.JPG?ad=18503&adview=true)
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biochemistry and Biophysics |
Institut de Biologie, INSERM U419, 44035 Nantes Cédex, France [F. G., M. G., K. M., J. H.] and Department of Geriatrics, University of Geneva, CH-1226 Chêne-Bourg, Switzerland [I. I-F.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
97,000 in PROb parental cells. We now
show that these autoantibodies are directed against BARD1, originally
identified as a protein interacting with the product of the breast
cancer gene 1, BRCA1. We demonstrate that the
Mr 67,000 antigen is a cleaved form of BARD1
present in apoptotic bodies derived from rat and human colon and
mammary carcinoma cell lines. Moreover, we show that the cleavage site
of BARD1 is located NH2 terminally but downstream of the RING domain
essential for BARD1 and BRCA1 protein interaction. In
vitro studies using [35S]methionine-labeled human
BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells
indicate that BARD1 proteolysis occurs at an early stage of apoptosis
and in a cell cycle-dependent manner. This hydrolysis is inhibited by
EGTA, and the calpain inhibitor I,
N-acetyl-leu-leu-norleucinal, but not by several
caspases inhibitors, suggesting that BARD1 is hydrolyzed by the
calcium-dependent cysteine proteases, calpains. Thus, the highly
immunogenic form of cleaved BARD1 could contribute to the antitumoral
response mediated by apoptotic bodies. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our laboratory has developed a strategy for the treatment of rats with induced peritoneal colon carcinomatosis, aimed at increasing the immunogenicity of colon cancer cells and providing greater stimulation of the immune system. Combined i.p. injection of IL3 -2 and the differentiation agent NaB cured rats with peritoneal carcinomatosis induced by a poorly immunogenic colon carcinoma (PROb cells), whereas IL-2 or NaB alone were inefficient (7) . We hypothesized that the increase of NaB-induced tumor immunogenicity could be attributable to its apoptotic effect (8) . In fact, the clustering and marked concentration of the self-molecules on the surface blebs of apoptotic cells and their modification by apoptosis-specific proteolytic cleavage represent a potential source of immunocryptic epitopes conducive to breaking self-tolerance (9) and stimulating the autoantibody response, as observed in systemic autoimmune diseases such as systemic lupus erythematosus (10, 11, 12) .
We provide support for our hypothesis by the treatment of rats with induced peritoneal carcinomatosis with injections of a large quantity of apoptotic bodies derived from PROb cells alone (13) or in combination with antigen-presenting cells having engulfed apoptotic bodies (14) . This treatment led to tumor regression and induced a long-term specific protection from parental tumoral challenge. In comparison, injections of necrotic cells had no therapeutic effect. This curative vaccine was accompanied by a tumor-specific cytotoxic T-cell response (14) and the production of autoantibodies that strongly recognized a Mr 67,000 antigen specifically expressed in apoptotic bodies and weakly reacted with a Mr 97,000 protein present in parental PROb cells (13) .
In this study, we demonstrated that the Mr 67,000 antigen is a cleaved form of the Mr 97,000 protein generated during apoptosis. We report the immunoscreening of a PROb cDNA library with sera from cured rats that led to the identification of the Mr 67,000 protein as a proteolytic fragment of BARD1, a protein interacting with the tumor suppressor gene product BRCA1 (15) , and the mechanism of the cleavage of BARD1 during apoptosis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immunological Screening and Cloning of the Rat BARD1 cDNA.
A cDNA library of the PROb rat carcinoma cell line was constructed in
the expression vector 
TriplEx (Clontech, Palo Alto, CA). One million
of plaques were screened with sera from cured rats after vaccination
with apoptotic bodies/IL-2 (13)
. Antibodies against
Escherichia coli were removed from rat antiserum by
incubating sonicated E. coli with serum diluted to one-tenth
in PBS + 5% nonfat milk dry for 4 h at room temperature
and then centrifuged at 13,000 x g for 10
min. The insert of 456 bp (F1 fragment) was sequenced, and the sequence
was submitted to the National Center for Biotechnology Information
(NCBI) gene bank analysis. It presented a strong homology with
the human protein BARD1. Cloning of the complete cDNA of rat BARD1 was
achieved using the PROb cDNA library constructed in SMART PCR kit
(Clontech). Internal primers for the rapid amplification of cDNA ends
PCR were chosen from the cloned insert as recommended by the
manufacturer.
