irradiation as a
modality for cancer therapy.
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Ricin is a cytotoxin that inactivates mammalian
ribosomes by catalyzing the cleavage of the N-glycosidic
bond of 28S rRNA (Endo & Tsurngi, 1987). This enzyme is
extremely toxic when given systemically, but may be
localized to tumor through the use of radiation targeting
of the gene encoding ricin.
The transforming growth factor type alpha gene
has been fused to modified Paeudomonas toxin gene from
which the cell-recognition domain has been deleted
(Chaudhary, et al., 1987). The chimeric gene has been
expressed in Escherichia coli, and the chimeric protein,
PE40-TGF-alpha, has been highly purified. PE40-TGF-alpha
kills cells expressing epidermal growth factor receptors
and has little activity against cells with few receptors.
This chimeric protein might be useful in treating cancers
that contain high numbers of epidermal growth factor
receptors. The gene encoding pseudomonas toxin or its
chimeric may be targeted by radiation to eliminate the
potential systemic sequelae of this toxin.
An extremely wide variety of genetic material
can be transferred to cancer cells or tissues using the
compositions and methods of the invention. For example,
the nucleic acid segment may be DNA (double or single-
etranded) or RNA (e.g., mRNA, tRNA, rRNA); it may also be
a "coding segment", i.e., one that encodes a protein or
polypeptide, or it may be an antisense nucleic acid
molecule, such as antisense RNA that may function to
disrupt gene expression. The nucleic acid segments may
thus be genomic sequences, including exons or introns
alone or exons and introns, or coding cDNA regions, or in
fact any construct that one desires to transfer to a
cancer cell or tissue. Suitable nucleic acid segments
35may also be in virtually any form, such as naked DNA or
RNA, including linear nucleic acid molecules and
plasmids, or as a functional insert within the genomes of
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various recombinant viruses, including viruses with DNA
genomes and retroviruses.
Delivering is preferably injecting the DNA
molecule into the tumor. tnThere the tumor is in a subject
delivering is preferably administering the DNA molecule
into the circulatory system of the subject. In a more
preferred embodiment, administering comprises the steps
(a) providing a vehicle that contains the DNA
molecule; and
(b) administering the vehicle to the subject.
A vehicle is preferably a cell transformed or
transfected with the DNA molecule or a transfected cell
derived from such a transformed or transfected cell. An
exemplary and-preferred transformed or transfected cell
is a leukocyte such as a tumor infiltrating lymphocyte or
a T cell or a tumor cell from the tumor being treated.
Means for transforming or transfecting a cell with a DNA
molecule of the present invention are set forth above.
The transfer of nucleic acids to mammalian
cells has been proposed a method for treating certain
diseases or disorders. Nucleic acid transfer or delivery
is often referred to as "gene therapy". Initial efforts
toward postnatal (somatic) gene therapy relied on
indirect means of introducing genes into tissues, e.g.,
target cells were removed from the body, infected with
viral vectors carrying recombinant genes, and implanted
into the body. These type of techniques are generally
referred to as ex vivo treatment protocols. Direct in
vivo gene transfer has recently been achieved with
formulations of DNA trapped in liposomea (Ledley et al.,
1987); or in proteoliposomes that contain viral envelope
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receptor proteins (Nicolau et al., 1983); calcium
phosphate-coprecipitated DNA (Benvenisty & Reshef, 1986);
and DNA coupled to a polylysine-glycoprotein carrier
complex (Wu & Wu, 1988). The use of recombinant
replication-defective viral vectors to infect target
cells is vivo has also been described (e.g., Seeger et
al., 1984).
In recent years, Wolff et a1. demonstrated that
direct injection of purified preparations of DNA and RNA
into mouse skeletal muscle resulted in significant
reporter gene expression (Wolfe et al., 1990). This was
an unexpected finding, and the mechanism of gene transfer
could not be defined. The authors speculated that muscle
cells may be particularly suited to take up and express
polynucleotides in vivo or that damage associated with
DNA injection may allow tranafection to occur.
Wolff et a1. suggested several potential
applications of the direct injection method, including
(a) the treatment of heritable disorders of muscle, (b)
the modification of non-muscle disorders through muscle
tissue expression of therapeutic transgenes, (c) vaccine
development, and (d) a reversible type of gene transfer,
in which DNA is administered much like a conventional
pharmaceutical treatment. In an elegant study Liu and
coworkers recently showed that the direct injection
method can be successfully applied to the problem of
influenza vaccine development (Ulmer et al., 1993).
Human lymphocytes can also be transfected with
radiation-inducible plasmid constructs using existing
technology including retroviral mediated gene transfer
(Overell, et al., 1991; Fauser, 1991). In an exemplary
embodiment, LAK cells which tend to home in on the tumor
site in question with some degree of preference though as
is well known, they will also distribute themselves in
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the body in other locations, may be used to target
tumors. Indeed, one of the most important advantages of
the radiation inducible system is that only those LAK
cells, which are in the radiation field will be activated
and will have their exogenously introduced lymphokine
genes activated. Thus, for the case of LAK cells, there
is no particular need for any further targeting.
Alternatively, the vehicle is a virus or an
l0 antibody that specifically infects or immunoreacts with
an antigen of the tumor. Retroviruses used to deliver
the constructs to the host target tissues generally are
viruses in which the 3' LTR (linear transfer region) has
been inactivated. That is, these are,enhancer-less 3'
LTR's, often referred to as SIN (self-inactivating
viruses) because after productive infection into the host
cell, the 3' LTR is transferred to the 5' end and both
viral LTR's are inactive with respect to transcriptional
activity. A use of these viruses well known to those
skilled in the art is to clone genes for which the
regulatory elements of the cloned gene are inserted in
the space between the two LTR's. An advantage of a viral
infection system is that it allows for a very high level
of infection into the appropriate recipient cell, e.g.,
LAK cells.
For purposes of this invention, a radiation
responsive enhancer-promoter which is 5' of the
appropriate encoding region may be cloned into the virus
0 using standard techniques well known in the art.
A variety of viral vectors, such a retroviral
vectors, herpes simplex virus (U. S. Patent 5,288,641),
cytomegalovirus, and the like may be employed, as
described by Miller (1992); as my recombinant adeno-
as ociated viru (AAV vectors), such as those
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de cribed by U.S. Patent 5,139,941; and, particularly,
recombinant adenoviral vectors. Techniques for preparing
replication-defective infective viruses are well known in
the art, as exemplified by Ghosh-Choudhury & Graham
(1987); McGrory et a1. (1988); and Gluzman et a1. (1982).
The viral constructs are delivered into a host
by any method that causes the constructs to reach the
cells of the target tissue, while preserving the
characteristics of the construct used in this invention.
By way of example, a rat glioma cell line, C6-BU-1,
showed differential susceptibility to herpes simplex
virus type 1 (HSV-1) and type 2 (HSV-2), namely, all the
HSV-1 strains tested so far persisted in this cell-line
but the HSV-2 strains did not (Sakihama, et al., 1991).
C5-BU-l cells consist of subpopulations heterogeneous in
susceptibility to HSV-1 which may be possibly
interchangeable. Furthermore, growth of tumors produced
from C6-derived cells bearing the HSV-1 tk gene, but no
parental C6 cells, could be inhibited by intraperitoneal
administration of ganciclovir (Ezzeddine; et al., 1991 ).
This work demonstrated the effectiveness of the thymidine
kinase expressed by the HSV-1 tk gene in sensitizing
brain tumor cells to the toxic effects of nucleoside
analogs. Retrovirus vectors should thus prove useful in
the selective delivery of this killer gene to dividing
tumor cells in the nervous system, where most endogenous
cells are not dividing. Radiation will be used to
enhance the specificity of delivery or activation of
transcription of the tk gene only in irradiated areas.
Antibodies have been used to target and deliver
DNA molecules. An N-terminal modified poly(L-lysine)
(NPLL)-antibody conjugate readily forms a complex with
plasmid DNA (Trubetskoy et al., 1992). A complex of
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monoclonal antibodies against a cell surface
thrombomodulin conjugated with NPLL was used to target a
foreign plasmid DNA to an antigen-expressing mouse lung
endothelial cell line and mouse lung. Those targeted
endothelial cells expressed the product encoded by that
foreign DNA.
In a preferred embodiment exposing comprises
the steps of:
a) providing a radiolabeled antibody that
immunoreacts with an antigen of the tumor; and
b) delivering an effective expression
inducing of the radiolabeled antibody to the tumor.
The efficacy of using antibodies to target
radiotherapy has been demonstrated including the modeling
of dose to tumor and normal tissue from intraperitoneal
radioimmunotherapy with alpha and beta emitters4. This
technology has been applied to in vivo experiments.
Astatine-211 labeling of an antimelanoma antibody and its
Fab fragment using N-auccinimidyl p-astatobenzoate:
comparison in vivo with the p-[125]iodobenzoyl
conjugates.
Alternatively, a process of inhibiting growth
of a tumor comprises the steps of:
a) delivering to the tumor a therapeutically
effective amount of
(1) a first DNA molecule compr'lsing a
radiation responsive enhancer-promoter operatively linked
to an encoding region that comprises
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(i) a first encoding sequence that
encodes a DNA binding domain of a first transcription
factor;
(ii) a second encoding sequence that
encodes an activation or repression domain of a second
transcription factor;
(iii) a third encoding sequence that
encodes a nuclear localization signal, whereby the first,
second and third encoding sequences are operatively
linked in frame to each other in any order with the
proviso that the third encoding sequence need be present
only if the first or second encoding sequence does not
encode a nuclear localization signal; and
(iv) a transcription-terminating
region that is operatively linked to any of the first,
second or third encoding sequences such that the
transcription-terminating region is located 3' to all of
the first, second and third encoding sequences; and
(2) a second DNA molecule comprising a
binding region that is capable of binding the DNA binding
domain of the first transcription factor, which binding
region is operatively linked to a minimal promoter that
is operatively linked to an encoding region that encodes
a polypeptide having tumor cell cytotoxic activity, which
encoding region is operatively linked to a tranacription-
terminating region; and
b) exposing the cell to an effective
expression-inducing dose of ionizing radiation.
Preferably, a radiation responsive enhancer-
promoter comprises a CArG domain of an Egr-1 promoter or
an AP-1 binding domain of a c-Jun promoter and the
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polypeptide having tumor cell cytotoxic activity is a
cytokine, a dominant negative, a tumor suppressing
factor, or an angiogenesis inhibitor as set forth above.
Delivering is preferably the same as set forth
above.
TNF-a is increased after treatment with x-rays
in certain human sarcoma cells. The increase in TNF-a
mRNA is accompanied by the increased production of TNF-a
protein. The induction of a cytotoxic protein by
exposure of cells containing the TNF gene to x-rays was
suspected when medium decanted from irradiated cultures
of some human sarcoma cell lines was found to be
cytotoxic to those cells as well as to other tumor cell
lines. The level of TNF-a in the irradiated tumor
cultures was elevated over that of nonirradiated cells
when analyzed by the ELISA technique (Sariban, et al.,
1988). Subsequent investigations showed that elevated
TNF-a protein after irradiation patentiates x-ray killing
of cells by an unusual previously undescribed mechanism
(see Example 1).
