[GRAPHIC]
US005482833A
United States Patent Patent Number: 5,482,833
Pero et al. Date of Patent: Jan. 9, 1996
_______________________________________________________________________________
TEST TO DETERMINE PREDISPOSITION OR SUSCEPTIBILITY TO DNA ASSOCIATED DISEASES
Inventors: Ronald W. Pero, New York; Daniel G. Miller, Scarsdale, both
of N.Y.
Assignee: Preventive Medicine Institute, New York, of N.Y.
Appl. No.: 430,326
Filed: Apr. 26, 1995
Related U.S. Application Data
Continuation of Ser. No. 869,823, Apt. 15, 1992, abandoned, which is a
continuation of Ser. No. 333,841, Apr. 3, 1989, abandoned, which is a
continuation of Ser. No. 820,203, Jan. 17, 1986, abandoned.
Int. Cl(6)...............................................C12Q 1/68; A61K 48/00;
A61K 49/00
U.S. Cl...................................................435/6; 435/4; 435/15;
435/29; 435/173.1; 435/820; 436/501; 436/63;
436/64; 436/813; 436/815; 514/2; 514/44;
536/22.1; 536/25.3; 935/77
Field of Search...............................................435/4, 6, 15, 29,
435/173.1, 820; 436/501, 63, 64, 813, 815;
514/2, 44; 536/22.1, 25.3; 935/77
References Cited
U.S. PATENT DOCUMENTS
4,585,736 4/1986 Dolbeare et al. ....................................435/6
FOREIGN PATENT DOCUMENTS
3336786 3/1985 Germany............................................435/15
OTHER PUBLICATIONS
Pero et al. (1985) Carcinogenesis, vol. 6, No. 7, pp. 1055-1058.
Pero et al. (1985) Mutation Res., vol. 142, pp. 69-73.
Biological Abstracts 78 (7) 55503.
Biological Abstracts 80 (2) 13747.
Chemical Abstract 105: 19647.
Chemical Abstract 105: 95311.
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Davis et al., Microbiology including Immunology and Molecular Genetics, 3rd
ed., pp. 186-189 (1980).
Robbins, Pathologic Basis of Disease, pp. 148-152 (1974).
Simic et al., Mechanisms of DNA Damage and Repair, pp. 164-165 and 357-363
(1986).
Weisburger, J. "Bioassays and Tests for Chemical Carcinogens," Chemical
Carcinogens, 1976, chapter 1, pp. 1-23.
Primary Examiner -- W. Gary Jones
Assistant Examiner -- Ardin H. Marschel
Attorney, Agent, or Firm -- Cooper & Dunham
ABSTRACT
Cellular DNA repair enzyme activity has been found to be an indicator of
susceptibility or predisposition of an individual to DNA associated diseases.
The activity of the enzyme adenosine diphosphate ribosyl transferase (ADPRT)
has been found to be a good indicator as to the susceptibility of an individual
to DNA associated diseases, such as cancer.
23 Claims, 1 Drawing Sheet
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TEST TO DETERMINE PREDISPOSITION OR
SUSCEPTIBILITY TO DNA ASSOCIATED DISEASES
This is a continuation of application Ser. No. 869,823 filed Apr. 15,
1992, which is a continuation of Ser. No. 333,841 filed Apr. 3, 1989, which is
a continuation of application Ser. No. 820,203, filed Jan. 17, 1986 (all now
abandoned).
BACKGROUND OF THE INVENTION
This invention relates to DNA associated diseases, such as cancer. In one
aspect of this invention, this invention relates to a method for determining
the level of cellular DNA repair enzyme activity. In another aspect, this
invention relates to a method for monitoring the level of activity of cellular
DNA repair enzymes in response to a stress. In another aspect, this invention
relates to a method for screening individuals for the predisposition to cancer
or other diseases associated with DNA damage. In yet another aspect, this
invention relates to a method for screening therapeutic agents which may be
useful for treating individuals with a predisposition to or a disease
associated with DNA damage.
