U.S. patent application number 09/181027 was filed with the patent office on 2003-11-06 for methods and compositions utilizing rad51.
Invention is credited to GOLUB, EFIM ILYA, HAAF, THOMAS, RADDING, CHARLES MEYER, REDDY, GURUCHARAN.
Application Number | 20030208053 09/181027 |
Document ID | / |
Family ID | 27358244 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030208053 |
Kind Code |
A1 |
HAAF, THOMAS ; et
al. |
November 6, 2003 |
METHODS AND COMPOSITIONS UTILIZING RAD51
Abstract
In accordance with the objects outlined above, the present
invention provides methods of diagnosing individuals at risk for a
disease state which results in aberrant Rad51 loci. The methods
comprise determining the distribution of Rad51 foci in a first
tissue type of a first individual, and then comparing the
distribution to the distribution of Rad51 foci from a second normal
tissue type from the first individual or a second unaffected
individual. A difference in the distributions indicates that the
first individual is at risk for a disease state which results in
aberrant Rad51 loci. Preferred disease states include cancer and
disease states associated with apoptosis. In an additional aspect,
the present invention provides methods for identifying apoptotic
cells and cells under stress associated with nucleic acid
modification. The methods comprise determining the distribution of
Rad51 foci in a first cell, and comparing the distribution to the
distribution of Rad51 foci from a second non-apoptotic cell. A
difference in the distributions indicates that the first cell is
apoptotic or under stress.
Inventors: |
HAAF, THOMAS; (BERLIN,
DE) ; GOLUB, EFIM ILYA; (NEW HAVEN, CT) ;
REDDY, GURUCHARAN; (REDWOOD CITY, CA) ; RADDING,
CHARLES MEYER; (HAMDEN, CT) |
Correspondence
Address: |
FLEHR HOHBACH TEST
ALBRITTON & HERBERT
FOUR EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
|
Family ID: |
27358244 |
Appl. No.: |
09/181027 |
Filed: |
October 27, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09181027 |
Oct 27, 1998 |
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09007020 |
Jan 14, 1998 |
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6090539 |
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60035834 |
Jan 30, 1997 |
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60045668 |
May 6, 1997 |
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Current U.S.
Class: |
536/23.1 ;
435/325 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/57415 20130101; G01N 33/5743 20130101; G01N 33/574
20130101 |
Class at
Publication: |
536/23.1 ;
435/325; 514/2; 514/44 |
International
Class: |
C07H 021/00; A01N
037/18; A01N 043/04; C12N 005/00 |
Claims
We claim:
1. A method of diagnosing individuals at risk for a disease state
comprising a) determining the distribution of Rad51 foci in a first
tissue type of a first individual; and b) comparing said
distribution to the distribution of Rad51 foci from a second normal
tissue type from said first individual or a second unaffected
individual; wherein a difference in said distributions indicates
that the first individual is at risk for a disease state which
results in aberrant Rad51 loci.
2. A method according to claim 1 wherein said disease state is
cancer.
3. A method of diagnosing individuals at risk for cancer comprising
a) determining the distribution of Rad51 foci in a potential
cancerous tissue type of a first individual; and b) comparing said
distribution to the distribution of Rad51 foci from a second normal
tissue type from said first individual or a second unaffected
individual; wherein a difference in said distributions indicates
that the first individual is at risk for a cancer which results in
aberrant Rad51 loci.
4. A method according to claim 3 wherein the cancer is selected
from breast cancer and skin cancer.
5. A method of diagnosing individuals at risk for a disease state
associated with apoptosis, said method comprising a) determining
the distribution of Rad51 foci in a first tissue type of a first
individual; and b) comparing said distribution to the distribution
of Rad51 foci from a second normal tissue type from said first
individual or a second unaffected individual; wherein a difference
in said distributions indicates that the first individual is at
risk for a disease state associated with apoptosis which results in
aberrant Rad51 loci.
6. A method according to claim 1 wherein the extent of aberrent
distribution indicates the severity of the disease state.
7. A method according to claim 1 wherein said distribution is
determined through the use of polyclonal antibodies.
8. A method according to claim 1 wherein said distribution is
determined through the use of monoclonal antibodies.
9. A method according to claim 7 or 8 wherein said antibodies are
raised against eukaryotic Rad51.
10. A method according to claim 9 wherein said eukaryotic Rad51 is
mammalian Rad51.
11. A method for identifying an apoptotic cell comprising a)
determining the distribution of Rad51 foci in a first cell; and b)
comparing said distribution to the distribution of Rad51 foci from
a second non-apoptotic cell; wherein a difference in said
distributions indicates that the first cell is apoptotic.
12. A method according to claim 11 wherein said distribution is the
association of Rad51 with DNA fibers.
13. A method according to claim 11 wherein said distribution is the
association of Rad51 into micronuclei.
14. A method for identifying a cell under stress associated with
nucleic acid modification comprising a) determining the
distribution of Rad51 foci in a first cell; and b) comparing said
distribution to the distribution of Rad51 foci from a second
non-affected cell; wherein a difference in said distributions
indicates that the first cell is under stress associated with
nucleic acid modification.
15. A method according to claim 14 wherein said stress is oxidative
or hypoxic stress.
16. A method according to claim 14 wherein said stress is heat
shock.
17. A method according to claim 14 wherein said stress is cold
shock.
18. A method for identifying a cell containing a mutant Rad51 gene
comprising determining the sequence of all or part of at least one
of the endogenous Rad51 genes.
19. A method of identifying the Rad51 genotype of an individual
comprising determining all or part of the sequence of at least one
Rad51 gene of said individual.
20. A method according to claim 18 or 19 further comprising
comparing the sequence of said Rad51 gene to a known Rad51
gene.
21. A method according to claim 20 wherein a difference in the
sequence between the Rad51 gene of said individual and said known
Rad51 gene is indicative of a disease state or a propensity for a
disease state.
22. A method for screening for a bioactive agent capable of binding
to Rad51 comprising: a) adding a candidate bioactive agent to a
sample of Rad51; and b) determining the binding of said candidate
agent to said Rad51.
23. A method for screening for a bioactive agent capable of
modulating the activity of Rad51, said method comprising the steps
of: a) adding a candidate bioactive agent to a sample of Rad51; and
b) determining an alteration in the biological activity of
Rad51.
24. A method according to claim 23 wherein said biological activity
is DNA dependent ATPase activity.
25. A method according to claim 23 wherein said biological activity
is nucleic acid strand exchange.
26. A method according to claim 23 wherein said biological activity
is DNA binding.
27. A method according to claim 23 wherein said biological activity
is filament formation.
28. A method according to claim 23 wherein said biological activity
is DNA pairing.
29. A method for screening for a bioactive agent capable of
modulating the activity of Rad51, said method comprising the steps
of: a) adding a candidate bioactive agent to a cell; and b)
determining the effect on the formation or distribution of Rad51
foci in said cell.
30. A method according to claim 25 further comprising subjecting
said cell to conditions which induce nucleic acid damage.
31. A method of inducing apoptosis in a cell comprising increasing
the activity of Rad51 in said cell.
32. A method according to claim 31 wherein said increasing
comprises overexpression of endogenous Rad51.
33. A method according to claim 31 wherein said increasing
comprises administration of a gene encoding Rad51.
34. A method according to claim 31 wherein said increasing
comprises administration of Rad51 protein.
35. A method according to claim 31 wherein said cell is a cancer
cell.
36. A method according to claim 31 further comprising subjecting
said cell to conditions which induce nucleic acid damage.
37. A method according to claim 36 wherein said conditions comprise
the administration of a chemical agent which causes nucleic acid
damage.
38. A method according to claim 36 wherein said conditions comprise
subjecting said cell to radiation.
39. A method according to claim 31 further comprising increasing
the activity of p53 in said cell.
40. A composition comprising: a) nucleic acid encoding a Rad51
protein; and b) nucleic acid encoding a tumor suppressor
protein.
41. A composition according to claim 38 wherein said tumor
suppressor protein is p53.
42. A composition according to claim 38 wherein said tumor
suppressor protein is BRCA1.
43. A composition according to claim 38 wherein said tumor
suppressor protein is BRCA2.
44. A composition according to claim 38 comprising: a) nucleic acid
encoding a Rad51 protein; b) nucleic acid encoding a BRCA1 protein;
c) nucleic acid encoding a BRCA2 protein; and d) nucleic acid
encoding a p53 protein.
45. A composition comprising: a) a recombinant Rad51 protein; and
b) a recombinant tumor suppressor protein.
46. A kit for detecting the distribution of Rad51 foci in a cell or
tissue comprising: a) binding agent for Rad51 foci; and b) a
detectable label.
Description
[0001] This is a continuing application of 60/035,834, filed Jan.
30, 1997 and 60/045,668, filed May 6, 1997, both of which are
expressly incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods of diagnosis and screening
utilizing Rad51molecules.
BACKGROUND OF THE INVENTION
[0003] Homologous recombination is a fundamental process which is
important for creating genetic diversity and for maintaining genome
integrity. In E. coli RecA protein plays a central role in
homologous genetic recombination in vivo and promotes homologous
pairing of double-stranded DNA with single-stranded DNA or
partially single-stranded DNA molecules in vitro. Radding, C. M.
(1988). Homologous pairing and strand exchange promoted by
Escherichia coli RecA protein. Genetic Recombination. Washington,
American Society for Microbiology. 193-230; Radding, C. M. (1991).
J. Biol. Chem. 266: 5355-5358; Kowalczykowski, et al., (1994).
Annu. Rev. Biochem. 63: 991-1043. In the yeast Saccharomyces
cerevisiae there are several genes with homology to recA gene;
Rad51, Rad57 and Dmc1. Rad51 is a member of the Rad52 epistasis
group, which includes Rad50, Rad51, Rad52, Rad54, Rad55 and Rad57.
These genes were initially identified as being defective in the
repair of damaged DNA caused by ionizing radiation and were
subsequently shown to be deficient in both genetic recombination
and the recombinational repair of DNA lesions. Game, J. C. (1983).
Yeast Genetics: Fundamental and applied aspects. J. F. T. Spencer,
D. H. Spencer and A. R. W. Smith, eds (New-York:springer-verlag):
109-137; Haynes, et al., (1981). The molecular biology of the yeast
Saccharomyces cerevisiae: Life cycle and inheritance. J. N.
Strathern, E. W. Jones and J. M. Broach, eds (Cold Spring harbor,
New York:Cold Spring Harbor laboratory press): 371-414; Resnick, M.
A. (1987). Meiosis, P. B. Moens, ed. (New York: Academic Press):
157-210. During meiosis Rad51 mutants accumulate DNA double-strand
breaks at recombination hot spots (Shinohara, et al., (1992). Cell
69: 457-470). Yeast rad51 gene was cloned and sequenced (Basile, et
al., (1992). Mol. Cell. Biol. 12: 3235-3246; Aboussekhara, et al.,
(1992) Mol. Cell. Biol. 12: 3224-3234). Although yeast Rad51 gene
shared homology with E. coli recA gene, the extent of homology was
not very strong (27%). However, the extent of structural
conservation between RecA protein and Rad51 protein became apparent
when the yeast Rad51 protein was isolated and was shown to form
nucleoprotein filaments that were almost identical to the
nucleoprotein filaments formed by RecA protein (Ogawa, et al.,
(1993). CSH Symp. Quant. Biol. 58: 567-576; Ogawa, T., et al.,
(1993). Science 259: 1896-1899; Story, et al., (1993). Science 259:
1892-1896). Recently genes homologous to E. coli recA and yeast
rad51 were isolated from all groups of eukaryotes, including
mammals (Morita, et al., (1993). Proc. Natl. Acad. Sci. USA 90,
6577-6580; Shinohara, et al., (1993). Nature Genet. 4, 239-243;
Heyer, W. D. (1994). Experientia 50, 223-233; Maeshima, et al.,
(1995). Gene 160: 195-200). Phylogenetic analysis by Ogawa and co
workers suggested the existence of two sub-families within
eukaryotic RecA homologs: the Rad51-like (Rad51 of human, mouse,
chicken, S. cerevisiae, S. pombe and Mei3 of Neurospora crassa) and
the Dmc 1-like genes (S. cerevisiae Dmc1 and Lilium longiflorum
LIM15) (Ogawa, supra). All these Rad51 genes share significant
homology with residues 33-240 of the E. coli RecA protein, which
have been identified as a `homologous core` region.
[0004] Yeast and human Rad51 proteins have been purified and
characterized biochemically. Like E. coli RecA protein, yeast and
human Rad51 protein polymerizes on single-stranded DNA to form a
right-handed helical nucleoprotein filament which extends DNA by
1.5 times (Story, supra; Benson, et al., (1994) EMBO J. 13,
5764-5771). Moreover like RecA protein Rad51 protein promotes
homologous pairing and strand exchange in an ATP dependent reaction
(Sung, P. (1994). Science 265, 1241-1243; Sung, P. and D. L.
