Gene amplification in human cells knocked down for RAD54.

Abstract

In mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs). The repair of DSBs mainly occurs through two mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). We previously showed that defects in the repair of DSBs via NHEJ could increase the frequency of gene amplification. In this paper we explored whether a single or a combined defect in DSBs repair pathways can affect gene amplification.
We constructed human cell lines in which the expression of RAD54 and/or DNA-PKcs was constitutively knocked-down by RNA interference. We analyzed their radiosensitivity and their capacity to generate amplified DNA. Our results showed that both RAD54 and DNA-PKcs deficient cells are hypersensitive to γ-irradiation and generate methotrexate resistant colonies at a higher frequency compared to the proficient cell lines. In addition, the analysis of the cytogenetic organization of the amplicons revealed that isochromosome formation is a prevalent mechanism responsible for copy number increase in RAD54 defective cells.
Defects in the DSBs repair mechanisms can influence the organization of amplified DNA. The high frequency of isochromosome formation in cells deficient for RAD54 suggests that homologous recombination proteins might play a role in preventing rearrangements at the centromeres.

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RESEARCHOpen Access
Gene amplification in human cells knocked down
for RAD54
Aurora Ruiz-Herrera1,3, Alexandra Smirnova1, Lela Khouriauli1, Solomon G Nergadze1, Chiara Mondello2and
Elena Giulotto1*
Abstract
Background: In mammalian cells gene amplification is a common manifestation of genome instability promoted
by DNA double-strand breaks (DSBs). The repair of DSBs mainly occurs through two mechanisms: non-homologous
end-joining (NHEJ) and homologous recombination (HR). We previously showed that defects in the repair of DSBs
via NHEJ could increase the frequency of gene amplification. In this paper we explored whether a single or a
combined defect in DSBs repair pathways can affect gene amplification.
Results: We constructed human cell lines in which the expression of RAD54 and/or DNA-PKcs was constitutively
knocked-down by RNA interference. We analyzed their radiosensitivity and their capacity to generate amplified
DNA. Our results showed that both RAD54 and DNA-PKcs deficient cells are hypersensitive to g-irradiation and
generate methotrexate resistant colonies at a higher frequency compared to the proficient cell lines. In addition,
the analysis of the cytogenetic organization of the amplicons revealed that isochromosome formation is a
prevalent mechanism responsible for copy number increase in RAD54 defective cells.
Conclusions: Defects in the DSBs repair mechanisms can influence the organization of amplified DNA. The high
frequency of isochromosome formation in cells deficient for RAD54 suggests that homologous recombination
proteins might play a role in preventing rearrangements at the centromeres.
Background
Different pathways, mainly controlling either the cell
cycle in response to DNA damage or the repair of the
damage itself, maintain genome stability in mammalian
cells. Mutations in genes implicated in these pathways
cause genetic lesions that can give rise to cellular trans-
formation. Gene amplification, the increase in the copy
number of a portion of the genome, is a common mani-
festation of genome instability in tumour cells and an
important mechanism of oncogene activation as well as
drug resistance, since it leads to over-expression of rele-
vant genes. Amplification of DNA sequences containing
cancer genes has been described in several types of solid
tumours and lymphomas [1,2]. The fact that gene ampli-
fication has never been detected in cells of normal
origin [3,4] suggests that either control mechanisms that
prevent the occurrence of gene amplification are active
(such as the p53-mediated damage-sensing pathway), or
cells carrying gene amplifications do not survive.
Cytogenetic manifestations of amplified DNA include
self-replicating extrachromosomal elements called double
minutes (DMs), amplified regions on a single chromo-
some (homogeneously staining regions, HSRs) or ampli-
fied regions distributed throughout the genome [5]. The
existence of specific regions of the genome that are hot-
spots for amplification in cancers with similar cell of ori-
gin suggests that they contain genes relevant for tumour
formation and progression [6,7]. In addition, the genomic
context where the amplified DNA is embedded [8] and
its proneness to breakage [9] seem to contribute to the
propensity to amplify of specific genomic territories.