RNA from untreated cells (control) and
irradiated cells was size-fractionated and hybridized to
32P-labeled TNF-a cDNA (STSAR-13) and PE4 plasmid
containing TNF-a cDNA (STSAR-48). Autoradiograma showed
increased expression of TNF-a mRNA 3 hours after
irradiation in cell line STSAR-13 and at 6 hours in cell
line STSAR-48. These data show that TNF-a gene
expression is increased after radiation.
As can be seen from these results and from
information discussed in Example 1, the tumor necrosis
factor a is increased after treatment with x-rays. Both
mRNA and TNF-a proteins were increased.
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Although DNA-damaging agents other than
ionizing radiation have been observed to induce
expression of variety of prokaryotic and mammalian genes,
the TNF-a gene is the first mammalian gene found to have
increased expression after exposure to ionizing
radiation. This gene is not categorized as a DNA repair
gene.
A DNA molecule of the present invention has
uses other than inhibition of tumor growth. Exemplary
such uses are summarized below in Table 3.
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Use Encoded PolypeptideApplication to Disease
Kill tumor cells Toxins Solid &
TNF Hematologic
Growth Factors Malignancies
fIL~1-6, PDGF,FGF)
Protect normal tissuesLymphokines GCSF Solid & Hematologic
s from radiation and CMCSF Malignancies
other
cytotoxins during Erythropoietin
cancer
therapy Aplastic Anemic
Inhibit Metastasis NM23 Cancer
Metastasis
Tumor Suppressor Rb p53 Prevention of
to Gene Products Malignancy
Following Standard
Radio therapy and
Chemotherapy
Radiosensitization TNF Solid &
Chemosensitization Hematologic
(enhance routine Malignancies
treatment effects)
1s Correct Defects Factor 8 Clotting
in
Clotting Factors Disorders
Introduce Streptokinase Myocardial
Anticlotting Urokinase Infarction
Factors CNS Thrombosis,
Pheripheral
Thrombosis
2o Correct defects Normal Hemoglobin Sickle Cell
Characterizing Anemia
Hemoglobinopathy
Correct DeficienciesNerve Growth FactorAlzheimer's
Leading to Disease
2s Disease Neurodegenerative
Provide Treatment Insulin Diabetes
Component for
Diabetes
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Use Encoded PolypeptideApplication to Disease
Disease of DNA ERCC~1, XRCC~1 Ataxia
Repair Telangiectasia
Abnormalities Xeroderma
Pigmentosum
s
The following examples are included to
demonstrate preferred embodiments of the invention. It
should be appreciated by those of skill in the art that
the techniques disclosed in the examples which follow
represent techniques discovered by the inventor to
function well in the practice of the invention, and thus
can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are
disclosed and still obtain a like or similar result
without departing from the spirit and scope of the
invention.
EXAMPLE 1: Increased Tumor Necrosis Factor a mRNA
After Cellular Exposure to Ionizing
Radiation
A. Protein Products
To investigate TNF-a protein production after
x-irradiation, the levels of TNF-a in the medium of human
tumor cell lines and fibroblasts were quantified by the
ELISA technique (Sariban, et al., 1988) before and after
exposure to 500-cGy x-rays. Five of 13 human bone and
soft tissue sarcoma cell lines (STSAR-5, -13, -33, -43,
and -48) released TNF-a into the medium after
irradiation, whereas TNF-a levels were not elevated in
supernatant from normal human fibroblast cell lines (GM-
1522 and NHF-235) and four human epithelial tumor cell
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lines (HN-SCC-68, SCC-61, SCC-25, and SQ-20B) after
exposure to radiation. The assay accurately measures
TNF-a levels between 0.1 and 2.0 units per ml (2.3 x 106
units/mg) (Saribon, et al., 1988). Tumor cell line
STSAR-13 produced undetectable amounts of TNF-a before x-
irradiation and D.35 units/ml after x-ray exposure. Cell
lines STSAR-5 and -33 responded to x-irradiation with
increases in TNF-a concentrations of >5- to 10-fold;
however quantities above 2 unita/ml exceeded the range of
the assay (Saribon, et al., 1988). Cell lines STSAR-43
and -48 demonstrated increases in TNF-a of 1.5- to 3-fold
(Table 4, below). TNF-a protein in the medium was first
elevated at 20 hr after x-ray treatment, reached maximal
levels at 3 days, and remained elevated beyond 5 days.
Furthermore, supernatant from irradiated, but not control
STSAR-33, was cytotoxic to TNF-a-sensitive cell line SQ-
20B.
2D
Table 4
TI~TF-a level funits/ml)
Cell Line Oriain Control X-rav
STSAR-5 MFH 0.4 >2.0
STSAR-13 Liposarcoma 0.0 0.34
STSAR-33 Ewing sarcoma 0.17 >2.0
STSAR-43 Osteosarcoma 0.41 1.3
STSAR-48 Neurofibrosarcoma 0.28 0.43
TNF-a levels were measured in medium from confluent
cell cultures (control) and in irradiated confluent cells
(x-ray). TNF-a levels increased as measured by the ELISA
technique. MFH, malignant fibrous histiocytoma.
B. -g).7A Analysis
Increased levels of TNF-a mRNA were detected in
the TNF-a-producing sarcoma cell lines after irradiation
relative to unirradiated controls. For example, TNF-~
transcripts were present in unirradiated STSAR-13 and -48
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cell lines. TNF-a mRNA levels in cell line STSAR-13
increased by >2.5-fold as measured by densitometry 3 hr
after exposure to 500 cGy and then declined to baseline
levels by 6 hours. These transcripts increased at 6
hours after irradiation in cell line STSAR-48, thus
indicating some heterogeneity between cell lines in terms
of the kinetics of TNF-a gene expression. In contrast,
irradiation had no detectable effect on 7S RNA levels or
expression of the polymerase ,Q gene.
-
C. Interaction Between TNF-a and X-
Irradiation
To investigate the influence of TNF-a on
radiation-induced cytotoxicity in TNF-a-producing cell
lines, recombinant human TNF-cr was added to cultures
before irradiation. Recombinant human TNF-a (1000
units/ml) (2.3 x 106 units/mg) was cytotoxic to four of
five TNF-a-producing sarcomas (STSAR-5, -13, -33, and -
43). The plating efficiency (PE) was reduced by 60-90%
at 1000 units/ml in these lines. Radiation-survival
analysis of cell line STSAR-33 was performed with TNF-a
(10 units/ml). The radiosensitivity (DO), defined as the
reciprocal of the terminal slope of the survival curves
was 80.4 cGy for cell line STSAR-33. When TNF-a was
added 20 hr before irradiation, the DO was 60.4 cGy.
Surviving fractions were corrected for the reduced PE
with TNF-a. Thus, the interaction between TNF-a and
radiation in STSAR-33 cells was synergistic (Dewey,
1989).
Sublethal concentrations of TNF-a (10 units/ml)
enhanced killing by radiation in cell line STSAR-33,
suggesting a radiosensitizing effect of TNF-a The
surviving fraction of cell line STSAR-5 at 100-700 cGy
was lower than expected by the independent killing of -
TNF-a and x-rays, although the DO values were similar.
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Thus, the interaction between TNF-a and radiation is
additive (Dewey, 1979) in STSAR-5 cells. Cell lines
STSAR-13 and STSAR-43 were independently killed with x-
rays and TNF-a, and no interaction was observed.
To determine the possible interactions between
TNF-a and x-rays in non-TNF-a producing cells, human
epithelial tumor cells (SQ-20B and HNSCC-68) were
irradiated 20 hr after TNF-a was added. These cell lines
do not produce TNF-a in response to ionizing radiation.
TNF-a (1000 units/ml) was cytotoxic to SQ-20B and SCC-61
cells, reducing the PE by 60-80%. The DO for cell Line
SQ-20B is 239 cGy. With TNF-a (1000 units/ml) added 24
hr before x-rays, the DO was 130.4 cGy. Therefore, a
synergistic interaction (Dewey, 1979) between TNF-a and
x-rays was demonstrated in this cell line. TNF-a added
after irradiation did not enhance cell killing by
radiation in cell lines SQ-20B. Nonlethal concentrations
of TNF-a (10 units/ml) resulted in enhanced radiation
killing in cell line HNSCC-68, providing evidence that
TNF-a may sens~.tize some epithelial as well as
mesenchymal tumor cell lines to radiation..
The following specific methods were used in
Example 1.
Cell Lines. Methods of establishment of human
sarcoma and epithelial cell lines have been described
(Weichselbaum, et al., 1986; 1988). Culture medium for
epithelial tumor cells was 72.5% Dulbecco's modified
Eagle's medium/22.5% Ham's nutrient mixture F-12 [DMEM/F-
12 (3:1)]5% fetal bovine serum (FBS), transferrin at 5
ug/ml/10-10 M cholera toxin/1.8 x 10-4 M adenine,
hydrocortisone at 0.4 ~g/ml/2 x 10-11 M triodo-L-
thyronine/penicillin at 100 units/ml/streptomycin at 100
~g/ml. Culture medium for sarcoma cells was DMEM/F-12
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(3:1)/20% FBS, penicillin at 100 units/ml/streptomycin at
100 ~g/ml.
TNF-a Protein Assay. Human sarcoma cells were
cultured as described-above and grown to confluence. The
medium was analyzed for TNF-a 3 days after feeding and
again 1-3 days after irradiation. Thirteen established
human sarcoma cell lines were irradiated with 500-
centigray (cGy) x-rays with a 250-kV Maxitron generator
(Weichselbaum, et al., 1988). TNF-a was measured by
ELISA with two monoclonal antibodies that had distinct
epitopes for TNF-a protein (Sariban, et al., 1988); the
assay detects TNF-a from 0.1 to 2.0 units/ml.
RNA Isolation and RNA Blot Analysis. Total
cellular RNA was isolated from cells by using the
guanidine thiocyanate-lithium chloride method (Cathala,
et al., 1983). RNA was size-fractionated by
formaldehyde-1% agarose gel electrophoresis, transferred
to nylon membranes (GeneScreenPlus, New England Nuclear),
hybridized as previously described to the 1.7-kilobase
(kb) BamFiI fragment of the PE4 plasmid containing TNF-a
cDNA (19, 23), and autoradiographed for 16 days at -85°C
with intensifying screens. Northern blots were also
hybridized to 7S rRNA and ~i-polymerase plasmids as
described (FOrnace, et al., 1989). Ethidium bromide
staining revealed equal amounts of RNA applied to each
lane. RNA blot hybridization of TNF-a was analyzed after
cellular irradiation with 500 cGy. Cells were washed
with cold phosphate-buffered saline and placed in ice at
each time interval. RNA was isolated at 3, 6, and 12 hr
after irradiation.
3~-Irradiation and TNF-a. Exponentially growing
cells were irradiated by using a 250-kV x-ray generator.
The colony-forming assay was used to determine cell
survival (Weichselbaum, et al., 1988). The multitarget __
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model survival curves were fit to a single-hit
multitarget model [S = 1 - (-e-D~DO)n~. Concentrations of
recombinant human TNF-a (10 units/ml) (2:3 x 106
units/mg) and (1000 units/ml) (Asahi Chemical, New York)
were added 24 hr before irradiation.