Robberson (1995). Cell 82: 453-461; Baumann, et al., (1996) Cell
87, 57-766; Gupta, et al., (1997) Proc. Natl. Acad. Sci. USA 94,
463-468). Surprisingly, polarity of strand exchange performed by
Rad51 protein is opposite to that of RecA protein (Sung and
Robberson supra) and the relevance of this observation remains to
be seen.
[0005] Surprisingly, studies with mouse models show that targeted
disruption of the Rad51 gene leads to an embryonic lethal phenotype
(Tsuzuki, et al., (1996). Peoc. Natl. Acad. Sci. USA 93:
6236-6240). Moreover attempts to generate homozygous
rad51-/-embryonic stem cells have not been successful. These
results show that Rad51 plays an essential role in cell
proliferation, a surprise in view of the viability of S. cerevisiae
carrying rad51 deletions. It is also interesting to note that Rad51
was found to be associated with RNA polymerase II transcription
complex (Maldonado, et al., (1996). Nature 381, 86-89), the
specificity and functional nature of these interactions remains to
be seen but all these observations point to a pleitropic role of
hsRad51 in DNA metabolism.
[0006] While Rad51 transcripts and protein are present in all the
cell types examined thus far, the highest transcript levels are
found in tissues active in recombination, including spleen, thymus,
ovary and testis (Morita, supra). Rad51 is specifically induced in
murine B cells cultured with lipopolysaccharide, which stimulates
switch recombination and Rad51 localizes to nuclei of switching B
cells (Li, et al., (1996). Proc. Natl. Acad. Sci. USA 93:
10222-10227). These findings are consistent with the view that
Rad51 plays an important role in lymphoid specific recombination
events such as V(D)J recombination and immunoglobulin heavy chain
class switching. In spermatocytes undergoing meiosis, Rad51 is
enriched in the synaptonemal complexes, which join paired
homologous chromosomes (Haaf, et al., (1995) Proc. Natl. Acad. Sci.
USA 92, 2298-2302; Ashley, et al., (1995) Chromosoma 104: 19-28;
Plug, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 5920-5924). In
cultured human cells, Rad51 protein is detected in multiple
discrete foci in the nucleoplasm of a few cells by
immunofluorescent antibodies. After DNA damage, the localization of
Rad51 changes dramatically when multiple foci form in the nucleus
and stain vividly with anti-Rad51 antibodies (Haaf, supra, 1995).
After DNA damage the percentage of cells with focally concentrated
Rad51 protein increases; the same cells show unscheduled DNA-repair
synthesis.
[0007] Micronuclei (MN) originate from chromosomal material that is
not incorporated into daughter nuclei during cell division.
Different chemicals and treatment of cells induce qualitatively
different types of micronuclei. MN caused by ionizing radiation or
clastogens (i.e. 5-azacytidine) mostly contain acentric chromosome
fragments (Verhaegen, F., and Vral, A. (1994). Radiation Res. 139,
208-213; Stopper, et al., (1995). Carcinogenesis 16, 1647-1650). In
contrast, MN induced by aneuploidogens (i.e. colcemid) result from
lagging whole chromosomes and stain positively for the presence of
kinetochores/centromeres (Marrazini et al., 1994; Stopper, et al.,
(1994). Mutagenesis 9, 411-416). Determination of MN frequencies
represents a good assay to measure genetic damage in cells, since
it is much faster and simpler than karyotype analyses. In this
light, the MN test has been widely used as a dosimeter of human
exposure to radiation or clastogenic and aneugenic chemicals, and
for the detection and risk assessment of environmental mutagens and
carcinogens (Heddle, et al., (1991) Environmental Mol. Mutagenesis
18, 277-291; Norppa, et al., (1993). Environmental Health Perspect.
101, Supp. 3, 139-143; Hahnfeldt, et al., (1994) Radiation Res.
138, 239-245). However, although the MN assay is a convenient in
situ method to monitor cytogenetic effects, the understanding of
the connection between initial DNA damage and formation of MN is
still poor.
[0008] The tumor suppressor p53 prevents tumor formation after DNA
damage by halting cell cycle progression to allow DNA repair or by
inducing apoptotic cell death. Loss of wild-type p53 function
renders cells resistant to DNA damage induced cell cycle arrest and
ultimately leads to genomic instabilities including gene
amplifications, translocations and aneuploidy. Some of these
chromosomal lesions are based on mechanisms that involve
recombinational events (Lane, D. P. (1992). Nature 358: 15-16;
Lane, D. P. (1993). Nature 362: 786-787; Sturzbecher, et al.,
(1996). EMBO J. 15: 1992-2002) reported that wild-type tumor
suppressor protein p53 interacts physically with human Rad51
protein and it inhibits the biochemical functions of Rad51 like
ATPase and strand exchange. In vivo temperature sensitive mutant
p53 formed complexes with Rad51 only in wild type but not in mutant
conformation. They suggested that gene amplifications and other
types of chromosome rearrangements involved in tumour progression
might occur not only as a result of inappropriate cell
proliferation but as a direct consequence of a defect in p53
mediated control of homologous recombination processes due to
mutations in the p53 gene. (Meyn, et al., (1994). Int. J. Radiat.
Biol. 66: S141-S149) showed that normal cells transfected with a
dominant-negative p53 mutant acquired interference with the G1-S
cell cycle checkpoint and showed up to an 80-fold elevation in
RAD51 mediated homologous DNA recombination rates compared with the
normal parental control cells. Thus, loss of normal p53 function
may cause a loss in control of normal DNA repair, recombination,
and ultimately replication, resulting in inappropriate cell
division and neoplastic growth. Breast tumour cells have mutated
p53 genes and proteins and have various types of chromosomal
aberrations like insertions, deletions, rearrangements,
amplifications etc., indicative of abnormally controlled
recombination.
[0009] Accordingly, it is an object of the invention to provides
methods of diagnosis and screening which focus on Rad51.
SUMMARY OF THE INVENTION
[0010] In accordance with the objects outlined above, the present
invention provides methods of diagnosing individuals at risk for a
disease state which results in aberrant Rad51 loci. The methods
comprise determining the distribution of Rad51 foci in a first
tissue type of a first individual, and then comparing the
distribution to the distribution of Rad51 foci from a second normal
tissue type from the first individual or a second unaffected
individual. A difference in the distributions indicates that the
first individual is at risk for a disease state which results in
aberrant Rad51 loci. Preferred disease states include cancer and
disease states associated with apoptosis.
[0011] In an additional aspect, the present invention provides
methods for identifying apoptotic cells and cells under stress
associated with nucleic acid modification. The methods comprise
determining the distribution of Rad51 foci in a first cell, and
comparing the distribution to the distribution of Rad51 foci from a
second non-apoptotic cell. A difference in the distributions
indicates that the first cell is apoptotic or under stress.
[0012] In a further aspect, the present invention provides methods
for identifying a cell containing a mutant Rad51 gene comprising
determining the sequence of all or part of at least one of the
endogenous Rad51 genes.
[0013] In an additional aspect, the invention provides methods of
identifying the Rad51 genotype of an individual comprising
determining all or part of the sequence of at least one Rad51 gene
of the individual. The method may include comparing the sequence of
the Rad51 gene to a known Rad51 gene.
[0014] In a further aspect, the present invention provides methods
for screening for a bioactive agent capable of binding to Rad51.
The methods comprise adding a candidate bioactive agent to a sample
of Rad51, and determining the binding of the candidate agent to the
Rad51.
[0015] In an additional aspect, the invention provides methods for
screening for a bioactive agent capable of modulating the activity
of Rad51. The method comprises the steps of adding a candidate
bioactive agent to a sample of Rad51, and determining an alteration
in the biological activity of Rad51. The method may also comprise
adding a candidate bioactive agent to a cell, and determining the
effect on the formation or distribution of Rad51 foci in the
cell.
[0016] In a further aspect, the invention provides methods of
inducing apoptosis in a cell comprising increasing the activity of
Rad51 in the cell. This can be done by overexpressing an endogenous
Rad51 gene, or by administration of a gene encoding Rad51 or the
protein itself.
[0017] In an additional aspect, the present invention provides
composition comprising a nucleic acid encoding a Rad51 protein, and
a nucleic acid encoding a tumor suppressor protein. The tumor
suppressor protein may be p53 or a BRCA protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a digital image of photographs of cells that
depict type I and type II Rad51 foci, respectively.
[0019] FIGS. 2A and 2B are digital images of photographs of two
different breast cancer cells from a breast cancer cell line (BT20)
that show Rad51 foci. The staining is localized to the nucleus, and
does not occur in either the cytoplasm or the nucleolus.
[0020] FIGS. 3A, 3B, 3C and 3D show dynamic changes in the
higher-order nuclear organization of Rad51 foci after DNA damage
and cell-cycle arrest. (a-c) TGR-1 fibroblasts were irradiated with
a lethal dose (900 rad) of .sup.137Cs and then allowed to recover
for various times. Rad51 protein is stained (light), nuclei are
counterstained with DAPI. Three hours after irradiation (a), Rad51
foci are distributed throughout the entire nuclear volume. Many
foci have a double-dot appearance. After 16 hrs (b), clusters of
Rad51 foci and linear higher-order structures are formed. Somatic
pairing of linear strings of Rad51 foci is observed. After 30 hrs
(c), Rad51 clusters move towards the nuclear periphery and are
eliminated into micronuclei. (d) Simultaneous staining of Rad51
protein (light) and replicating DNA (dark) in an exponentially
growing, XPA fibroblast culture. BrdU was incorporated into DNA for
30 hrs and detected with red anti-BrdU antibody. Note that the
Rad51-positive cell is devoid of BrdU label. Magnification
1000.times..
[0021] FIG. 4 depicts the exclusion of Rad51-protein in micronuclei
after DNA damage. TGR-1 fibroblasts, two days after .sup.137Cs
irradiation with a dose of 900 rad. Rad51 protein is stained by
(light), nuclei are counterstained with DAPI. Note the complete
absence of Rad51-protein staining in nuclei. All Rad51 foci are
excluded into micronuclei. Most micronuclei exhibit paired
Rad51-protein structures. Magnification 1000.times..
[0022] FIGS. 5A, 5B, 5C and 5D illustrates that apoptotic bodies
(micronuclei) contain Rad51 protein and fragmented DNA. (a and b)
TGR-I nuclei, 3 hrs (right), 16 hrs (middle), and 30 hrs (left)
after .sup.137Cs irradiation. Rad51-protein foci show light
staining. The repair proteins Rad52 (a) and Gadd45 (b) are detected
by antibody probes (darker staining). Nuclei are counterstained
with DAPI. Note that neither Rad52 nor Gadd45 foci co-localize with
Rad51. Only the Rad51 foci segregate into micronuclei. (c and d)
Micronuclei induced by treatment of TGR-1 cultures with colcemid
(c) and etoposide (d) contain Rad51 protein (light staining, left
nucleus) and fragmented DNA (darker staining, right nucleus).
Magnification 1000.times..
[0023] FIGS. 6A, 6B and 6C show the association of Rad51 protein
with linear DNA molecules. (a) Mechanically stretched chromatin
prepared from a .sup.137Cs-irradiated cell culture and stained with
light anti-HsRad51 antibodies. The Rad51 signals appear as
beads-on-a-string on the linearly extended chromatin fibers. (b and
c) DNA fibers excluded from TGR-1 nuclei, one day after .sup.137Cs
irradiation. Preparations are not experimentally stretched.
Chromatin is counterstained with DAPI. The DNA fibers are covered
with Rad51 protein (c, light staining), whereas the remaining
nuclei are devoid of detectable Rad51 foci. DNA-strand breaks in
chromatin fibers are end labeled with fluorescent nucleotides (c,
darker staining co-localizing with the Rad51 staining). Some fibers
appear to form micronuclei. Magnification 1000.times..
[0024] FIGS. 7A, 7B, 7C, 7D, 7E and 7F show the linear higher-order
structures of Rad51 protein in overexpressing nuclei and in
colcemid-induced micronuclei. Rad51 protein is stained with
anti-Rad51 antiserum, detected by green FITC fluorescence (light
staining). Preparations are counterstained with DAPI, except the
nucleus in b. (a and b) Human 710 kidney cells overexpressing Rad51
fused to a T1-tag epitope. Nuclei are filled with a network of
linear Rad51 structures. Magnification 1000.times.. (c)
Subconfluent rat TGR 928.1-9 cells overexpressing HsRad51. Nuclear
staining is most prominent in cells during G.sub.0 and G.sub.1
phase of the cell cycle. Magnification 1000.times.. (d) TGR 928.1-9
nucleus filled with linear Rad51 structures. Magnification
1000.times.. (e and f) Linear Rad51 structures in colcemid-induced
micronuclei. TGR-1 fibroblasts were treated with colcemid for one
day and then allowed to recover for two days. Note the absence of
Rad51 staining in the nuclei. Magnification 1000.times..
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is directed to a series of discoveries
relating to the pivotal role that Rad51 plays in a number of
cellular functions, including those involved in disease states.
