Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR MODULATING CYTOKINE
RELEASE IN RESPONSE TO GENOTOXIC AGENTS
CROSS REFERENCE TO RELATED PROVISIONAL APPLICATION
This application claims the benefit under 35 USC ~119(e) of U.S.
Provisional Application No. 60/073,640, filed February 4, 1998.
FIELD OF THE INVENTION
This invention relates to cytokines and genotoxic agents. More
particularly, the invention relates to compositions and methods which can
control, e.g., modulate, the release of cytokines by cells in response to
exposure to one or more genotoxic agents.
BACKGROUND OF THE INVENTION
A. Genotoxic Aeents
As known in the art, genotoxic agents are those chemicals or
treatments, such as heat or radiation, that cause or induce damage to DNA,
either directly or indirectly. Such damage can lead to mutations, the
stoppage of cell cycling, and/or cell death. The damage may be to the
nucleic acid bases or to the sugar-phosphate backbone, or may be single- or
double-stranded breaks in the DNA chain. The mutations, when they
occur, are heritable changes in the DNA sequence or DNA modification
patterns that lead to heritable changes in cell function.
Genotoxic agents are found in the environment as natural
components, such as ultraviolet or ionizing radiation, or as natural
contaminants in food, such as aflotoxin, or as man-made pollution such as
benzo[a]pyrenes in cigarette smoke or industrial emissions. Genotoxic
agents are also used for pharmaceutical and health-related purposes. For
example, many anti-cancer radiotherapies and chemotherapeutics are
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genotoxic agents. Similarly, ultraviolet light, the genotoxic agent to which
humans and animals are most often exposed, can be used for beneficial
purposes such as tanning. In addition, light or ionizing radiation can be
combined with light-sensitizing drugs for dermatological and anti-cancer
treatments and to sterilize blood.
One of the biological responses to genotoxic agents is the cessation of
cell cycling in order to allow time for DNA repair to be undertaken. Such
repair may or may not be successful depending on such factors as the level
of DNA repair enzymes within the cell, the extent and type of DNA damage,
and the like. The cessation of cell cycling serves the important function of
preventing acute damage to the genetic material that would result from cell
division without repair. If the damage is irreparable then the cell invokes
the apoptosis response, that is, pathways of programmed cell death. This
general process of molecular signaling within a cell leading to cessation of
cell cycling and/or apoptosis has been recently reviewed by P. Herrlich, C.
Blattner, A. Knebel, K. Bender and H. Rahmsdorf, "Nuclear and non-
nuclear targets of genotoxic agents in the induction of gene expression.
Shared principles in yeast, rodents, man and plants," Biological Chemistry,
volume 378, pages 1217-1229, 1997; and J.Y.J. Wang, in "Cellular
20 responses to DNA damage," Current Opinion in Cell Biology, volume 10,
pages 240-247, 1998.
B. Lon,~ Term Effects of Genotoxic Agents -- Mutations of DNA
The long-term effects of genotoxic agents are mutations in DNA,
which occur if DNA repair is unsuccessful. These mutations are heritable
changes in the DNA sequence and are an essential element in the process of
carcinogenesis in humans. Not all the steps are understood that lead from
mutation fixation, that is, permanent establishment of DNA changes, to the
development of cancers. It is a characteristic of most human cancers that
there is a long, multi-year, latency period between the time of exposure to a
30 genotoxic agent and the development of cancer. This is true despite the
fact
that the mutations are fixed soon after the genotoxic exposure. Ongoing
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changes to tissue containing mutated cells, called tumor promotion, is
facilitated by the release of cytokines induced by genotoxic agents and leads
to the appearance of tumors.
C. Short Term Effects of Genotoxic Agents -- The Release of
C o ' es
One of the important short-term effects of genotoxic agents in
animals and man is systemic and involves tissue responses that include
erythema, inflammation, fever, antigen-specific immune suppression, and
other physiological effects. These effects are mediated by cytokines (for
review see T. Luger and T. Schwarz, "Epidermal cell-derived cytokines," in
Skin Immune System, ed. J.D. Bos, CRC Press Inc., Boca Raton, Fla., 1990,
pp25?-291). The present invention is concerned with these cytokine-based
responses to genotoxic agents.
As known in the art, cytokines are a large and varied family of
proteins that are released by one cell to influence the activity of other
cells
and/or itself. Cytokine levels are often modulated in response to
perturbations of cell functioning and serve to mediate the response and
homeostasis of tissues, organ systems, and whole organisms following
exposure to genotoxic agents.
Cytokines are not known to be released individually. Rather, they
are released as a group that produces a menu of characteristic responses to
a genotoxic agent. For example, sunburn caused by solar LTV (the genotoxic
agent) simultaneously induces: (a) the expression of interleukin-1 (IL-1)
that causes fever, (b) interleukin-6 (IL-6) that mobilizes liver function, (c)
tumor necrosis factor a (TNFa) that induces inflammation, contributes to
local antigen-specific immune suppression and activates latent viruses, (d)
interleukin-10 (IL-10) that induces suppresser T-cells, (e) a transient
decrease followed by an increase in intercellular adhesion molecule 1
(ICAM-1) that controls infiltration of lymphocytes, and (f) a decline in
interferon y (IFNY) that modulates immune response, as well as many other
cytokines.
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In terms of extracellular signaling, the most important cytokines are
TNFa and IL-1, since they are capable of not only producing physiological
responses themselves but also of inducing the expression of cytokines from
other distant cells. Controlling this cytokine-based extracellular signaling
caused by genotoxic agents is one of the primary objects of the present
invention.
The most common mechanism of action of cytokines is through their
release from a cell and migration to another cell. However, in some cases, a
cytokine can affect extracellular signaling by being displayed on the surface
of the damaged cell. Accordingly, as used herein, the phrases "cytokine
release" and "cytokine production" are intended to include extracellular
signaling both by actual release of a cytokine from a cell and by
extracellular display of a cytokine at the cell surface. These terms are also
intended to be interpreted broadly to include any cellular mechanism which
results in increased extracellular cytokine signaling, including, without
limitation, de novo synthesis, precursor processing, intracellular transport,
extracellular discharge, and surface display of a cytokine.
D. Misunderstandines in the Art Reeardine the Mechanism of
Action of Genotoxic A. eats
(1) The Erroneous Strict Dichotomy Theory
To date, those skilled in the art have believed that damage to DNA is
responsible for the immediate cessation of cell cycling that can lead to the
fixation of mutations in DNA or the apoptotic cell death response following
genotoxic exposure. This theory is reviewed by L.H. Hartwell and M.B.
Kastan in "Cell cycle control and cancer," Science, volume 266, pages 1821-
1828, 1994.
The induction of cytokines, on the other hand, has been thought to
occur as a result of damage to the cell membrane, that is, damage to targets
removed from the cell nucleus. This theory is reviewed by P. Barnes and M.
Karin in "Nuclear factor xB: a pivotal transcription factor in chronic
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inflammatory diseases," New England Journal of Medicine, volume 336,
pages 1066-10?1.
As discussed below, in accordance with the invention, it has been
found that this view, i.e., that there is a strict dichotomy between (a) the
effects of DNA (nuclear) damage that leads to intracellular signaling and
(b) membrane (cytoplasmic) damage that leads to extracellular signaling, is
incorrect. In fact, DNA damage caused by genotoxic exposure also leads to
extracellular signaling through the production of cytokines.
Moreover, and also in accordance with the invention, it has been
found that the production of cytokines by this mechanism requires DNA-
protein kinases. As a result, cytokine production arising from genotoxic
exposure can be controlled by controlling the levels and/or the activity of
DNA-protein kinases. This control mechanism, which is highly effective,
has previously been unknown and therefore unutilized.
(2) The Evidence Which Led to the Erroneous Strict Dichotomy
Theory
The evidence for the conventional belief that the induction of
cytokines is due solely to changes at the cell membrane is very strong. The
pathways leading to activation of several proteins capable of activating
cytokine gene expression have been established. All these pathways begin
with protein kinases that are located at the cell's outer membrane, and
most of these protein kinases are related to the inner cytoplasmic portion of
a transmembrane protein whose external portion is a cell receptor
(reviewed in Y. Devary, ft. Gottlieb, T. Smeal and M. Karin, "The
mammalian ultraviolet response is triggered by activation of Src tyrosine
kinases," Cell, volume ?1, pages 1081-1091, 1992).
These protein kinases are activated by events at the cell membrane,
distant from the cell nucleus, and activate additional cytoplasmic kinases
that culminate in the phosphorylation of systems of gene-activating factors
such as AP-1 and NFxB. DNA binding sites for these modified or released
gene-activating factors are often found in the promoter regions of genes
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coding for cytokines. For example, many of these binding sites are found in
the promoter sequence of the TNFa gene, as described by S. Takashiba, L.
Shapira, S. Amar, and T. Van Dyke, in "Cloning and characterization of
human TNFa promoter region," Gene, volume 131, pages 307-308, 1993. It
has thus been presumed that the binding of these membrane-activated
factors to their binding sites in the promoter regions of the cytokine genes
is
the pathway by which changes at the cell membrane result in changes in
expression of cytokines.
Further evidence for the strict dichotomy theory has come from
recent reports which have demonstrated that genotoxic agents such as UV
can trimerize cell surface receptors directly and activate kinases that begin
cascades presumably leading to signal transduction and gene expression.