EXAMPLE 2: - Increased c-Jun Expression After
F~r,~~a"re to Ionizina Radiation
Another embodiment of a DNA molecule derives
from the c-Jun protooncogene and related genes. Ionizing
radiation regulates expression of the c-Jun
protooncogene, and also of related genes c-fos and Jun-B.
The protein product of c-Jun contains a DNA binding
region that is shared by members of a family of
transcriptionfactors. Expression level after radiation
is dose dependent. The c-Jun gene encodes a component of
the AP-1 protein complex and is important in early
signaling events involved in various cellular functions.
AP-1, the product of the protooncogene c-Jun recognizes
and binds to specific DNA sequences and stimulates
transcription-of genes responsive to certain growth
factors and phorbol esters (Bohmann, et al., 1987; Angel,
et al., 1988). The product of the c-Jun protooncogene
contains a highly conserved DNA binding domain shared by
a family of mammalian transcription factors including
Jun-B, Jun-D, c-fos, fos-B, fra-1 and the yeast GCN4
protein.
In addition to regulating expression of the c-
Jun gene, c-Jun transcripts are degraded-
posttranscriptionally by a labile protein in irradiated
cells. Posttranscriptional regulation of the gene's
expression is described in Sherman, et al., 1990.
Contrary to what would be expected based on
previous DNA damage and killing rates for other agents,
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decreasing the dose rate, for example, from 14.3 Gy/min
to 0.67 Gy/min. was associated with increased induction
of c-Jun transcripts.
Maximum c-Jun mRNA levels were detectable after
50 Gy of ionizing radiation. Similar kinetics of c-Jun
induction were observed in irradiated human U-937
monocytic leukemia cells and in normal human AG-1522
diploid fibroblasts. Treatment of AG-1522 cells with
ionizing radiation was also associated with the
appearance of a minor 3.2-kb c-Jun transcript.
The following methods were used in Example 2.
Cell Culture. Human HL-60 promyelocytic
leukemia cells, U-937 monocytic leukemia cells (both from
American Type Culture Collection), and AG-1522 diploid
foreskin fibroblasta (National Institute of Aging Cell
Repository, Camden, NJ) were grown in standard fashion.
Cells were irradiated using either Philips RT 250
accelerator at 250 kV, 14 mA equipped with a 0.35-mm Cu
filter or a Gammacell 1000 (Atomic Energy of Canada,
Ottawa) with a l3~Cs source emitting at a fixed dose rate
of 14.3 Gy/min as determined by dosimetry. Control cells
were exposed to the same conditions but not irradiated.
Northern Blot Analysis. Total cellular RNA was
isolated as described (29). RNA (20 ~g per lane) was
separated in an agarose/formaldehyde gel, transferred to
a nitrocellulose filter, and hybridized to the following
s2P-labeled DNA probes: (i) the 1.8-kilobase (kb)
BamHI/EcoRI c-Jun cDNA (30); (ii) the 0.91-kb Sca I/Nco I
c-fos DNA consisting of exons 3 and 4 (31); (iii) the
1.8-kb EcoRI Jun-B cDNA isolated from the p465.20 plasmid
(32); and (iv) the 2.0-kb PstI (3-actin cDNA purified from
pAl (33). The autoradiograms were scanned using an LKB
UltroScan XL Iaser densitometer and analyzed using the
wo 9s~aiss9 219 2 813 PCT~S9510s9s9
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LKB GelScan XL software package. The intensity of c-Jun
hybridization ivas normalized against (3-actin expression.
Rnn-nn Transcriptional Analysis. HL-60 cells
were treated with ionizing radiation and nuclei were
isolated after 3 hours. Newly elongated 32P-labeled RNA
transcripts were hybridized to plasmid DNAs containing
various cloned inserts after digestion with restriction
endonucleases as follows: (i) the 2.0-kb Pat I fragment
of the chicken ~i-actin pAl plasmid (positive control);
(ii) the 1.1-kb BamHI insert of the human ~-globin gene
(negative control); and (iii) the 1.8-kb BamF3I/EcoRI
fragment of the human a-Jun-cDNA from the pBluescript
SK(+) plasmid. The digested DNA was run in a 1% agarose
gel and transferred to nitrocellulose filters by the
method of Southern. Hybridization was performed with 10~
cpm of 32P-labeled RNA per ml of hybridization buffer for
72 h at 42~C.- Autoradiography was performed for 3 days
and the autoradiograma were scanned as already described.
EXAMPLE 3: Radiation Induced Transcription of Jurt and
Ear-1
There was increased mRNA expression for
different classes of immediate early response to
radiation genes (Jun, Egr-1) within 0.5 to 3 hours
following cellular x-irradiation. Preincubation with
cycloheximide was associated with superinduction of Jun
and Egr-1 in x=irradiated cells. Inhibition of protein
kinase C (PKC) activity by prolonged stimulation with TPA
or the protein kinase inhibitor H7 prior to irradiation
attenuated the increase in Egr-1 and Jun transcripts.
These data implicated Egr-1 and Jun as signal transducers
during the cellular response to radiation injury and
suggested that this effect is mediated in part by a
protein kinaee C (PKC) dependent pathway.
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Jun homodimers and Jun/fos heterodimers
regulate transcription by binding to AP1 sites in certain
promoter regions (Curran and Franza, 1988). The Jun and
fos genes are induced following x-ray exposure in human
myeloid leukemia cells suggests that nuclear signal
transducers participate in the cellular response to
ionizing radiation.
The Egr-1 and Jun genes are rapidly and
transiently expressed in the absence of de novo protein
synthesis after ionizing radiation exposure. Egr-1 and
Jun are most likely involved in signal transduction
following x-irradiation. Down-regulation of PKC by TPA
and H7 is associated with attenuation of Egr-1 and Jun
gene induction by ionizing radiation, implicating
activation of PKC and subsequent induction of the Egr-1
and Jun genes as signaling events which initiate the
mammalian cell phenotypic response to ionizing radiation
injury.
Control RNA from unirradiated cells
demonstrated low but detectable levels of Egr-1 and Jun
transcripts. In contrast, Egr-1 expression increased in
a dose dependent manner in irradiated cells. Levels were
low but detectable after 3 Gy and increased in a dose
dependent manner following 10 and 20 Gy. Twenty Gy was
used in experiments examining the time course of gene
expression so that transcripts were easily detectable.
Cells remained viable as determined by trypan blue dye
exclusion during this time course. A time dependent
increase in Egr-i and Jun mRNA levels was observed. SQ-
20B cells demonstrated coordinate increases in Egr-1 and
Jun expression by 30 minutes after irradiation that
declined to baseline within 3 hours. In contrast, Egr-1
transcript levels were increased over basal at 3 hours
while Jun was increased at one hour and returned to basal
at 3 hours in AG1522. Jun levels were increased at 6
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hours in 293 -cells while Egr-1 was increased at 3 hours
and returned to basal levels by 6 hours.
To determine whether Egr-1 and Jun participated
as immediate early genes after x-irradiation, the effects
of protein synthesis inhibition by cycloheximide were
studied in cell lines 293 and SQ-20B after x-ray
exposure. Cycloheximide treatment alone resulted in a
low but detectable increase in Egr-1 and Jun transcripts
normalized to 7S. In the absence of CHI, the level of
Egr-1 and Jun expression returned to baseline. In
contrast, SQ-20B cells pretreated with CHI demonstrated
persistent elevation of Egr-1 at 3 hours and 293 cells
demonstrated persistent elevation of Jun mRNA at 6 hours
after irradiation thus indicating superinduction of these
transcripts.
mRNA levels of transcription factors Egr-1 and
Jun increased following ionizing radiation exposure in a
time and dose dependent manner. The potential importance
of the induction of Egr-1 and Jun by ionizing radiation
is illustrated by the recent finding that x-ray induction
of the PDGFa chain stimulates proliferation of vascular
endothelial cells (Witte, et al., 1989). PDGF has AP-1
and Egr-1 binding domains while TNF has elements similar
to AP-1 and Egr-1 target sequences (Rorsman, et al.,
1989; Economou, et al., 1989). X-ray induction of PDGF
and TNF are likely regulated by Egr-I and Jun.
The following is a method used in EXAMPLE 3:
Kinase Inhibitors
Cell-line SQ-20B was pretreated with 1 ~M TPA
for 40 hours to down regulate PKC and then stimulated
with TPA, serum, or x-ray (20 Gy). Controls included x-
ray without TPA pretreatment, TPA (50 nM) without TPA
pretreatment and untreated cells. RNA was isolated after
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one hour and hybridized to Egr-1. SQ-2oB cells were
preincubated with 100 uM H7 (1-(5-isoquinolinylsulfonyl)-
2-methyl piperazine) or 100 ~M HA1004 (N-[2-methyl-amino]
ethyl)-5-isoquino-linesulfonamide) (Seikagaku America,
Inc., St. Petersberg; FL) for 30 minutes or TPA
pretreatment (1 uM) for 40 hours and followed by exposure -
to 20 Gy x-irradiation. RNA was extracted one hour after
irradiation. Positive control cells treated under the
same conditions but in the absence of inhibitor also
received 20 Gy, while negative control cells received
neither H7 nor X-ray. RNA was extracted at one hour
after 20 Gy without inhibitor. Northern blots were
hybridized to Egr-1 or 7S. 293 cells pretreated with the
above inhibitors were irradiated, RNA was extracted after
3 hours and the Northern blot was hybridized to Jun and
7S probes.
ESAMPLE 4: Ionizing Radiation Activates Transcription
9f the Ear-1 Gene via OArG Domains
The cellular response to ionizing radiation
includes cell cycle-specific growth arrest, activation of
DNA repair mechanisms and subsequent proliferation of
surviving cells. However, the events responsible for the
control of this response remain unclear. Recent studies
have demonstrated that ionizing radiation exposure is
associated with activation of certain immediate-early
genes that code for transcription factors. These include
members of the Jun/foa and early growth response (Egr)
gene families (Sherman, et al., 1990; Hallahan, et al,
1991). Other studies have demonstrated that x-rays
induce expression and DNA binding activity of the nuclear
factor xB (NF-xB; Brach, et al., 1991).
The activation of these transcription factors
may represent transduction of early nuclear signals to
longer term changes in gene expression which constitute
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the response to ionizing radiation. In this context,
irradiation of diverse cell types is also associated with
increased expression of the TNF, PDGF, FGF and
interleukin-1 genes (Hallahan, et al., 1989; Witte, et
al, 1989; Woloschak, et al., 1990; Sherman, et al.,
1991). Expression of cytokines is conceivably involved
in the repair and repopulation associated with x-ray-
induced damage to tissues, and may explain some of the
organiamal effects of ionizing radiation (Hall, 1988).
Moreover, it is possible that immediate-early
transcription factors serve to induce these changes in
gene expression.