Cloning of the Human BARD1 cDNA.
Three fragments of human BARD1 cDNA were amplified from total RNA
extracted from SW48 human colonic carcinoma cell line. Fragments A, B,
and C were obtained using the following primers: fragment A: sense
primer, R135S/antisense primer, B202N (17)
;
fragment B: sense primer, B202A (17)
/antisense primer,
5'-CACCAATGCCTTATGCTGGAGC-3'; fragment C: sense primer,
5'-GAAGTAGTGACTCCTGAGAAGG-3'/antisense primer,
5'-TCAGCTGTCAAGAGGAAGCAACTC-3'. Each fragment was cloned into the
pGEM plasmid (Promega Corp., Madison, WI) and then excised using
NotI-PstI/PstI-HindIII/HindIII-BstXI,
respectively, purified, and then ligated together into the
NotI/BstXI sites of pGEM.
Apoptosis Induction and Purification of Apoptotic Bodies.
Apoptosis was induced by NaB treatment. Cells at different stages of
confluence were treated in complete medium at 37°C with 5
mM NaB (Sigma, St. Quentin Fallavier, France) for different
time periods as indicated in the figure legends. Apoptotic bodies were
purified as described previously (18)
.
Production and Purification of Rat BARD1 F1 Fragment.
The F1 fragment of the rat BARD1 was excised from the plasmid
(TriplEx) derived from the cDNA library and inserted in-frame into
the PstI site of pQE32 plasmid (Qiagen, Courtaboeuf,
France). The resulting fusion protein, containing a 6xHis tag placed at
the NH2 terminus of the BARD1 F1 fragment, was
expressed in E. coli and then purified by affinity
chromatography on Ni-NTA resin using the manufacturer’s
recommendations for the QIAexpressionist kit (Qiagen).
Mouse Immunization and Monoclonal Antibody Production.
Balb-c mice (Iffa-Credo, l’Arbresle, France) received s.c. injections
of 100 µg of rat BARD1 F1 fragment in 0.1 ml of Freund’s incomplete
adjuvant (Life Technologies, Inc.) emulsified in 0.1 ml of sterile PBS,
0.5% Triton X-100, in intervals of 3 weeks. Splenocytes from one mouse
were fused with mouse SP2O myeloma (ECACC) in the presence of
polyethylene glycol 1500 (Boehringer Mannheim, Meylan, France).
Hybridomas were plated in 96-well plates in complete medium
supplemented with 20% FCS, hypoxanthine-aminopterin-thymidine (Sigma),
and 1.5 ng/ml of recombinant IL6 (RD Systems, Minneapolis, MN).
Hybridoma supernatants were tested by ELISA using purified BARD1 F1
fragment as antigen.
Immunoprecipitation.
Apoptotic bodies were extracted on ice with 2% Triton X-100 in PBS
supplemented with an EDTA-free protease inhibitor cocktail (Boehringer
Mannheim) for 30 min. The extract was centrifuged for 15 min at
13,000 x g, and the supernatant was
incubated with rabbit polyclonal antibodies directed against human
BARD1 (669D; Refs. 15
and 19
) diluted to
1:1000. After 4 h incubation, with constant end-over-end mixing,
immune complexes were immunoprecipitated by adding 50 µl of
antirabbit IgG agarose. The agarose-bound immune complexes were washed
with 1% Triton X-100 in PBS containing protease inhibitors and
extracted from the agarose beads by boiling in reducing sample buffer
for electrophoresis and immunoblotting as described below.
Western Blots.