See I. Warmuth, H. Harth; M. Matsui, N. Wang and V. De Leo, "Ultraviolet
radiation induces phosphorylation of the epidermal growth factor receptor,"
Cancer Research, volume 54, pages 374-376, 1994; and C. Rosette and M.
Karin, "Ultraviolet light and osmotic stress: activation of the JNK cascade
through multiple growth factor and cytokine receptors," Science, volume
274, pages 1194-1197, 1996.
The strongest evidence, however, has come from the well-studied
system of cytokine release following ultraviolet (UV) irradiation. See T.
Schwarz and T. Luger, "Effect of UV irradiation on epidermal cell cytokine
production," Journal of Photochemistrv and Photobioloev. B: Biology,
volume 4, pages 1-13, 1989. Many cytokines, such as IL-1, IL-6, IL-10, TNF
a, ICAM-1 and IFNy are known to change levels of gene expression
(transcription) and release concurrently following exposure of cells to UV.
Using this system, experiments were performed which were thought
to (a) definitively demonstrate that gene activation is entirely dependent on
events at the membrane and (b) exclude DNA as a target for cytokine gene
activation. See Y. Devary, C. Rosette, J. DiDonato and M. Karin, "NFxB
activation by ultraviolet light is not dependent on a nuclear signal,"
Science, volume 261, pages 1442-1445, 1993 and M. Simon, Y. Aragane, A.
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Schwarz, T. Luger and T. Schwarz, "UVB light induces nuclear factor xB
(NFxB) activity independently from chromosomal DNA damage in cell-free
cytosolic extracts" Journal of Investieative Dermatolosv, volume 102, pages
422-427, 1994.
5 In some of these widely cited experiments, cells were enucleated
(chemically and physically treated to remove the nucleus) and the resulting
cytoplasts were irradiated with LTV. Despite the absence of nuclei (and
genomic DNA), activation of the gene-transcription enhancer NFxB was
detected.
10 Perhaps not surprisingly, these experiments led to the widespread
belief among those skilled in the art that the induction of cytokine gene
expression following genotoxic treatment is independent of DNA damage
and dependent entirely on events at the cell membrane. For example, G.
Vile, A. Tanew-Ilitschew and R. Tyrrell summarized the field in "Activation
15 of NFxB in human skin fibroblasts by the oxidative stress generated by
LTVA radiation", Photochemistrv and Photobioloy, volume 62, pages 463-
468, 1995: "However, LJVC radiation-dependent activation of NFxB is
evident in enucleated cells and t7VB radiation-dependent activation was
shown to occur in nuclear-free cell extracts. Thus it appears that, at least
20 with these two agents, the nucleus is not involved in the activation
p athway."
There has been some evidence presented that DNA damage may be
an initiating event fox induction of cytokine gene expression. These reports
have shown that the dose-response relation for cytokine gene expression is
25 shifted to lower genotoxic doses in cells that are deficient in DNA repair
(see B. Stein, H. Rahmsdorf, A. Steffen, M. Litfin and P. Herrlich, "W-
induced DNA damage is an intermediate step in IJV-induced expression of
human immunodeficiency virus type 1, collagenase c-fos and
metallothionein," Molecular and Cellular Biology, volume 9, pages 5169-
30 5181, 1989), and that increasing DNA repair by delivery of exogenous DNA
repair enzymes reduces cytokine expression or release (D. Yarosh, L. Alas,
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J. Kibitel, A. O'Connor, F. Carrier and A. Fornace, "Cyclobutane pyrimidine
dimers in W-DNA induce release of soluble mediators that activate the
human immunodeficiency virus promoter," Journal of Investigative
Dermatoloev, volume 100, pages 790-794, 1993).
However, prior to the present invention, there has been no
recognized biochemical mechanism by which DNA damage produced by
genotoxic agents could be translated into activation of cytokine gene
expression, a deficit readily identified by those skilled in the art. For
example, Herrlich, Blattner, Knebel, Bender and Rahmsdorf wrote in 1997
in Biological Chemistry, supra, at page 1223: "In conclusion, yeast share a
DNA damage dependent pathway of transcriptional regulation with
mammalian cells. The components are better characterized due to yeast
genetics. The nature of damage recognition and signaling is nevertheless
not understood." Similarly, Stephen Jackson wrote in 1997 in "DNA-
dependent protein kinase," International Journal of Biochemistrv and Cell
Biolo , volume 29, pages 935-938, at page 937: "Furthermore, by
triggering protein kinase phosphorylation cascades, it is possible that DNA-
PK activation could induce cellular DNA damage signaling pathways that
impinge on the transcription, apoptotic and cell cycle machineries.
However, no direct evidence for a role in signaling has so far been
demonstrated." Even more recently, Jean Wang wrote in 1998 in Current
Opinion in Cell Bioloev, supra, at page 242: "The UV response can be
mediated by the plasma membrane. Whether UV-induced lesions (the
cyclobutane dimers and other photoadducts) can generate signals to
activate a Rad3/ATM-like protein in mammalian cells is not known." The
Herrlich and Rahmsdorf laboratory repeated their 1997 view of the state of
the art in 1998 in C. Blattner, Klaus Bender, Peter Herrlich and Hans
Rahmsdorf, "Photoproducts in transcriptionally active DNA induce signal
transduction to the delayed U.V.-responsive genes for collagenase and
metallothionein," Oncoeene, volume 16, pages 2827-2834, 2832, 1998: "It is
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yet unknown how DNA lesions in transcribed genes are processed to elicit
signal transduction."
Without a biochemical mechanism -- the key link -- the data that
DNA damage may be an initiating event for induction of cytokine gene
expression has largely been ignored. Moreover, without knowledge of a
mechanism, control of the mechanism, and thus control of cytokine release,
was clearly impossible. The present invention provides these missing
elements in the art.
SUMMARY OF THE INVENTION
In view of the foregoing, it is an object of the invention to provide
methods and compositions for controlling (modulating) the release of
cytokines upon exposure to genotoxic agents.
A particular object of the invention is to provide such methods and
compositions where the genotoxic agent is ultraviolet light, the most
common genotoxic agent to which humans and animals are exposed, and
the control (modulation) involves reducing the release of cytokines in
response to the genotoxic agent.
It is a further particular object of the invention to provide such
methods and compositions where the genotoxic agent is a chemotherapy or
radiotherapy agent used in cancer treatment and again, the control
(modulation) involves reducing the release of cytokines.
It is a further object of the invention to reduce the susceptibility to
genotoxic agents (e.g., ultraviolet radiation) of individuals who have had an
organ transplant and are receiving immunosuppressive drugs to prevent
rejection of the transplant.
It is an additional particular object of the invention to provide such
methods and compositions where the genotoxic agent is an
immunosuppressive agent, and the control (modulation) involves increasing
the release of cytokines in response to the genotoxic agent.
It is an additional object of the invention to identify individuals who
need to have the level and/or activity of one or more of their DNA-protein
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kinases modulated to modulate the individual's response (sensitivity) to one
or more genotoxic agents. In connection with this object, it is a further
object of the invention to identify the particular DNA-protein kinase or
kinases whose level and/or activity is in need of such modulation.
It is a further object of the invention to provide procedures for
determining whether or not an immunosuppressive agent is being
administered at a level which will provide a desired reduction or increase in
DNA-protein kinase activity sufficient to affect cytokine release in response
to genotoxic agents.
10 It is an additional object of the invention to provide improved assays
for levels of DNA-protein kinase activity.
The invention achieves the foregoing and other objects through the
discovery that DNA-protein kinases, a class of enzymes which recognize
changes, e.g., double-stranded breaks, in DNA and phosphorylate other
15 proteins andlor themselves, play a role in the release of cytokines in
response to genotoxic agents. In particular, it has been discovered that one
or more DNA-protein kinases are required for transcription andlor
translation of cytokine genes after exposure to genotoxic agents.
As used herein, a "DNA-protein kinase" is a member of the family of
20 proteins and protein complexes that respond to changes in DNA structure
or conformation by phosphorylating other proteins and/or themselves. The
characteristics of this family of enzymes has been reviewed by S. Jin, S.
Inoue and D. Weaver in "Functions of the DNA Dependent Protein Kinase,"
Cancer Surveys, volume 29, pages 221-261, 1997.
25 Heretofore these enzymes have only been implicated in (1) the
formation of immunocompetent cells especially those that require gene
rearrangements involving double-stranded DNA breaks, (2) repair of double
stranded breaks in DNA, and (3) regulation of cell cycling and the apoptosis
response following DNA damage. These connections have been reviewed by
30 M. Hoekstra in "Responses to DNA damage and regulation of cell cycle
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checkpoints by the ATM protein kinase family," Current Ouinion in
Genetics & Development, volume 7, pages 170-175, 1997.
Significantly, with regard to W light, the most ubiquitous of the
genotoxic agents, no member of the family of DNA-protein kinases has been
5 related to W induced release of cytokines from cells. As noted above, the
view of those skilled in the art that cytokine gene expression was controlled
by membrane interactions eliminated these DNA-protein kinases from
consideration as participants in the signal transduction cascade leading to
cytokine release.