Most living cells possess systems for recognizing and eliminating DNA
damage. As used herein, the term "DNA damage" refers to strand breaks,
dimerization, unpaired bases, modified bases, conversion of one base into
another resulting in unpaired bases, chromatin unwinding or other
modifications, etc. For example, E coli possesses a variety of enzymes for
responding to DNA damage, such as enzymes of the SOS repair system and the
various Rec proteins. These enzymes, and others, respond to DNA damage caused
by U.V. radiation, chemical mutagens and the like. However, little is known
about the mechanism by which the repair systems are activated by DNA damage.
In addition to the prokaryotic enzymes discussed above, eucaryotic and
mammalian cells are also known to possess DNA repair enzymes. These enzymes are
important in controlling diseases associated with DNA damage, as is clearly
shown in the disease Xeroderma pigmentosum (Xp). This recessive disease results
in hypersensitivity to sunlight, particularly to ultraviolet radiation. The
disease is the result of a faulty excision repair system. Fibroblasts from Xp
patients are deficient in the ability to excise and correct thymine dimers and
other adducts. The deficit has been shown to be in enzymes that function at the
excision step of repair. Another disease correlated with faulty DNA repair is
Bloom's disease, in which an increased frequency of chromosomal aberrations is
seen.
DNA repair synthesis has been studied in cancer patients, see particularly
the article by R.W. Pero et al. entitled "Reduced Capacity for DNA Repair
Synthesis in Patients with or Genetically Predisposed to Colorectal Cancer",
JNCI, Vol. 70, No. 5, pp. 867-875, May, 1983, and the article by R.W. Pero et
al. entitled "Unscheduled DNA Synthesis in Mononuclear Leukocytes from Patients
with Colorectal Polyps", Cancer Research, Vol. 45, pp. 3388-3391, July, 1985.
These articles are of interest to the practices of this invention as applied to
the measurement of the activity of DNA repair enzymes as being an indicator of
the predisposition or susceptibility of an individual to colorectal cancer.
DNA damage, as indicated herein, may be caused by a number of agents. For
example, oxygen supplied at concentrations greater than those of normal air has
long been known to damage plants, animals and aerobic bacteria, see J.D.
Balantine, Pathology of Oxygen Toxicity, 1982, Academic Press, New York. It has
been proposed that many of the damaging effects of O2 could be attributed to
the formation of O2 radicals, see D.L. Gilbert (ed) Oxygen and Living
Processes: An Interdisciplinary Approach, 1981, Springer Verlag, New York. The
reactive oxygen species are superoxide, H2O2 and a hydroxyl radical. These are
generated in vivo, i.e. endogenously in the body, as a consequence of normal
metabolism; see B. N. Ames, Science, 221: 1256-1264, 1983. The oxidation of
certain cellular components by these oxygen species could, in turn, contribute
both to aging and to age-dependent diseases, such as cancer; see P.A. Cerutti,
Science, 227:375-380, 1985.
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H2O2 is produced by all viable cells; see Romasarma, Biochem Biophysica
Acta, 694:69-93, 1982, and it can be both a mutagen/carcinogen or promoter; see
Troll & Wiesner, Ann. Rev. Pharmocol. Toxicol. 25:509-528, 1985, depending upon
the cell type. The molecular response of a cell to stress whether it be induced
by hyperthermia or by H2O2 is very similar; see Christman et al, Cell
41:753-762, 1985.
The practice of this invention in one embodiment employs H2O2 as an agent
for oxidative stress to produce cellular DNA damage, thereby to induce a
cellular DNA repair enzyme response, such as a response of the DNA repair
enzyme adenosine diphosphate ribosyl transferase (ADPRT).
ADPRT is a nuclear enzyme which covalently attaches ADP-ribose moieties
derived from NAD to chromatin proteins; see Hayoishi and Ueda, Ann. Rev.