Moreover, the instability of amplified DNA further
increases the extent of amplification. A large body of evi-
dence indicates that DNA double-strand breaks (DSBs)
can promote gene amplification through different pro-
cesses such as successive breakage-fusion-bridge (BFB)
cycles, unequal sister chromatid exchange, rolling circle
replication or fold-back priming (for a review see [10]).
* Correspondence: elena.giulotto@unipv.it
1Dipartimento di Genetica e Microbiologia “Adriano Buzzati-Traverso”,
Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy
Full list of author information is available at the end of the article
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GENOME INTEGRITY
© 2011 Ruiz-Herrera et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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Mammalian cells repair DSBs through two main mechan-
isms: (1) homologous recombination (HR), which requires
large regions of homology, and (2) non-homologous end
joining (NHEJ), which does not require extended homolo-
gies [11]. NHEJ is an error prone process that dominates
during the G1 to early S phase of the cell cycle whereas
HR is mainly used in the late S and G2 phases. The NHEJ
pathway requires the activity of several proteins, including
the DNA-PK complex, which is composed of a heterodi-
meric subunit with DNA end-binding activity (Ku) and a
catalytic subunit, the DNA-dependent protein kinase
(DNA-PKcs). The Ku proteins bind the ends of the DSB
and recruit DNA-PKcs, whose kinase activity is essential
for the activation of other repair factors. DNA-PKcs is
therefore a key player in NHEJ and cells defective in the
DNA-PKcs gene are hypersensitive to ionizing radia-
tions [12-14]. In addition to DNA breaks, DNA-PKcs
binds to telomeres and is involved in telomere mainte-
nance; in fact, defective cells show an increased fre-
quency of telomeric fusions [15-17]. Homologous
recombination is also a complex mechanism requiring
several proteins among which, RAD51 and RAD54
represent the key players. These proteins are members
of the RAD52 epistatic group of genes that codify the
enzymes implicated in the homologous recombination
process and the repair of DSBs [18]. RAD51 is the
recombinase that recognizes the region of homology
and promotes strand exchange. The RAD54 protein is a
dsDNA-dependent ATPase that interacts physically and
functionally with RAD51 performing several important
functions in HR; it translocates along the dsDNA indu-
cing topological changes, binding Holliday junctions
and driving their migration (for a review see [19]). The
interaction of RAD54 with different repair proteins dur-
ing the HR process indicates that it is an important
player in this pathway [20,21]. Moreover, RAD54 defects
can cause sensitivity to ionizing radiations [22].
The first line of evidence showing a link between gene
amplification and DSBs repair mechanisms was obtained
by Mondello and collaborators [23], who showed that a
defect in DNA-PKcs increases the frequency of gene
amplification by one order of magnitude both in Chi-
nese hamster cells and in immortal mouse embryonic
fibroblasts. In addition, pre-treatment of DNA-PKcs
deficient cells with ionizining radiation further increases
the frequency of the process [24]. These observations
have been confirmed in human cells in which DNA-
PKcs expression was constitutively inhibited by RNA
interference [14]. These cells are more radiosensitive
and prone to gene amplification than parental cell lines.
A defect in the DNA-PKcs may facilitate gene amplifica-
tion by delaying the joining of broken ends, by altering
the equilibrium between NHEJ and HR or by increasing
the frequency of telomeric fusions. A defect in the ATM
pathway also causes an increased propensity to gene
amplification [25]. DNA-PKcs and ATM defective cells
possess the so called “amplificator” phenotype [26]. On
the contrary, immortalized mouse embryonic fibroblasts
derived from animals knocked-out for mTERC (the telo-
merase RNA component) did not show amplification
capacity [27], suggesting that telomerase may be
required for the stabilization of chromosomes carrying
amplified DNA. In the light of these data, we can specu-
late that altered expression of other genes that partici-
pate in processes such as repair, recombination, cell
cycle checkpoint control and telomere maintenance
could affect the propensity or permissiveness to gene
amplification.