10 For example, in the above cited review, M. Hoekstra wrote on page
170: "In this article, I discuss the PIK kinase (P13-kinase-related protein
kinase) family members properties as sensors for cell cycle regulation." In
another example, Hosoi et al., in their paper entitled "A
phosphotidylinisitol 3-kinase inhibitor wortmannin induces radioresistant
15 DNA synthesis and sensitizes cells to bleomycin and ionizing radiation,"
International Journal of Cancer, volume 78, pages 642-647, 1998,
demonstrated that wortmannin is an inhibitor of DNA-protein kinases but
ascribed any effects of wortmannin on cytokines to inhibition of the
membrane-bound phosphotidylinisitol 3-kinase (PI-3). At page 645 of their
20 article, these authors wrote: "Wortmannin inhibits cytokine/chemokine-
mediated signal transduction pathways by inactivation of PI3-kinase...."
This state of the prior art is represented schematically in Figure 1,
where the upper dotted box shows the traditional pathway of damage to
DNA leading to activation of DNA-protein kinases, which phosphorylate
25 key protein substrates, which then leads to cessation of cell cycling and
apoptosis due to intracellular signaling. Cytokine release, on the other
hand, was ascribed in the prior art to the pathway of the lower dotted box,
where the genotoxic agent affects the cell membrane andlor cell membrane
receptors, which activate a lipid and protein kinase cascade, which then
30 leads to cytokine gene expression and extracellular cytokine release.
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Figure 2 schematically shows the genotoxic response pathways in
accordance with the invention. A primary difference between Figure 1 and
Figure 2 is that the prior art scheme of Figure 1 did not distinguish
between early and late events, i.e., events occurring within 3 hours of
exposure to a genotoxic agent (early events) and those occurring 6 hours or
more after exposure (late events). This time dimension is an important
aspect of the discovery which forms the basis of the present invention.
An even more fundamental difference between the pathways of
Figure 2 and those of Figure 1 is the fact that Figure 2 includes the
production of cytokines as one of the effects arising from damage to DNA by
genotoxic agents, such production leading to such biological effects as
erythema, antigen-specific immunosuppression, melanogenesis and
tanning. This cytokine-production effect of genotoxic agents is shown at the
lower right of the right hand box of Figure 2, and as indicated in that box,
the effect depends on the action of at least one DNA-protein kinase.
Different types of DNA damage produced by different genotoxic
agents activate different types of DNA-protein kinases. For example,
double-stranded breaks activate the DNA-protein kinases known generally
as DNA-PK and ATM, while UV-induced photoproducts in DNA activate
the DNA-protein kinase known generally as FR.AP. On-going research in
various laboratories throughout the world is expected to identify other
forms of DNA damage and other members of the DNA-protein kinase family
that fit this pattern and the present invention shall be equally applicable to
these subsequent DNA damageIDNA-protein kinase combinations.
As illustrated in Figure 2, the common features upon which the
invention is based are that (1) a DNA-protein kinase is central to the
recognition of altered DNA and (2) the phosphorylation of downstream
proteins results in cytokine gene expression, i.e., cytokine gene
transcription and/or translation. Based on this discovery, cytokine
production in response to genotoxic agents can be controlled by modulating
the levels or activity of one or more DNA-protein kinases.
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Such modulation can be achieved by: (1) inhibiting the activity of the
one or more DNA-protein kinases using one or more inhibitors, (2)
increasing or decreasing the transcription and/or translation of genes
involved in the production of the one or more DNA-protein kinases, andlor
5 (3) enhancing the activity of the one or more DNA-protein kinases by, for
example, providing the kinase with enhanced levels of the damaged DNA
which it responds to and/or with enhanced levels of the substrate which it
phosphorylates.
In one specific application of the invention, compounds that inhibit
10 DNA-protein kinases, such as rapamycin, are used to block induction of
cytokines by genotoxic agents. Other inhibitors of DNA-protein kinases
include pyrophosphate, wortmannin, 6-dimethylaminopurine, the pyridone
derivative OK-1035, and single-stranded DNA, as described by S.P. Lees-
Miller, "The DNA-dependent protein kinase, DNA-PK: 10 years and no ends
15 in sight," Biochemistry and Cell Bioloev, volume 74, pages 503-512, 1996.
The exposure to the genotoxic agent may be unintentional, as in
exposure to environmental pollution or exposure to sunlight during day-to-
day activities. The exposure may also be intentional, and the side-effects
undesirable, as in the case of intentional sun tanning or cancer
20 radiotherapy or chemotherapy. Induction of cytokines may be unwanted
because they are immunosuppressive, inflammatory, activate viruses, cause
unwanted pigmentation, keloids, adhesions or scarring, or other primary or
side-effects of exposure to genotoxic agents.
Specific examples of these aspects of the invention include the
25 incorporation of one or more DNA-protein kinase inhibitors, e.g., rapamycin
or rapamycin-like compounds, in skincare and suncare cosmetics and
pharmaceutical products to prevent unwanted side-effects of sun and
pollution damage to DNA, such as erythema, inflammation, immune
suppression, activation of latent herpes infections, activation of proteases
30 (e.g., collagenase and metallothionein proteases), and skin cancer.
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DNA-protein kinase inhibitors such as rapamycin or rapamycin-like
compounds may also be used in combination with cancer chemotherapy
drugs or radiotheraphy procedures to reduce the side-effects associated with
such treatments, such as, fever, erythema, nausea, vomiting, headaches,
chills and abnormal pigmentation. In these applications of the invention,
the DNA-protein kinase inhibitor or inhibitors are preferably administered
in close temporal proximity to the exposure to the genotoxic agent, such as
sunlight, air pollution, chemotherapy, or ionizing radiation, most preferably
30 minutes to one hour prior to exposure.
The invention can also be used to avoid the most deleterious side
effects of immunosuppressive therapy in transplantation. Organ
transplants have become quite common with the introduction of well-
tolerated immunosuppressive compounds such as cyclosporin. However, a
major side effect of this immunosuppressive therapy has been a rise in skin
15 cancers on sun exposed skin of these patients. See M. Glover, C. Proby and
I. Leigh, "Skin cancer in renal transplant patients," Cancer Bulletin,
volume 45, pages 220-224, 1993.
Because the mechanism by which cyclosporin provides
immunosuppression is by interfering with calcineurin, it does not block the
release of cytokines following LTV-B exposure. See A. Marionnet, Y.
Chardonnet, J. Viac and D. Schmitt, "Differences in responses of
interleukin-1 and tumor necrosis factor a and secretion to cyclosporin-A and
ultraviolet B-irradiation by normal and transformed keratinocyte cultures,"
Experimental Dermatology, volume 6, pages 22-28, 1997. As such,
25 cyclosporin does not have the beneficial effects of a DNA-protein kinase
inhibitor, such as rapamycin, in blocking induction of W-inducible
cytokines in sun-exposed skin.
The current invention teaches that genotoxic-exposed organs in
general, and sun-exposed skin in particular, should be treated with a DNA-
protein kinase inhibitor, such as rapamycin, at a time just prior to or at or
following the time of genotoxic exposure, in order to prevent the induction
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of cancers caused by the genotoxic agent while maintaining the generalized
state of immune suppression required to retain the organ transplant. When
the DNA-protein kinase inhibitor is itself an immunosuppressive agent
capable of suppressing rejection of a transplant (such as rapamycin), the
5 inhibitor may be used with or without other immunosuppressive agents
andlor other types of drugs or treatments. In such cases, the regimen of
administration and dosage levels of the immunosuppressive agent are
selected to take into account both its ability to suppress transplant
rejection
and its ability to suppress the production of cytokines in response to
genotoxic exposure.
Other uses of the invention include compounds that augment the
activity of DNA-protein kinases in order to enhance the induction of
cytokines following genotoxic treatment (hereinafter referred to as "DNA-
protein kinase enhancers"). Thus some genotoxic treatments are used to
15 induce immunosuppressive responses. For example, psoralen-plus-light is
used in skin grafts and psoriasis to induce immunosuppressive cytokines
and suppress antigen-specific autoimmune responses. In view of the
mechanism set forth in Figure 2, compounds that enhance the activity of
DNA-protein kinases can make these immune suppressing therapies more
brisk, stronger, andlor more uniform, thus increasing the efficacy of the
therapy. Thus, in accordance with the invention, compounds that increase
DNA-protein kinase activity are used in conjunction with, or in place of,
genotoxic agents to enhance the desired immunosuppressive response.
For example, in accordance with these aspects of the invention, one
or more compounds that act like tJV-irradiated DNA or short pieces of
duplex DNA and thus can stimulate DNA-protein kinase activity are
applied at the time of the genotoxic treatment, or in place of the genotoxic
treatment, to stimulate the release of immunosuppressive cytokines and
provide relief from diseases related to autoimmune responses. Examples of
such compounds include lipid or liposome bound duplex DNA andlor its
congeners.
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Another application of the invention is to identify individuals who
need to have the level andlor activity of one or more of their DNA-protein
kinases modulated to modulate the individual's response (sensitivity) to one
or more genotoxic agents. In accordance with these aspects of the invention,
the DNA-protein kinase activities and/or levels of a patient suffering from a
disease in which a genotoxic agent produces an insufficient or excessive
cytokine expression are screened to identify the specific DNA-protein
kinases responsible for the patient's symptoms. For example, some forms of
dermatitis, such as atopic dermatitis, lupus erythematosus and porphyria,
are caused by immune system over-reaction to environmental LTV light or
pollution. By screening such patients for DNA-protein kinase activity,
those patients who would benefit from inhibitors of specific DNA-protein
kinases can be identified. In these applications of the invention, cell
extracts are prepared from tissue samples, and a DNA-protein kinase assay
is performed.