Thus, it appears that the levels, function, and distribution of the
Rad51 protein within cells may be monitored as a diagnostic tool of
cellular health or fate. In addition, due to Rad51's essential role
in a number of cellular processes, Rad51 is an important target
molecule to screen candidate drug agents which can modulate its
biological activity.
[0026] Accordingly, in a preferred embodiment, the invention
provides methods of diagnosing individuals at risk for a disease
state. As will be appreciated by those in the art, "at risk for a
disease state" means either that an individual has the disease, or
is at risk to develop the disease in the future. By "disease state"
herein is meant a disease that is either caused by or results in
aberrant Rad51 distribution or biological activity. For example, as
is more fully described below, aberrant distribution of Rad51 foci
in a cell can be indicative of cancer, apoptosis, cellular stress,
etc., which can lead to the development of disease states.
Similarly, disease states caused by or resulting in aberrant Rad51
biological activity, including alterations caused by mutation,
changes in the cellular amount or distribution of Rad51, and
changes in the biological function of Rad51, for example altered
nucleic acid binding, filament formation, DNA pairing (i.e. D-loop
formation), strand-exchange, strand annealing or recombinagenicity,
are also included within the definition of disease states which are
related to or associated with Rad51.
[0027] Thus, disease states which may be evaluated using the
methods of the present invention include, but are not limited to,
cancer (including solid tumors such as skin, breast, brain,
cervical carcinomas, testicular carcinomas, etc.), diseases
associated with premature or incorrect apoptosis, including AIDS,
cancers (e.g. melanoma, hepatoma, colon cancer, etc.), liver
failure, Wilson disease, myelodysplastic syndromes,
neurodegenerative diseases, multiple sclerosis, aplastic anemia,
chronic neutropenia, Tupe I diabetes mellitus, Hashimoto
thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma,
leukemia, solid tumors, and autoimmune diseases), diseases
associated with cellular stress which is affiliated with nucleic
acid modification, including diseases associated with oxidative
stress such as cardiovascular disease, immune system function
decline, aging, brain dysfunction and cancer.
[0028] In one embodiment, the method comprises first determining
the distribution of Rad51 foci in a first tissue type of a first
individual, i.e. the sample tissue for which a diagnosis is
required. In some embodiments, the testing may be done on single
cells. The first individual, or patient, is suspected of being at
risk for the disease state, and is generally a human subject,
although as will be appreciated by those in the art, the patient
may be animal as well, for example in the development or evaluation
of animal models of human disease. Thus other animals, including
mammals such as rodents (including mice, rats, hamsters and guinea
pigs), cats, dogs, rabbits, farm animals including cows, horses,
goats, sheep, pigs, etc., and primates (including monkeys,
chimpanzees, orangutans and gorillas) are included within the
definition of patient.
[0029] As will be appreciated by those in the art, the tissue type
tested will depend on the disease state under consideration. Thus
for example, potentially cancerous tissue may be tested, including
breast tissue, skin cells, solid tumors, brain tissue, etc.
Similarly, cells or tissues of the immune system, including blood,
and lymphocytes; cells or tissues of the cardiovascular system (for
example, for testing oxidative stress).
[0030] In a preferred embodiment, the disease state under
consideration is cancer and the tissue sample is a potentially
cancerous tissue type. Of particular interest is breast, skin,
brain, colon, prostate, and other solid tumor cancers. As outlined
in the Examples, cultured breast cancer cells and primary invasive
breast cancer cells all demonstrate an increase in the presence of
Rad51 foci.
[0031] Similarly, several diseases caused by defective nucleotide
excision repair (NER) systems, including Xeroderma pigmentosium,
show increased Rad51 foci.
[0032] In a preferred embodiment, primary cancerous tissue is used,
and may show differential Rad51 staining. While the number of cells
exhibiting Rad51 foci may be less than for cell lines, primary
cancerous tissue shows an increase in Rad51 foci. Thus for example,
from 0.05 to 10% of primary cancerous cells exhibit differential
Rad51 foci, with from about 1 to about 5% being common.
[0033] It should be noted that not all cancer cell lines exhibit
aberrant Rad51 protein foci. For example, the ovarian cancer cell
line Hey does not show an increase in Rad51 foci. Similarly, as
outlined in the examples, transformed but non-malignant human cells
can show an increased percentage of Rad51-positive cells (compared
to non-transformed cells), although it is generally not as great as
in tumor cells.
[0034] In a preferred embodiment, the disease state under
consideration involves apoptosis, and includes, but is not limited
to, including AIDS, cancers (e.g. melanoma, hepatoma, colon cancer,
etc.), liver failure, Wilson disease, myelodysplastic syndromes,
neurodegenerative diseases, multiple sclerosis, aplasitic anemia,
chronic neutropenia, Tupe I diabetes mellitus, Hashimoto
thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma,
leukemia, solid tumors, and autoimmune diseases. This list includes
disease states that include too much as well as too little
apoptosis. See Peter et al., PNAS USA 94:12736 (1997), hereby
incorporated by reference.
[0035] In a preferred embodiment, the disease state under
consideration involves cellular stress associated with nucleic acid
modification, including aging, cardiovascular disease, declines in
the function of the immune system, brain dysfunction, and
cancer.
[0036] The distribution of Rad51 foci is determined in the target
cells or tissue. To date, two main types of Rad51 foci have been
identified. As reported earlier (Haaf, 1995, supra) in situ
immunostaining with Rad51 antibodies reveals three kinds of nuclei:
1) nuclei that did not show any staining at all (no foci); 2)
nuclei that showed weak to medium staining and showed only a few
foci (Type I nuclei); and 3) nuclei that showed strong staining and
showed many foci (Type II nuclei). In general, the staining is
excluded from the cytoplasm. Type I and Type II patterns of nuclei
staining are shown in FIG. 1; many of the foci have a double-dot
appearance, typical of paired DNA segments. In normal cells, type I
nuclei are found in 7-10% of cells and type II nuclei in less than
0.4 to 1% of cells, with generally about 90% of the cells showing
no foci. In contrast, some cells involved in disease states show a
marked increase in Rad51 foci. As outlined herein and shown in the
examples, the numbers of cells showing Rad51 foci in cells
associated with disease states is significantly increased. Thus, in
a preferred embodiment, the number of cells showing type I nuclei
is generally from about 5% to about 50% of the nuclei, with from
about 10% to about 40% generally being seen. Thus, in a preferred
embodiment, there is at least a 5% increase in the type I foci,
with at least about 10% being preferred, and at least about 30%
being particularly preferred. Generally, to see this effect, at
least about 100 cells should be evaluated, with at least about 500
cells being preferred, and at least about 1000 being particularly
preferred.
[0037] Similarly, the number of cells showing type II nuclei also
increases, with from about 1% to about 10% of the nucleic
exhibiting type II foci and from about 1% to about 5% being common.
Thus, in a preferred embodiment, there is at least a 5% increase in
type II foci, with at least about 10% being preferred, and at least
about 30% being particularly preferred. In a preferred embodiment,
both types of foci increase simultaneously. In alternate
embodiments, only one type of foci increases.
[0038] Similarly, an increase in both types of foci (i.e. an
increase in any foci, irrespective of type) can also be evaluated
using the same numbers.
[0039] The distribution of Rad51 foci can be determined in a
variety of ways. In a preferred embodiment, a labeled binding agent
that binds to Rad51 is used to visualize the foci. By "labeled"
herein is meant that a compound has at least one element, isotope
or chemical compound attached to enable the detection of the
compound. In general, labels fall into three classes: a) isotopic
labels, which may be radioactive or heavy isotopes; b) immune
labels, which may be antibodies or antigens; and c) colored or
fluorescent dyes. The labels may be incorporated into the compound
at any position. Preferred labels are fluorescent or radioactive
labels. The binding agent can either be labeled directly, or
indirectly, through the use of a labeled secondary agent which will
bind to the first binding agent. The cells or tissue sample is
prepared as is known for cellular or in situ staining, using
techniques well known in the art, as outlined in the Examples.
[0040] In a preferred embodiment, the binding agent used to detect
Rad51 foci is an antibody. The antibodies may be either polyclonal
or monoclonal, with monoclonal antibodies being preferred. In
general, it is preferred, but not required, that antibodies to the
particular Rad51 under evaluation be used; that is, antibodies
directed against human Rad51 are used in the evaluation of human
patients. However, as the homology between different mammalian
Rad51 molecules is quite high (73% identity as between human and
chicken, for example), it is possible to use antibodies against
Rad51 from one type of animal to evaluate a different animal (mouse
antibodies to evaluate human tissue, etc.). Thus, in a preferred
embodiment, antibodies raised against eukaryotic Rad51 are used,
with antibodies raised against mammalian Rad51 being especially
preferred. Thus, antibodies raised against yeast, human, rodent,
primate, and avian Rad51 proteins are particularly preferred. In
addition, as will be appreciated by those in the art, the protein
used to generate the antibodies need not be the full-length
protein; fragments and derivatives may be used, as long as there is
sufficient immunoreactivity against the sample Rad51 to allow
detection. Alternatively, other binding agents which will bind to
Rad51 at sufficient affinity to allow visualization can be
used.
[0041] Without being bound by theory, as outlined in the Examples,
it does not appear that the quantitative amount of Rad51 protein is
necessarily altered in cells exhibiting the presence or altered
distribution of foci. However, in some circumstances the
quantitative amount of Rad51 may be measured and correlated to the
presence or absence of Rad51 foci.
[0042] In addition, the appearance of the foci may be used in the
determination of the presence of aberrant Rad51 foci. As noted in
the Examples, in some cases linear "strings" of 5-10 Rad51 foci are
formed, with somatic association of "homologous" strings of similar
length, tightly paired at one of the ends. These structures are
generally associated with DNA fibers, as is shown in the Figures.
Thus, the formation of these types of structures can be indicative
of aberrant Rad51 foci.
[0043] Furthermore, in a preferred embodiment, particularly in
disease states involving apoptosis and DNA damage, aberrant Rad51
foci includes the development of micronuclei containing Rad51. As
shown in the Examples, evaluation of Rad51 foci over time, in
particular after cellular stress, can lead to the concentration and
exclusion of the Rad51 foci (which are associated with DNA) into
micronuclei, which frequently is accompanied by genome
fragmentation. This effect is seen in a wide variety of apoptotic
cells, as is shown in the Examples, even in the absence of induced
DNA damage, such as through the use of colcemid, a spindle poison,
thus indicating the role of Rad51 in normal apoptotic pathways.
[0044] In addition to the evaluation of the presence or absence of
Rad51 foci, the cells may be evaluated for cell cycle arrest, as is
outlined in the Examples.
[0045] Once the distribution of Rad51 foci has been determined for
the target sample, the distribution of foci is compared to the
distribution of Rad51 foci from a second cell or tissue type. As
will be appreciated by those in the art, the second tissue sample
can be from a normal cell or tissue from the original patient or a
tissue from another, unaffected individual, which has been matched
for correlation purposes. A difference in the distribution of Rad51
foci as between the first tissue sample and the second matched
sample indicates that the first individual is at risk for a disease
state which results in aberrant Rad51 loci.
[0046] In a preferred embodiment, the difference in Rad51 foci
distribution is an increase in Rad51 foci, of either type 1 or type
2 foci, as outlined above. In an alternate embodiment, the
difference in Rad51 foci distribution is a decrease in the number
of Rad51 foci.
[0047] In some embodiments, there need not be a direct comparison.
For example, having once shown that a particular normal tissue only
contains a small percentage of Rad51 foci, the tissue or cells
under evaluation may not need to be compared to a control sample;
the presence of a higher percentage allows the diagnosis. Thus, for
example, in breast cancer, the presence of at least 1% of the cells
containing Rad51 foci is indicative that the patient is at risk for
breast cancer or in fact already has it.
[0048] In a preferred embodiment, a difference in the distribution
of Rad51 foci, in particular an increase in Rad51 foci, indicates
that the cell or tissue is cancerous.
[0049] In a preferred embodiment, a difference in the distribution
of Rad51 foci, in particular an increase in Rad51 foci, indicates
that the cell or tissue is apoptotic. These differences can include
the association of Rad51 with DNA fibers, the association of Rad51
with damaged DNA in micronuclei, or the presence of Rad51 in
micronuclei.
[0050] In addition, in a preferred embodiment, the extent of
aberrant distribution indicates the severity of the disease state.
Thus, for example, high percentages of cells containing Rad51 foci
can be indicative of highly malignant cancer.
[0051] In addition to the evaluation of Rad51 foci, the presence or
absence of variant (mutant) Rad51 genes may also be used in
diagnosis of disease states. Mutant forms of p53 have been found in
roughly 50% of known cancers, and it is known that Rad51 and p53
can interact on a protein level. In addition, p53 and Rad51 have
somewhat similar biochemical functions. Thus, the present discovery
that Rad51 plays a pivotal role in some cancers and apoptosis thus
suggests that variant Rad51, or incorrectly controlled Rad51 levels
or functions may be important in some disease states.