In this paper we aimed at exploring whether a single
or a combined defect in the homologous recombination
and non-homologous end joining processes can affect
gene amplification. To this purpose, we constructed
human cell lines, derived from HeLa cells, in which the
expressions of RAD54 and/or DNA-PKcs were constitu-
tively knocked-down by RNA interference (KD cells).
We analyzed the radiosensitivity of the different cell
lines, together with their amplification ability. Moreover,
we investigated the cytogenetic organization of the
amplicons detected in clones isolated from cells
knocked-down for the different functions. Our results
suggest that defects in the DSBs repair mechanisms can
influence the organization of amplified DNA.
Results
Construction of HeLa cell lines with stable inhibition of
RAD54 and/or DNA-PKcs expression
To inhibit the expression of RAD54, HeLa cells were
transfected with the plasmids shRAD54p-1, shRAD54p-2,
shRAD54p-3 or shRAD54p-4, each containing the neo-
mycin resistance gene and different oligonucleotides for
the production of shRNA against human RAD54, or with
the plasmid p-scrambled, containing an oligonucleotide
that is not homologous to any human gene. Pools of
G418 resistant clones were isolated and the level of
RAD54 expression was measured by immunofluorescence
using an antibody specific for human RAD54. As
expected, the plasmid containing the scrambled sequence
did not induce any reduction in RAD54 expression; plas-
mids shRNAp-1 and shRNAp-2 did not produce a
detectable effect on RAD54 expression while plasmids
shRNAp-3 and shRNAp-4 caused a reduction in the
levels of the protein (results not shown).
Eleven clones containing the shRAD54p-3 and four
clones containing the shRAD54p-4 plasmids were iso-
lated and RAD54 expression was measured by immuno-
fluorescence (Figure 1). In four out of the eleven clones
containing the shRAD54p-3 plasmid (shRAD54 3.3a,
shRAD54 3.4d, shRAD54 3.1b and shRAD54 3.1c) the
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expression of RAD54 was reduced (Figure 1a): a mean of
~15 RAD54 foci per cell was detected in parental HeLa
cells while ~5 or less RAD54 foci per cell were detected
in these clones. In none of the four clones containing
the plasmid shRAD54p-4 inhibition of RAD54 expres-
sion was observed (data non-shown). The two clones
(shRAD54 3.1b and shRAD54 3.1c) showing the lowest
number of RAD54 foci (an average of 2.2 foci/cell, Fig-
ure 1a) were chosen for the long-term experiments
required for radiation sensitivity and gene amplification
analysis. In these clones, the residual level of the RAD54
protein, measured by western blotting, was about 40%
(Figure 1c).
In order to obtain HeLa cell lines in which both
RAD54 and DNA-PKcs expression were inhibited, the
shRAD54 3.1b clone was transfected with the plasmid
vector pCRPK1, which contains the puromycin resis-
tance gene and an oligonucleotide for the expression of
shRNA against DNA-PKcs [14]. Thirteen puromycin
resistant clones were isolated and the level of DNA-PKcs
expression was measured by immunofluorescence using
an antibody specific for human DNA-PKcs. The two
clones (shRAD54/CRPK 2b and shRAD54/CRPK 5a)
with the greatest reduction in DNA-PKcs levels were
chosen for further experiments. In these clones, DNA-
PKcs was undetectable both by immunofluorescence
Figure 1 Inhibition of RAD54 and DNA-PKcs expression in cell lines constitutively expressing shRNA. Indirect immunofluorescence
analysis of RAD54 and DNA-PKcs expression in cell lines constitutively expressing shRNA. (a) Distribution of the number of RAD54 foci detected
in 50 cells of the HeLa parental cell line and in four clones knocked down for RAD54 (clones 3.3a, 3.4d, 3.1b and 3.1c). The number of foci per
cell (X-axis) and the number of cells (Y-axis) are reported. Solid lines in red represent Normal distributions. The mean number of foci per cell is
indicated for each cell line. (b) Examples of immunofluorescence using antibodies against the human RAD54 (left section of the panel) and the
human DNA-PKcs (right section of the panel) proteins on the clones shRAD54 3.3a and shRAD54/CRPK 2b. In both cases the HeLa parental cell
line was used as positive control. (c) Western blot analysis of DNA-PKcs and RAD54 expression in single and double knocked down clones.