Biochem. 46:96-116, 1977 and Purnell et al., Biochem. Soc. Transa. 8:215-227,
1980. The enzyme is dependent on DNA and is strongly stimulated by DNA-strand
breaks; see Halldorsson et al., FEBS LETT. 85:349-352, 1978; Benjamin and Pill.
J. Biol. Chem. 255:10493-10508, 1980; Cohen and Berger, Biochem. Biophys. Res.
Commun. 98:268-274, 1981. Although the role of ADPRT in cells is not fully
understood, convincing data have been reported in its involvement in DNA
repair; see Durkacz et al., Nature 283:593-596, 1980; Zwelling et al., Biochem.
Biophys. Res. Commun. 104:897-902, 1982; Althaus et al, Biol. Chem.
257:5528-5535, 1982; Chreissen and Shall, Nature 296:271-272, 1982 and Pero et
al., Chem. Biol. Interact. 47:265-275, 1983. The involvement of this enzyme in
cellular differentiation is reported by Farzaneh et al., Nature 300:262-266,
1982; Johnstone and Williams, Nature 300:368-370, 1982); and Pero et al.
Carcinogenesis 6:1055-1058, 1985. The involvement of this enzyme in gene
expression is mentioned by Althaus et al., Nature 300:366-368, 1982 and in
connection with longevity by Pero et al., Mutation Res. 142:69-73, 1985. All
these cellular events are important to the process of carcinogenesis and thus
are important potential regulators of individual sensitivity or risk to develop
cancer.
Although, as indicated herein, H2O2 has been known to be produced by
viable cells and to have both carcinogenic and promoting properties, it has
never been shown to directly activate ADPRT in eucaryotic cells. Moreover,
interindividual variation in stress-induced ADPRT, such as oxidative, e.g. H2O2
stress-induced ADPRT, was not known nor was any link to cancer or DNA
associated disease susceptibility previously known. In the development of this
invention there has been observed in 100 uM H2O2-induced ADPRT measured values,
a greater than 50-fold variation in the cell population tested.
The disclosures of the above-identified publications are herein
incorporated and made part of this disclosure.
It is an object of this invention to provide a method whereby individuals
with a predisposition to diseases associated with DNA damage could be
recognized. Upon recognition, such individuals might then beneficially receive
more frequent diagnostic examinations, pretreatment with drugs and the like.
It is also an object of this invention to provide a method for measuring
the activity of DNA repair enzymes, particularly ADPRT activity.
It is also an object of this invention to provide a method for screening
agents for potential therapeutic value for the treatment of individuals
predisposed to diseases associated with the activity of DNA repair enzymes,
such as the activity of ADPRT.
How these and other objects of this invention are achieved will become
apparent in the light of the accompanying disclosure made with reference to the
accompanying drawing which graphically illustrates the relationship of cancer
patients and those with a positive family history of cancer or a negative
family history of cancer with the measured ADPRT activity of such individuals.
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SUMMARY OF THE INVENTION
In accordance with this invention a method has been developed
to determine the predisposition or susceptibility of an individual to DNA
associated disease. This method involves subjecting the cellular DNA of the
individual to stress to induce or bring about DNA damage. The activity of the
cellular DNA repair enzymes, particularly ADPRT activity, is then measured and
the measured enzyme activity is then compared against a given or predetermined
value to determine the relative predisposition or susceptibility of the tested
individual to DNA associated disease, such as cancer. A measured value of
cellular DNA repair activity, such as ADPRT activity, below said given value
would indicate a greater susceptibility or predisposition to DNA associated
diseases compared to an individual having a measured value above said given
value.
BRIEF DESCRIPTION OF THE DRAWING
The Figure illustrates patient or family history relationships of cancer
to measured ADPRT activity.
