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and out of the focus (reviewed in 375 ). The composition
of these repair centers depends on the type of DNA
damage and the cell cycle. Regulation of the components
of these centers is ensured through complex post-
transcriptional modifications.
The accumulation of DDR factors around a DSB is
thought to serve multiple purposes. 375 First of all, they
shelter the broken DNA ends from decay, and prevent
illegitimate repair processes or transcription of the
damaged gene. They also promote changes in chromatin
structure, which lead to chromatin relaxation and facili-
tated access. Furthermore, the gathering into foci
provides a means to rapidly increase local protein
concentrations at the site of damage without the need
of transcriptional induction. The higher concentration
of DDR proteins stimulates their activities and results
in an amplification of the DNA damage signal. Foci
also serve as a “toolbox” for repair, in which DDR
proteins assemble, promoting different repair pathways.
53BP1, for example, is responsible for NHEJ, whereas
BRCA1 facilitates HR. The competition and balance of
these proteins will
such as gene expression profiles or comparative
genome hybridization (CGH) have been employed in
breast and ovarian cancer to define a BRCAness pheno-
type (summarized by Yap et al. 370 ). While these
approaches appear promising, technical issues such as
the appropriateness of formalin-fixed paraffin-
embedded tissues and the need for validation studies
remain.
An instructive example of how determination of
single gene expression can impact clinical care involves
the predictive value of ERCC1 levels in NSCLC. Results
of the International Adjuvant Lung Cancer Trial (IALT)
Biology Study were published in 2006. 371 The study was
based on 761 evaluable patients with operable NSCLC
who had been randomized to receive postoperative
cisplatin-based doublet chemotherapy or observation
alone. ERCC1 staining in paraffin-embedded surgical
tumor specimens was evaluated by immunohistochem-
istry and graded from 0 to 3. ERCC1 staining in endo-
thelial cells in tonsil tissue was set as the reference at
2. A semiquantitative H score was calculated based on
the percentage of positive tumor nuclei as well as the
staining intensity of nuclei. Tumors with less than the
median H score were scored as “ERCC1 negative”
and those with an H score above the median as
“ERCC1 positive.” Only patients with ERCC1 negative
but not positive tumors derived a survival benefit
from chemotherapy. One implication from this is that
patients with ERCC1 positive tumors are better treated
with non-platinum regimens. 372 Despite of the apparent
success of the IALT Biology Study and other studies, 178
testing for ERCC1 levels has not yet entered routine
clinical testing, which likely is due to several factors
including: (a) the ambiguity of determining expression
levels as highlighted above, (b) the recognition that
ERCC1 status alone is insufficient to fully predict plat-
inum sensitivity given the multitude of other proteins
involved in ICL repair and HRR, and (c) variable anti-
body performance.
There is a rapidly enlarging literature attempting to cor-
relate the expression levels of various other DNA repair
and HRR genes with treatment outcome. 148,177,181,373
However, no expression marker thus far appears to be
robust enough to prove predictive in multiple studies.
In addition, as discussed above, expression levels of indi-
vidual genes or even sets of genes may not accurately
reflect the total repair pathway activity in a tumor.
influence the choice of
repair
pathway. 375
In the following, we will briefly review probably the
most important HRR foci, i.e., RAD51, BRCA1/2, and
FANCD2, and summarize the role of DSB markers
such as g -H2AX in monitoring the activity of HRR.
RAD51 foci form in response to collapsed replication
forks or DSB in the S- or G2-phase of the cell cycle.
RAD51 foci are commonly used as surrogate measure
of local HRR activity based on genetic analysis and
reporter substrate studies. 249,282,376 The proper forma-
tion of RAD51 foci is controlled by a multitude of
proteins, including BRCA2, PALB2, BRCA1, RAD54,
the RAD51 paralogs, RAD51AP1, but also upstream
regulators such as MDC1 or ATM. 90,280,282,334,377 e 380
Recently, a role of RAD52 in controlling RAD51
assembly in mammalian cells has also been uncov-
ered. 381 Thus, genetic or epigenetic defects anywhere
in the HRR pathway likely lead to an impairment or
even complete abrogation of RAD51 foci formation.
Whether defects in the FA pathway compromise
RAD51 foci formation, via impairment of FANCD2/
BRCA2 function, has been controversial. 104,108,152,164,382
Some of the conflicting data are likely related to the
ambiguity of what to consider a HRR “proficient”
versus “deficient” cell. In foci studies, cells that contain
five or more RAD51 foci are frequently regarded as
“positive”. However, this is a rather arbitrary endpoint,
and it is necessary to carefully estimate the number of
foci in every cell. For data presentation, the most appro-
priate “cut-off” for RAD51 foci positivity, such as
Basics of HRR Protein Foci Formation
Subnuclear foci are multiprotein complexes that are
organized into centers surrounding a DNA lesion and
can be visualized as “dots” using immunofluorescence
microscopy. 374 These complexes are highly dynamic
structures in order to coordinate repair and checkpoint
responses. Many DDR proteins shuttle transiently in
10,
>
20 foci per nucleus, may be chosen, but this
cut-off may well vary with the cell line under study and
the intensity of the damage signal among other factors.
15, or
>
>
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