Tubulin was used as loading control.
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and western blotting (Figure 1b and 1c) and RAD54
levels were similar to those observed in the single KD
parental cell line shRAD54 3.1b (Figure 1c).
Cellular sensitivity to ionizing radiation
Since it is known that RAD54 defects cause radiation
sensitivity [22], we measured the survival of the KD cell
lines shRAD54 3.1c and shRAD54 3.1b to g-irradiation
at five different doses (1, 2, 4, 6 and 8 Gy; Figure 2).
Both cell lines were hypersensitive to g-irradiation
(Lethal Dose or LD50~ 2 Gy) compared to the control
cell line containing the scrambled sequences and to
HeLa cells (whose LD50~ 4 Gy). The sensitivity to g-
rays in the RAD54 KD cell lines was less severe than in
the previously isolated DNA-PKcs defective cell line
CRPK1-4 (LD50~ 1Gy) [14] while in the double KD
clones (shRAD54/CRPK 2b and shRAD54/CRPK 5a)
sensitivity was higher than in single KD cells (LD50~
0.5 Gy). In conclusion, impairment in the HR repair
pathway causes hypersensitivity to g-rays, and defects in
both pathways further increase sensitivity.
Frequency of generation of methotrexate resistant clones
MTX is a potent competitive inhibitor of the dihydrofo-
late reductase (DHFR) enzyme, which is essential for
DNA synthesis and cell growth [28]. It is well known
that mammalian cells have the capacity to acquire resis-
tance to MTX through amplification of the DHFR gene,
among other mechanisms.
Preliminary experiments were carried out to deter-
mine the sensitivity to MTX of the cell lines by measur-
ing the inhibition of growth in massive cell cultures; the
dose inhibiting cell growth to 50% (DR50) was found to
be approximately 10 nM for all the cell lines (data not
shown).
The frequency of MTX resistant colonies was then
measured taking into account the plating efficiency of
each cell line. The results of four experiments per-
formed at two different MTX concentrations are shown
in Table 1 and in Additional file 1. We detected a mild
if any (2-3 fold) increment in the frequency of MTX
resistant colonies in the RAD54 defective cell lines com-
pared to the control cell lines (HeLa wt and pSSP-1,
Table 1). Moreover, and according to our previous stu-
dies [14], cell lines defective for DNA-PKcs (CRPK1.1
and CRPK1.4, Figure 1c) showed a greater increment in
the frequency of gene amplification (between 3.3 and 8
fold). Surprisingly and in spite of their radiosensitivity,
the double KD cell lines (shRAD54/CRPK 2b and
shRAD54/CRPK 5a) showed a frequency of MTX resis-
tant colonies similar, or even lower in some experi-
ments, compared to control cell lines (Table 1).
Figure 2 Sensitivity to g-irradiation. The results of three different
experiments (A, B and C) are reported in which the following cell
lines were analyzed: single RAD54 inhibited (shRAD54 3.1c and
shRAD54 3.1b), single DNA-PKcs inhibited (CRPK1-4) and double
RAD54/DNA-PKcs (shRAD54/CRPK 2b and shRAD54/CRPK 5a)
inhibited cell lines. The HeLa parental cell line and cell line stably
transfected with the scrambled plasmid (shRAD54 scrambled) were
used as control. Survival to irradiation for all cell lines was measured
in duplicate samples using a clonogenic assay.