Section (A) of the Figure is a graph illustrating the relationship of
cancer patients with the measured ADPRT activity of such individuals;
Section (B) of the Figure is a graph illustrating the relationship of
persons with a positive family history of cancer with the measured ADPRT
activity of such individuals; and
Section (C) of the Figure is a graph illustrating the relationship of
persons with a negative family history of cancer with the measured ADPRT
activity of such individuals.
DETAILED DESCRIPTION OF THE INVENTION
The method of this invention for identifying an individual with a
predisposition or susceptibility to diseases associated with the activity of
DNA repair enzymes comprises isolating a cell, such as a mononuclear leukocyte
or an epithelial or a fibroblast cell from the individual to be tested,
stressing the cell to damage the cellular DNA structure to produce a stressed
cell containing damaged cellular DNA and then determining a value for the ADPRT
activity in the stressed cell. The measured value of ADPRT is then compared to
the value of ADPRT activity in a cell from a so-called normal individual or a
given value of ADPRT activity. A significant decrease in the activity of the
enzyme ADPRT in the stressed cell would indicate a predisposition of the
individual from whom such cells were taken and tested to DNA associated
disease, such as cancer, relative to another individual whose cells, when so
tested, show a higher measured DNA repair enzyme activity, such as ADPRT
activity.
A variety of agents associated with causing DNA structural damage may be
used in the practice of this invention to stress the cell to be tested to
induce DNA damage. Those agents which cause oxidative stress are preferred,
such as hydrogen peroxide, cumene hydroperoxide and benzoyl peroxide. Other
agents usefully employed include xanthine, xanthine-oxidase, phorobol diesters
and bleomycin. Radiation, such as ultraviolet radiation, gamma radiation or
x-ray radiation, may also be employed to induce cellular DNA damage.
As indicated hereinabove, in the practices of this invention as an
indicator of DNA repair enzyme activity it is preferred to measure the value of
ADPRT activity in a cell containing damaged DNA. In the preferred practice of
this invention the activity of ADPRT is measured as counts per minute (cpm) of
3H-NAD per 1 x 106. The cells to be tested, as indicated herein, could be
isolated from a variety of tissues. Presently preferred cells for testing in
accordance with the practice of this invention are the mononuclear leukocytes,
fibroblasts or epithelial cells but other DNA containing cells may also be
employed. The activity of ADPRT in the cell is determined by contacting the
cell with hydrogen peroxide to produce a stressed cell
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containing damaged DNA, followed by measuring the activity of the ADPRT in the
stressed cell to obtain a value for ADPRT activity. The value so obtained is
compared with a predetermined value of at least about 1200 cpm 3H-NAD per 1 x
106 cells, such as a value in the range of 3500-4500 cpm 3H-NAD per 1 x 106
cells. A significant difference of the measured value from the predetermined
value would indicate that the cell so tested provides a modified cellular ADPRT
activity.
In another embodiment of the practice of this invention there is presented
a method for screening therapeutic agents for the treatment of individuals
predisposed to diseases associated with DNA or the activity of DNA repair
enzymes. The method comprises isolating a cell from a predisposed individual,
stressing the cell with an agent to produce cellular DNA damage and with a
therapeutic agent to produce a resulting stressed and treated cell. The
activity of the DNA repair enzymes, such as ADPRT activity in the stressed and
treated cell, is then determined to obtain a value of ADPRT activity and this
value is compared against a predetermined value or a value obtained from a
stressed cell which has not been treated with said therapeutic agent. If the
ADPRT activity value of the stressed but untreated cell is less than the ADPRT
value of the stressed and treated cell, this result would indicate that the
tested therapeutic agent may be effective for the treatment of the predisposed
individual.
The following is an example illustrative of the practice of this
invention:
EXAMPLE
ADPRT is the only known biological reactant that consumes the ADP moiety
of NAD. Accordingly, if NAD is radiolabeled in the adenine moiety, the
trichloracetic acid (TCA)-precipitable radioactive counts would reflect ADPRT
activity via (ADP-ribose) polymerization to chromatin proteins. The protocol
used to measure ADPRT activity is a modification of the procedure of Berger
(D.M. Prescott ed.), Methods in Cell Biology 20:325-400, 1978 and is published
in detail by Pero et al. in Chem Biol Interactions 147, 265-275, 1983.