Electrophoresis was conducted under denaturing conditions (SDS-PAGE;
Ref. 20
). The proteins were transferred to a
polyvinylidene difluoride 0.45 µM filter (Millipore,
Bedford, MA) and blotted with primary antibodies. Secondary antibodies
were conjugated to horseradish peroxidase and used diluted to 1:15,000
(Sigma). The immune complexes were visualized by chemiluminescence
using Super Signal kit (Pierce, Rockford, IL).
Coupled in Vitro Transcription/Translation and
in Vitro Protein Cleavage Assay.
[35S]Methionine-labeled human BARD1 was
in vitro transcribed and translated using the TNT coupled
reticulocyte lysate systems kit (Promega). One µg of plasmid was used
in a 50-µl transcription/translation reaction containing 4 µl of
translation grade [35S]methionine (DuPont NEN,
Le Blanc Mesnil, France). For the in vitro cleavage, 2 µl
of the transcription/translation product were incubated with apoptotic
or nonapoptotic cellular extracts prepared in DIV buffer [20
mM HEPES (pH 7.5), 10 mM
NaCl, 1.5 mM MgCl2, 0.1%
SB14, and 0.5 mM phenylmethylsulfonyl fluoride]
at 37°C during the indicated period of time. Hydrolysis products were
then separated by SDS-PAGE and revealed by autoradiography using
PhosphorImager 445SI (Molecular Dynamics, Sunnyvale, CA). The cleavage
inhibition was evaluated by adding caspases inhibitors or proteasome
inhibitor (lactacystin; Calbiochem, Meudon, France) or calpain
inhibitor I (ALLnL; Chemicon, Souffelweyersheim, France).
Cell Cycle Synchronization.
SW48 cells were arrested in G0 by contact
inhibition in 175-cm2
flasks. After 3 days of
confluence, the cells were split 1:10 in 75-cm2
flasks at a concentration of 3 x 106 cells/flask. Twelve, 20, 28, 36, and 44 h after seeding, cells were treated with 5 mM NaB during
24 h and harvested. To determine the cell cycle distribution at
each time point, the contents of each flask were trypsinized, washed
three times in 10 ml of ice-cold PBS, and fixed with 1 ml of ice-cold
70% ethanol added dropwise for 16 h at -20°C. The fixed cells
were pelleted, resuspended in 500 µl of PC buffer [96% 0.2
M Na2HPO4, 4%
0.1 M citric acid (pH 7.8)] and left 30 min at room
temperature. Cells were then washed and resuspended in 500 µl of
propidium iodide (50 µg/ml) in staining solution (PBS, 0.12%
Triton X-100, 0.12 mM EDTA, and 100 µg/ml RNase A),
incubated for 30 min at 37°C, and analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). For the in vitro
protein cleavage assay, cells were scraped, and cellular extracts were
prepared as described previously.
Caspase Activity Assay.
For caspase activity assays, 10 µg of cellular extracts were diluted
in 100 µl of DIV buffer.
Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin,
acetyl-Val-Glu-Ile-Asp-7-amino-4-methylcoumarin, and
acetyl-Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (Bachem, Bubendorf,
Switzerland) substrates for caspase 3, 6, and 8, respectively, were
added at a final concentration of 50 µM. Cleavage
activity was monitored on Fluorolite 1000 (Dynatech Laboratories).
Cell Fractionation.
