Biology Reference
In-Depth Information
In addition, FA pathway defects are expected to affect
only foci formation in S-phase, so that normal RAD51
foci formation in G2 phase can potentially mask any
foci alterations in S-phase in an asynchronous cell
population. This highlights the general need to assess
cell cycle distributions and cell cycle specificity of
RAD51 foci formation when conducting these kinds
of studies.
Scully et al. were the first to report the co-localization
of BRCA1 and RAD51 in S-phase nuclear foci, consistent
with a functional relationship between the two
proteins. 383 It was shown that BRCA1 foci are present
in undamaged S-phase cells, probably processing stalled
replication forks due to endogenous damage. After
inflicting exogenous damage, these foci disperse and
relocate at exogenous damage sites together with
BARD1 and RAD51. 384 The recruitment of BRCA1 into
foci is sophisticated and involves several protein
complexes with functions that extend beyond HRR
control (reviewed in 50 ). Mutations in BRCA1 will
disrupt BRCA1 foci formation and impair RAD51 foci
formation, while alterations in directly or functionally
interacting proteins, such as MDC1, may attenuate the
induction of BRCA1 foci in response to damage. 281,334
The recruitment of FANCD2 into subnuclear foci is
dependent upon mono-ubiquitination by the FA core
complex as well as BRCA1 function. 108 To which extent
FANCD2 is involved in the promotion of HRR is
unknown. 151,295 One study proposed that FANCD2
helps recruit BRCA2 to promote HRR, 106 though an
effect on RAD51 foci formation is not clear as discussed
above. The role of FANCD2 in foci may be to specifically
facilitate HRR needed for fork restart, an activity that
may be difficult to study in asynchronous cell popula-
tions. 112,113 Thus, attenuated or absent FANCD2 foci is
expected to predict sensitivity to crosslinking or other
DNA damaging agents. 385 For clinical application,
a challenge will be to determine what exactly constitutes
an attenuated FANCD2 (or BRCA1 or RAD51) foci
response to DNA damage without knowing the normal
kinetics of foci formation and the normal range of foci
per nucleus across a range of different cancer cell lines
or cancers.
One of the first biochemical responses to the presence
of a DSB is the phosphorylation of histone H2AX at
serine 139 by one or more kinases. 386,387 There is tremen-
dous interest in the use of g -H2AX foci as a functional
biomarker of DSB induction and repair (reviewed
in 388 e 391 ). g -H2AX foci also form at collapsed replica-
tion forks in response to DNA damage such as produced
by UV radiation, cisplatin, or topoisomerase I or PARP
inhibitors. 183,363,392 e 394 Foci have been shown to accu-
mulate from 6 hours to 18 e 24 hours following cisplatin
exposure as cells progress into S-phase. 183,394 Thus, HRR
defects are expected to increase the number of
replication fork-associated, unrepaired DSB at 18 e 24
hours, which correlates with reduced cell survival. 183,394
HRR Foci as Functional Biomarkers
The identification of HRR-deficient tumors is a major
challenge in cancer research, especially when taking into
account the complexity of the repair network with many
yet to be identified pathway components. Additionally,
assessing the expression of individual repair network
components is unlikely to reveal the overall incidence
of defects that can occur anywhere in the network.
Lastly, it is not established whether reduced gene
expression translates into functional repair defects.
Recently, there has been great interest in determining
HRR activities by assaying for the subnuclear location of
central pathway components, such as RAD51, BRCA1,
and FANCD2, as well as endpoints of unrepaired DSB,
such as g -H2AX and 53BP1. As outlined above, the
activity of the HRR pathway is less dependent on
protein expression levels than on the ability to localize
these proteins into foci, in order to coordinate and
execute repair. It is important to appreciate that the func-
tional status of HRR (and other DDR pathways) is typi-
cally revealed only when cells are exposed to DNA
damage. For example, in untreated repair-proficient
cells, BRCA1 and RAD51 foci may be visible in S-phase
even in the presence of pathway defects, and the fraction
of cells with foci and the number of foci per cells
increases post-irradiation, as described in the previous
section. 106,282,377 In contrast, BRCA1-deficient cells
have an impaired ability to mount a FANCD2 and
RAD51 foci response. 108,282 Thus, it should be possible
to use the ability of cells to form repair foci as a func-
tional biomarker of the integrity of the HRR pathway
and vice versa interpret the absence of repair foci induc-
tion, coupled with a persistence of DSB markers, as
indicative of chemosensitivity or PARP inhibitor sensi-
tivity (see Figure 7.11 ).
The advantage of using foci as biomarkers is that they
can detect repair defects due to several mechanisms
such as gene mutations, epigenetic events, or alterations
in signal transduction pathways. Moreover, they
provide a global measurement of network function
without needing to know the identities of all the compo-
nents, many of which are still unknown. One can envi-
sion developing mechanism-based “HRR foci
signatures” that reflect nodal points in the HRR pathway
or network of associated DDR proteins. Such a foci
signature likely will include at a minimum BRCA1,
FANCD2, and RAD51.
How can we translate into patients the requirement
for inducing a DNA damage signal, in order to measure
the cellular foci response? Assessing the foci response in
live tumors would require a repeat biopsy following
initial administration of treatment ( Figure 7.12 ). Such
Search WWH ::




Custom Search