[0052] Accordingly, in a preferred embodiment, the present
invention provides methods for identifying a cell containing a
mutant Rad51 gene comprising determining the sequence of all or
part of at least one of the endogenous Rad51 genes. By "variant
Rad51 gene" herein is meant any number of mutations which could
result in aberrant Rad51 function or levels. Thus, for example,
mutations which alter the biochemical function of the Rad51
protein, alter its half-life and thus its steady-state cellular
level, or alter its regulatory sequences to cause an alteration in
it's steady-state cellular level may all be detected. This is
generally done using techniques well known in the art, including,
but not limited to, standard sequencing techniques including
sequencing by PCR, sequencing-by-hybridization, etc.
[0053] Similarly, in a preferred embodiment, the present invention
provides methods of identifying the Rad51 genotype of an individual
or patient comprising determining all or part of the sequence of at
least one Rad51 gene of the individual. This is generally done in
at least one tissue of the individual, and may include the
evaluation of a number of tissues or different samples of the same
tissue. For example, putatively cancerous tissue of an individual
is the preferred sample.
[0054] The sequence of all or part of the Rad51 gene can then be
compared to the sequence of a known Rad51 gene to determine if any
differences exist. This can be done using any number of known
homology programs, such as Bestfit, etc.
[0055] In a preferred embodiment, the presence of a difference in
the sequence between the Rad51 gene of the patient and the known
Rad51 gene is indicative of a disease state or a propensity for a
disease state.
[0056] The present discovery relating to the role of Rad51 in
cancer and apoptosis thus provide methods for inducing apoptosis in
cells. In a preferred embodiment, the methods comprise increasing
the activity of Rad51 in the cells. By "biological activity" of
Rad51 herein is meant one of the biological activities of Rad51,
including, but not limited to, the known Rad51 DNA dependent ATPase
activity, the nucleic acid strand exchange activity, the formation
of foci, single-stranded and double-stranded binding activities,
filament formation (similar to the recA filament of yeast), pairing
activity (D-loop formation), etc. See Gupta et al., supra, and
Bauman et al., supra, both of which are expressly incorporated by
reference herein. As will be appreciated by those in the art, this
may be accomplished in any number of ways. In a preferred
embodiment, the activity of Rad51 is increased by increasing the
amount of Rad51 in the cell, for example by overexpressing the
endogenous Rad51 or by administering a gene encoding Rad51, using
known gene-therapy techniques, for example. In a preferred
embodiment, the gene therapy techniques include the incorporation
of the exogenous gene using enhanced homologous recombination
(EHR), for example as described in PCT/US93/03868, hereby
incorporated by reference in its entirety.
[0057] In a preferred embodiment, the cells which are to have
apoptosis induced are cancer cells, including, but not limited to,
breast, skin, brain, colon, prostate, testicular, ovarian, etc.
cancer cells, and other solid tumor cells.
[0058] In a preferred embodiment, the methods may also comprise
subjecting the cells to conditions which induce nucleic acid
damage, as this appears to provide a synergistic effect, as
outlined above.
[0059] In a preferred embodiment, the methods further comprise
increasing the activity of p53 in the cell, for example by
increasing the amount of p53, as outlined above for Rad51.
[0060] The present discoveries relating to the pivotal role of
Rad51 in a number of important cellular processes and disease
states also makes Rad51 an important target in drug screening.
Thus, in a preferred embodiment, the present invention provides
methods for screening for a bioactive agent which may bind to Rad51
and modulate its activity.
[0061] In a preferred embodiment, the methods are used to screen
candidate bioactive agents for the ability to bind to Rad51. In
this embodiment, the methods comprise adding a candidate bioactive
agent to a sample of Rad51 and determining the binding of the
candidate agent to the Rad51. By "candidate bioactive agent" or
"candidate drugs" or grammatical equivalents herein is meant any
molecule, e.g. proteins (which herein includes proteins,
polypeptides, and peptides), small organic or inorganic molecules,
polysaccharides, polynucleotides, etc., which are to be tested for
the capacity to bind and/or modulate the activity of Rad51.
Candidate agents encompass numerous chemical classes. In a
preferred embodiment, the candidate agents are organic molecules,
particularly small organic molecules, comprising functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more chemical
functional groups.
[0062] Candidate agents are obtained from a wide variety of
sources, as will be appreciated by those in the art, including
libraries of synthetic or natural compounds. Any number of
techniques are available for the random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications to produce structural
analogs.
[0063] In a preferred embodiment, candidate bioactive agents
include proteins, nucleic acids, and organic moieties.
[0064] In a preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue"
, as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes amino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration. In the preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents may be used, for example to
prevent or retard in vivo degradations.
[0065] In a preferred embodiment, the candidate bioactive agents
are naturally occurring proteins or fragments of naturally occuring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
may be used. In this way libraries of procaryotic and eukaryotic
proteins may be made for screening against Rad51. Particularly
preferred in this embodiment are libraries of bacterial, fungal,
viral, and mammalian proteins, with the latter being preferred, and
human proteins being especially preferred.
[0066] In a preferred embodiment, the candidate bioactive agents
are peptides of from about 5 to about 30 amino acids, with from
about 5 to about 20 amino acids being preferred, and from about 7
to about 15 being particularly preferred. The peptides may be
digests of naturally occuring proteins as is outlined above, random
peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents herein is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0067] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0068] In a preferred embodiment, the candidate bioactive agents
are nucleic acids. By "nucleic acid" or "oligonucleotide" or
grammatical equivalents herein means at least two nucleotides
covalently linked together. A nucleic acid of the present invention
will generally contain phosphodiester bonds, although in some
cases, as outlined below, nucleic acid analogs are included that
may have alternate backbones, comprising, for example,
phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein; Letsinger, J. Org. Chem. 35:3800 (1970);
Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al.,
Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805
(1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate
(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);
Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy et al., Proc. Natl. Acad.
Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)
pp169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occuring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xathanine
hypoxathanine, isocytosine, isoguanine, etc.
[0069] As described above generally for proteins, nucleic acid
candidate bioactive agents may be naturally occuring nucleic acids,
random nucleic acids, or "biased" random nucleic acids. For
example, digests of procaryotic or eucaryotic genomes may be used
as is outlined above for proteins.
[0070] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0071] The candidate agents are added to a sample of Rad51 protein.
As is outlined above, all or part of a full-length Rad51 protein
can be used, or derivatives thereof. Generally, the addition is
done under conditions which will allow the binding of candidate
agents to the Rad51 protein, with physiological conditions being
preferred. The binding of the candidate agent to the Rad51 sample
is determined. As will be appreciated by those in the art, this may
be done using any number of techniques.
[0072] In one embodiment, the candidate bioactive agent is
labelled, and binding determined directly.
[0073] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescent molecules, enzyme reporters,
calorimetric reporters, chemiluminescers, specific binding
molecules, particles, e.g. magnetic or gold particles, and the
like. Specific binding molecules include pairs, such as biotin and
streptavidin, digoxygenin and antidigoxygenin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule which provides for detection, in accordance with
known procedures.
[0074] In some embodiments, only one of the components is labeled.
For example, the Rad51 may be labeled at tyrosine positions using
.sup.125I. Alternatively, more than one component may be labeled
with different labels; using .sup.125I for the Rad51, for example,
and a fluorophor for the candidate agents.
[0075] In a preferred embodiment, the binding of the candidate
bioactive agent is determined directly. For example, the Rad51 may
be attached to a solid support such as a microtiter plate or other
solid support surfaces, and labelled candidate agents added under
conditions which favor binding of candidate agents to the Rad51
protein. Incubations may be performed at any temperature which
facilitates optimal activity, typically between 4 and 40.degree. C.
Incubation periods are selected for optimum activity, but may also
be optimized to facilitate rapid high through put screening.
Typically between 0.1 and 1 hour will be sufficient. Excess
reagents are washed off, the system is evaluated for the presence
of the label, which is indicative of an agent which will bind to
the Rad51. The agent which binds can then be characterized or
identified as needed.
[0076] In a preferred embodiment, the binding of the candidate
bioactive agent is determined through the use of competitive
binding assays. In this embodiment, the competitor is can be any
molecule known to bind to Rad51, for example an antibody to Rad51,
or one of the proteins known to interact with Rad51, including
Rad52, Rad54, Rad55, DMC 1, BRCA1, BRCA2, p53, UBC9, RNA polymerase
II, and Rad51 itself, any or all of which may be used in
competitive assays. Either the candidate agents or the competitor
may be labeled, or both may be labeled with different labels. In
this embodiment, either the candidate bioactive agent, or the
competitor, is added first to the Rad51 sample for a time
sufficient to allow binding, if present, as outlined above. Excess
reagent is generally removed or washed away. The second component
is then added, and the presence or absence of the labeled component
is followed, to indicate binding.
[0077] In a preferred embodiment, methods for screening for a
bioactive agent capable of modulating the activity of Rad51
comprise the steps of adding a candidate bioactive agent to a
sample of Rad51, as above, and determining an alteration in the
biological activity of Rad51. "Modulating the activity of Rad51"
includes an increase in activity, a decrease in activity, or a
change in the type or kind of activity present. Thus, in this
embodiment, the candidate agent should both bind to Rad51 (although
this may not be necessary), and alter its biological or biochemical
activity as defined above.
[0078] Thus, in this embodiment, the methods comprise combining a
Rad51 sample and a candidate bioactive agent, and testing the Rad51
biological activity as is known in the art to evaluate the effect
of the agent on the activity of Rad51.
[0079] In a preferred embodiment, the methods include both in vitro
screening methods, as are generally outlined above, and in vivo
screening of cells for alterations in the presence, distribution or
activity of Rad51. Accordingly, in a preferred embodiment, the
methods comprise the steps of adding a candidate bioactive agent to
a cell, and determining the effect on the formation or distribution
of Rad51 foci in the cell. The addition of the candidate agent to a
cell will be done as is known in the art, and may include the use
of nuclear localization signal (NLS). NLSs are generally short,
positively charged (basic) domains that serve to direct the entire
protein in which they occur to the cell's nucleus. Numerous NLS
amino acid sequences have been reported including single basic
NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro
Lys Lys Lys Arg Lys Val), Kalderon (1984), et al., Cell,
39:499-509; the human retinoic acid receptors nuclear localization
signal (ARRRRP); NF.kappa.B p50 (EEVQRKRQKL; Ghosh et al., Cell
62:1019 (1990); NF.kappa.B p65 (EEKRKRTYE; Nolan et al., Cell
64:961 (1991); and others (see for example Boulikas, J. Cell.
Biochem. 55(1):32-58 (1994), hereby incorporated by reference) and
double basic NLS's exemplified by that of the Xenopus (African
clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp), Dingwall, et
al., Cell, 30:449-458, 1982 and Dingwall, et al., J. Cell Biol.,
107:641-849; 1988). Numerous localization studies have demonstrated
that NLSs incorporated in synthetic peptides or grafted onto
reporter proteins not normally targeted to the cell nucleus cause
these peptides and reporter proteins to be concentrated in the
nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell
Biol., 2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci.
USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci.
USA, 87:458-462, 1990. In general, the Rad51 foci will be evaluated
as is generally discussed above.
[0080] In a preferred embodiment, the methods comprise adding a
candidate bioactive agent to a cell, and determining the effect on
double strand break repair, homologous recombination, sensitivity
to ionizing radiation, and class switch recombination. Assays are
detailed in Park, J. Biol. Chem. 270(26):15467 (1995) and Li et
al., PNAS USA 93:10222 (1996), Shinohara et al., supra, 1992, all
of which are hereby incorporated by reference.
[0081] In a preferred embodiment, the cells to which candidate
agents are added are subjected to conditions which induce nucleic
acid damage, including the addition of radioisotopes (I.sup.125,
Tc, etc., including ionizing radiation and uv), chemicals (Fe-EDTA,
bis(1,10-phenanthroline), etc.), enzymes (nucleases, etc.).
[0082] a variety of other reagents may be included in the screening
assays or kits, below. These include reagents like salts, neutral
proteins, e.g. albumin, detergents, etc which may be used to
facilitate optimal protein-protein binding and/or reduce
non-specific or background interactions. Also reagents that
otherwise improve the efficiency of the assay, such as protease
inhibitors, nuclease inhibitors, anti-microbial agents, etc., may
be used. In general, the mixture of components may be added in any
order that provides for the requisite binding.
[0083] Once identified, the compounds having the desired
pharmacological activity may be administered in a physiologically
acceptable carrier to a host, as previously described. The
inhibitory agents may be administered in a variety of ways, orally,
parenterally e.g., subcutaneously, intraperitoneally,
intravascularly, etc. Depending upon the manner of introduction,
the compounds may be formulated in a variety of ways. The
concentration of therapeutically active compound in the formulation
may vary from about 0.1-100 wt. %.