The present studies relate to mechanisms
responsible for x-ray-induced activation of the Egr-1
gene (also known as zif/268, TIS-8, NFGI-A and Krox-24;
Sukhatme, et al., 1988; Christy, et al., 1988; Milbrandt,
1987; Lemaire, et al., 1988; Lim, et al., 1987). The
Egr-1 gene encodes a 533-amino acid nuclear
phosphoprotein with a Cysa-His2 zinc finger domain that
is partially homologous to the corresponding domain in
the Wilms tumor-susceptibility gene (Gessler, 1990). The
Egr-i proteinbinds to the DNA sequence CGCCCCCGC in a
zinc-dependent manner and functions as a regulator of
gene transcription (Christy, et al, 1989; Cao, et al.,
1990; Gupta, et al, 1991). Both mitogenic and
differentiation signals have been shown to induce the
rapid and transient expression of Egr-1 in a variety of
cell types. For example, the Egr-1 gene is induced after
mitogenic stimulation of Balb/c-3T3 cells by serum, PDGF
or FGF (Lau, -et al, 1987; Sukhatme, et al., 1987). The
Egr-1 gene is also induced during: 1) cardiac and
neuronal differentiation of the pluripotent EC line
(Sukhatme, et al., 1988); and 2) monocytic
differentiating of human myeloid leukemia cell lines
(Kharbanda, et al., 1991; Bernstein, et al., 1991).
While Egr-1 transcription is activated by the protein
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tyrosine kinase activity of v-src and v-fps (Gius, et
al., 199D; Christy, et al., 1989; Homma, et al., 1986),
additional work indicates that the serine/threonine
kinase activity of c-raf-1 is also involved in mediating
inducibility of this gene (Chirgwin, et al., 1979).
Other studies have demonstrated that induction of Egr-1
expression is similar to that of c-fos in many situations
and that this coordinate regulation is mediated by the
presence of serum response elements in both promoters
(Sukhatme, et al., 1988; Cleveland, et al., 1980; Wilson,
et al., 1978).
Although previous work has demonstrated that
ionizing radiation treatment is associated with increases
in Egr-1 mRNA levels (Hallahan, et al., 1991), the
mechanisms responsible for this effect are unclear. The
present studies demonstrate that x-rays activate
transcription of the Egr-1 gene. Serum response or CArG
domain [CC(A/T)6GG] domains in the 5' promoter of the
Egr-1 gene are functional in this response. This is the
first report of specific DNA sequences involved in
regulating gene transcription by ionizing radiation.
A low but detectable level of 3.4-kb Egr-1
transcripts were present in untreated HL-525 cells. In
contrast, treatment with ionizing radiation was
associated with an increase in Egr-1 expression that was
detectable at 1 hour. Maximal increases (18-fold) in
Egr-I mRNA levels were obtained at 3 hours, while longer
intervals were associated with down-regulation to nearly
that in control cells. This transient induction of Egr-1
expression occurred in the absence of significant changes
in actin mRNA levels.
Nuclear run-on assays were performed to
determine whether x-ray-induced increases in Egr-1
transcripts are controlled at the transcriptional level.
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There was no detectable transcription of the fi-globin
gene (negative control) in HL-525 cells, while the actin
gene (positive control) was transcribed constitutively.
Moreover, treatment with ionizing radiation had little
effect on transcription of these genes. However, while
transcription of the Egr-1 gene was detectable at low
levels in control cells, treatment with ionizing
radiation resulted in a 30-fold increase in this rate.
Taken together, these findings indicated that x-ray-
induced Egr-1 expression is controlled substantially by
transcriptional mechanisms.
In order to identify cis elements responsible
for x-ray-induced Egr-1 transcription, the Egr-1 promoter
region extending from position -957 upstream to the
transcription start site to position +248 was ligated to
the CAT reporter gene -(plasmid pEgr-1 P1.2). This region
contains several putative cis elements including two AP-1
sites and six CArG domains (Christy, et al., 1989; Gius,
et al., 1990). Treatment of the pEgr-iPl.2 transfected
cells with ionizing radiation was associated with a 4.1-
fold increase-in CAT activity as compared to tranafected
but unirradiated cells. In contrast, similar studies
performed with plasmid pOEgr-1 P1.2 (-550 to -50 deleted)
demonstrated little if any inducibility by x-rays. These
data suggested that x-ray inducibility of Egr-1 is
mediated by sequences present between -550 and -50 of the
Egr-1 promoter. Indeed, irradiation of pE425 transfected
cells was associated with a 3.6-fold induction of CAT
activity compared to that in non-irradiated cells
transfected with this construct.
A series of deleted Egr-1 promoter constructs
was next used-to further define the x-ray responsive
elements in pE425. These constructs have been previously
described and are shown schematically in Scheme 1.
Sequential deletion of the three distal CArGa
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progressively decreased CAT activity. pE395 (first CArG
deleted) conferred x-ray inducibility to a lesser extent
than pE425. Deletion of the first and second (pE359)
CArGs resulted in further decreases in CAT activity,
while deletion of the first three CArG domains (pE342)
was associated with little if any increases in CAT
activity. Taken together, these findings supported the
hypothesis that the three distal CArG elements confer x-
ray inducibility of the Egr-1 gene.
Other studies were performed with fragments of -
the Egr-1 promoter linked to HSV-TK and the CAT gene.
There was no detectable inducibility of pTK35CAT by
x-rays. In contrast, pE425/250TK, which contains the
four distal CArG domains, was more responsive to x-ray
treatment than pTK35CAT. The region from -395 to =250,
which excludes the first CArG element, was also
functional in conferring x-ray inducibility to the
heterologous promoter, but to a lesser extent than
pE425/250TK. While these findings provided further
support for the involvement of CArG domains in x-ray
induced Egr-I transcription, other sequences between
these domains could be the functional cis elements.
X-ray inducibility of pTK35CAT transcription was also
demonstrated with the first CArG with seven base pairs of -
the 5' and 3' flanking sequences (pSREITK).
Since the results of the transient expression
assays indicated that the CArG domain is the target
sequence for x-ray-mediation activation of the Egr-1
gene, other studies were performed to determine whether
nuclear proteins interact with this element and whether
x-ray treatment alters this interaction. Nuclear
extracts from untreated and x-ray-treated cells were
incubated with a 32P-labeled probe encompassing the first
CArG domain. Nuclear proteins from control cells
resulted in the formation of several DNA-protein
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complexes. A-similar pattern was obtained with nuclear
proteins from x-ray-treated cells. In order to determine
which complexes reflected CArG-protein binding, increased
amounts of unlabeled probe were preincubated with the
nuclear proteins. In these experiments, a 100-fold
excess of unlabeled probe completely inhibited formation
of the complex with least mobility. In contrast,
addition of an oligonucleotide containing the unrelated
NF-xB consensus sequence had no effect on the intensity
of this complex. These findings and the results of
similar gel retardation studies with this labeled
fragment (Cleveland, et al., 1980) indicate that the
upper complex represents specific CArG-protein
interaction.
The following methods were used in Example 4.
Cell Cultures. Human HL-525 myeloid leukemia
cells (Jamal, et al., 1990) were maintained in RPMI 1640
medium containing 20% fetal bovine serum (FBS) with i mM
L-glutamine, 100 U/ml penicillin and 100 ~.g/ml
atreptomycin_- Irradiation (20 Gy) was performed at room
temperature using a Gammacell 1000 (Atomic Energy of
Canada Ltd_, Ontario) with a l3~Ca source emitting at a
fixed dose rate of 13.3 Gy/min as determined by
dosimetry.
Isolation and analvsis of RNA. Total cellular
RNA was purified by the guanidine isothiocyanate-cesium
chloride technique (Grosschedl, et al., 1985). The RNA
was analyzed by electrophoresis through I% agarose
formaldehyde gels, transferred to nitrocellulose filters,
and hybridized to the following 32p-labeled DNA probes:
1) the 0.7-kb non-zinc finger insert of a murine Egr-I
cDNA (9); and 2) the 2.0-kb PstI insert of a chicken /3-
actin gene purified from the pAl plasmid (Dignam, et al.,
1983). Hybridizations were performed at 42'C for 24 h in
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50% (v/v) formamide, 2x SSC, 1X Denhardt's solution, 0.1%
SDS, and 200 ~.g/ml salmon sperm DNA. The filters were
washed twice in 2x SSC-0.1% SDS at room temperature and
then in O.lx SSC-0.1% SDS at 60'C for 1 h. Signal
intensity was determined by laser densitometry and
normalized to that for the actin control.
uclear run-on assays. Nuclei were isolated
from 10a cells and suspended in 100 ~,1 glycerol buffer
(50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, and
0.1 mM EDTA). An equal volume of reaction buffer (10 mM
Tris-HC1, pH 8.0, 5 mM MgCl2, 100 mM KC1, 1 mM ATP, 1 mM
CTP, 1 mM GTP, and 5 mM dithiothreitol) was added to the
nuclei in suspension and incubated at 26'C for 45 min
with 250 ~Ci [a-32p] UTP (3000 Ci/mmol; Dupont, Boston,
MA). The nuclear RNA was isolated as described
(Kharbanda, et al., 1991) and hybridized to the following
DNAs: 1) a 1.1-kb BamHI insert of a human ~i-globin gene
(negative control) (Hallahan, et al., 1991); 2) a PstI
digest of the pAl plasmid containing a fragment of the
chicken (3-actin gene (positive control) (Dignam, et al.,
1983); and 3) the 0.7-kb insert of the murine Egr-1 cDNA
(Sukhatme, et al., 1988). The digested DNAe were run in
1% agarose gels and transferred to nitrocellulose
filters. Hybridizations were performed with 10~ cpm of
32P-labeled RNA/ml in 10 mM Tris-HC1, pH 7.5, 4x SSC, 1
mM EDTA, 0.1% SDS, 2x Denhardt's solution, 40% formamide,
and 100 ~.g/ml yeast tRNA for 72 h at 42'C. The filters
were washed in: a) 2x SSC-0.1% SDS at 37'C for 30 min; b)
200 ng/ml RNase A in 2x SSC at mom temperature for 5
min; and c) O.lx SSC-0.1% SDS at 42'C for 30 min.
Reporter assays. The pEgr-IP1.2, pE425, pE395,
pE359, pE342, pE125, pE98 and pE70 constructs were
prepared as described (26). pE425/250TK was constructed
by cloning a HindIII-SmaI fragment from pE425, spanning
the region -425 to -250 of the Egr-1 promoter, upstream
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of the herpes-simplex virus thymidine kinase (HSV-TK)
promoter in plasmid pTK35CAT (Homma, et al., 1986).
pE395/250TK was constructed in the same manner using a
HindIII-SmaI fragment from pE395. pSREITK contains the
5'-most distal or first CArG domain in the Egr-1 promoter
along with seven base pairs of the 5' and 3' flanking
sequences cloned into the SalI-BamHI site of pTK35CAT
(Homma, et al_, 1986). The constructs were transfected
into cells using the DEAF-dextran technique (Treisman, et
al., 1990). Calls (2x107) were incubated in 1 ml of
Tris-buffered saline solution (25 mM Tris-HC1, pH 7.4,
137 mM NaCl, 5 mM KC1, 0.6 mM NaaHP04, 0.7 mM CaCl2, and
0.7 mM MgCl2 containing 0.4 mg DEAF-dextran and 8 ~.g
plasmid, at 37'C for 45 min. The cells were washed with
media containing 10% FBS, resuspended in complete media
and then incubated at 37°C. Thirty-six h after
transfection, one aliquot of the cells served as a
control and the other was treated with ionizing
radiation. The cells were harvested after 8 hours and
lyaed by three cycles of freezing and thawing in 0.25 M
Tris-HC1, pH 7.8, and 1 uM phenylmethylaulfonyl-fluoride.