Table 1 Relative gene amplification ability
Ratio versus HeLa parental line
Cell lines45 nM MTX 55 nM MTX
1234Mean1234Mean
Hela1111
1
11 n.a1
1
pSSP12.80.6 1.71.1
1.5
2.0 0.7 n.a. 0.9
1.2
CRPK 1.1 6.43.0 17.3 n.a.
8.9
3.5 3.1 n.a. n.
a.
3.3
CRPK 1.4 11.4 2.9 18.3 2.1
8.7
4.5 5.1 n.a. 4.5
4.7
shRAD54 3.1b 2.22.82.0n.a.
2.3
2.0 2.1 n.a. n.
a
2.0
shRAD54 3.1c 5.01.4 3.03.4
3.2
3.0 1.1 n.a. 5.3
3.1
shRAD54/CRPK
2b
n.a. 0.31.10.8
0.7
n.
a
0.3 n.a. 1.3
0.8
shRAD54/CRPK
5a
0.40.30.6 1.4
0.7
0.5 0.3 n.a. 1.2
0.4
Cell lines with different repair function impaired were analyzed together with
their respective controls. The values represent the ratio between the
frequency of MTX-resistant clones in each cell line versus the HeLa parental
cell line in four different experiments and at two different MTX
concentrations. n.a.: not-analyzed.
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Cytogenetic characterization of MTX-resistant clones
It is known that the DHFR gene is located on human
chromosome 5q14. Since HeLa cells have an aneuploid
and rearranged karyotype, we first characterized the
chromosomes bearing the DHFR gene in the cell line
used for the present studies. FISH analysis using a
human chromosome 5 painting probe and a BAC con-
taining the DHFR gene (CTC-325J23) revealed the pre-
sence of seven chromosomes (A, B, C, D, E, F and G)
with complete or partial homology to human chromo-
some 5 (Figure 3); two of these chromosomes (A and C)
carry one copy of the DHFR gene located in the proximal
region of the long arm, corresponding to the 5q14 band,
whereas the other markers derived from chromosome 5
(B, D, E, F and G) do not contain the DHFR gene. A BAC
clone (RP11- 297G19), which maps to 5q15, was then
hybridized with the chromosomes of HeLa cells together
with the BAC containing the DHFR gene. The results
showed that the RP11- 297G19 sequence is located on
chromosomes A and C, near the DHFR gene, and
on chromosome D, where the DHFR gene is absent
(Figure 3).
To test whether MTX resistance was due to amplifica-
tion of the DHFR gene, we isolated a total of 56 MTX
resistant independent clones from different cell lines: six
clones from parental HeLa cells, twelve from shRAD54
3.1c, twelve from shRAD54 3.1b, five from CRPK1.1, ten
from CRPK1.4, six from shRAD54/CRPK 5a and five
from shRAD54/CRPK 2b cells. All the clones were
expanded to obtain clonal populations. Additionally,
fourteen clones (five clones from parental cells and nine
clones defective in DNA-PKcs) previously isolated in
our laboratory [14] were included in the present study.
Chromosome spreads were prepared at initial passages
(passages 4-6) following clonal isolation and FISH
experiments were performed hybridizing the DHFR
BAC and the RP11- 297G19 BAC probes with all the
clones. A detailed cytogenetic analysis revealed that
gene amplification was the main mechanism responsible
for MTX resistance in cell lines defective for DNA DSB
repair mechanisms (Table 2). 67% (16 out 24) of the
clones isolated from DNA-PKcs defective cells, 67% (16
out of 24) of those obtained from RAD54 defective cell
lines and 64% (7 out of 11) of those obtained from the
double defective RAD54/DNA-PKcs cells contained
amplified DNA, while in only 36% (4 out of 11) of the
clones isolated from parental HeLa cells gene amplifica-
tion was detected. In order to shed light into the
mechanisms responsible for amplification, we character-
ized in more details the chromosomal organization of
the different amplicon structures observed. Regardless of
the type of amplicon detected, all the clones analyzed
contained two or more chromosomes carrying a single
copy of the DHFR gene; when more than two DHFR
carrying chromosomes were detected, the extra-chromo-
somes probably resulted from non-disjunction and
might contribute to MTX resistance.