The assay can, for example, be of the type described below in
Example 4, in which antibodies are used to immunoprecipitate the DNA-
protein kinase, and then the precipitated DNA-protein kinase is exposed to
varying types of DNA damage together with its substrate, such as p53
protein. By measuring the degree of p53 phosphorylation one determines
the DNA-protein kinase activity. When compared to normal controls, these
studies determine if there is more DNA-protein kinase expressed in the
diseased tissue or if the DNA-protein kinase activity is greater in such
tissue. This information can then be used in diagnosing disease and
selecting therapeutic treatment.
In accordance with various of the foregoing aspects of the invention,
assays for DNA-protein kinase levels/activities are required to determine,
for example, if a drug is being administered at a level sufficient to inhibit
or
induce cytokine release. As illustrated in Example 4 below, the invention
provides effective assays for this purpose in which: {1) a sample of cells is
obtained from the subject, (2) a preparation containing DNA-protein
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kinase(s) is obtained from the sample, (3) the preparation is exposed to
DNA damage of the types) the kinase(s) is (are) sensitive to together with
the appropriate substrates) for the kinase(s), and (4) the level of
substrates) phosphorylation is used as a measure of the level/activity of the
kinase(s).
The foregoing and other aspects of the invention are discussed in
further detail below in connection with the detailed description of the
invention and its preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
10 Figure 1 is flow diagram showing the understanding in the prior art
of the pathways involved in the response of cells to genotoxic agents. As
shown therein, DNA damaged by genotoxic agents was thought to activate
DNA-protein kinases that then produced phosphorylated substrates, which
then triggered intracellular events of cessation of cell cycling and
apoptosis.
I5 Figure 2 is a flow diagram showing the pathways involved in the
response of cells to genotoxic agents in accordance with the present
invention. As shown therein, DNA damaged by a genotoxic agent activates
DNA-protein kinases that then phosphorylate substrates which trigger
extracellular release of cytokines.
20 Figure 3 is a family tree of DNA-protein kinases. The relationships
of the known lipid and DNA-protein kinases are shown based on amino acid
sequence homologies. The proteins are divided into those with lipid kinase
activity and those with protein kinase activity. For each protein the name
and the organization of the protein is shown. The thin solid bars indicate
25 regions of non-homology, while the thick solid bars represent regions where
small subunits, such as the Ku or FKBP proteins, bind. The open thick
bars represent the region of the kinase active site, and the vertically
striped
region is the carboxy terminus that shows homology among the DNA-
protein kinases. For each protein the number of amino acids (where
30 known) and the percent similarity (identical or conserved amino acid
substitutions) to the kinase region amino acid sequence of the ATM protein
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are shown. This figure is drawn from V. Zakian, "ATM-related genes: what
do they tell us about functions of the human genes?" Cell. volume 82, pages
685-687, 1995, and S. Jin, S. Inoue and D. Weaver, "Functions of the DNA
Dependent Protein Kinases", Cancer Surveys, volume 29, pages 221-261.
Figure 4 is a Western blot of TNFa protein expression in human
keratinocytes. Cells from the human line HaCat were irradiated with 200
J/m2 LTV-B or treated with 1 pglml LPS and incubated for 24 hours at
37°C.
Extracts were prepared, electrophoresed in a 15% polyacrylamide gel,
transferred to nitrocellulose, and blotted with antibodies against human
TNFa followed by secondary antibodies linked to horseradish peroxidase
and exposed using the ECL chemiluminescence system. The lanes are: (1)
irradiated; {2) irradiated and treated with 2 ng/ml rapamycin beginning 30
minutes prior to irradiation; (3) treated with LPS; (4) treated with LPS and
rapamycin; (5) authentic TNFa standard.
Figure 5 shows the induction of chloramphenicol acetyltransferase
(CAT) from the TNFa promoter following UV exposure in the presence and
absence of various DNA-protein kinase inhibitors. XP12BE cells, deficient
in nucleotide excision repair, were transfected with the TNFcat transgene
to form the XPTNF2 cell line that expresses CAT from the TNFa promoter.
The cells were treated with DNA-protein kinase inhibitors beginning 30
minutes prior to irradiation and then exposed to 100 J/m2 W-B. After 18
hours, extracts were prepared and assayed for CAT activity using
fluorescent chloramphenicol substrate. The reaction products were
separated by thin layer chromatography and the fluorescence visualized by
W-A light. The samples are: (1) substrate alone; (2) untreated cells; (3)
ITV irradiated cells; (4) cells W irradiated and treated with rapamycin; (5)
cells treated with rapamycin alone; (6) untreated cells; (7) W irradiated
cells; (8) cells UV irradiated and treated with wortmannin; (9) untreated
cells; (10) W irradiated cells; (11) cells UV irradiated and treated with
staurosporine; (12) LPS treated cells; and (13) cells treated with LPS and
rapamycin.
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Figure 6 shows induction of chloramphenicol acetyltransferase (CAT)
from the TNFa promoter following LPS treatment. XPTNF2 cells were
treated as described in Figure 5, except for exposure to 1 p.glml LPS instead
of UV. CAT activity was calculated from the amount of protein extract and
the amount of product formed in 30 minutes, quantified by computerized
image analysis of the fluorescent thin layer chromatography plate.
Figure 7 is a Western blot of p70ssx phosphorylation in human
keratinocytes. Cells from the human line HaCat were irradiated with 200
JIm2 UV-B or treated with 1 ~g/ml LPS and incubated for 24 hours at
37°C.
10 Extracts were prepared, electrophoresed in a 10% polyacrylamide gel,
transferred to nitrocellulose and blotted with antibodies against the serine
and threonine phosphorylated form of human p70ssx followed by secondary
antibodies linked to horseradish peroxidase and exposed using the ECL
chemiluminescence system. The lanes are: (1) unirradiated; (2) irradiated;
15 (3) irradiated and treated with 2 ag/ml rapamycin beginning 30 minutes
prior to irradiation; (4) treated with LPS; and (5) treated with LPS and
rapamycin.
Figure 8 shows FRAP and ATM kinase activity on p53 peptide.
Extracts were prepared from the human keratinocyte line HaCat. The
20 extracts were incubated with antibody against FR.AP (black bars) or ATM
(gray bars) and the bound antibody-kinase product was precipitated with
Protein G agarose beads by centrifugation. The bead-bound FR,AP kinase
was mixed with the FKBP protein and both FRAP and ATM were incubated
with a peptide portion of the p53 protein. To this mixture were added
25 various DNAs and inhibitors, as shown. After 2 hours at 30°C, the
reaction
products were diluted eight-fold, added to ELISA plates and developed with
antibodies against phosphoserine-modified protein and phosphothreonine-
modified protein, and alkaline phosphatase secondary antibodies with
nitrophenyl phosphate substrate. Controls included phosphoserine- and
30 phosphothreonine-bovine serum albumin. Phosphorylated proteins were
measured by optical density at 405 nm.
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Figure 9 is a dose response curve showing the level of inhibition of
TNFcat expression for different concentrations of rapamycin. The CAT
activity was calculated from the fraction of acetylated chloramphenicol as
described in Figure 5 and compared to the activity in the absence of
rapamycin.
The foregoing drawings, which are incorporated in and constitute
part of the specification, illustrate various embodiments of the invention,
and together with the description, serve to explain the principles of the
invention. It is to be understood, of course, that both the drawings and the
description are explanatory only and are not restrictive of the invention.
DETAILED DESCRIPTION OF THE INVENTION
AND ITS PREFERRED EMBODIMENTS
As discussed above, key aspects of the present invention are: (1) the
discovery that DNA-protein kinases play a central role in the release of
cytokines by cells in response to genotoxic agents, and (2) the application of
that discovery to modulate cytokine release through the administration of
DNA-protein kinase inhibitors (if cytokine release is to be reduced) or
enhancers (if cytokine release is to be increased).
A. DNA-Protein Kinases
DNA-protein kinases were originally recognized by animal and
human mutants that lacked activity in one member of this family of
enzymes.
Mice with SCID {severe combined immunodeficiency disease) failed
to generate a complete immune system due to failure of immunoglobulin
gene rearrangements. These mice were found to have a genetic mutation
that inactivated a DNA protein kinase activity essential to a process of
immunoglobulin gene rearrangement involving double stranded DNA
breaks. In this way, an intermediate in development of immune cells,
double-stranded breaks, resembles DNA damage. See the review by S.
Jackson, "DNA-dependent protein kinase," in the International Journal of
Biochemistry and Cell Biol~, volume 29, pages 935-938, 1997.
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Patients with the genetic disease AT (ataxia telangiectasia) have loss
of muscle control (ataxia), and abnormal blood vessels in the eye
(telangiectasia), as well as a predisposition to cancers of the blood such as
lymphomas. These patients have an abnormal gene referred to as ATM (AT
mutant).
When the SCID and ATM genes, respectively, of these diseases were
cloned and their nucleotide sequences analyzed they revealed greatest
homology to 3'-phosphotidylinositol (3'-Pn kinases, a family of lipid, not
protein, kinases, as reviewed in S. Jackson, supra. This at first was quite
10 puzzling, as no lipid kinase activity could be detected biochemically. It
is
now recognized that despite the sequence homology with 3'-PI kinases,
these enzyme are true protein kinases.