[0084] The pharmaceutical compositions can be prepared in various
forms, such as granules, aerosols, tablets, pills, suppositories,
capsules, suspensions, salves, lotions and the like. Pharmaceutical
grade organic or inorganic carriers and/or diluents suitable for
oral and topical use can be used to make up compositions containing
the therapeutically-active compounds. Diluents known to the art
include aqueous media, vegetable and animal oils and fats.
Stabilizing agents, wetting and emulsifying agents, salts for
varying the osmotic pressure or buffers for securing an adequate pH
value, and skin penetration enhancers can be used as auxiliary
agents.
[0085] In a preferred embodiment, kits are provided. The kits can
be utilized in a variety of applications, including determining the
distribution of Rad51 foci, diagnosing an individual at risk for a
disease state, including cancer, diseases associated with
apoptosis, and diseases associated with stress (including oxidative
stress, hypoxic stress, osmotic stress or shock, heat or cold
stress or shock). The kits include a Rad51 binding agent, that will
bind to the Rad51 with sufficient affinity for assay. Antibodies
are preferred binding agents. The kits further include a detectable
label such as is outlined above. In one embodiment, the Rad51
binding agent is labeled; in an additional embodiment, a secondary
binding agent or label is used. Thus for example, the binding agent
may include biotin, and the secondary agent can include
streptavidin and a fluorescent label. Additional reagents such as
outlined above can also be included. Furthermore, the kit may
include packaging and instructions, as required.
[0086] The identification of the crucial role of Rad51 in a number
of cellular processes and disease states also identifies a number
of methods and compositions relating to combinations of Rad51 and
other tumor suppressor genes. Thus, Rad51 may function
interactively with a number of tumor suppressor genes and thus
compositions comprising combinations of these genes may be useful
in methods of gene therapy treatment and diagnosis.
[0087] Accordingly, in a preferred embodiment, compositions
comprising a nucleic acid encoding a Rad51 protein and at least one
nucleic acid encoding a tumor suppressor gene are provided.
Suitable tumor suppressor genes include, but are not limited to,
p53, and the BRCA genes, including BRCA1 and BRCA2 genes. Thus,
preferred embodiments include compositions of nucleic acids
encoding a) a Rad51 gene and a p53 gene; b) a Rad51 gene and a
BRCA1 gene; c) a Rad51 gene and a BRCA2 gene; d) a Rad51 gene, a
p53 gene, and a BRCA gene; and e), a Rad51 gene, a p53 gene, a
BRCA1 gene and a BRCA2 gene.
[0088] In an additional embodiment, the compositions comprise
recombinant proteins. By "recombinant" herein is meant a protein
made using recombinant techniques, i.e. through the expression of a
recombinant nucleic acid as depicted above. a recombinant protein
is distinguished from naturally occurring protein by at least one
or more characteristics. For example, the protein may be isolated
or purified away from some or all of the proteins and compounds
with which it is normally associated in its wild type host, and
thus may be substantially pure. For example, an isolated protein is
unaccompanied by at least some of the material with which it is
normally associated in its natural state, preferably constituting
at least about 0.5%, more preferably at least about 5% by weight of
the total protein in a given sample. a substantially pure protein
comprises at least about 75% by weight of the total protein, with
at least about 80% being preferred, and at least about 90% being
particularly preferred. The definition includes the production of a
protein from one organism in a different organism or host cell.
Alternatively, the protein may be made at a significantly higher
concentration than is normally seen, through the use of a inducible
promoter or high expression promoter, such that the protein is made
at increased concentration levels. Alternatively, the protein may
be in a form not normally found in nature, as in the addition of an
epitope tag or amino acid substitutions, insertions and deletions,
as discussed below.
[0089] In a preferred embodiment, these compositions can be
administered to a cell or patient, as is outlined above and
generally known in the art for gene therapy applications.
[0090] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are specifically incorporated by reference.
EXAMPLES
Example 1
Immunofluorescent Staining of Human Breast Cancer Cells
[0091] Breast tumour cells have mutated p53 and have various types
of chromosomal aberrations like insertions, deletions,
rearrangements, amplifications etc. Recombination proteins such as
Rad51 could evidently participate in such processes. In order to
better understand the role of uncontrolled recombination and its
role in tumour formation and progression, the status of Rad51
protein in breast tumour cells by staining them with anti Rad51
antibodies was done.
[0092] Detailed methods of cloning and expression of HsRad51 gene
in E. coli, purification of recombinant HsRad51 protein with six
histidine residues at it's aminoterminal end and preparation of
ployclonal antibodies against HsRad51 protein were described
previously by Haaf, Golub et al. 1995, supra, which is expressly
incorporated herein by reference.
[0093] Immunofluorescent staining with anti-Rad51 protein
antibodies. Monolayer cultures of different cell substrates (see
table 1) were grown in Dulbecco's MEM medium supplemented with 10%
fetal bovine serum and antibiotics. The cells were detached from
culture flasks by gentle trypsinization, pelleted and resuspended
in phosphate buffered saline (PBS; 136 mM NaCl, 2 mM KCl, 10.6 mM
Na.sub.2HPO.sub.4, 1.5 mM KH.sub.2PO.sub.4 [pH 7.3]) prewarmed at
37.degree. C. For immunofluorescence staining standard protocols
were used (Haaf 1995, supra). Cultured cells were washed and
resuspended in PBS. The density of somatic cells was adjusted to
about 10.sup.5 cells per ml in PBS. Aliquots (0.5 ml) of the cell
suspension were centrifuged onto clean glass slides at 800 rpm for
4 min, in a Cytospin (Shandon, Pittsburg). Immediately after
cytocentrifugation, the slides were fixed in -20.degree. C.
methanol for 30 min and then immersed in ice-cold acetone for a few
seconds to permealize the cells for antibody staining. Following
three washes with PBS, the preparations were incubated at
37.degree. C. with rabbit anti-HsRad51 antiserum, diluted 1:50 with
PBS containing 0.5% bovine serum albumin, in a humidified incubator
for 30 min. The slides were washed three times for 10 min each and
then incubated for 30 min with fluorescein-isothiocyanate
(FITC)-conjugated anti-rabbit IgG diluted 1:20 with PBS. After
three washes with PBS, the preparations were counterstained with
4',6-diamidino-2- phenylindole (DAPI; 0.1 ug/ml for 1 min) and
mounted in antifade {90% (vol/vol) glycerol/0.1 m tris-HCl pH
8.0)/2.3% 1,4-diazabicyclo[2.2.2]octane (DABCO)}.
[0094] Digital Imaging Microscopy. Images were taken with a Zeiss
epifluorescence microscope with a thermo-electronically cooled
charge coupled device (CCD) camera (model PM512; Photometrics,
Tucson, Ariz.) which was controlled by an Apple Macintosh computer.
Grey scale source images were captured separately with filter sets
for fluorescein and DAPI. Gray scale source images were
pseudocolored and merged using ONCOR Image and ADOBE Photoshop
software. It is worth emphasizing that although a CCD imaging
system was used, all antibody signals were clearly visible by eye
through the microscope.
[0095] To study the possible involvement of Rad51 in tumorigenesis
we compared the the in situ localization of of Rad51 protein
homologs in different cell substrates i.e. mortal fibroblast
strains, virus-transformed non-malignant cell lines and tumor cell
lines (see table). a specific rabbit antiserum raised against human
Rad51 protein was used in these studies. These antibodies reacted
mainly with Rad51 protein in mammalian cell extracts as judged by
Western blotting (see FIG. 2 in (Haaf, Golub et al. 1995).
Immunostaining of different cells showed that HsRad51 is
concentrated in small and discrete sites (foci) through out
nucleoplasm and is largely excluded from nucleoli and cytoplasm. At
least 250 nuclei of exponentially growing cultures were analyzed
for each experiment. As reported earlier (Haaf, Golub et al. 1995)
immunostaining revealed three kinds of nuclei: 1) nuclei that did
not show any staining at all ( no foci), 2) nuclei that showed weak
to medium staining and showed only a few foci (Type I nuclei) 3)
nuclei that showed strong staining and showed many foci (Type II
nuclei). In normal fibroblast control cells, we found type I nuclei
in about 10% of cells and type II nuclei in less than 0.4 to 1% of
cells and about 90% of the cells showed no foci. Use of preimmune
serum, as well as omission of either the primary or secondary
antibody, resulted in the absence of focally concentrated nuclear
immunofluorescence.
[0096] As reported earlier (Haaf, Golub et al. 1995) in normal
(mortal) fibroblast control cells (Hs68) we found type I nuclei in
7% -10% of cells and type II nuclei in less than 0.4% of cells,
where as 90% or more of the cells showed no foci (Table 1). In
contrast all breast tumor cell lines tested (BT20, SrBr3, MoF7)
exhibited 1-5% of type II nuclei and 10-38% of type I nuclei (Table
1). Transformed but non-malignant human cells, i.e. SV 40
transformed fibroblasts (LNL8, 63L7), EBV-transformed lymphoblasts
(GM 01194), and adenovirus-transformed kidney cells (293) also
showed an increased percentage of Rad51-positive cells (compared to
normal fibroblasts), however the numbers observed were lower than
in tumor cells. Interestingly, some tumor substrates i.e. the
ovarian cancer line Hey; did not show a significant increase of
Rad51-positive cells.
[0097] As demonstrated earlier (Haaf, Golub et al. 1995), when the
normal fibroblast cells were exposed to DNA damaging agents like
137Cs, there was a significant increase of cells containing type I
and type II nuclei (Table 2). It is worth emphasizing that
non-irradiated breast tumor cells show approximately the same
percentage of Rad51-positive nuclei as Hs68 fibroblasts exposed to
900 rad Cs.sup.137 which kills 99% of cells (Table 2). The
immunofluorescent patterns of (non-irradiated) breast cancer cells
(FIG. 1) and fibroblasts that were exposed to DNA damaging agents
are identical.
[0098] When the breast cancer cells were exposed to Cs137, the
increase in the number of cells with type I and type II nuclei was
even more dramatic than in normal (Hs68) or transformed (LNL8)
fibroblasts (Table 2). Up to 40% of irradiated breast cancer cells
showed type I nuclei and 11%-18% showed type II nuclei.
[0099] In order to rule out any artifacts that would arise due to
the examination of cultured breast cancer cells, we then examined
the breast tissue obtained directly from the patient for Rad51
positive staining. Immunohistochemical evaluation revealed definite
nuclear staining of invasive breast carcinoma cells. Specifically,
nuclear reactivity could be demonstrated in sections obtained from
three paraffin-embedded samples. The nuclear staining appeared
granular in some areas, and in others, occupied the entire nucleus.
The actual number of invasive carcinoma cells that fluoresced was
quite small, and estimated to be less than 5% of the nuclei seen in
three samples with definite reactivity FIG. 2). Nuclear staining
was not identical in normal breast epithelium or lactating breast
tissue. Bright nuclear reactivity was seen in positive control
testicular tissue, specifically, in the cells lining the
seminiferous tubules. Background staining did not appear to be
problematic.
[0100] Increase in immunofluorescence of HsRad51 in breast cancer
cells can result from either increase in the amount of Hsrad51 in
these cells or it could be seen as a result of re-organization of
Hsrad51 in these nuclei in response to damage related activities.
We think that the latter is true because there was no apparent
increase in the amount HsRad51 in breast cancer cells as shown by
the Western blots (data not shown).
[0101] The molecular basis and the consequence of the increase in
HsRad51 in breast cancer cells is not clear. Since Rad51 protein
interacts with other proteins of the Rad52 epistasis group and
these multiprotein complexes are involved in the recombinational
repair of double-strand breaks (Hays, et al., (1995). Proc. Natl.
Acad. Sci. USA 92: 6925-6929; Johnson, R. D. and L. S. Symington
(1995). Mol. Cell. Biol. 15: 4843-4850), it is tempting to
speculate that these foci are the sites where repair/recombination
events are taking place. Since p53 is known to interact with Rad51
it iwill be interesting to see the colocalization of p53 and Rad51
protein in these complexes. It is quite possible that these foci
contain either wild type or mutant p53 and other breast cancer
related proteins like BRCA1, BRCA2 or the newly discovered STG1
protein. We propose that the increase in the immunofluorescence of
Rad51 in the breast cells can be used as an important cytological
marker for cell proliferation and malignant cell growth. Further
experimentation will be done to validate this proposal and to
understand the role of increase in Rad51 foci and
carcinogenesis.