Cells were cultured in a 75-cm2
flask,
trypsinized, and resuspended in 100 µl of CEB buffer [50
mM HEPES (pH 7.4), 50 mM KCl, 2 mM
MgCl2, 1 mM DTT, and 10
µM cytochalasin B]. The resuspended cells were left on
ice for 30 min and then homogenized by 50 strokes in an ice-cold Dounce
homogenizer. Nuclear fraction was prepared by centrifugation at
800 x g for 10 min at 4°C. The pellet was
resuspended in CEB buffer and stored at -80°C. Mitochondrial and
postmitochondrial fractions were obtained after centrifugation at
13,000 x g for 10 min at 4°C. Both the
mitochondrial pellet resuspended in CEB and the postmitochondrial
fraction were aliquoted and stored at -80°C until required.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
TriplEx bank with rat sera cured of
carcinoma by treatment with apoptotic bodies/IL-2 led to the
identification of a positive insert of 456 bp of the rat
BARD1 gene (fragment F1). This fragment is spanning
the ankyrin repeats and the BRCT domain encoding amino acids 460
through 611 (Fig. 1)
. The complete rat
BARD1 cDNA revealed an open reading frame coding for 768 amino acids,
slightly longer than mouse (765 amino acids) but shorter than the human
sequence (777 amino acids; Fig. 1
). Table 1
shows that the rat and mouse
orthologues of BARD1 share 88% identity, whereas rat and human
proteins share 65% identity. This last value is similar to that
observed for mouse and human BARD1 (67%). Higher levels of
conservation are found within the conserved domains (RING finger,
ankyrin repeats, and BRCT domain) of BARD1, 94–95% between rat and
mouse BARD1. This percentage was lower between rat and human or mouse
and human proteins, 91% within the ankyrin repeats, 86% within the
RING finger, and 80% within the BRCT domain.
|
|
67,000 in apoptotic bodies
derived from these cells after NaB treatment (Fig. 2A)
. This result was confirmed
by using the 669D polyclonal antibody (15
, 19)
against
human BARD1 (data not shown). Treatment of human colonic SW48 or
mammary MCF7 carcinoma cells with NaB showed a similar result after
blotting with the 669D polyclonal antibody (Fig. 2B)
.
Finally, the immunoprecipitation of BARD1 from human or rat
carcinoma-derived apoptotic bodies with the 669D polyclonal antibody,
followed by immunoblotting with cured rat serum, detected a protein of
Mr 67,000 (Fig. 2D)
.
Together, these results prove the identity of the p67 kDa protein as
BARD1.
|
. This strongly suggests that the
cleavage site of BARD1 is located NH2 terminally
but downstream of the RING domain (amino acids 40–84; Fig. 1
)
essential for BARD1 and BRCA1 protein interaction (15)
.
Cell Cycle-dependent Cleavage of BARD1 during Apoptosis.
We examined the effect of NaB treatment on BARD1 cleavage in adherent
SW48 cells or in the apoptotic bodies recovered from the supernatant.
Fig. 3A
shows that the lysates
of adherent cells completely cleaved the radiolabeled hBARD1 after
4 h of incubation, because the full-length hBARD1 protein
completely disappeared and a protein of
Mr 67,000 appeared (Fig. 3
A,
lines 2). However, the incubation with lysates from apoptotic
bodies had no effect on hBARD1 hydrolysis (Fig. 3
A, lines
1). These results indicate that proteolytic activity involved in
BARD1 cleavage occurred before the ultimate step of apoptosis, which
leads to the formation of apoptotic bodies and cell detachment.
|
. Further analysis of the kinetic induction of this
cleavage by NaB showed that the p67 protein appears after 4 h of
NaB cell treatment (Fig. 3C)
, and the cleavage was nearly
complete within 12 h (Fig. 3C)
.
Interestingly, the hydrolysis of hBARD1 is regulated in a cell
cycle-dependent manner and was predominantly observed during the
G0-G1 state (Fig. 4
, top). This was demonstrated
by the addition of NaB 32 h after of cell plating, at which point
80% of the cells were in
G0-G1 and only 6% in
G2-M phase, which led to a complete conversion of
hBARD1 to p67 (Fig. 4
, bottom).
|
. The
effector protease cascade of the apoptotic process comprises cysteine
proteases such as caspases or calpains. Therefore, we determined the
activities of caspases during the treatment of SW48 cells with NaB by
using specific substrates, and we found that caspases 3, 6, and 8
activities progressively increased (Fig. 3D)
, caspase 3
being the most active after 6 and 12 h of NaB treatment. The use
of peptide inhibitors for caspases showed that the
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a pan inhibitor
of caspases, slightly inhibited hBARD1 hydrolysis at high concentration
(100 µM; Fig. 6A
). However, specific
inhibitors of caspase 3, the
benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone or caspase 6,
benzyloxycarbonyl-Val-Glu-Ile-Asp-fluoromethylketone, had no effect
(not shown). This phenomenon was confirmed by the fact that purified
caspase 3 had no effect on hBARD1 hydrolysis (Fig. 6B)
.