Interestingly, amplification of the DHFR gene was
observed in different cytogenetic configurations depend-
ing of the genetic background of the cell line (Table 2
and Figure 4). We detected homogeneous staining
regions (Figure 4a), extra-chromosomal double minutes
(Figure 4b), isochromosomes 5q [i(5q)] (Figure 4c), as
well as additional labeled chromosomes. DMs were the
predominant amplified structures observed in MTX-
resistant HeLa parental cell (100% of the clones ana-
lyzed) and DNA-PKcs defective clones (87.5%). Both
DMs and HSR were observed in clones defective for
RAD54 and/or DNA-PKcs proteins, while the i(5q) was
observed almost exclusively in MTX-resistant clones
defective for RAD54 (Table 2).
By double colour FISH with the DHFR and the RP11-
297G19 BAC clones we could detect different types of
HSRs (Figure 4 d-g). The majority of these structures
were clearly organized as inverted repeats (Figure 4 d-f),
suggesting that successive BFB cycles, initiated by a DSB
downstream from the 5q15 chromosomal band and fol-
lowed by sister chromatid fusion, were the main
mechanism of formation. In the unique DNA-PKcs
defective clone with HSRs (CRPK1.1-45nM-9), as well
as in one of the RAD54 deficient clones (shRAD54 3.1c-
55nM-4), HSRs containing multiple copies of the DHFR
gene organized as compact ladder-like structures
Figure 3 Chromosome 5 complement of the HeLa parental cell
line used for the construction of defective cell lines. In all
instances, the DHFR probe is labeled in green, whereas whole
chromosome 5 painting and the BAC clone RP11-297G19 are
labeled in red. FISH using the human chromosome 5 painting
revealed the presence of seven chromosomes (A, B, C, D, E, F and
G) with complete or partial homology to human chromosome 5.
Chromosome A corresponds to a normal human chromosome 5
with a deletion of the chromosomal band 5q35; this organization
was demonstrated by the lack of hybridization signal using the BAC
clone RP11-117L6 (data not shown). Chromosome C corresponds to
a translocated derivative chromosome between 5q and the short
arm of chromosome 3 [der(3p5q)]. Chromosome B contains the
region corresponding to 5q22.2-q35 translocated with an unknown
chromosome, whereas chromosome D corresponds to 5q14-qter
also translocated with another chromosome. In addition,
chromosome F is an isochromosome of the p arm [i(5p)].
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without any hybridization signal for BAC RP11- 297G19
were observed (Figure 4g), suggesting that these struc-
tures could be composed by short inverted repeats,
deriving from BFB cycles starting from a break very
close to the DHFR gene, or by direct repeats. Both
clones contained, either this type of HRS (22.7% of the
cells analyzed in CRPK1.1-45nM-9 and 3.4% in
shRAD54 3.1c-55nM-4) or DMs (60.6% of the cells ana-
lyzed in the clone defective for DNA-PKcs and 91.5% in
the clone defective for RAD54) or both structures (4.5%
in CRPK1.1-45nM-9 and 5.1% shRAD54 3.1c-55nM-4).
The observation of HSRs of variable length suggests that
chromosomal breakage occurred at different positions
along the 5q arm; the presence of HRSs and DMs in dif-
ferent cells from the same clone and, in a few cases, in
the same cell, suggests that breaks occurring within the
amplified region following its formation could be
responsible for the generation of DMs. To test this
hypothesis, we propagated one of the cell lines
(shRAD54 3.1c-55nM-4) for 30 additional passages and
performed cytogenetic analysis again. We found that
100% of the cells contained only DMs, supporting the
hypothesis that DMs derived from multiple breakages
within HSR after long periods in culture.