1TABLE 1 Percentage of nuclei containing discrete foci enriched
with HsRad51 protein. No Cell Substrate foci Type I Type II Hs68
Normal fibroblasts 90% 10% 0% 93% 7% 0% LNL8 (NI 00847) Transformed
fibroblasts 90% 9% 1% (SV 40) 90% 8% 2% 63L7 Transformed
fibroblasts 94% 6% 0% (SV40) 94% 3% 3% GM01194 Transformed
lymphoblasts 91% 7% 2% (EBV) 90% 9% 1% 92% 8% 0% 80% 18% 2% 80% 19%
1% 293 Cells Transformed kidney cells 75% 23% 2% (Adenovirus) 83%
15% 2% 82% 17% 1% BT20 Breast cancer line 86% 10% 4% 82% 13% 5% 78%
17% 5% SrBr3 Breast cancer line 74% 25% 1% MoF7 Breast cancer line
57% 38% 5% 88% 10% 2% Tera2 Testicular teratoma 76% 23% 1% 77% 22%
1% Hey Ovarian cancer line 94% 5% 1% 98% 2% 0% HeLa Cervix (?)
tumor cells 67% 31% 2%
[0102]
2TABLE 2 Percentage of nuclei containing discrete foci enriched
with HsRad51 protein. No Type Type Cell substrate Treatment foci I
II Hs68 None 90% 10% 0% (normal None 93% 7% 0% fibroblasts) 6 hrs
after 10 rad Cs137 96% 4% 0% 6 hrs after 50 rad Cs137 96% 4% 0% 6
hrs after 150 rad Cs137 92% 7% 1% 6 hrs after 450 rad Cs137 88% 8%
4% 6 hrs after 900 rad Cs 137 91% 4% 5% LNL8(NI 00847) None 90% 9%
1% (SV40-transformed None 90% 8% 2% fibroblasts) 6 hrs after 150
rad Cs137 88% 11% 1% 6 hrs after 300 rad Cs137 76% 19% 5% 6 hrs
after 900 rad Cs137 78% 17% 5% BT20 None 86% 10% 4% (breast cancer
None 82% 13% 5% cells) None 78% 17% 5% 6 hrs after 300 rad Cs137
44% 41% 11% 6 hrs after 900 rad Cs137 52% 30% 18%
Example 2
Nuclear Foci of Human Recombination Protein Rad51 in Nucleotide
Excision Repair Defective Cells
[0103] Eurkaryotic cells have several different mechanisms for
repairing damaged DNA (for review see R. Wood, 1996). One of the
major pathway is nucleotide excision repair (NER), which excises
damage within oligomers that are 25-32 nucleotides long. Patients
with recessive heredity disorder XP have defects in one of several
enzymes, which participate in ER. There are seven XP groups (XP-A
to XP-G), which have defects in the initial steps of the DNA
excision repair.
[0104] DNA damage is removed several-fold faster from transcribed
genes than from non-transcribed, mainly due to preferential NER of
the transcribed strand (for review see Hanawalt, 1994). This
mechanism does not function in Cockayne's syndrome (CS)
patients.
[0105] NER defective cells, evidently, sustain increased amount of
DNA damage. Thus we evaluated NER defective cells from XP and CS
cells for an increased amount of Rad51 protein foci.
[0106] To study possible effect of NER on localization of HsRad51
in somatic tissue culture cells, we compare in situ localization of
the protein in normal fibroblasts, different XP cells and CS-B
cells. A policlonal rabbit antiserum raised against human Rad51
protein was used in this study. These antibody reacted in mammalian
cell extract mainly with Rad51 protein as judged by Western
Blotting (see FIG. 2 in Haaf et al., 1995). Immunostaining of
different cell lines showed that HsRad51 is concentrated in small
and discrete sites (foci) throughout nucleoplasm and is largely
excluded from nucleoli and cytoplasm. As discussed above,
immunostaining revealed three kinds of nuclei, types I, II and III.
The results are shown in Table 3.
3TABLE 3 Percentage of nuclei containing discrete foci enriched
with HsRad51 protein No Type Type Cell substrate foci I* II* Hs68
Normal fibroblasts 90% 10% 0% 63L7 Normal fibroblasts 94% 6% 0%
63L7 (confluent) FA fibroblasts 94% 3% 3% 6935 FA fibroblasts 92%
6% 2% 6914 Normal 72% 21% 7% 6914 lymphoblasts 72% 25% 3% 6914 67%
24% 9% GM01194 Normal 91% 7% 2% GM01194 lymphoblasts 90% 9% 1%
GM01194 92% 8% 0% GM07063 FA lymphoblasts 90% 8% 2% GM07063 96% 4%
0% GM13020 FA lymphoblasts 92% 7% 1% GM13022 FA lymphoblasts 86%
13% 1% GM13022 78% 20% 2% GM13023 94% 5% 1% GM13071 81% 15% 4%
GM13071 74% 23% 3% *Type I nuclei show only few (<15) foci
and/or weak to medium HsRad51 immunofluorescence, whereas Type II
cells show many and/or strongly fluorescing foci. 250 nuclei were
analyzed for each experiment.
[0107] In normal (mortal) fibroblast control cells, LNL8 and NF, we
found type I nuclei in 5-9% cells and type II nuclei in 1-7% cells,
where as 88-90% of the cells showed no foci (Table 3). Use of
preimmune serum, as well as omission of either the primary or
secondary antibody, resulted in the absence of focally concentrated
nuclear immunofluorescence.
[0108] XP-V cells are normal in NER, but have defect in
postreplication repair process (Boyer et al., 1990; Griffiths et
al., 1991; Wang et al., 1991, 1993). As we expected, these cells
showed the same distribution pattern of nuclear HsRad51 as control
cell lines (Table 3).
[0109] Distribution of HsRad51 foci in CS-B cells also was similar
to the cells with normal NER (Table 3). This result was also
anticipated. CS-B cells are defective in NER which is coupled with
transcription (Venema et al., 1990). Transcribed genes, evidently,
comprise only a small part of the whole genomic DNA and damage in
transcribed genes, therefore, should be accounted for only a very
small fraction of the damage in genomic DNA.
[0110] XP-A, XP-B, XP-F and XP-G cells are all defective in NER.
XP-A cells have defect in XPA protein, which carries out a crucial
rate-limiting step in NER-recognition of DNA lesion (Jones and
Wood, 1993). The protein makes a ternary complex with ERCC 1
protein and XPF protein, which is defective in XP-F cells (Park and
Sankar, 1994). XP-B and XP-G cells are defective in different steps
of NER which follow damage recognition (Reviewed in Ma et al.,
1995).
[0111] XP-A and XP-F cell lines have increased amount of cells with
HsRad51 protein foci (Table 3). In contrast, XP-B and XP-G cells
have about the same level of HsRad51 protein foci, as cells with
normal NER (Table 3). This result could be easily understood if we
assume, that 1) formation of HsRad51 foci is caused by DNA damage,
b) DNA lesion is excluded from the pool of damage DNA which cause
Rad51 foci formation as soon as XPA/XPF/ERCC 1 complex binds to the
lesion. DNA damage in XP-Band XP-G cells is recognized by NER
system, but the damage cannot be proceeded and removed by the
system. Such unremoved damage, evidently, is not considered as a
substrate for Rad51 protein involved repair as soon as the damage
is recognized by NER complex XPA/XPF/ERCC1 as a substrate for NER,
even if defect in subsequent steps of NER makes its removing
impossible.
[0112] Induction of principal DNA repair system (SOS respond) in E.
coli is, assumed to be triggered by formation of single-stranded
DNA (ssDNA) which results from DNA damage (reviewed in Little and
Mount, 1982). DNA damage in XP-A cells is not recognized by NER
and, therefore, at least a considerable part of DNA damage is not
proceeded to formation of ssDNA regions. Nevertheless, Rad51 foci
are effectively formed in XP-A cells and their amount could be
further increased by UV or -irradiation (Tables 4 and 5).
Evidently, ssDNA is not a primary signal for HsRad51 protein foci
formation.
4TABLE 4 Percentage of nuclei containing discrete foci enriched
with HsRad51 protein Cell substrate Treatment No foci Type I* Type
II* LNL8 No treatment 90% 9% 1% (control) " 90% 8% 2% NF (control)
No treatment 88% 5% 7% " 89% 5% 6% XPA No treatment 51% 39% 10% "
72% 20% 8% " 55% 34% 7% XPB No treatment 86% 11% 3% " 86% 11% 3%
XPD No treatment 87% 8% 5% " 63% 28% 9% XPF No treatment 48% 41%
11% " 64% 25% 8% XPG No treatment 88% 7% 5% " 85% 9% 6% XPV No
treatment 94% 5% 1% " 89% 11% 0% CBS None 87% 8% 5% *Type I nuclei
show only a few (<15) foci and/or weak to medium HsRad51
immunofluorescence, whereas type II cells show many and/or strongly
fluorescing foci. 250 nuclei were analyzed for each experiment.
[0113]
5TABLE 5 Percentage of nuclei containing discrete foci enriched
with HsRad51 protein No Type Cell substrate Treatment foci I* Type
II* LNL8 No treatment 90% 9% 1% (control) No treatment 90% 8% 2% 6
hrs after 150 rad Cs137 88% 11% 1% 6 hrs after 300 rad Cs137 76%
19% 5% 6 hrs after 900 rad Cs137 None 78% 17% 5% 3 hrs after 300
rad .sup.137Cs XPA** 51% 39% 10% None 61% 24% 15% 6 hrs after 900
rad .sup.137Cs None 72% 20% 8% 5 hrs after 5 J/m.sup.2 UV 59% 25%
16% 5 hrs after 15 J/m.sup.2 UV 59% 34% 7% None 53% 31% 16% 5 hrs
after 800 rad .sup.137Cs 27 hrs after 800 rad .sup.137Cs 55% 26%
19% CBS** 87% 8% 5% 60% 21% 19% 77% 6% 17% *Type I nuclei show only
a few (<15) foci and/or weak to medium HsRad51
immunofluorescence, whereas Type II cells show many and/or strongly
fluorescing foci. 150 nuclei were analyzed for each experiment.
**Induction of HsRad51 foci in Xeroderma pigmentosum (Type A)
implies that single stranded DNA molecules are not the primary
signal. ***Induction of HsRad51 foci in cells from patients with
Cockayne's syndrome implies that the induction is not dependent on
transcription.
[0114] In conclusion, human recombination protein HsRad51 is
concentrated in multiple discrete foci in nucleoplasm of cultured
human cells. After treatment of cells with DNA damaging agents, the
percentage of cells with HsRad51 protein immunofluorescence
increases. Xeroderma pigmentosum (XP) cells XP-A with inactive
protein XPA, responsible for lesion recognition by nucleotide
excision repair (NER) system have increased percentage of cells
with HsRad51 protein foci. XP-F cells, defective in XPF protein,
which forms complex with XPA protein, also have increased level of
the HsRad51 protein foci. In contrast, XP-B and XP-G cells with
defects in different steps ER, which follow the damage recognition,
as well as XP-V cells (normal level of NER) and Cockayne's syndrome
(CS) cells (defect in NER, responsible for preferential repair of
the transcribed DNA strand) have normal level of HsRad51 protein
foci. Evidently, formation of HsRad51 protein foci is caused by DNA
damages. DNA damages, however, do not participate in causing
formation of HsRad51 protein foci, as soon as they are recognized
by NER system, even if the system is blocked on one of the step,
leading to DNA repair.
Example 3
Higher Order Nuclear Structures of Rad51 and its Exclusion Into
Micronuclei After Cell Damage
[0115] Previous studies have revealed a time- and dose-dependent
increase of nuclear HsRad51 protein foci after DNA damage
introduced into the genome by various agents (Haaf et al., 1995,
supra). Here we show that when the damaged cells are allowed to
recover, these Rad51 foci form specific higher-order nuclear
structures. Finally, all the focally concentrated Rad51 protein is
eliminated into micronuclei that undergo apoptotic genome
fragmentation. Treatment of cells with clastogens and
aneuploidogens implements a mechanism that affects the nuclear
distribution of Rad51 protein and targets Rad51 foci, most likely
along with irreversibly damaged DNA into micronuclei. To examine
the role of Rad51 protein in DNA repair and cell proliferation, we
have analyzed the intranuclear distribution of overexpressed Rad51
protein during the cell cycle and in cell populations proceeding
through apoptosis.
[0116] Experimental Procedures
[0117] Cell Culture. The sources of the cell lines were as follows.
Rat TGR-I cells, J. Sedivy, Brown University; mouse 3T3-Swiss
cells, ATCC; human 293 kidney cells, ATCC; human teratoma cells, B.
King, Yale University; human LNL8 fibroblasts, S. Meyn, Yale; human
XPA and XPF fibroblasts, P Glazer, Yale.
[0118] Monolayer cultures were grown in D-MEM medium supplemented
with 10% fetal bovine serum and antibiotics. The cells were
detached from culture flasks by gentle trypsination, pelleted and
resuspended in phosphate-buffered saline (PBS; 136 mM NaCl, 2 mM
KCI, 10.6 mM Na.sub.2HPO.sub.4, 1.5 mM KH.sub.2PO.sub.4, pH 7.3)
prewarned at 37.degree. C.
[0119] To induce DSBs in DNA and recombinational repair, cell
cultures were exposed to a .sup.137Cs irradiator at doses of 900
rad and then allowed to recover for various time spans. In another
experiment, cells were treated with 10 .mu.M 5-aza-dC for 24 hrs.