Equal amounts of the cell extracts were incubated with
0.025 uCi [14C]chloramphenicol, 0.25 M Tris-HC1, pH 7.8
and 0.4 mM acetyl-coenzyme A for 1 hour at 37'C. The
enzyme assay was terminated by addition of ethyl acetate.
The organic Layer containing the acetylated
[14C]chloramphenicol was separated by thin-layer
chromatography using chloroform: methanol (95%:5%; v/v).
Following autoradiography, both acetylated and
unacetylated forms of [14C]chloramphenicol were cut from
the plates, and the conversion of chloramphenicol to the
acetylated form was calculated by measurement of
radioactivity in a ~i-scintillation counter.
~;el retardation assava. Nuclear extracts were
prepared as described (Filch, et al_-, 1987). A 40 by
synthetic double-stranded oligonucleotide containing the
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first CArG element (Cleveland, et al., 1980) was end-
labeled with [a-32p] dCTP using T4 polynucleotide kinase
and then purified in a 5% polyacrylamide gel. The
purified end-labeled probe (1 ng; approx. 1x105 cpm) was
incubated with varying amounts of nuclear protein extract
for 15 min at 20'C in a buffer containing 12 mM HEPES, pH
7.9, 60 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 6% (v/v)
glycerol, 1 ~.g bovine serum albumin, and 0.25 ~,g of
sonicated salmon sperm DNA. Competition experiments were
performed with either unlabeled probe or a 22 by
oligonucleotide containing the NF-xB binding site.
Competing oligonucleotides were preincubated with the
nuclear protein extract for 20 min at 4°C before adding
the labeled probe. The final reaction products were
analyzed by electrophoresis in a 5% polyacrylamide gel
containing :4x TBE (22 mM Tris, 22 mM boric acid, 0.5 mM
EDTA) and subsequent autoradiography.
In the present studies, HL-525 cells responded
to ionizing radiation with induction of Egr-I mRNA
levels. These findings indicated that ionizing radiation
increases Egr-I expression through signaling pathways
distinct from those activated during induction of this
gene in TPA-treated cells. Furthermore, the finding that
x-ray-induced TNF gene expression is attenuated in the
HL-525 line {Gilman, 1988) suggests that ionizing
radiation induces the Egr-1 and TNF genes by distinct
signaling pathways in these cells.
These studies further demonstrate that x-ray-
induced Egr-I expression is regulated at least in part by
transcriptional mechanisms. Nuclear run-on assays
demonstrated an increase in the rate of Egr-1 gene
transcription following ionizing radiation. Moreover,
analysis of the full length Egr-I promoter (pEgr-IP1.2)
in transient expression assays demonstrated inducibility
by ionizing radiation. Transfection of p~Egr-1P1.2 (-550
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to -50 deleted) and pE425 provided additional evidence
that the promoter region containing the six CArG elements
was responsible for conferring x-ray inducibility. These
findings were supported by the use of several other
deleted promoter constructs which indicated that the
region encompassing the first three CArG elements is
functional in the x-ray response.
In this context, sequential deletion of these
distal CArGs progressively eliminated the x-ray response.
The four distal CArGs also conferred x-ray inducibility
to a heterologous promoter and this effect was decreased
by deleting the first CArG domain. More importantly,
studies with the first CArG domain demonstrated that this
element was sufficient to confer the x-ray response.
Taken together, these findings strongly support the CArG
domain as the radiation responsive element.
EXAMPLE 5: Radiation Sianallina Mediated by Jun _
Activation
Ionizing radiation produces a wide range of
effects on cells which include induction of mutations,
lethality, malignant transformation in some surviving
cells, cell cycle arrest, and subsequent proliferation of
cells. Jun, a transcription factor that is central to
tumor promotion, proliferation and cell cycle regulation,
is activated by DNA damaging agents in mammalian cells
(Devary, 1991 and Bernstein, 1989). One proposed
mechanism of Jun activation is through dissociation of
Jun from an inhibitor of Jun transcription (Baichwal,
1990).
To investigate protein binding to the AP-1
sequence following irradiation, nuclear proteins were
extracted from irradiated human sarcoma cell line RIT-3
cells at 5 minutes intervals for 30 minutes following
W O 95/31559 PCTIUS95I05959
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exposure to 10 Gy. The AP-1, NFxB, SP-1 and CTF binding
sequences labeled with 32P were incubated with cell
extracts. DNA-protein mixtures were then separated by
electrophoresis. An increase in nuclear protein binding
to AP-1 DNA sequences was found at 10 to 20 minutes
following irradiation as compared to untreated control
cells in electrophoretic mobility shift assay, whereas
there was no increase in nuclear protein binding to NF-
xB, SP-l, Oct-1 or CTF following irradiation of RIT-3
cells.
DNA-protein complexing was not prevented by
adding the inhibitor of protein synthesis cycloheximide,
to cells prior to irradiation. AP-1 binding was
eliminated when non-labeled AP-1 consensus sequence
(Rauscher, 1988) competed for nuclear protein when added
to extracts prior to the addition of labeled AP-1 and
eliminated the banding produced by extracts from
irradiated RIT-3 cells. In contrast, a nonspecific DNA
sequence (Oct-1) did not compete for nuclear protein
binding. These data indicated that nuclear protein -
binding to AP-1 following irradiation is specific. - -
To determine whether AP-1 binding nuclear
proteins from irradiated RIT-3 cells share epitopes with
known transcription factors, Jun and fos antisera
(Chiles, 1991 and Stopera, 1992) were added to nuclear
extracts prior to the addition of labeled AP-1 DNA
sequences. The addition of antiserum to the DNA binding
domains of fos (Ab-2) and Jun (CRB) resulted in a
reduction in protein complexing to the AP-1 sequence
following irradiation. Increasing concentrations of -
antiserum progressively reduced protein-DNA complexes
accordingly. These data indicate that proteins that bind
the AP-1 sequence following irradiation have epitopes
recognized by antiserum to the DNA binding domains of Jun
and fos.
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To determine whether activated Jun results in
increased transcriptifln of the AP-1 binding site
following ionizing radiation exposure, the plasmid
(p3xTRE-CAT) containing three AP-1 sites upstream of the
minimal tk promoter (pBLCAT2) was transfected into RIT-3
cells. Irradiation of p3xTRE-CAT transfectants resulted
in a 3 fold increase in CAT expression. To determine
whether the c-Jun promoter is induced by radiation in a
manner analogous to serum and phorbol esters, the 1840-
base pair segment of the c-Jun promoter placed upstream
of the chloramphenicol acetyl transferase (CAT) gene
[Angel, 1988]was transfected into RIT-3 cells.
Following transfection, cells were maintained in 0.2%
fetal calf serum (FCS) and irradiated (10 Gy, 2 Gy/min)
40 hrs post-transfection and CAT was extracted 5 hours
after irradiation.
Transfection of the -l.lkb to +740-by region of
the c-Jun promoter (c-Jun-CAT) demonstrated a 3-fold
increase in gene expression following exposure to
ionizing radiation. Tranafection of the plasmid with a
deletion of the AP-1 site located at +150-by (-132/+170 D
AP-1CAT) resulted in a lose of x-ray-mediated induction.
These results suggest that activated AP--1 participates in
the transcription of c-Jun and that the AP-1 DNA sequence
is sufficient and necessary to confer x-ray-mediated gene
induction.
Several regulatory and DNA binding domains
exist within the Jun protein. Close to the DNA binding
domain is a region designated as A2, which is required to
activate transcription (reviewed in (Lewin, 1991). A1,
an additional transcriptional activation domain is found
near the N terminus adjacent to a region termed Delta (D)
which is proposed to bind a cellular protein that
inhibits the transcriptional activating properties of Jun
(Baichwal, 1990 and Baichwal, 1991). Jun transcriptional
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activity can be conferred through either or both
activation domains A1 and A2. Phorbol ester treatment
results in the modification of the Jun protein by a
protein kinase C (PKC)-dependent phosphorylation of the
A1 region and thereby auto-induces transcription of c-Jun
(Binetruy, 1991 and (Pulverer, 1991).
Increased Jun binding to AP-1 sequences
following irradiation indicate that Jun protein is
modified following irradiation. Taken together with the
recent findings that PKC is activated following
irradiation of cells and that PKC depletion suppress
c-Jun induction by irradiation (Hallahan, 1992), the data
suggest that X-ray exposure activates Jun through PKC
modification of the A1 domain (Binetruy, 1991 and
Pulverer, 1991).
Two plasmids were transfected into HeLa and
RIT-3 cells to study activation of the transcriptional
potential of Jun protein following irradiation.
pSG-JunS-253 contains the SV40 promoter, which is not
transcriptionally responsive to radiation, upstream of
the coding sequence for D, A1, and A2 (Baichwal, 1990).
The DNA binding domain of Jun was replaced with the DNA
binding domain of the yeast GAL4 gene which encodes a
protein involved in yeast transcriptional regulation
(Baichwal, 1990). A second plasmid, GSBCAT contains the
DNA sequence which binds Gal4 protein placed 5' of the
Elb TATA box upstream of the CAT reporter gene (Baichwal,
1990). When the activation domain of Jun protein becomes
transcriptionally active, the chimeric Gal-Jun protein,
initiates CAT transcription following binding to the Gal4
binding sequence.
These plasmids were co-transfected by calcium
phosphate precipitation into HeLa and RIT-3 cells.
Transfectants were irradiated with 20 Gy and demonstrated
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a three-fold increase in CAT activity as compared to
untreated controls. This level of expression was
comparable to that observed following TPA stimulation
which produced a 3.5 fold increase in CAT activity.
Irradiation of RIT-3 cells transfected with GSBCAT alone
demonstrated no increase in CAT activity. Similar
results were.obtained in HeLa cells which contain the Jun
inhibitor. However, Hep G2 cells which do not contain
the Jun inhibitor [Baichwal, 1990] demonstrated no
x-ray-induced activation of the Gal4-Jun chimeric. These
data suggest that the Gal-Jun chimeric protein is
activated following irradiation resulting in DNA binding
to accelerate transcription of CAT.
Because x-ray induced c-Jun gene expression is
attenuated when PKC is depleted or inhibited, the PKC
inhibitor H7 was added to RIT-3 cells transfected with
pSG-JunS-235 and GSBCAT. H7 treatment abrogated the
x-ray induced-increase in CAT activity. This finding is
consistent with previous results that demonstrated
X-ray-induced PKC activation is required for gene
expression (Hallahan, 1991).