ADPRT activity was measured as follows: Peripheral blood samples (20 ml)
were collected by venous puncture into heparinized tubes (10-20 USP units/ml)
from 24 individuals with diagnosed cancer of the lung, colon or pancreas, from
25 individuals with at least a first degree relative having either lung, colon
or pancreas cancer and from 21 individuals with no family history of cancer.
The mononuclear leukocyte fraction was isolated from the whole blood samples by
density gradient centrifugation at 400XG for 20 minutes after layering on top
of an Isopaque Ficoll cushion at a density of 1.077 gm/ml.
Duplicate cultures of 1-5x106 cells were incubated with or without either
a standardized dose of either 100 uM H2O2 in 1.0% autologous plasma
supplemented physiological saline for 60 minutes at 37(degree) C. The resulting
mixtures were removed at the end of the incubation period by centrifugation.
The cells (+) and (-) H2O2 treatment were permeabilized, adjusted to 0.5x106
cells per treatment and ADPRT activity estimated after 15 minutes at 30(degree)
C. in a reaction mixture containing 175 uM (161.6 uCi/mmol) of [3H]
adenine-labeled NAD. The data were recorded as TCA precipitable [3H]-NAD per
1x106 cells which were collected onto nitrocellulose filters. The (-) H2O2
ADPRT values were then subtracted from the (+) H2O2 values.
The results of these tests are graphically indicated in the accompanying
drawing. As shown in the drawing, it can be seen that the frequency
distribution of individual values for 100 uM H2O2 induced ADPRT varied in
accordance with either the occurrence or the genetic predisposition to develop
cancer. For example, when 100% of the values for the cancer patients were below
ADPRT values of 2300, 72% of the individuals with a positive family history of
cancer were below 2300 while the corresponding value for the group with no
family history of cancer was 38%, all as indicated in the accompanying drawing.
These results, as shown and quantified in the drawing, can usefully predict an
individual's risk or predisposition or susceptibility to DNA associated
disease, such as cancer.
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Although in the practices of this invention it is preferred to measure
directly ADPRT activity by the technique disclosed in the Example described
hereinabove involving the measurement of TCA precipitated radiolabeled protein,
the measurement of ADPRT activity can also be carried out indirectly through
its effect or influence upon other DNA repair enzymes, such as topoisomerase,
ligase and endonuclease and other related DNA associated enzymes, such as
polymerase and exonuclease. The activities of these enzymes as affected by the
activity of ADPRT can be separately measured by suitable techniques involving,
as may be appropriate, radiolabeled components or monoclonal antibodies to
components or products of the activity of such enzymes, particularly as may be
influenced or effected by the activity of ADPRT.
As will be apparent to those skilled in the art in the light of the
foregoing disclosure, many modifications, alterations and substitutions are
possible in the practices of this invention without departing from the spirit
or scope thereof.
What is claimed is:
1. A method for identifying an individual with a predisposition to
diseases associated with the activity of DNA repair enzymes which comprises
stressing cells of said individual to produce stressed cells containing damaged
DNA, thereby to cause induced activity of adenosine diphosphate ribosyl
transferase (ADPRT) in the stressed cells, determining a value for the induced
activity of the ADPRT in the stressed cells, and comparing the value so
determined with a reference value of the activity of ADPRT to ascertain whether
said value so determined is higher or lower than said reference value, a
determined value lower than said reference value identifying said individual as
having said predisposition.
2. A method in accordance with claim 1 wherein the cell is subjected to
stress by exposure to radiation.
3. A method in accordance with claim 1 wherein said cells are stressed by
ultraviolet radiation.
4. a method in accordance with claim 1 wherein said cells are stressed by
x-ray radiation.