DNA-protein kinases are found in all eucaryotic organisms from
yeast to humans. Each cell has more than one type of DNA-protein kinase,
15 drawn from this large family of similar enzymes. The family of amino acid
sequence related DNA-protein kinases includes, among others, the original
DNA-PK~ and Ku subunits related to the SCID mutation, ATM, ATR,
TELL, MEC1, MEI41, FR.AP, TORT, TOR2, and RAD3, as described in Jin,
Inoue and Weaver, 1997, supra. This family of DNA-protein kinases and its
20 sequence homology are shown in Figure 3. Research is on-going to identify
additional members of the family.
DNA-protein kinases are in general comprised of two subunits, one
much larger than the other. The smaller subunit is not shown in Figure 3.
Both the SCID and AT diseases result from mutations in the gene coding
25 for the smaller subunit. These genetic mutants, as well as inhibitors that
block DNA-protein kinase activity, have been used to analyze the functions
which DNA-protein kinases perform.
B. DNA-Protein Kinase Inhibitors
As discussed above, in accordance with certain of its aspects, the
30 present invention is concerned with compounds that interfere with the
activity of one or more DNA-protein kinases, whether by directly
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inactivating the active site or sites of the DNA-protein kinase, by directly
interfering with binding of the DNA-protein kinase to DNA and/or the
substrate or substrates which the DNA-protein kinase phosphorylates, by
competing with the DNA-protein kinase for binding to DNA and/or the
substrate(s), or by interfering with assembly of the subunits of the DNA-
protein kinase.
Examples of DNA-protein kinase inhibitors which can be used in the
practice of the invention include rapamycin, pyrophosphate, wortmannin,
6-dimethylaminopurine, OK-1035, staurosporine, and single-stranded DNA.
These inhibitors, when combined with suitable vehicles known in the art,
can be administered to a subject in various forms, including orally, by
injection, and topically. The level of administration will depend on the
specific subject, inhibitor, and genotoxic agent, and can be determined in
accordance with standard medical practices for therapeutic treatments.
In particular, a level of inhibitor administration is selected which can
be tolerated by the patient and which achieves a reduction in DNA-protein
kinase activity su~cient to reduce the expression of cytokines in response
to one or more genotoxic agents. Cytokine expression levels can be
measured using standard techniques known in the art. Suitable inhibitor
dose levels and administration regimens can thus be selected by monitoring
cytokine expression in response to genotoxic agents as the doselregimen is
varied. For example, levels of TNF-a can be monitored after UV exposure
for subjects receiving one or more DNA-protein kinase inhibitors. It should
be noted that whereas primary cytokines, like TNF-a increase with
exposure to genotoxic agents, secondary cytokines can have more complex
kinetics, e.g., levels of interferon-y can fall and levels of ICAM-1 can fall
and
then rise in response to a genotoxic agent, such as ITV. If such a secondary
cytokine is used to determine doses/regimens for a DNA-protein kinase
inhibitor, these more complex kinetics need to be taken into account, e.g., a
level of inhibitor may be selected which maintains the level of interferon-y
activity at a predetermined value after genotoxic exposure.
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In certain preferred embodiments of the invention, the levellregimen
of inhibitor administration is selected by assaying for DNA-protein kinase
activity, e.g., by assaying for DNA-protein kinase activity using the assays
discussed herein. In accordance with these embodiments, a levellregimen of
inhibitor administration is selected which produces a substantial decrease
(e.g., a decrease of 10%, preferably 50%) in the activity of one or more DNA-
protein kinases. For example, levels of FRAP activity can be monitored and
selected so as to reduce the release of cytokines upon exposure to one or
more genotoxic agents. Again, W exposure can be the genotoxic agent,
with the level of FRAP being selected to minimize TNF-a release.
The DNA-protein kinase inhibitor can be administered either
continuously or, preferably, in connection with the exposure of the patient
to the genotoxic agent(s). Most preferably, the DNA-protein kinase
inhibitor is administered in advance of exposure to the genotoxic agent(s),
e.g., 30 minutes before exposure, with the administration being continued
through exposure and for a period thereafter, e.g., 24 hours after exposure.
Administration at less than before, during, and after exposure can also be
used in the practice of the invention, but is less preferred.
C. Ranamvcin
A particularly important inhibitor of DNA-protein kinase activity is
rapamycin. This inhibitor specifically inhibits the assembly of the large
and small subunits of FRAP, as described by E. Brown, P. Beal, C. Keith, J.
Chen, T. Shin and S. Schreiber, "Control of p70 S6 kinase by kinase activity
of FRAP in uiuo," Nature, volume 277, pages 441-446, 1995. These authors
have identified mutant FRAPs that confer resistance to rapamycin,
demonstrating the specificity of the drug.
In contrast to rapamycin, wortmannin, another important inhibitor
of DNA-protein kinase activity, blocks FR,AP by altering the key amino
acids involved in phosphorylation, as described by M. Wymann, G.
Bulgarelli-Leva, M. Zvelebil, L. Pirola, B. Vanhaesebroeck, M. Waterfield,
and G. Panayotou, "Wortmannin inactivates phosphoinisitide 3-kinase by
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covalent modification of lys-802, a residue involved in the phosphate
transfer reaction," Molecular and Cellular Biolo~v, volume 16, pages 1722-
1733, 1996.
Rapamycin is used clinically today to suppress the immune system
during grafting and transplantation because it blocks the proliferation of -
traditional immune cells, e.g. T cells, as described in M. Suthanthiran and
T Strom, "Immunoregulatory drugs: mechanistic basis for use in organ
transplantation", Pediatric Nenhmloey, volume 11, pages 651-657, 1997. It
has not been used in conjunction with short term exposure to genotoxic
agents, but rather has been used for extended periods in order to retain
transplants. Significantly, patients receiving immunosuppressive therapy,
including patients receiving rapamycin, are explicitly directed to avoid
genotoxic exposure such as sunlight during the entire extended periods of
immunosuppressive therapy which can and usually does continue for years.
See M. Glover, C. Proby and I. Leigh, "Skin cancer in renal transplant
patients", Cancer Bulletin, volume 45, pages 220-224, 1993.
Currently, rapamycin is in the late stages of clinical testing, and is
commercially produced by Wyeth-Ayerst Laboratories, a division of
American Home Products, as Rapamune~.(sirolimus/rapamycin). It is used
either alone or in combinations with low doses of cyclosporin A in treatment
of transplant patients.
When delivered by the oral route, typical dose ranges for rapamycin
are 2 to 5 mg per day. When delivered by the topical route, typical
concentrations are in the range of 0.2% wlv. When delivered by the
intravenous route, maximally tolerated doses are in the range of 25 mg per
kg of body weight. Clinically, doses for immunosuppressive purposes are in
the range of 0.5 to 25 mg per square meter of skin surface per day.
In particular, a loading dose of rapamycin of approximating 21 to 24
mg per m2 body surface area is initially delivered intravenously, as
described by C.G. Groth, C. Brattstrom, and L. Backman, "New trails in
transplantation: how to exploit the potential of sirolimus in clinical
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transplantation", Transplantation Proceedines, volume 30, pages 4064-
4065, 1998, and B. Kahan, "Rapamycin: personal algorithms for use based
on 250 treated renal allograft recipients", ansplantation Proceedines,
volume 30, pages 2185-2188, 1988. This is followed by doses of about 1- ?
5 mglm2 adjusted to yield trough concentrations of 15-30 nglml in whole blood
for 2-3 months, and then further reduced to achieve trough concentrations
of about 10-15 nglml in whole blood thereafter. However, because most
rapamycin is sequestered in red blood cells, the plasma and tissue
concentrations of rapamycin and metabolites are less than 2 ng/ml, as
described by D. Trepanier, H. Gallant, D. Legatt and R. Yatscoff,
"Rapamycin: distribution, pharmacokinetics and therapeutic range
investigations: an update", Clinical Biochemistry, volume 31, page 345-
351, 1998.
Significantly, such tissue levels are insufficient to modulate DNA-
protein kinase activities after genotoxic exposure and thus modulate
cytokine release in response to such exposure. This is because
concentrations higher than 2 ~,glml (or about 2 nlV1) are necessary in order
to achieve substantial inactivation of DNA-protein kinases, specifically,
FR.AP kinase. As shown below in Example 5 below, UV induction of TNFoc
is insensitive to rapamycin at doses less than 2 pg/ml. Thus, the current
practice of administering rapamycin to achieve whole blood levels of 10-30
pg/ml do not achieve rapamycin levels in the plasma or tissues that are
sufficient to inactivate FRAP according to the invention.
To date, the recommended doses of rapamycin have been determined
solely by the ability to prevent rejection of transplanted organs, and not on
the doses required to inhibit FR,AP activity in genotoxically-exposed non-
immune cells. In addition, the doses are prescribed for extended, long-term
use and are not modulated for exposure to genotoxic agents.
In contrast to these previous uses of rapamycin, in accordance with
the invention, rapamycin is used to reduce the release of
immunosuppressive cytokines, such as, IL-1, IL-6, IL-10, ICAM-1 and
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TNFcc, through administration immediately before and shortly after
exposure to a genotoxic agent, such as UV. Moreover, rapamycin is used at
dosage levels high enough to achieve such reduction in the release of
cytokines, i.e., at levels higher than those used to prevent transplant
rejection. Thus, in accordance with the invention, rapamycin is used to
preserve, instead of suppress, the immune system, specifically, to preserve
the immune system after genotoxic exposure.