The-inhibitor of Jun transcription that binds
to the A/A1 domains represses the transcriptional
activity of A1. The inhibitor of Jun transcription was
originally defined in experiments where an excess of Jun
competed for inhibitor and thereby allowed uninhibited
Jun to increase transcription of the G5-CAT construct
when compared to untreated control cells. Based on these
results, HeLa yells are reported to contain the Jun
inhibitor (Baichwal, 1990) whereas, in HepG2 cells do
not. To determine whether the Jun inhibitor is present
in RIT-3 cells, the expression vector CMV-Jun, which
constitutively expresses c-Jun, was co-transfected with
pSGJunS-235 an3 GSBCAT. Basal expression of CAT
increased in-RIT-3 and HaLa cells but not HepG2 when
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CMV-Jun was added. These results confirm and extend the -_
results of Tijan et al. in that Hela and RIT-3 cells
contain the Jun inhibitor but not HepG2 cells. Cells
that contain the Jun inhibitor demonstrate an increase in
radiation mediated transcription of GSBCAT, whereas cells
that do not contain the inhibitor of Jun show no increase
in transcription of GSBCAT.
RIT-3 cells co-transfected with CMV-Jun,
pSGJunS-235 and GSBCAT do not demonstrate an increase in
transcription as compared to these tranafectanta treated
with identical conditions, but no irradiation. These
data suggest that dissociation from the Jun inhibitor may
be one mechanism of regulating radiation-mediated
transcription.
The following methods were used in Example 5.
uclear Extracts. Nuclear extracts were
prepared according to previously described methods
(Schreiber, 1989) at 10,20,30, and 60 min. after
irradiation. RIT-3 cells (106) were washed in 10 ml
PBS, scraped, and pelleted by centrifugation at 1500 g
for 5 min. The pellet was resuapended in 1 ml PBS,
transferred into an Eppendorf tube and pelleted again for
15 sec. PBS was removed and the cell pellet resuapended
in 400 ~1 of cold buffer A (10 mM HEPES pH 7.9; 10 mM
RC1; 0.1 mM EDTA; 1 mM DTT; 0.5 mM PMSF). The cells were
allowed to swell on ice for 15 min, followed by the
addition of 25 ml of 10% NP-40. The mixture was
centrifuged for 30 sec and the nuclear pellet resuapended
in 50 ul ice-cold buffer B (20 mM HEPES pH 7.9; 0.4 M
NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF) for 15
min and the nuclear extract centrifuged for 5 min.
Protein content was determined by the Bradford method
(Bio-Rad). The AP-1 consensus sequence DNA (BRL-GIBCO)
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was end-labeled with [-32P]dATP using DNA polymerase I
Klenow fragment.
Binding Assava. Binding assays were performed
by incubating the end-labeled DNA (1 ng) with 10 ~Cg
nuclear protein, 75 mM KC1 and 1 ug/ml poly (dI-dC) in a
20 ul reaction for 20. min at room temperature.
Competition Assays. Competition studies were
performed using oligonucleotides corresponding to known
cis-acting elements AP-1, and Oct-1 (BRL-GIBCO) at a 100-
fold molar excess as compared to the labeled fragments.
The reaction products were separated by 5% polyacrylamide
gel electrophoresis, dried and analyzed by
autoradiography.
Antisera Studies. Antisera-to Jun and fos
proteins reduced radiation-induced AP-1 binding. A.
Antiserum to human transcription factors c-Jun (amino
acids 73-87, Ab-2, Oncogene Sci.), c-Jun, Jun-B, Jun-D
(Cambridge Research, log OA-11-837), and c-fos (amino
acids 4-17, Ab-2, Oncogene Sci and (amino acids 1-14, Lot
OA-li-823, Cambridge Research) were added to l0 ~Cg of
nuclear extracts at a 1:200 dilution and incubated at
24°C with rocking for one hour. DNA segments were added
as described above followed by separation using
electrophoretic mobility shift assays.
Transfection Assays., Plasmids contain c-Jun-.
CAT, 3xTRE-CAT, and oAP-1-CAT (2 fig) were co-transfected
with a plaemid containing a CMVpromoter linked to the
i3-galactosidase gene (1 fig) and 12 ~Cg of carrier DNA into
RIT-3 cells. Cells were transfected using Lipofectin
Reagent (BRL-GIBCO) for 20 hrs., followed by the addition
of medium with 0.2% fetal bovine serum. Transfectants
were incubated for 4~ h after transfection followed by
treatment with 10 Gy (1 Gy/min, GE Maxitron) ionizing
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radiation and harvested by scraping 6 h later. The cells
were lyaed and extracts were incubated with
(1aC]chloramphenicol and acetyl coenzyme A for one hour-
at 37~C. CAT activity was determined by separating
acetylated-(14C]chloramphenicol by ascending
chromatography. Scintillation counting of both the non-
acetylated and acetylated forms of [14C]chloramphenicol
was used to quantify CAT activity. shown above are the
results of: The 1.1 Kb segment of the c-Jun promoter
linked to CAT (c-Jun-CAT). The 3xTAE-CAT. The DAP-1-CAT
plasmid in which the AP-1 sequence has been deleted from
the c-Jun promoter. CAT activity is compared to
nonirradiated and TPA treated transfectants. The mean
and standard errors of 3 experiments are presented.
Plasmida pSG-JunS-253 (2 Pg) and GSBCAT (4 fig)
were co-transfected with a plasmid containing a CMV
promoter linked to the i3-galactosidase gene (1 fig) and
12 ~g of carrier DNA into RIT-3 and HeLa cells using
lipofectin. CAT activity is compared to nonirradiated
and TPA treated transfectants. The mean and standard
errors of 3 experiments are presented.
EgAMPLE 6: Involvement of Reactive Intermediates in
the Induction of c-Jun Gene Transcription
by Ionizing Radiation
Ionizing radiation has been postulated to
induce activation of DNA repair mechanisms, cell cycle
arrest in G2 phase and lethality by either direct
interaction with DNA or through the formation or reactive
oxygen intermediates (ROI) which damage DNA. Recent
studies have further suggested a role for the activation
of immediate-early genes in the response to ionizing
radiation. For example, exposure of cells to x-rays is
associated with activation of the c-Jun/c-fog and Egr-1
gene families which code for transcription factors.
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Other studies have demonstrated that ionizing radiation
induces expression and DNA binding activity of the
nuclear factor xB (NF-xB). The activation of
transcription factors likely represents a critical
control point in transducing early nuclear signals to
longer term changes in gene expression that reflect the
response to x-ray-induced damage. Studies have
demonstrated that radiation treatment is associated with
increased expression of certain cytokines, including TNF,
platelet-derived growth factor, fibroblast growth factor
and interleukin-1. The increase in TNF expression
following exposure to ionizing radiation is regulated by
transcriptional mechanisms, although it is not known
which DNA binding proteins confer this inducibility.
The--c-Jun gene codes for the major form of the
40-44 kD AP-1 transcription factor. As observed in
irradiated cells, this gene is induced as an immediate
early event in response to phorbol esters and certain
growth factors. The Jun/AP-1 complex binds to the
heptomeric DNA consensus sequence TGAG/~TCA. The DNA
binding domain of c-Jun is shared by a family of
transcription factors, including Jun-B, Jun-D and c-fos.
Moreover, the affinity of c-Jun binding to DNA is related
to the formation of homodimers or heterodimers with
products of the foa gene family.
Jun-B also forms dimers and binds to the AP-1
element, although the trans-acting properties of Jun-B
differ from those of c-Jun. While the product of the
Jun-D gene also interacts with c-fos and has similar
binding properties to that of c-Jun, the function of
Jun-D is unknown. Certain insights are available
regarding the signals which contribute to the regulation
of these genes. For example, the finding that phorbol
esters activate c-Jun transcription in diverse cell types
has implicated the involvement of a phosphorylation-
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dependent mechanism. A similar pathway appears to play a
role, at least in part, in the induction of c-Jun
expression by ionizing radiation. In this regard,
prolonged treatment with phorbol esters to down-regulate
PKC is associated with decreases in the effects of x-rays
on c-Jun transcription. Furthermore, non-specific
inhibitors of PKC, such as the isoquinolinesulfonamide
derivative H7, block x-ray-induced c-Jun expression.
Taken together with the demonstration that ionizing
radiation induces an activity with characteristics of
PKC, these findings have suggested that PKC or a related
kinase transducer signals which confer x-ray inducibility
of the c-Jun gene.
The present studies examined the effects of
ionizing radiation on c-Jun expression in an HL-60 cell
variant, designated HL-525, which is deficient in
PKC-mediated signal transduction. This variant is
resistant to both phorbol ester-induced differentiation
and x-ray-induced TNF gene expression. The present
results demonstrate that HL-525 cells are also resistant
to the induction of c-Jun expression by phorbol esters.
The results also demonstrate that treatment of these
cells with ionizing radiation is associated with a
superinduction of c-Jun mRNA levels compared to phorbol
ester-responsive HL-60 cells. The findings indicate that
this effect of ionizing radiation is related at least in
part to the formation of reactive oxygen intermediates.
Previous studies have demonstrated that
treatment of HL-205 cells with TPA is associated with
translocation of PKC activity from the cytosolic fraction
to the cell membrane, while no cellular redistribution of
PKC is detectable during similar exposures of HL-525
cells. Because previous work has suggested that ionizing
radiation induces early response gene expression by a
PKC-dependent mechanism, studies were performed to
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determine the effects of x-rays on a cell, such as
HL-525, which is deficient in PKC-mediated signal
transduction. A low level of c-Jun transcripts was
detectable in untreated HL-205 cells, while treatment
with ionizing radiation was associated with a transient
increase which was maximal at 3 hours. The kinetics and
intensity of this response were identical to that
reported for the parent HL-60 cells. Expression of the
c-Jun gene was-also low in untreated HL-525 cells.
However, exposure of these cells to ionizing radiation
resulted in c-Jun mRNA levels which at 3 h were 3.5-fold
higher than that obtained in HL-205 cells. Higher levels
of c-Jun expression were similarly detected in HL-525
cells at 6 and S h after x-ray exposure. Expression of
the Jun-B and Jun-D genes was also transiently increased
following x-irradiation of the HL-205 line. Similar
findings were obtained with HL-525 cells, although mRNA
levels for Jun=B and Jun-D at 3 hours were 2.0- and
2.5-fold higher at 3 hours in this variant as compared to
that in HL-205 cells.
Proteins encoded by members of the Jun gene
family can fox~n heterodimers with fos gene products.
Consequently, the effects of x-rays on c-fos and fos-B
expression were also studied. c-fos transcripts were
present at low levels in HL-205 cells and there was
little if..any effect of ionizing radiation on expression
of this gene. -Similar findings were obtained for fos-B.
In contrast, while expression of c-fos and fos-B was also
low in HL-525 cells, x-ray exposure was associated with
transient increases in transcripts for both of these
genes. The kinetics of these increases in fos gene
expression were similar to that obtained for members of
the Jun gene family. Thus, activation of multiple Jun
and fos genes could contribute to diverse nuclear signals
in the response of cells to x-rays.
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Treatment of HL-60 cells and other myeloid
leukemia cells with TPA is associated with induction of
the c-Jun gene. Similar effects were obtained in TPA-
treated HL-205 cells. The response of these cells to TPA
was associated with increases in c-Jun expression that
were detectable at 6 hours and reached maximal levels by
24 hours. In contrast, similar exposures of HL-525 cells
to TPA resulted in an increase in c-Jun expression which
was transient at 12 hours and attenuated compared to that
in the HL-205 link. These findings indicated that HL-525
cells are resistant at least in part to the effects of
TPA on Jun/AP-1-mediated signaling events. Since TPA
activates PKC and translocation of this enzyme is
undetectable in HL-525 cells, the expression of PKC in
the HL-205 and HL-525 lines was compared. HL-60 cells
have been shown to express the a- and i3-PKC isozymes.