This hypomethylating base analog is a potent DNA-strand breaker
(Snyder, et al., (1989). Mutation Res. 226, 185-190; Haaf, 1995).
Incubation of cells with the spindle poison colcemid (1 .mu.g/ml
for 24 hrs) resulted in the formation of multinuclei and
micronuclei containing entire chromosomes. Under the experimental
conditions chosen, colcemid does not cause chromsome breakage.
Treatment with etoposide (Sedivy), a drug that inhibits DNA
topoisomerase II, is a classic system for inducing apoptosis in
cells (Mizumoto, et al., (1994). Mol. Pharmac. 46, 890-895).
[0120] Antibody Probes. HsRad51 protein, expressed in E. coli, was
isolated and used for preparation of rabbit polyclonal antibodies.
Westem blotting experiments revealed that rabbit antiserum does not
react significantly with any other proteins in mammalian cells
except Rad51 (Haaf et al., 1995). Similarly, polydonal antibodies
against HsRadS2, a structural homolog of yeast Rad52, were raised
in the rat, as is known in the art. Mouse monoclonal antibody 30T14
recognizes Gadd45, a ubiquitously expressed mammalian protein that
is induced by DNA damage (Smith, et al., (1994). Science 266,
1376-1380). Monoclonal antibodies H4 and H14 bind specifically to
the large subunit of RNAPII (Bregman et al., (1995) J. Cell Biol.
129, 287-298). Monoclonal antibody Pab246 against amino acids 88-93
of mouse p53 was purchased from Santa Cruz Biotechnology, Inc.
[0121] Immunofluorescent Staining. Harvested cells were washed and
resuspended in PBS. Cell density was adjusted to .about.10.sup.5
cells/ml. 0.5 ml aliquots of this cell suspension were centrifuged
onto clean glass slides at 800 rpm for 4 min, using a Shandon
Cytospin. Immediately after cytocentrifugation, the preparations
were fixed in absolute methanol for 30 min at -20.degree. C. and
then rinsed in ice-cold acetone for a few seconds. Following three
washes with PBS, the preparations were incubated at 37.degree. C.
with rabbit anti-HsRad51 antiserum, diluted 1:100 with PBS, in a
humidified incubator for 30 min. For some experiments, the slides
were simultaneously labeled with rat anti-HsRad52 antiserum or
mouse monoclonal antibody. The slides were then washed in PBS
another three times for 10 min each and incubated for 30 min with
fluorescein-isothiocyanate (FITC)-conjugated anti-rabbit laG,
appropriately diluted with PBS. Rad52, Gadd45, p53, and RNAPII were
detected with rhodamine, conjugated anti-rat IgG or anti-mouse
IgG+IgM. After three further washes with PBS, the preparations were
counterstained with 1 .mu.g/ml 4,6-diamidino-2-phenylindole (DAPI)
in 2.times.SSC for 5 min. The slides were mounted in 90% glycerol,
0.1 M Tris-HCI, pH 8.0, and 2.3% 1,4-diazobicyclo-2,2,2-octane
(DABCO).
[0122] For preparation of chromatin fibers, cells were centrifuged
onto a glass slide and covered with 50 .mu.l of 50 mM Tris-HCI, pH
8.0, 1 mM EDTA, and 0.1% SDS. The protein-extracted chromatin was
mechanically sheared on the slide with the aid of another slide
(Heiskanan, et al., (1994) BioTechniques 17, 928-933) and then
fixed in methanol/acetone.
[0123] Fluorescence In Situ End Labeling (FISEL). FISEL detects
cell death (apoptosis) in situ by quantitating DNA strand breaks in
individual nuclei. It uses terminal transferase (TdT) to label the
3'-ends in fragmented genomic DNA with biotinylated nucleotide. 100
.mu.l of reaction mix contain 1 .mu.l (25 Units) TdT (Boehringer
Mannheim), 20 .mu.l 5.times.TdT buffer (supplied with the enyzme),
1 .mu.l 0.5 mM biotin-ll-dUTP, 3 .mu.l 0.5 mM dTTP, and 75 .mu.l
ddH.sub.20. Cytological preprations are incubated at 37.degree. C.
for 1 hr with this reaction mix. Washing the slides for 3.times.5
min in PBS is sufficient to terminate the reaction. The
incorporated biotin-dUTP is detected with rhodarnine-conjugated
avidin.
[0124] In Situ Labeling of DNA-Replication Synthesis. The base
analog BrdU is incorporated in place of thymidine into the DNA of
replicating cells. In order to mark cycling cells, 10 .mu.g/ml BrdU
were added to the culture medium 30 hrs before cell harvesting.
Depending on the cell substrate, this corresponds to one or two
population doublings. At the end of the labeling period, slides
were prepared as described above. After Rad51-protein staining, the
preparations were again fixed in a 3:1 mixture of methanol and
acetic acid for several hours at -20.degree. C. Since the anti-BrdU
antibody only recognizes BrdU incorporated into chromosomal DNA if
the DNA is in the single-stranded form, the slides were denatured
in 70% formamide, 2.times.SSC for 1 min at 80.degree. C. and then
dehydrated in an alcohol series. BrdU incorporation was visualized
by indirect anti-BrdU antibody staining. First, the preparations
were incubated with mouse monoclonal anti-BrdU antibody (Boehringer
Mannheim), diluted 1:50 with PBS, for 30 min. The slides were
washed with PBS and then incubated with rhodamine-conjugated
anti-mouse IgG, diluted 1:20 with PBS, for another 30 min. Only
cells with intense BrdU labeling of the entire nucleus were
considered BrdU-positive and scored as cycling cells.
[0125] Overexpression of HsRad51 Protein in Mammalian Cells. Human
kidney cells (line 293, ATCC CRL1573) were stably transformed by
plasmid pEG9 15. This plasmid carries the whole coding sequence of
the HsRad51 gene inserted in frame with the 5'-end terminal
sequence of vector pEBVHisB (Invitrogen). The resulting cell lines
710 and 717 constitutively express Rad51 protein fused to a T7-tag
epitope (Haafet al., 1995).
[0126] Digital Imaging Microscopy. Images were taken with a Zeiss
epifluorescence microscope equipped with a thermoelectronically
cooled charge coupled device (CCD) camera (Photometries CH250),
which was controlled by an Apple Macintosh computer. Gray scale
source images were captured separately with filter sets for FITC,
rhodamine, and DAPI. Gray scale images were pseudocolored and
merged using ONCOR Image and ADOPE Photoshop software. It is worth
emphasizing that although a CCD imaging system was used, the
immunofluorescent signals described here were clearly visible by
eye through the microscope.
[0127] Dynamic Nuclear Distribution of Rad51 Protein after DNA
Damage Nuclear foci of mammalian Rad51-recombination protein can be
induced significantly after irradiation of cell cultures with
Cesium (.sup.137Cs). Since Western blots have not shown a dramatic
net increase in Rad51 protein in irradiated cells, we conclude that
DNA damage mainly affects its nuclear distribution (Haaf et al.,
1995). To gain insight into the radiation-induced perturbations in
nuclear organization and the possible role of Rad51 protein in
repair processes, we have analyzed the topological rearrangements
of Rad51-protein foci in rat TGR-I fibroblasts that have sustained
DNA damage. TGR-I is an immortal rat cell line with a stably
diploid karyotype. After .sup.137Cs irradiation with a dose of 900
red which kills 99% of cells, rat Rad51 protein was visualized in
situ using polyclonal antibodies raised against HsRad51. The
percentage of cells with cytologically detectable Rad: 1-protein
foci started to increase in the first three hours (Table 6).
Rad51-positive nuclei contained up to several dozen discrete foci
throughout their nucleoplasm. Immunofluorescence staining was
largely excluded from the cytoplasm. Many of these nuclear Rad51
foci had a double-dot appearance, typical of paired DNA segments
(FIG. 3a).
6TABLE 6 Induction of Rad51 Foci after.sup.137Cs Irradiation of
TGR-1 cells and Their Elimination into Micronuclei Percentage of
Percentage of Cells with Cells with Percentage Type I.sup.a Foci
Type II.sup.a Foci of Cells in in without in Micro- in Micro-
Treatment Foci Nuclei nuceli Nuclei nuclei None 93% 6% 0% 1% 0% 3
hrs after 80% 8% 0.4% 11% 0.6% 900 rad .sup.137Cs 16 hrs after 73%
9% 8% 1% 9% 900 rad .sup.137Cs 30 hrs after 72% 1% 13% 1% 13% 900
rad .sup.137Cs 4 days after 90% 0% 4% 0% 6% 900 rad .sup.137Cs
.sup.aType I nuclei and micronuclei show weak to medium HsRad51
immuno- fluorescence, whereas type II cells show strongly
fluorescing foci. 1000 cells were anlayzed for each experiment.
[0128] When irradiated cells were then cultured for various times
to allow repair of induced DNA damage and apoptosis to occur,
significant changes in the distribution of Rad51-protein foci were
detected. Nuclear foci coalesced into larger clusters with
extremely high immunofluorescence intensity after 6-20 furs. Only a
few discrete foci remained singly in the nucleoplasm. In a
percentage of nuclei linear strings of 5-10 Rad51-protein foci were
formed (FIG. 3b). Immediately striking was the somatic association
of "homologous" strings of similar length. These strings were
always tightly paired at one of their ends. The dynamics of the
Rad51-protein foci after induction of DNA damage are clear evidence
for a higher-order organization of nuclear structure that
accompanies DNA repair and/or programmed cell death.
[0129] One to two days after .sup.137Cs irradiation with a lethal
dose the coalesced Rad51 clusters showed a highly non-random
localization towards the nuclear periphery (FIG. 3c). Finally, the
Rad51 structures were excluded into micronuclei. The nucleoplasm
was virtually cleared of Rad51 protein and only aggregated Rad51
foci in MN were remaining (FIG. 4;Table 6). Similar to the
situation seen earlier in interphase nuclei, many MN displayed
paired Rad51 foci and higher-order structures. The highest number
of MN (approximately three per cell) as well as the highest number
of Rad51-positive MN(approximately 30%) were observed 16 hrs after
irradiation (Table 7). However, at each time point analyzed the
majority of radiation-induced MN did not show detectable
Rad51-protein foci.
7TABLE 7 Rad51 Foci in Micronuclei of Different Cell Substrates
Number of Percentage of Percentage of Cell substrate Micronuclei in
Rad51-Positive Rad51-Negative Treatment 1000 Cells Micronuclei
Micronuclei TGR01 None 93 14% 86% 3 hrs after 900 279 22% 78%
rad.sup.137Cs 16 hrs after 900 2719 28% 72% rad.sup.137Cs 4 days
after 900 1040 20% 80% rad.sup.137Cs LNL8 None n.d. 23% 77% None
n.d. 26% 74% XPA None n.d. 18% 82% Teratoma None n.d. 10% 90%
3T3-Swiss None 472 125% 88% 1000 cells were analyzed for each
experiment
[0130] hypomethylating base analog induces inhibition of chromatin
condensation, leading to instability of the affected chromosome
regions (Haaf, 1995). Its cytotoxic effects are at least partially
due to the induction of single- and double-strand breaks in DNA.
Like .sup.137Cs irradiation, 5-aza-dC can induce the formation of
Rad51-protein foci in nuclei and its elimination into MN. Rat TGR-1
and human LNL8 fibroblast cultures treated with non-lethal doses of
5-aza-dC displayed MN with focally concentrated Rad51 protein in
5-10% of their cells (Table 8).
8TABLE 8 Induction of Rad51 Foci by 5-Azadeoxycytidine Percentage
of Percentage of Cells with Cells with Type I.sup.a Foci Type
II.sup.a Foci Percentage of in in Cell type Cells without in Micro-
in Micro- Treatment Foci Nuclei nuclei Nuceli nuclei TGR-1 None 93%
6% 0% 1% 0% 5-aza-dC.sup.b 86% 5% 4% 1% 4% LNL8 None 92% 6% 1% 1%
0% 5-aza-dC.sup.b 89% 3% 1% 2% 5% .sup.aType I nuclei and
micronuclei show weak to medium HsRad51 immunofluorescence, whereas
type II cells show strongly fluorescing foci. 500 cells were
analyzed for each experiment. .sup.b10.sup.-5M 5-aza-dC were added
to the culture medium 24 h rs before cell harvest.
[0131] Rapidly dividing cell cultures always exhibit a baseline MN
frequency even without exposure to clastogens or aneuploidogens. In
five different substrates studied, LNL8, XPA, teratoma, 3T3-Swiss,
and TGR-1 cells, 10-30% of these spontaneously occuring,
non-induced MN exhibited Rad51-protein foci (Table 7). This further
links Rad51-protein foci and MN formation.