This idea of using an immunosuppressive drug to protect the immune
system is clearly counterintuitive and runs opposite to the understanding
and practice of the existing art.
D. DNA-Protein Kinase Enhancers
Although the most common applications of the invention will involve
reductions in cytokine release, in some cases it is desirable to increase such
release. In particular, certain therapies involve using genotoxic treatments
to abrogate antigen-specific immune responses {see below).
Enhancers of DNA-protein kinase activity include (1) short segments
(e.g., segments having a length of less than about 25 thousand base pairs)
of double-stranded DNA, i.e., segments of DNA which have ends to which
the DNA-protein kinase can bind, (2) damaged double-stranded DNA which
can be short or long strands, (3) DNA-protein kinase subunits in situations
where such subunits have been depleted, (4) the substrate or substrates
which the DNA-protein kinase phosphorylates, and (5) ATP.
The DNA-protein kinase enhancers can be administered in the same
manner as discussed above in connection with DNA-protein kinase
inhibitors except that, since the enhancers are biologicals, vehicleslcarriers
which preserve the enhancer's biological activity and promote its delivery to
target tissue should be used. For example, in the case of DNA, cationic
lipids can be used to deliver the DNA into cells, and in the case of proteins,
topical administration can be performed using a liposome delivery system.
See Yarosh, U.S. Patent No. 5,190,762.
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Doseslregimens for enhancers can similarly be determined using the
procedures discussed above with regard to inhibitors. Thus, the enhancer
will normally be administered prior to the exposure to the genotoxic agent,
with the administration being continued during the exposure and for a
5 period of time thereafter. Again, monitoring of cytokine andlor DNA-
protein kinase levels can be used to determine appropriate doses and
regimens for particular subjects, enhancers, and genotoxic agents.
E. Modulation of Cvtokine Release
As discussed above, in accordance with the invention, cytokine
10 release following genotoxic exposure can be either decreased or increased
by
inhibiting or enhancing DNA-protein kinase activity. In moat cases, a
decrease in cytokine release will be desired, the most common example
being reducing cytokine release as a result of UV exposure.
Another example where reduction is desired is in connection with
15 cancer chemotherapy and radiotherapy. Many chemotherapy drugs, such
as carmustine and mitomycin C, and many radiotherapies, such as
treatments with x-rays, are genotoxic. In accordance with the invention,
one or more DNA-protein kinase inhibitors are administered to a patient
undergoing such therapy to reduce the side-effects of the therapy.
20 As with other applications of the invention, the one or more
inhibitors are preferably administered in advance of a therapy session, with
the administration being continue for a period after the session, e.g., for a
day or so. Preferably, the DNA-protein kinase inhibitor or inhibitors are
delivered in such a manner that they reach the tissues that have suffered
25 the undesired genotoxic damage from the therapy. In the case of
chemotherapy, the DNA-protein kinase inhibitor or inhibitors are typically
administered in the same manner as the chemotherapy agent, but they may
also be specifically directed to, for example, the gastrointestinal track
(oral
administration), the scalp (topical administration), andlor the site of
30 injection of the chemotherapy agent (topical or subcutaneous
administration) where the side-effects of chemotherapy are most common.
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The same types of specific administration can be used in the case of
radiotherapy.
Patients undergoing transplant rejection therapy are a particularly
in need of protection from genotoxic agents because their immune systems
are suppressed. As discussed above, these patients commonly suffer from
skin cancer on sun exposed skin, the onset of such cancers often being
within a few years of the beginning of therapy. Although DNA-protein
kinase inhibitors, specifically, rapamycin, are used in such transplant
rejection therapy, these uses do not achieve protection from genotoxic
exposure. The present invention teaches that the DNA-protein kinase
inhibitors should be delivered in different forms and in different ways from
the forms and ways in which they have been used in the transplant field.
Specifically, these inhibitors, such as rapamycin, should be given
immediately before and for a short time after genotoxic exposure. In those
cases where the systemic use of immunosuppressive drugs is limited by
toxicity, additional localized application of DNA-protein kinase inhibitors
should be used to alleviate the effects of genotoxic exposure. In particular,
in the case of sun exposure, one or more inhibitors should be applied
topically to those areas of the skin which have been exposed to the sun
light. The doses should be adjusted to achieve inhibition of cytokine release
as a result of DNA damage. This means that for short periods of time
surrounding the genotoxic exposure the dosage level of one or more DNA-
protein kinase inhibitors, such as rapamycin, will be higher than at other
times during transplant rejection therapy.
In some cases, in accordance with the invention, one or more
transplant rejection drugs which are DNA-protein kinase inhibitors, e.g.,
rapamycin and its analogs (such as SDZ R,AD), are used in conjunction with
one or more transplant rejection drugs which are not such inhibitors, e.g.,
cycloaporin A or ascomycin. Since drugs of these two types generally do not
have overlapping toxicities, this combination allows for greater flexibility
in
achieving the goal of immunosuppression, while at the same time, allowing
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for protection of the patient from cytokine production as a result of exposure
to one or more genotoxic agents.
In accordance with these aspects of the invention, the transplant
rejection drug or drugs which are DNA-protein kinase inhibitors are used at
a level which contributes at least some immunosuppression, but more
importantly, are used at a level and/or in a manner which inhibits cytokine
release in response to genotoxic agents. The relative amounts of the two
types of immunosuppressive drugs are determined for each patient based
on the patient's sensitivities to the drugs and on the amount of DNA-
protein kinase inhibitors) required to achieve a desired level of protection
from cytokine release.
Although the most common application of the invention is in
connection witb the reduction of cytokine production, in some cases, the
invention can be used to enhance cytokine production in response to
genotoxic agents. In particular, the invention can be used in the treatment
of a variety of diseases, including autoimmune diseases, which respond to
immunosuppressive genotoxic agents.
For example, enhanced cytokine production can be used in the
treatment of psoriasis, a skin disease characterized by keratinocyte
hyperproliferation and T-cell infiltration into the skin. Two common
genotoxic agents used to treat this disease are coal tar and psoralen-plus-
ligbt. In each case, the genotoxic treatment causes cytokines to be released
which suppress the T-cell activation and thus alleviate the disease
symptoms. In accordance with the invention, DNA-protein kinase
enhancers are used to increase the level of cytokine release in response to
the genotoxic agent.
In particular, one or more DNA-protein kinase enhancers are
administered to the patient just before or, preferably, at the time of
administration of the immunosuppressive genotoxic agent(s). The use of
these enhancers can permit reduction in the amount of genotoxic agent
which needs to be administered and, in some cases, the enhancer(s) alone or
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with only a minor amount of genotoxic agent can achieve the desired
release of immunosuppresive cytokines.
Other diseases which can be addressed in this manner include atopic
dermatitis, lupus erythematosus, arthritis, and porphyria. Each of these
diseases produces inflammation through T-cell activation. Such T-cell
activation can be suppressed by the appropriate immunosuppressive
cytokines, and the DNA-protein kinase enhancers of the invention serve to
augment the formation of those immunosuppressive cytokines upon
exposure to genotoxic agents.
F. Assays for Levels of DNA-Protein Kinase Activity
The discovery that DNA-protein kinases are a central biological link
between genotoxic agents and cytokine release allows those kinases to serve
as measurement points (biological endpoints) for (1) sensitivity of
individuals to genotoxic agents and (2) the effectiveness of modulators of
cytokine release.
Thus, by measuring the level of a DNA-protein kinase activity of an
individual, one can determine the level of sensitivity of that individual to
genotoxic agents) which produce the type of DNA damage to which the
DNA-protein kinase responds. For example, to measure the sensitivity to
UV, one would measure the level of FRAP activity, while to measure the
sensitivity to ionizing radiation (x-rays), one would measure the level of
ATM activity. A high measured level of DNA-protein kinase activity
indicates an individual who will respond to the genotoxic agents) with a
high level of cytokine release, and a low measured level indicates an
individual who will respond to the agents) with low levels of cytokine
release.
Either case is undesirable and its identification allows suitable
diagnostic and/or therapeutic procedures to be undertaken. In particular,
for individuals with high levels of a particular DNA-protein kinase activity,
an inhibitor or inhibitors for that DNA-protein kinase can be used as
described above to modulate the individual's cytokine response to the
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genotoxic agents) associated with that kinase. For individuals with low
levels of DNA-protein kinase activity, further diagnostic procedures can be
undertaken to determine the source of the low level of activity, e.g., a
genetic screening can be performed. Such a DNA-protein kinase assay can
provide a basis for undertaking a screening which otherwise would not be
conducted.
With regard to the effectiveness of modulators of cytokine release, by
conducting an assay for DNA-protein kinase activity, one can determine if a
sufficient amount of a DNA-protein kinase inhibitor or enhancer has been
administered to a patient and adjustments in the dose, mode of delivery, or
delivery schedule can be made as appropriate.
In the case of patients undergoing immunosuppressive therapy using
an immunosuppressive agent which is a DNA-pmtein kinase inhibitor, e.g.,
rapamycin, assays for DNA-protein kinase activity can be used to obtain a
desired level of the immunosuppressive agent. In particular, because of the
complex metabolism and biodistribution of immunosuppressive drugs such
as rapamycin, blood levels of the parent compound may not be
representative of the biological effectiveness of the parent compound and its
metabolites. An assay for DNA-protein kinase activity more directly
monitors the biological effectiveness of the compound and any of its
metabolites.