5. A method in accordance with claim 1 wherein the cells are subjected to
oxidative stress.
6. A method in accordance with claim 5 wherein said oxidative stress
involves exposure to hydrogen peroxide.
7. A method in accordance with claim 5 wherein said oxidative stress
involves exposure to cumene hydroperoxide.
8. A method in accordance with claim 5 wherein said oxidative stress
involves exposure to benzoyl peroxide.
9. A method in accordance with claim 5 wherein said oxidative stress
involves exposure to xanthine-xanthine oxidase.
10. A method in accordance with claim 1 wherein the cells are subjected to
stress by contact with phorbol esters.
11. A method in accordance with claim 1 wherein the cells are subjected to
stress by contact with bleomycin.
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12. A method in accordance with claim 1 wherein the activity of ADPRT is
measured as cpm 3H-NAD per 1x106 cells.
13. A method in accordance with claim 1 wherein said cells are mononuclear
leukocytes.
14. A method in accordance with claim 1 wherein said cells are
fibroblasts.
15. A method in accordance with claim 1 wherein said cells are epithelial
cells.
16. A method for screening therapeutic agents suitable for the treatment
of individuals predisposed to diseases associated with DNA which comprises
stressing cells of an individual to produce DNA damage, thereby to cause
induced activity of adenosine diphosphate ribosyl transferase (ADPRT) in the
stressed cells, contacting or treating the resulting stressed cells with a
therapeutic agent to produce resulting stressed and treated cells, determining
a value for the induced activity of the ADPRT in the resulting stressed and
treated cells, and comparing the value so determined with a predetermined value
to evaluate the effectiveness of the therapeutic agent for the treatment of
said individual by ascertaining whether said obtained value is higher or lower
than said predetermined value, an obtained value higher than said predetermined
value indicating that the therapeutic agent is effective for the treatment of
said individual.
17. A method of testing an individual for a predisposition to a disease
associated with DNA damage, comprising stressing DNA-containing cells of the
individual to produce stressed cells having damaged cellular DNA, thereby to
cause induced activity of adenosine diphosphate ribosyl transferase (ADPRT) in
the stressed cells, determining a value for the induced activity of ADPRT in
the stressed cells, and comparing the value so determined with a reference
value of ADPRT activity to ascertain whether said value so determined is higher
or lower than said reference value, wherein said predisposition is indicated if
said value so determined is lower than said reference value.
18. A method according to claim 17, wherein said cells are mononuclear
leukocytes, epithelial cells, or fibroblast cells.
19. A method according to claim 18, wherein said cells are mononuclear
leukocytes.
20. A method according to claim 17, wherein said disease is cancer.
21. A method according to claim 20, wherein said disease is cancer of the
colon, liver or pancreas.
22. A method for testing the immune competency of an individual,
comprising stressing DNA-containing cells of the individual to produce stressed
cells having damaged cellular DNA, thereby to cause induced activity of
adenosine diphosphate ribosyl transferase (ADPRT) in the stressed cells,
determining a value for the induced activity of ADPRT in the stressed cells,
and comparing the value so determined with a reference value of ADPRT activity
to ascertain whether said value so determined is higher or lower than said
reference value, wherein a low immune competency with respect to a disease
associated with cellular DNA damage is indicated if said value so determined is
lower than said reference value.
23. A method for testing the efficacy of a therapeutic agent for treating
an individual for a disease associated with DNA damage, comprising stressing
DNA-containing cells of the individual to produce stressed cells having damaged
cellular DNA, thereby to cause induced activity of adenosine diphosphate
ribosyl transferase (ADPRT) in the stressed cells, determining a value for the
induced activity of ADPRT in the stressed cells, and comparing the value so
determined with a reference value of ADPRT activity to ascertain whether said
value so determined is higher or lower than said reference value, wherein said
cells are cells
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treated with said agent, and wherein efficacy of said agent is indicated when
said determined value is higher than said reference value.
* * * * *
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