Indeed, transcripts for PKCa and PKCi3 were detectable in
HL-205 cells. However, constitutive expression of both
genes was decreased by over 75% in HL-525 cells.
These results suggested that the relative
resistance of HL-525 cells to TPA-induced c-Jun
transcription could be attributable to low levels of PKC
expression. Taken together with the finding that c-Jun -
expression is superinduced by ionizing radiation in
HL-525 cells, these results also suggested that x-ray
induced c-Jun expression may be mediated by events
independent of PKCa and PKC13.
3o In order to further define the mechanisms
responsible for induction of c-Jun expression in HL-525
cells, nuclear run-on assays were performed to determine
the effects of x-rays on rates of c-Jun transcription.
Similar studies were conducted in HL-205 cells for
comparative purposes. The actin gene (positive control)
was constitutively transcribed in HL-205 cells, while
there was no detectable transcription of the i3-globin
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gene (negative control). Similar patterns were observed
in HL-525 cells. Transcription of the c-Jun gene was
detectable at low levels in both cell types. Moreover,
x-ray treatment of the HL-205 and HL-525 lines resulted
in a 6- and 5-fold stimulation in the rate of c-Jun
transcription,respectively. These findings suggested
that other mechanisms, perhaps at the posttranscriptional
level, were responsible for the higher levels of c-Jun
expression following treatment of HL-525 cells.
In order to address this issue, the stability
of c-Jun mRNA after treatment with actinomycin D to
inhibit further transcription was studied. The half-life
of c-Jun transcripts in x-ray-treated HL-205 cells was 31
minutes. In contrast, stability of these transcripts is
irradiated HL-525 cells was increased 3-fold with a half-
life of 106 minutes. Taken together, these results
indicated that tranacriptional activation of the c-Jun
gene by x-rays is similar in both cell types and that
higher levelsof d-Jun expression in the HL-525 variant
are related to differences in posttranscriptional
control.
The finding that ionizing radiation induces
c-Jun expression in the HL-525 cells which exhibit an
alternated response of these gene to TPA suggested that
PKC-independent pathways may mediate this effect of
x-rays. in this context, ionizing radiation is known to
induce the formation of ROIs. Moveover, recent studies
have demonstrated that H202, another agent that acts
through production of ROIs, activates the c-Jun gene in
HeLa cells. The effects of ROIs in cells is counteracted
by the well characterized antioxidant N-acetyl-L-cysteine
(NAC). The exposure of HL-205 cells to 30 mM NAC had
little if any effect on constitutive levels of c-Jun
transcripts. However, this agent inhibited x-ray-induced
increases in c-Jun expression by over 90-%. Similar
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findings were obtained in the HL-525 cells. These
inhibitory effects of NAC on x-ray-induced c-Jun
expression were mediated by a block in transcriptional
activation of the c-Jun gene. While ionizing radiation
alone increased the rate of c-Jun transcription in HL-525
cells by 4-fold, the addition of NAC inhibited this
response by nearly 90%.
In contrast, NAC had no detectable effect on
induction of the c-Jun gene in HL-525 cells by 1-Q-D-
arabinofuranosylcytosine (era-C; data not shown), another
DNA-damaging agent which incorporates into the DNA
strand. The findings indicated that NAC is a specific
inhibitor of x-ray-induced c-Jun transcription,
presumably through its effects of ROIs.
Previous studies have demonstrated that the
cellular response to other diverse claaaea of DNA-
damaging agents, including era-C, W light, alkylating
agents and etoposide, includes the induction of c-Jun
expression. These findings have suggested that DNA
damage per se is the signal responsible for activation of
this gene. This response also appears to involve a
protein kinase down-regulated by prolonged exposure to
TPA. Since ROIs damage DNA, studies were performed to
determine whether the response to these intermediates
also includes a TPA-sensitive mechanism. Treatment of
the HL-525 cells with TPA alone for 36-39 hours had no
detectable effect on c-Jun mRNA levels. However,
pretreatment with this agent blocked the x-ray induced
increases in c-Jun expression by over 75%.
Similar studies were performed with bryostatin,
an agent distinct from TPA which also transiently
activates PKC. Treatment of HL-525 cells with bryostatin
for 36 hours had little if any effect on the induction of
c-Jun transcripts by ionizing radiation. Since H20a also
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acts as a DNA-damaging agent through the production of
ROIs, similar-experiments were performed in HL-525 cells
treated with this agent. H202 transiently induced c-Jun
expression in these cells and this effect was inhibited
by NAC.
Pretreatment with TPA blocked H202-induced
increases in c-Jun expression by 80%, while a similar
exposure to bryostatin had no detectable effect. Taken
together, these findings indicated that agents, such as
ionizing radiation and H202 which produced ROIs, induce
c-Jun expression by a mechanism down-regulated by TPA and
not bryostatin.
NAC counteracts the effects of oxidative stress
by scavenging ROIs and increasing intracellular
glutathione (GSH). Previous studies have demonstrated
that NAC is a potent inhibitor of phorbol ester-induced
activation of the HIV-1 long terminal repeat. This
antioxidant has also been found to inhibit activation of
the nuclear factor KB (NF-rcB) by phorbol esters and other
agents such as H202. The available findings suggest that
ROIS activate NF-xB by induced the release of tl2e
inhibitory subunit IrcB. ROIs are also formed during the
treatment of cells with ionizing radiation. Thus, the
cellular response to this agent may involve the ROI-
induced activation of transcription factors and thereby
longer term e~~ects on gene expression. Indeed, recent
work has demonstrated that DNA binding activity and
expression of NF-xB is induced following exposure to
ionizing radiation. However, ROIs have extremely short
half-lives and while DNA-binding of NF-xB is rapidly
increased in x-ray-treated cells, it is not clear whether
this effect is directly or indirectly related to the
formation of oxygen radicals.
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The results of the present studies suggest that
ROIs also contribute to the activation of c-Jun
transcription by ionizing radiation. This event was
inhibited by NAC. Moreover, H202 increased c-Jun
expression and this effect was similarly inhibited by
NAC. While these findings might reflect a nonspecific
inhibition of c-Jun expression, NAC had no detectable
effect on ara-C-induced increases in c-Jun tranacripts~.
Ara-C damages DNA by incorporation into elongating
strands and is not known to mediate its cytotoxic effects
through ROIs. Of interest, the cellular response to ara-
O also includes activation of NF-xB. Since ROIs damage
DNA in irradiated and H202-treated cells, this damage may
represent the event in common with agents such as ara-C.
Other studies have suggested that the induction
of c-Jun expression by ionizing radiation is mediated by
a PKC-dependent mechanism. Prolonged treatment with TPA
to down-regulate PKC results in marked attenuation of c-
Jun gene by x-rays is also inhibited by H7, a nonspecific
inhibitor of PKC, but not by HA1004, a more selective
inhibitor of cyclic nucleotide-dependent protein kinases.
Consequently, cell lines such as HL-525, which are
deficient in PKC-mediated signaling, would likely respond
to ionizing radiation with an attenuated induction of c-
Jun expression. Indeed, the activation of TNF expression
in x-xay-treated HL-525 cells is diminished compared to
TPA-responsive HL-60 lines. Furthermore, the present
finding that treatment of HL-525 cells with TPA is
associated with an attenuated c-Jun response supports a
defect in PKC-mediated events that control c-Jun
expression. Nonetheless, HL-525 cells responded to
ionizing radiation with an increase in c-Jun expression
which was in fact more pronounced than that obtained in
HL-205 cells.
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The results demonstrate that this increase in
c-Jun mRNA levels is regulated by activation of c-Jun
transcription, as well as a prolongation in the half-life
of these transcripts. Other members of the Jun/fos
family (Jun-B, Jun-D, c-fos, fos-B) were also induced
following x-ray exposure of the HL-525 variant, while
treatment of these cells with TPA resulted in little if
any effect on expression of these genes. These findings
indicated that ionizing radiation increases Jun/foa
expression through signaling pathways distinct from those
activated during induction of these genes in TPA-treated
cells.
The basis for the lack of PKC redistribution in
TPA-treated HL-525 cells is unclear. Nonetheless,
translocation of PKC from the cytosol to the cell
membrane may be necessary for certain TPA-induced
signaling events, such as induction of c-Jun expression.
However, it is not known whether translocation to the
cell membrarie is necessary for activation of each of the
different PKC isoforms. The present results demonstrate
that prolonged treatment of the HL-525 variant with TPA
blocks x-ray-induced increases in c-Jun expression. This
finding lends support to the involvement of a PRC-
dependent mechanism.
The HL-525 variant expresses relatively low
levels of PKCa and PKC(3 compared to HL-205 cells. Low to
undetectable levels of PKCy mRNA were also found in both
the HL-205 and HL-525 lines (data not shown). Thus,
other PKC isozymes which are sensitive to PKC down-
regulation may be responsible for transducing signals
which confer x-ray inducibility of the c-Jun gene.
Alternatively, prolonged TPA treatment could cause down-
35. regulation of other PKC-independent signaling pathways
involved in induction of c-Jun by ionizing radiation. X-
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ray treatment was previously shown to be associated with
activation of a PKC-like activity.
The following methods were used in Example 6.
Cell culture. Clone HL-205 was isolated from
the human HL-6D myeloid leukemia cell line. The phorbol
ester-resistant variant of HL-60 cells, designated
HL-525, was isolated by exposing wild-type cells to low
l0 concentrations of 12-0-tetradecanoylphorbol-13-acetate
(TPA; 0.5 to 3 nM) for 102 passages. These cells were
maintained in RPMI 1640 medium containing 20% fetal
bovine serum (FBS) with 1 mM L-glutamine, 100 U/ml
penicillin and 100 ~.g/ml streptomycin. Irradiation was
performed at room temperature using a Gamma cell 1000
(Atomic Energy at Canada Ltd., Ontario) with a 137Cs
source emitting at a fixed dose rate of.14.3 Gray
(Gy)/min as determined by dosimetry.
Isolation and analysis of RNA. Total cellular
RNA was purified by the guanidine isothiocyanate-cesium
chloride technique. The RNA was analyzed by
electrophoresis through 1% agaroae-formaldehyde gels,
transferred to nitrocellulose filters, and hybridized to
the following 32P-labeled DNA probes: 1) the 1.8-kb
BamHI/EcoRI insert of a human c-Jun gene purified from a
pBluescript SK(+) plasmid; 2) the 1.5-kb EcoRI fragment
of the murine Jun-B cDNA from the p465.20 plasmid; 3)
Jun-D; 4) the 0.9-kb ScaI/NcoI insert of human c-fos gene
purified from the pc-fos-1 plasmid; 5) the 2.0-kb PstI
insert of a chicken LS-actin gene purified from the pAl
plasmid; and 6) the 1.9-kb BamHI/Psti insert of a human
TNF cDNA purified from the pE4 plasmid. Hybridizations
were performed at 42°C for 24 h in 50% (v/v) formamide,
2x SSC, lx Denhardt's solution, 0.1% SDS, and 200 ICg/ml
salmon sperm DNA. The filters were washed twice in 2x
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SSC-0.1% SDS at room temperature and then in O.lx
SSC-0.1% SDS at 60°C for 1 hour.