Assays for levels of DNA-protein kinase activity which can be used in
the practice of the invention include those which employ a radiolabeled ATP
substrate, e.g., ~P-ATP, a peptide substrate, gel electrophoresis, and an
autoradiographic readout. See, for example, D. Price, J. Grove, V. Calvo, J.
Avruch and B. Bierer, "Rapamycin-Induced Inhibition of the 70-Kilodalton
S6 Pmtein Kinase," Science, volume 257, pages 973-9??, 1992.
A preferred assay which avoids the use of radiolabeled reactants and
which can simultaneously process many more samples than the Price et al.
procedure is illustrated in Example 4 below. When used to determine a
DNA-protein kinase level of a subject, the assay comprises the steps of (1)
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a sample of cells is obtained from the subject, (2) a preparation containing
DNA-protein kinase(s) is obtained from the sample using anti-DNA-protein
kinase antibodies, (3) the preparation is exposed to DNA damage of the
types) the kinase(s) is (are) sensitive to together with the appropriate
substrates) for the kinase(s), and (4) the level of phosphorylation of the
substrates) is used as a measure of the level/activity of the kinase(s). The
level of substrate phosphorylation is quantified using standard ELISA
methods with antibodies specific to the substrate when phosphorylated by
the DNA-protein kinase under assay.
The specificity and sensitivity of the assay can be modified by
changing or combining the types andlor concentrations of the anti-DNA-
protein kinase antibodies andlor the substrates used in performing the
assay. In particular, levels of DNA-protein kinase activity for a plurality of
kinases can be determined simultaneously by forming mixtures of anti-
DNA-protein kinase antibodies and substrates in steps (2) and (3),
respectively. The sensitivity of the assay can be modified by increasing
antibody and/or substrate concentrations and/or reaction times.
G. Treatment Compositions
The DNA-protein kinase inhibitors of the invention may be
formulated alone with suitable vehicles or they can be combined with each
other andlor with other pharmaceutical ingredients, e.g., genoprotective
agents which are not DNA-protein kinase inhibitors. One such example is
a combination of rapamycin with one or more sunscreens, e.g., titanium
dioxide, andlor one or more DNA repair enzyme(s), e.g., T4 endonuclease V,
in topical formulations, to be used prior to, during or after exposure to one
or more genotoxic agents, such as solar UV. In the case of DNA repair
enzymes, encapsulation of the DNA repair enzyme in liposomes is a
preferred method of administration. See Yarosh, U.S. Patents Nos.
5,077,211, 5,296,231, and 5,272,079.
The levels of DNA-protein kinase inhibitors in such formulations are
selected as described above, e.g., using a DNA-protein kinase activity assay.
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The levels of the other active ingredients in the formulation will generally
correspond to the levels of the ingredient when used by itself.
In the case of patients undergoing chemotherapy, the DNA-protein
kinase inhibitors of the invention, e.g., wortmannin, can be combined with
a genotoxic chemotherapy agent, e.g., mitomycin C, for contemporaneous .
administration.
The DNA-protein kinase enhancers of the invention may also be
formulated alone with suitable vehicles or they can be combined with each
other and/or with pharmaceutical agents which are not DNA-protein kinase
10 enhancers, e.g., with genotoxic agents. One example is a combination of
damaged DNA, psoralen, and a suitable vehicle for application to the skin
of a psoriasis patient.
Without intending to limit it in any manner, the present invention
will be more fully described by the following examples. The materials and
methods which are common to the examples are as follows.
MATERIALS AND METHODS
Cell Culture
The human immortalized HaCat cell line was from Dr. Jonathan Garlick,
State University of New York at Stony Brook. The XPTNF2 cell line was
20 prepared by transfection of the XP group A SV-40-transformed fibroblast
cell line XP12BE with pCATTr~, as described by J. Kibitel, V. Hejmadi, L.
Alas, A. O'Connor, B. Sutherland and D. Yarosh in "UV-DNA Damage in
mouse and human cells induces the expression of tumor necrosis factor a",
Photochemistrv and Photobiologv, volume 67, pages 541-546, 1998. This
cell line expresses the chloramphenicol acetyltransferase gene from the
mouse TNFa promoter. These transformed cell lines were grown in
Dulbecco's modified Eagle's medium with 10% newborn calf serum and
antibiotics at 37°C in a humidified, 5% COz incubator. Non-transformed
human keratinocytes were purchased from Clonetics Corporation, San
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Diego, California, and grown in the protein-free keratinocyte growth media
supplied with the cells, at 3?°C in a humidified, 5% COa incubator.
Drugs and genotoxic treactment
Rapamycin, wortmannin, staurosporine and lipopolysaccharide (LPS) were
from Sigma Chemical Company. The drugs were prepared at 1,000-fold
concentration and diluted into media just before use. Rapamycin-,
wortmannin- or staurosporine- treated cells were pretreated for 30 minutes
before UV irradiation, and for 18 hours after irradiation. For LPS
treatment, the LPS was added to the cells for one hour at 37°C, and
then
the cells were refed with fresh media for 18 hours.
The W was delivered from a Westinghouse FS-40 unfiltered sunlamp at
3.2 JIm2lsec. For UV irradiation, the media was removed, and the cells
were irradiated and then refed with the same media for 18 hours.
I5 Cell extracts and Western blots
Normal human epidermal keratinocytes were grown to 90% confluence in
10-cm plates, and then treated with drugs and genotoxic agents. After
incubation, the cells were collected with a standard running buffer
containing sodium dodecyl sulfate (SDS), sonicated for 2 seconds with a
Heat Systems Ultrasonic Sonicator at 70% power, boiled in water for 5
minutes and stored at -?0°C.
For Western blots, 10 8.g of cell extracts were mixed with 5 p,l of sample
buffer containing running dye and the mixture boiled for 2 minutes and
loaded into a standard SDS-PAGE gel with a stacking buffer (Biorad mini
Protean II apparatus). For the p70~K Western, a 10% SDS-PAGE was
used, and for the TNFa Western, a 15% SDS-PAGE was used. The gels
were run at 25 mAmps per gel until the running dye reached the bottom of
the gel. The proteins were transferred to Immobilon P membrane by
~
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electrophoretic transblotting in a Semi-phor apparatus (Hoefer Scientific
Instruments, San Francisco, California) at 0.83 mAmpslcm2 for 45 min.
The blots were blocked with 5% non-fat dry milk, and probed with primary
antibody at 4°C overnight. For the p70~K blot the antibody was
specific for
the form phosphorylated at threonine-421 and serine-424 (New England
Biolabs). The blot was then washed and incubated with horse-radish
peroxidase linked anti-rabbit antibody. The blot was developed with the
ECL kit (Amersham) using Hyperfilm ECL for exposure. For the TNFa
blot the monoclonal antibody against human TNFa was from Boehringer
Mannheim Biochemicals. The blots were then washed and incubated with
goat anti-mouse IgG linked to biotin and then avidin-horse radish
peroxidase, and then developed with the ECL kit as above.
TNFcat assay
The CAT assays were performed as described in Kibitel et al., 1998.
Briefly, the XPTNF2 cells were treated with drugs and genotoxic agents
and incubated for 18-24 hours. Extracts were prepared by three rounds of
freezing and thawing and centrifugation, and 50 ~g of each extract was
mixed with 5 nmoles of BODIPY-chlorampenicol (Molecular Probes,
Eugene, Oregon) and 0.5mM acetyl-coA (Sigma Chemical Company). After
30 minutes incubation at 37°C, the reaction products were extracted
with
cold ethyl acetate, and analyzed by thin layer chromatography. The
fluorescent substrate and the acetylation products were visualized by ITV-A,
and the digitized image was analyzed by computerized image analysis to
calculate the fraction of acetylated chloramphenicol and thus the CAT
activity.
Immunoprecipitation and DNA protein kinase activity assay
HaCat cells were grown in 10-cm dishes to near confluence. They were
then sonicated for 10 sec with the Heat Systems Ultrasonicator microtip at
70% power in TGN Buffer (50 mM Tris, pH ?.5, 50 mM glycerophosphate,
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150 mM NaCI, 10% glycerol, 1°/ Tween 20, 1 mM DTT, 0.5 ~l/ml Sigma
P8340 protease inhibitor cocktail, 2 nM microclystin LR). After
centrifugation, the extract was adjusted to 1 mg/ml protein with the same
buffer and stored at -70°C. To assay DNA protein kinase activity, 1 mg
of
HaCat extract was incubated at 4°C far 2 hours with 2 ~g of goat
anti-
FRAP or anti-ATM antibody (Santa Cruz Biotechnology), then mixed with
40 ~l of Protein G PLUS-agarose (Santa Cruz Biotechnology) and rolled at
4°C overnight.