These results clearly showed that hBARD1 is not a direct substrate for
caspases. Moreover, the proteasome inhibitor lactacystin
(22)
did not block hBARD1 proteolysis, even at
concentrations of 100 µM (Fig. 6A)
,
thus excluding that the proteasome could be involved in this mechanism.
|
|
; and (b) the calpain inhibitor I ALLnL
and EGTA strongly inhibited hBARD1 hydrolysis in a dose-dependent
manner (Fig. 6A)
. Altogether, these results strongly suggest
that hBARD1 was hydrolyzed by calpains. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In this paper, we report that the p67 kDa protein recognized by the sera of rats cured and subsequently immunized against cancer is a proteolytic fragment of BARD1. This identity was confirmed by: (a) the cloning of the rat BARD1 by immunoscreening with the sera of cured rats; (b) the demonstration that the full-length BARD1 (Mr 97,000) is expressed in colon and mammary cancer cells, whereas the truncated form (Mr 67,000) is expressed in apoptotic bodies; and (c) the immunoprecipitation of the p67 kDa with specific antihuman BARD1 antibodies followed by Western blotting with the sera of cured rats.
BARD1 was reported to play a critical role in BRCA1-mediated tumor suppression. Both proteins possess NH2-terminal RING finger motifs and COOH-terminal BRCT domains, with the former responsible for the BARD1/BRCA1 heterodimer formation (15) . The cloning of the rat BARD1 gene confirms the expected high homology (88%) with the mouse molecule (29) . However, the homology with the human protein is only 65%, similar to the identity between mouse and human molecules. These comparisons suggest that the primary sequences of BRCA1 and BARD1 have diverged at a similar rate during mammalian evolution, consistent with the notion that both proteins function as components of the same heteromeric complex (29) .
BARD1, in association with BRCA1, colocalizes with proliferating cell
nuclear antigen, a protein involved in DNA replication
(30)
, and with Rad51 (31)
, a protein involved
in eukaryotic double strand break repair (32)
. This
dynamic colocalization is consistent with a role of the BRCA1/BARD1
complex in DNA replication checkpoint response (33, 34, 35)
.
Moreover, the colocalization of BARD1 and BRCA1 is cell cycle dependent
(19)
. Our data indicate that the maximal apoptotic
proteolysis of BARD1 can occur in confluent cell cultures with most
cells in the G0-G1 state.
However, when cells are in a proliferating state, with a high
percentage of cells in S phase, a low degree of BARD1 proteolysis was
observed (Fig. 5)
. These results suggest that the interaction of BARD1
and BRCA1 in S-phase could protect BARD1 from hydrolysis, probably
because of a low accessibility of the molecule by the effector
proteases. In fact, Meza et al. (36)
have shown
that BRCA1/BARD1 heterodimers are less susceptible to proteolysis than
the homodimers.
Our results clearly demonstrate that a proteolytic form of BARD1 is
present in apoptotic bodies derived from rat and human colon or breast
cancer cells. An antibody (WFS) specific for the
NH2 terminal side of mouse BARD1
(21)
failed to recognize p67 in apoptotic bodies, thus
suggesting that this form lacks the NH2 terminus.