Nuclear run-on assays., Nucle~.were isolated
from 108 cells and suspended in 100 ~1 glycerol buffer
(50 mM Tris-HC1, pH 8.3, 40% glycerol, 5 mM MgCl2, and
0.1 mM EDTA). An equal volume of reaction buffer (10 mM
Tris-HCl, ph8.0, 5 mM MgCl2, 100 mM KCl, 1 mM ATP, 1 mM
CTP, 1 mM GTP, and 5 mM dithiothreitol) was added to the
nuclei in suspension and incubated at 26°C for 30 min
with 250 ~Ci [a-32P7UTP (3000 Ci/mmol; Dupont, Boston,
MA). The nuclearRNA was isolated as described and
hybridized to_the following DNAs: 1) a PstI digest of
the pA1 plasmid containing a fragment of the chicken fi-
action gene; and 2) a BamHI/EcoRI digest of the
pBluescript SK(+) plasmid containing a fragment of the
human c-Jun gene. The digested DNAs were run in 1%
agarose gels and transferred to nitrocellulose filters.
Hybridizations were performed with 107 cpm of 32P-labeled
RNA/m1 in 10 mM Tris-HC1, ph 7.5, 4x SSC, 1 mM EDTA, 0.1%
SDS, 2x Denhardt's solution, 40% formamide, and 100 ~g/ml
yeast tRNA for 72 h at 42°C. The filters were washed in:
a) 2x SSC-0.1% SDS at 37°C for 30 min; b) 200 ng/ml RNase
A in 2x SSC at room temperature for 5 min; and c) O.lx
SSC-0.1% SDS at 42°C for 30 min.
iPXAATPLE VII
TnnrumawA fTT(' Prnrj~nt~nn gollowina Exposure to Tc99m
Radiation
The production of a marker protein in cells
following transfection and irradiation was investigated
in Human Pancreas Cancer Cell Lines AsPC-1 (ATCC No.
CRL1682), PANC-1 (ATCC No. CRL1469), and MIA PaCa-2 (ATCC
No. CRL1420).
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In this example, Egr-1 enhancer-promoter was
cloned into the XhoI and SacI restriction endonuclease
sites of the luciferase reporter vector (Promega). This
construct is illustrated in FIG. 1. Activation of the
luciferase gene produces luminescence in extracts of
transfected cells.
Procedure for Tra_nsfection
1. Seed 1-2 X 105 cells/flask in 4m1 DMEM
medium supplemented with 2% FBS.
2. Incubate the cells at 37°C in a C02
incubator for 24 hrs.
3. The DNA constructs were transfected into
cells using Lipofectin (GIBCO BRL). DNA (Egr-1-LUC) is
mixed at a concentration of l~Cg/10u1 in Lipofectin
solution (GIBCO BRL).
4. Add the DNA/lipofectin solution to 3 ml of
cell culture medium, mix gently, and incubate at room
temperature for 10-15 min.
5. Place the DNA/lipofectin medium on the
cells and allow to incubate 6 hrs at 37°C in a C02
incubator.
6. Replace the DNA containing medium with 4
ml of growth medium and incubate the cells at 37°C in a
C02 incubator for 48 hrs.
Alternatively, the following procedure may be
employed for stable transfection of cells.
Prepare the following solutions in 12 x 75 mm polystyrene
cubes. Solution A: For each plate to be transfected
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dilute 5-10 ~.l DNA into a final volume of 100 ul Opti-MEM
1 Medium. Solution B. For each plate to be transfected
dilute 5-501 of Lipofectin Reagent into a " final volume
of 100 wl Opti-MEM 1 Medium.
1. Seed 1/2 X 105 cells/60 min. tissue
culture plate-in 4 ml of the appropriate growth medium
supplemented with serum.
2. Incubate the cells at 37°C in a C02
incubator for 18-24 hrs. Cells should be 30-50%
confluent.
3. DNA (Egr-1-LUC) is mixed at a
concentration of l~Cg/10~.1 in Lipofectin solution (GIBCO
BRL). Prepare a tube with 3 ml of cell culture medium.
4. Combine the two solutions, mix gently, and
incubate at mom temperature for 10-15 min.
5. While the Lipofectin Reagent-DNA complexes
are forming, wash cells twice with 2 ml of Opti-MEM 1
Medium.
6.-- Add I_8 ml Opt-Mem 1 Medium to each cube
containing the Lipofectin Reagent-DNA complexes. Mix
gently and overlay onto cells. Swirl the plates gently.
7. Incubate the cells for 5-24 hrs at 37°C in
a C02 incubator.
8. Replace the DNA containing medium with 4
ml of growth medium supplemented with the normal
percentage of serum and incubate the cells at 37°C in a
C02 incubator for another 18 hrs.
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9. Split cells at desired ratios, at least
1:5, into medium designed to select for stable colonies.
Procedure for adding Radioactive Isotope
1. In a twelve-well tissue culture plate,
seed 1 X 104 cell per well in growth medium supplemented
with 2% FBS.
2. Incubate the cells at 37°C in a C02
incubator for 24 hrs.
3. Add Radioactive Isotope (1311 or Tc99m) to
growth medium.
4. The treated cells were harvested from four
wells for each time point.
procedure for LBC Assav
1. Remove the growth media from the cells to
be assayed.
2. Rinse the cells twice in PBS buffer.
3. Add 250 ~1 of Cell Lysis Reagent to cover
the cells.
4. Incubate at 4° C for 20 minutes.
5. Transfer the cells and solution to a
microcentrifuge tube; if necessary store the samples at -
20° C (frozen) until assayed.
6. Mix 20 ~1 of cell extract with 100 ~l of
Luciferase Assay Reagent (Promega).
WO 95131559 PCTIU595/05959
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7. Place the reaction in a luminometer (Lumat
LB 9501 Berthold).
S. Measure the light producec'i for a period of
30 seconds.
S~esults and 77iscusaion
8gr-1-LUC Gene Expression
The stimulation of Erg-I promoters by
radioactive isotopes was first tested using 131I.
Radioactive 1311 was chosen because it is high energy
beta emitting isotope, and also yields accessibility of
radio-labeling to many proteins such as tumor specific
monoclonal antibodies.
The results indicate that this isotope
stimulates Erg-2 activity with results comparable to X-
ray irradiation (4-10 Gy) in all three human pancreatic
cancer cell lines. Results of the human Pancreatic
cancer cell line AaPC-1 are shown in FIG. 2. Utilizing 6
MBq of 1311 exposure for 60 min. results-in 11,706 RLU
(relative light unit), at 3 hrs 35,648 RLU, and at 6 hrs
the highest values of 81,508 RLU, while at 12 hra there
was a decline to 47,490 RLU. All PBS and NaOH controls
ranged from 4224 to 9678 RLU. These results demonstrated
that 1311 can activate the Egr promoter nearly ten-fold
above background.
EXAMPLE vIII
Technetium-99 Induction of Geae Expression
Because of a greater potential for clinical
use, the radioisotope Technetium (Tc99m) was employed as
a gamma source. Utilizing 6 MBq of Tc99m for 60 min.
WO 95131559 PCT/US95/05959
~ 2192813
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resulted in 4,350 RLU, at 3 hrs. 8,441 RLU, at 6 hrs.
27,945 RLU, at 12 hrs 29,288 RLU and 24 hrs. 46,207. All
PBS and NaOH controls ranged from 4224 to 9678 RLU.
These results indicate that Tc99m is capable of inducing
Egr-1-LUC gene expression, and is more gradual and
consistent compared to that obtained with X-rays.
FIG. 3 shows the effects of higher doses of
Tc99m on the human Pancreatic cancer cell line AsPC-1. A
dose of 14.3 MBq is lower than the amount applied for
clinical imaging. Adding 14.3 MBq of Tc99m to culture
plate was associated with an RLU of 8,408 at 24 while
backgrounds were 500 to 600 RLU for this experiment.
Again, these results indicate that Tc99m can activate the
Egr-i promoter to express the LUC gene nearly ten to
fifteen fold above background.
The effects of Tc99m (14.3 MBq) on the all
three human Pancreatic cancer cell lines (ASPC-l, MIA
PaCa-2 and Panc-1) transfected with the are listed in
Table 1. The results indicate that Tc99m stimulates the
Egr-1-LUC gene at 24 hours in all three cell lines. The
increase in gene expression for AsPC-1 was 14-fold, for
MIA PaCa-2 18 times and for Panc-1 was 26 times above
background.
WO 95131559 219 2 813 PCT~S95105959
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Table 5
Effects of Radioactive Isotope Tc99m is Human
Pancreatic Cell Lines oa Egr-1-LUC Geae Expression
Time AsPC-1 MIA PaCa-2 PANC Control
45 min 1,797 2,401 2,895 521
90 min 1,640 3,738 1,345 536
3 hr 1,636 1,690 2,859 603
6 hr 2,920 4,341 1,550 629
12 hr 1,699 4,866 2,079 505
24 hr 8,408 10,934 15,951 612
48 hr 3,990 3,188 4,618 543
EXAMPLE IX
Protocol for Treatmeat of Boae Caacer with X-ray Iaduced
'~'hTR and Techaetium 99m Sodium Methvlene Diphosphonate
For treatment of patients with bone cancer, the
following steps are followed:
1. Prepare a DNA molecule (genetic construct
or vector) comprising a radiation responsive enhancer-
promoter operatively linked to an encoding region that
encodes a polypeptide or any other useful component, such
as an antisenae molecule with a sequence that is the
antiaense version to an oncogene. This is exemplified by
a construct that comprises a CArG domain of an Egr-I
promoter and the gene for tumor necrosis factor.
2. The construct is then administered to the
patient to be treated. This administration may be in the
form of intravenous infusion of naked DNA, or through the
use of viral delivery vectors. Exemplary viral vectors
are retrovirus that is self-inactivating, adenovirus, or
R'O 95131559 PCT/US95/05959
2192813
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adeno-associated virus. If a retrovirus is employed,
lymphokine-activated killer (LAK) cells are first
infected with the retrovirus containing the construct,
then administered to the patient.
3. The radionuclide, such as Technetium-99m
Sodium Methylene Diphosphonate, is given intravenously to
the patient, supplying ionizing radiation preferentially
to areas of altered osteogenesis. The preferred -
intravenous dosage is up to 4 mCi.
* * *
All of the compositions and methods disclosed
and claimed herein can be made and executed without undue
experimentation in light of the present disclosure.
While the compositions and methods of this invention have
been described in terms of preferred embodiments, it will
be apparent to those of skill in the art that variations
may be applied to the composition, methods and in the
steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and
scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically
and physiologically related may be substituted for the
agents described herein while the same or similar results
would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are
deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
CA 02192813 2004-10-22
WO 95131559 PCTlUS95l05959 '
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