The agarose beads were collected by centrifugation, washed with TGN
buffer, then high salt buffer (100 mM Tris, pH ?.4, 500 mM LiCl), then PK-
buffer (25 mM HEPES-KOH, pH 7.9, 50 mM KCI, 10 mM MgCl2, 1 mM
DTT, 0.5 ~I/ml Sigma P8340 protease inhibitor cocktail, 2 nM microclystin
LR, 200 uM ATP), and resuspended in 50 ~1 PK- buffer. Modifiers of the
reaction included 0.5 pg ~,-DNA or ~,-DNA irradiated with 250 J/m2 W-C .
from a G15T germicidal lamp, and rapamycin at 40 nglml. To initiate the
reaction, 2.6 pg FKBP (Sigma Chemical Company) and was added to the
FRAP reactions, and then 50 ~,g p53 peptide (amino acids 1-393, Santa
Cruz Biotechnology) and 1 nmol ATP were added to all the DNA protein
kinase reactions. After 2 hours incubation at 30°C, the reaction
products
were separated from the agarose beads by centrifugation.
The reaction products were diluted 8-fold into standard ELISA Coating
Buffer (1.59 g/L NazCOs, 2.93 gIL NaHCOs, 0.1 g/L thimerisol), and 200 pl
were placed in wells of a Immulon 2HB 96-well ELISA plate (Dynex
Technologies Inc., Chantilly, Virginia), and incubated at 4°C,
overnight.
The wells were washed and blocked with 5% bovine serum albumin for 60
minutes at room temperature. The wells were then incubated with a
mixture of 2.5 ~1 anti-phosphoserine-BSA antibody linked to biotin and 2.5
~l antiphosphothreonine-BSA antibody linked to biotin (Sigma Chemical
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Company) in 10 ml TBS/NonI (25 mUL 2M Tris pH 8, 30 mUL 5 M NaCl,
0.1% Nonidet P-40) for 60 minutes at room temperature. The wells were
washed and incubated with 100 ~,1 of a mixture of 10 ~1 avadin-alkaline
phosphatase (Sigma Chemical Company) in 10 ml TBS/NonI for 60 minutes
at room temperature. The plates were washed, developed with phospho-
nitrophenylphosphate in 0.1 M diethanolamine pH 10, and read at 405"m by
a microtiter plate reader.
Example 1
This example demonstrates that TNFa is expressed by normal
human keratinocytes after UV exposure and that this expression is
inhibited by rapamycin.
Human keratinocytes from the HaCat cell line were irradiated with
200 JIm2 W-B from an FS40 sunlamp. Parallel cultures were treated with
rapamycin at 2 ng/ml for 30 minutes prior to and then 60 minutes after
irradiation. After 24 hours incubation at 37°C, extracts were prepared
from
these cells by scraping them into a phosphate-saline buffer and sonicating
them with a 2 sec exposure to the microtip of an ultrasonicator {Heat
Systems Ultrasonicator) at 70% maximum energy.
Ten micrograms of protein were then loaded in each well of a 15%
polyacrylamide gel and electrophoresed. The separated proteins were
eluted onto nitrocellulose filter paper by semi-dry transblotting and probed
with antibody against human TNFa. A sample of human TNFa served as a
standard.
As shown in Figure 4, UV induces TNFa, as can be seen in lane 1.
Rapamycin at 2 ng/ml inhibited the expression of TNFa, as seen by the
great reduction in this band in lane 2. In contrast, lipopolysaccharide (LPS)
induces TNFa without DNA damage by binding to the cell surface
membrane receptor CD14, Cells treated with 1 ~g/ml LPS induced TNFa
(see lane 3) and rapamyciu had no effect on this LPS induction of TNFa
(compare lane 3 to lane 4).
_. ____ _ _ ___ _ ~ - _ _ __
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Example 2
This example demonstrates that the induction of TNFa mRNA
expression by W is inhibited by rapamycin.
To illustrate this principle, human cells were used that carried a
transgene composed of the chloramphenicol acetyltransferase (CAT) gene
under the control of the tumor necrosis factor a (TNFa) promoter. This
system has been used to investigate those stimuli that cause transcription
of the TNFa gene. Transcription of the CAT gene is readily measured by a
simple enzymatic assay that measures the formation of acetylated forms of
chloramphenicol by thin layer chromatography. Examples of these assays
are shown in Figure 5.
Substrate alone is shown in lane 1 and background levels of
acetylation by untreated cell extracts is shown in lanes 2, 6 and 9. CAT
expression from the TNFa promoter is reflected by increasing acetylation of
the fluorescent chloramphenicol substrate, resulting in faster migrating
species (from bottom to top) in the thin layer chromatography assay.
TNFoc is induced by genotoxic treatments, such as W (lanes 3,7 and
10), and by non-genotoxic treatments, such as treatment with LPS (lane
12). W induction of TNFa is inhibited by rapamycin (lane 5),
demonstrating that the DNA-protein kinase FRAP is required for
transduction of the signal of DNA damage into expression of the cytokine
TNFa gene.
Rapamycin alone has no effect compared to untreated cells (compare
lane 4 with lane 2). TNFa induction is also inhibited by wortmannin at 500
nM, a dose that inhibits DNA-protein kinases (lane 8), and also by
staurosporine at 200 nM (lane 11), a dose that specifically inhibits serine
phosphorylation, a characteristic type of phosphorylation by DNA-protein
kinases. These results clearly show that UV induction of expression from
the TNFa promoter requires DNA-protein kinases in general, and FR,AP
kinase in specific, and involves phosphorylation at serine resides.
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In contrast, induction of TNFa by the non-genotoxic agent LPS was
not inhibited by rapamycin as shown in lanes 12 and 13 and in Figure 6.
In sum, this example demonstrates that the pathway leading from
W-DNA damage to TNFa transcription requires rapamycin-sensitive
DNA-protein kinases, and that non-DNA damage events at the cell
membrane or elsewhere do not involve such kinases.
Examt~le 3
As known in the art, downstream in the pathway following activation
of the FR.AP kinase is phosphorylation of the p?OS6K kinase, which
phosphorylates ribosomal proteins and alters translation of gene
transcripts. One measure of W-specific activation of the FRAP kinase is
thus phosphorylation of p70S6K. This example uses this measure to
demonstrate such activation.
Human keratinocytes were pre-treated with 2 ng/ml rapamycin, then
treated with W-irradiation or LPS and extracted as described above (see
Example 1 and Materials and Methods). The extracts were electrophoresed
in a 10% polyacrylamide gel and then probed with antibodies specific for
the serinelthreonine ghosphorylated forms of p70S6K in a Western blot. As
a loading control, the polyacrylamide gel was stained with Coomassie blue
to identify total protein loaded in each lane.
As shown in Figure ?, LTV irradiation increased phosphorylation of
p70~K (compare lanes 1 and 2) and this phosphorylation was blocked by
pre-treatment with rapamycin (lane 3). In contrast, the LPS induced
phosphorylation of p70S6K (lane 4) was comparatively insensitive to
rapamycin (compare lanes 4 and 5). The loading control bands shown at
the bottom of this figure demonstrate the equivalent loading of protein in
the gel.
Example 4
This example demonstrates the direct activation of DNA-protein
kinase activity by damaged or broken DNA.
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ATM or FRAP in extracts of the human keratinocyte cell line HaCat
were immunoprecipitated by incubation with (1) antibodies against either
ATM or FR,AP and (2) immunoprecipitating anti-antibodies linked to
agarose beads.
The bound ATM or FR,AP was collected by centrifugation, and then
mixed with a phosphorylation substrate polypeptide derived from the p53
protein, and in the case of FR.AP, with its small subunit protein FKBP. In
some cases bacteriophage ~. DNA, W-irradiated DNA, or rapamycin were
added to the reactions. Adenosine triphosphate was then added and the
reactants incubated for 2 hours at 30°C. The reaction products were
then
bound to an ELISA plate and probed with antibodies specific for
phosphoserine and phosphothreonine. The binding of these antibodies was
detected by (1) secondary antibodies linked to alkaline phosphatase and {2)
nitrophenyl phosphate substrate. The resulting yellow color was measured
by optical density at 405 nm using a multiwell plate reader.
The results are shown in Figure 8. As shown therein, FR.AP
phosphorylation of the p53 peptide was stimulated by the addition of
~. DNA, whose short size resembled mammalian DNA with many broken
ends (compare the FRAP/FKBPIp53 bar with the +DNA bar). However, the
FR.AP activity was much greater when the DNA that was added was first
irradiated with IJV to induce photoproducts (compare the +DNA bar with
the +L1V-DNA bar). This LTV-DNA enhanced phosphorylation was
completely abrogated by coincubation with 2 ng/ml rapamycin (+rapamycin
bar). Similarly, ATM phosphorylated the p53 peptide (ATM/p53 bar), and
its phosphorylation was stimulated by addition of ~, DNA (+DNA bar),
whose short size resembles broken DNA.
Example 5
This example shows that rapamycin's ability to reduce induction of
TNFa by W is dose dependent.
XPTNF2 cells irradiated with 100 JIm2 LJV-B to induce expression of
the CAT gene from the TNFa promoter were incubated with increasing
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concentrations of rapamycin. The levels of inhibition of expression from the
promoter are shown in Figure 9.
As can be seen therein, at levels of rapamycin of less than 2 nglml,
the inhibitory effect of this DNA-protein kinase inhibitor was undetectable.
The effect became detectable at 2 ng/ml, and higher doses were increasingly
inhibitory.
Although specific embodiments of the invention have been described
and illustrated, it is to be understood that modifications can be made
without departing from the invention's spirit and scope.
The contents of the various literature citations referred to above are
hereby incorporated herein by reference.