[0132] Rad52 and Other Repair Proteins Are Not Excluded into
Micronuclei Studies in yeast (Shinohara et al., 1992, supra; Milne,
G., and Weaver, D. (1993). Genes Dev. 7, 1755-1765) and humans
(Shen, et al., (1996). J. Biol. Chem. 271, 148-152) have shown
physical interaction between Rad51 and Rad52 proteins both in vitro
and in vivo. Double immunofluorescence with rabbit anti-Rad51 and
rat anti-Rad52 antibodies on .sup.137CS irradiated TGR-1 cells
showed that both proteins are enriched in nuclear foci but they do
not co-localize. Rad52-protein foci remained in the nucleus
throughout the entire time course, while Rad51-protein foci were
segregated into MN (data not shown). The same holds true for Gadd45
(data not shown) an inducible DNA-repair protein that is stimulated
by p53 (Smith et al., 1994, supra). Biochemical evidence further
suggests specific protein-protein association between HsRad51 and
p53 (Sturzbecher et al., 1996, supra). However, after anti-p53
antibody staining the Rad51 foci were not particularly enriched
with p53 protein (data not shown). In addition, HsRad51 was
reported to be associated with a RNA polymerase II (RNAPII)
holoenyme (Maldonado et al., 1996, supra). Afthough RNAPII was
immunolocalized in discrete discrete nuclear foci, as reported
previously (Bregman et al., 1995, supra), transcription complexes
did not coincide with Rad51 foci (data not shown).
[0133] Association of Rad51 Protein with DNA Fibers In a few
(<1%) cells of irradiated and drug-treated cultures, we observed
very elongated Rad51 structures, up to several hundred micrometer
io length, that were eliminated from the nuclei. Since these
fiber-like structures stained DAPI-positively, they are thought to
contain single DNA molecules of several megabases covered with
Rad51 (data not shown). Fluorescence in situ end labeling (FISEL)
demonstrated that these DNA fibers contain fragmented DNA typical
of apoptosis (data not shown). Sometimes the DNA fibers appeared to
leak out of the nucleus through holes in the nuclear membrane and
coodense into micronuclei. In all cell substrates studied, a high
percentage of MN displayed genome fragmentation (data not
shown).
[0134] The association of Rad51 protein with DNA was also visible
on experimentally extended chromatin fibers from irradiated cells.
SDS lysis and mechanical stretching of nuclear chromatin across the
surface of a glass slide can cause complete deattachment of DNA
loops from the nuclear matrix, producing highly elongated, linear
chromatin fibers (Haaf, T., and Ward, D. C. (1994). Hum. Mol.
Genet. 3, 629-633.; Heiskanen et al., 1994, supra).
Immunofluorescence staining revealed linear strings of Rad51 label
on these stretched DNA fibers (data not shown). By comparison with
YAC hybridIzat.about.on signals on similar preparations (Haaf and
Ward, 1994, supra), the length of the Rad51 fibers was estimated
1-2 Mb.
[0135] Rad51-Protein Foci and Apoptosis To determine whether
Rad51-positive MN specifically detect exposure to clastogens,
analyses were performed in rat TGR-I cells with the aneuploidogen
colcemid. This mitotic spindle poison causes lagging of whole
chromosomes that are excluded into MN. Surprisingly, when
colcemid-treated cells were allowed to recover for 24 hrs in
drug-free medium, over 30% of the induced MN contained very
brightly fluorescing Rad51 foci (Table 9). Some MN contained
rod-like linear structures (data not shown) similar to those
observed in Rad51-overexpressing cells. Most of these
Rad51-positive MN, 24 hrs after colcemid, did not contain
fragmented DNA, as determined by simultaneous FISEL (Table 9). When
cells were grown for one or two more days in the absence of the
drug, the percentage of Rad51-containing MN decreased dramatically.
In addition, the Rad51 protein was no longer concentrated in
discrete foci but appeared to disperse throughout the entire MN
volume. At the same time most MN became apoptotic and by FISEL
their degraded DNA showed incorporation of fluorescent nucleotides.
Thus, we conclude that mitotic arrest after colcemid triggers a
cascade that induces the elimination of Rad51 protein into MN and
drives apoptotic events. Our results seem to be consistent with the
hypothesis that apoptosis is a special form of aberrant mitosis
(Ucker, D. S. (1991). New Biologist 3, 103-1009; Shietal., 1994,
supra).
9TABLE 9 Rad51 Foci and Apoptosis in Colcemid-Induced Micronuclei
of TGR-1 Cells Number of micro- Percentage of nuclei Cells
Showing.sup.a in 1000 Rad51-/ Rad51+/ Rad51+/ Rad51-/ Treatment
cells FISEL- FISEL- FISEL+ FISEL+ None 93 75% 12% 2% 11%
Colcemid.sup.b n.d. 85% 6% 0% 9% 1 day of 1293 54% 31% 1% 14%
recovery 2 days of 1061 45% 45% 6% 40% recovery 3 days of 769 43%
7% 4% 46% recovery
[0136] .sup.aApoptotic cells show fluorescence in situ end labeling
(FISEL+), while cells without genome fragmentation show absence of
labeling (FISEL-). "Rad51+" cells with Rad51 foci, "Rad51-+ cells
without foci.
[0137] .sup.bTGR-1 cells were grown for 24 hrs in medium contaiing
0.1 .mu.g/ml colcemid to induce micronucleus formation (without
inducing DNA damage). 185 of the colcemid-treated cells were
arrested at metaphase, 17% showed multinuclei (>10 micronuclei),
and 65% had no micronuclei. The cells were then allowed to recover
for various times in the absence of the drug. 500 micronuclei were
analyzed for each experiment.
[0138] Another more classical way for inducing apoptosis in vitro
is the exposure of TGR-1 cells to the topoisomerase II inhibitor
etoposide. After adding etoposide to the culture medium, the
percentage of apoptotic cells steadily increased (Table 10). After
36 hrs half of the cells showed genome fragmentation and stained
FISEL-positively. The nuclear events of apoptosis were accompanied
by the appearance of Rad51 protein in nuclei and MN. These results
indicate that different stimuli (e.g., irradiation and DNA-damaging
drugs, topisomerase inhibitors, and aneuploidogens) that condem
cells to apoptosis can induce focal concentration of Rad51 protein
and its exclusion into MN.
10TABLE 10 Induction of Rad51 Foci and Apoptosis by Etoposide
Treatment of TGR-1Cells Percentage Percentage Percent- of Cells of
Cells age with Type with Type of Percentage I.sup.a Foci II.sup.a
Foci Apop- of Cells in in totic without in Micro- in Micro-
Treatment Cells.sup.b Foci Nuclei nuclei Nuclei nuclei None 6% 93%
6% 0% 1% 0% 2 hrs after n.d. 92% 4% 1% 1% 2% etoposide.sup.c 5 hrs
after n.d 92% 3% 2% 1% 2% etoposide 12 hrs after 17% 87% 8% 2% 1%
2% etoposide 18 hrs after 24% 79% 3% 8% 1% 9% etoposide 24 hrs
after 33% 82% 2% 2% 6% 8% etoposide 36 hrs after 47% 83% 2% 5% 1%
9% etoposide .sup.aType I nuclei and micronuclei show weak to
medium HsRad51 immunofluorescence, whereas type II cells show
strongly fluorescing foci. 500 cells were analyzed for each
experiment. .sup.bDetected by fluorescence in situ end labeling
(FISEL+). .sup.cCells were grown in medium containing etoposide for
the indicated times.
[0139] Higher-Order Nuclear Organization of Overexpressed Rad51
Protein Human 293 cells were transfected with the HsRad51 gene. The
resulting cell lines 710 and 717 constitutively expressed a
HsRad51-fusion protein. This overexpressed protein formed brightly
fluorescing linear structures inside the nucleus (FIG. 7a). Some
nuclei were completely filled with a network of rod-like structures
(FIG. 7b). Identical Rad51 structures were observed in transformed
rat TGR 928.1-9 cells, stably expressing the HsRad51 protein
without a tag epitope (data not shown). This suggests that Rad51
protein is able to assemble into higher-order structures within the
highly ordered interphase nucleus. The linear nature of Rad51
structures in overexpressing cells is reminscent of the strings of
Rad51-protein foci after DNA damage and colcemid treatment and in
meiotic cells (Haaf et al., 1995).
[0140] However, in contrast to the situation after DNA damage, the
overexpressed HsRad51 protein is not eliminated into MN. The
numbers of Rad51-positive MN were not radically different in
Rad51-overexpressing human 717 cells versus in 293 control cells
and in rat 928.1-9 overexpressers versus in parental TGR-1 cells.
This means that Rad51 overexpression alone does not cause
apoptosis. In exponentionally growing unsynchronized cultures, 14%
of both 717 and 293 cells (500 cells were analyzed for each
experiment) and 8% of both 928.1-9 and TGR-1 cells showed cleavage
of the cell's DNA by FISEL. We conclude that the segregation of
Rad51 into MN is a specific behavior of apoptotic cells and
precedes genome fragmentation.
[0141] Cell-Cycle Arrest of Cells with Focally Concentrated Rad51
Protein Simultaneous Rad51-protein immunofluorescence and
antibromodeoxyuridine (BrdU) antibody staining demonstrated that
nuclei with focally concentrated Rad51 protein do not undergo
DNA-replication synthesis (data not shown). BrdU was incorporated
into replicating DNA of unsynchronized cell cultures for 30 hrs.
Rapidly growing transformed cell lines (293, LNL8, XPA, and XPF)
which showed detectable Rad51 immunolabling in a percentage of
nuclei even without induction of DNA damage as well as Rad51
overproducers (928.1-9 and 717) were analyzed. For each experiment,
100 nuclei with prominent Rad51 foci and 100 nuclei without
detectable Rad51 foci were stained with fluorescent anti-BrdU
antibody. In the widely different substrates tested, 80%-100% of
the cells with focally concentrated Rad51 protein were found to be
BrdU-staining negative (Table 11). In contrast, 30%-90% of the
cells without Rad51 foci from the same cultures showed BrdU
incorporation, indicative of cycling cells. The BrdU-substituted
DNA was located in discrete replication sites throughout the
nucleus as reported previously (Nakayasu, H., and Berezney, R.
(1989). J. Cell Biol. 108, 1-11; Fox, et al., (1991) J. Cell Sci.
99, 247-253). This suggests that even without induction of DNA
damage the cells with Rad51 foci are arrested during the cell cycle
or enter S phase delayed of the Rad51-foci negative cells.
11TABLE 11 Induction of Rad51 Foci after.sup.137Cs Irradiation of
TGR-1 cells and Their Elimination into Micronuclei Percentage of
Percentage of Cells with Cells with Type I.sup.a Foci Type II.sup.a
Foci Percentage of in in Cells without in Micro- in Micro-
Treatment Foci Nuclei nuceli Nuclei nuclei None 93% 6% 0% 1% 0% 3
hrs after 80% 8% 0.4% 11% 0.6% 900 rad .sup.137Cs 16 hrs after 73%
9% 8% 1% 9% 900 rad .sup.137Cs 30 hrs after 72% 1% 13% 1% 13% 900
rad .sup.137Cs 4 days after 90% 0% 4% 0% 6% 900 rad .sup.137Cs
.sup.aType I nuclei and micronuclei show weak to medium HsRad51
immuno- fluorescence, whereas type II cells show strongly
fluorescing foci. 1000 cells were anlayzed for each experiment.
[0142] Rat TGR-1 cells are capable of normal physiological
withdrawal into the quiescent (Go) phase of the cell cycle as well
as resumption of growth following the appropriate stimuli (Prouty,
et al., (1993). Oncogene 8, 899-907). In TGR 928.1-9 cells
o.noteq.erexpressing a HsRad51 transgene(s), Go arrest upon serum
starvation dramatically induced HsRad: 1-protein foci (Table 12).
Synchronous re-entry into the cell cycle after feeding reduced the
percentage of HsRad51-foci positive cells to very low levels.
However, new Go arrest upon contact inhibition following three
population doublings increased the number of cells with nuclear
Rad51 foci again. We therefore conclude that cells with prominent
nuclear Rad51 foci are most likely in Go or G1 phase of the cell
cycle.
12TABLE 12 Rad51 Foci in Micronuclei of Different Cell Substrates
Number of Percentage of Percentage of Cell substrate Micronuclei in
Rad51-Positive Rad51-Negative Treatment 1000 Cells Micronuclei
Micronuclei TGR01 None 93 14% 86% 3 hrs after 900 279 22% 78%
rad.sup.137Cs 16 hrs after 900 2719 28% 72% rad.sup.137Cs 4 days
after 900 1040 20% 80% rad.sup.137Cs LNL8 None n.d. 23% 77% None
n.d. 26% 74% XPA None n.d. 18% 82% Teratoma None n.d. 10% 90%
3T3-Swiss None 472 125% 88% 1000 cells were analyzed for each
experiment
[0143] Other references specifically incorporated by reference are
Haaf, T. (1995). Pharmac. Ther. 65, 19-46; Haaf, T., and Schmid, M.
(1991). Exp. Cell Res. 192, 325-332; and Owaga, et al, (1993)
Science 259, 1896-1899
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