Moreover, the analysis of the mechanism leading to BARD1 proteolysis
during apoptosis indicates that it occurs before the formation of
apoptotic bodies, which is the ultimate step within the apoptosis
pathway. Several lines of evidence suggest that the protease
responsible for cleavage of BARD1 is not a caspase: (a) the
cleavage activity is associated with cell fractions enriched with
mitochondria and nucleus but not with cytosol, whereas caspases are
found primarily in the cytosol and nucleus (37)
and
calpains have been isolated from the mitochondrial matrix of the cells
(38)
; (b) purified caspase-3 failed to cleave
BARD1 in vitro (Fig. 6)
and
benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone failed to block
BARD1 cleavage in vitro assays, indicating that caspase-3 is
not responsible for BARD1 cleavage in SW48 cells; (c) known
caspase cleavage sites are Asp-X sequences and require P1 and P4 for
cleavage specificity (39)
. However, mutations of
susceptible cleavage sites from Asp residues to Ala still resulted in a
proteolytic BARD1 fragment of Mr
67,000 (data not shown), confirming that BARD1 is not a direct
substrate for caspases; and (d) the protease inhibitor
profile of BARD1 hydrolysis is consistent with that of calpains in that
it was inhibited by ALLnL, EGTA but not by the specific proteasome
inhibitor lactacystin (22)
. In addition, our results show
that calpastatin, an in vivo calpain inhibitor
(23)
, was hydrolyzed by apoptotic SW48 cell lysates, thus
activating BARD1 proteolysis by calpains, as it was observed in other
systems (40
, 41)
.
Several proteins such as poly(ADP-ribose) polymerase, lamin, U1–70 kDa, DNA-dependent protein kinase (DNA-PKcs), and nuclear mitosis antigen (NuMA) are fragmented during apoptosis and have been reported to become autoantigenic targets in systemic autoimmune diseases (reviewed in Ref. 12 ). Our previous results have shown clearly that only rats cured from carcinomatosis after treatment with apoptotic bodies/IL-2 developed antibodies against the cleaved form of BARD1 (p67; Ref. 13 ). This allows two nonexclusive explanations: anti-BARD1 antibodies contribute to the tumoral rejection; or their appearance is associated with treatment efficiency. Recent results from our laboratory indicate that the vaccination of rats with rat BARD1 F1 fragment (amino acids 460–611) leads to a 2-fold reduction of the tumor growth rate (data not shown). Although these results are preliminary, they illustrate the potential of BARD1 for the stimulation of an antitumoral immune response. Vaccination with p67 BARD1 cleavage product generated in vitro and fine mapping of sequences contributing to this antitumoral effect are the focus of ongoing research and should confirm our observations.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 F. G. had a fellowship from the Ligue Contre le
Cancer de Loire Atlantique et de Vendée. 
2 To whom requests for reprints should be
addressed, at Institut de Biologie, INSERM U419, 9 Quai Moncousu, 44035
Nantes Cédex, France. Phone: 33-240-08-40-43; Fax:
33-240-08-40-82; E-mail: jharb{at}nantes.inserm.fr
3 The abbreviations used are: IL, interleukin;
BARD1, BRCA1-associated RING domain protein; hBARD1, human BARD1;
BRCA1, breast cancer gene 1 product; NaB, sodium butyrate; ECACC,
European collection of cell cultures; ALLnL,
N-acetyl-leu-leu-norleucinal.
Received 2/ 2/00. Accepted 10/16/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med., 189: 371-380, 1999.This article has been cited by other articles:
|
|
 |
|
  L. Li, S. Ryser, E. Dizin, D. Pils, M. Krainer, C. E. Jefford, F. Bertoni, R. Zeillinger, and I. Irminger-Finger Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers Cancer Res., December 15, 2007; 67(24): 11876 - 11885. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Tembe and B. R. Henderson BARD1 Translocation to Mitochondria Correlates with Bax Oligomerization, Loss of Mitochondrial Membrane Potential, and Apoptosis J. Biol. Chem., July 13, 2007; 282(28): 20513 - 20522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Feki, C.-E. Jefford, P. Durand, J. Harb, H. Lucas, K.-H. Krause, and I. Irminger-Finger BARD1 Expression During Spermatogenesis Is Associated with Apoptosis and Hormonally Regulated Biol Reprod, November 1, 2004; 71(5): 1614 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lafarge and M.-H. Montane Characterization of Arabidopsis thaliana ortholog of the human breast cancer susceptibility gene 1: AtBRCA1, strongly induced by gamma rays Nucleic Acids Res., February 15, 2003; 31(4): 1148 - 1155. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