i(5q) chromosomes carrying amplified DNA on both
arms represented an additional type of marker chromo-
some indicating that isochromosome formation is an
important mechanism by which HeLa cell lines devel-
oped resistance to MTX. FISH analyses revealed that,
in all these chromosomes, the DHFR gene and the
BAC clone RP11- 297G19 were organized symmetri-
cally relative to the centromere (Figure 4h), as expected
for isochromosomes. A remarkable result was the fact
that isochromosomes were observed in MTX-resistant
clones defective for RAD54 (with the exception of one
clone defective for DNA-PKcs with only15% of the
metaphases showing this structure). Six out of the six-
teen (37.5%) amplified clones defective for RAD54 and
four out of seven (57.1%) amplified clones derived
from double defective cells presented the i(5q) (Table
2). In the majority of these clones, the i(5q) was the
only structure bearing additional DHFR copies and was
detected in 100% of the metaphases analyzed; in a few
cases, it was detected in combination with DMs and/or
HSR in the same cells (Table 2). It is important to note
that i(5q) could undergo intra-chromosomal reorgani-
zations in one of the double defective MTX-resistant
clones, reflecting the dynamic nature of this structure.
In Figure 4i an example of an i(5q) with a duplication
of the chromosomal region bearing the DHFR gene is
shown.
Discussion
It is well known that several molecular mechanisms are
implicated in gene amplification [5,29,30], all of which
involve DNA DSBs as a primarily event (reviewed in
[10]). We have previously shown that gene amplification
occurs at higher frequency both in rodent [23] and
HeLa [14] cells defective for NHEJ than in proficient
cells. A possible explanation for this finding is that, in
the absence of a functional NHEJ, the repair of broken
DNA molecules is delayed, allowing misrepair of DNA
ends and the initiation of gene amplification. Hinz and
collaborators [31] have shown that also a defect in HR
genes, such as RAD51D, xrcc2 and xrcc3, leads to an
increased gene amplification ability in Chinese hamster
cell lines (CHO).
Figure 4 Cytogenetic structure of DHFR gene amplicons. Upper
section of the panel: metaphase spreads hybridized with the BAC
clone containing the DHFR gene (red signal) showing (a) a DHFR
gene amplicon organized as a ladder, (b) DMs and (c) i(5q). Lower
section of the panel: examples of different ladder-like structures (d-g)
and i(5q) (h-i) found in MTX-resistant clones analyzed using double-
colour FISH with the BAC containing the DHFR gene (green signal)
and the BAC RP11-297G19 (red signal).
Table 2 Gene amplification analysis in MTX-resistant clones
N° (%) MTX-resistant clones with
Knocked-down
gene
N° clones
analyzed
N° amplified
clones (%)
HSRDMsHSR DMsi(5q)DMs i(5q)HSR DMs i(5q)
None (HeLa wt)
DNA-PKcs
RAD54
RAD54 and DNA-PKcs
11
24
24
11
4 (36%)
16 (67%)
16 (67%)
7 (64%)
0
0
4 (100%)
14 (87.5%)
6 (37.5%)
1 (14.3%)
00
0
0
0
0
1 (6.2%)
3 (18.7%)
0
1 (6.2%)
0
0
1 (6.2%)
2 (28.6%)
4 (25%)
3 (42.8%)
2 (12.5%)
1 (14.3%)
The clones analyzed derived from cell lines with different repair functions impaired. The organization of the DHFR gene was analyzed on mitotic chromosomes
by FISH with a specific DHFR probe. HSR: homologous staining regions, DMs: double minutes, i(5q): isochromosome.
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In this study, we investigated the consequence of a
defect in the RAD54 HR repair gene on gene amplifica-
tion ability of human cells; in addition, we tested the
effect of a combined deficiency in both HR and NHEJ.
To this purpose, we constructed HeLa cell lines in which
either RAD54, or both RAD54 and the NHEJ gene DNA-
PKcs, had been stably knocked down by RNA inter-
ference. The DNA-PKcs knocked down cells were
previously described in [14]. The successful impairment
of RAD54 expression was demonstrated by the lower
levels of the protein and by the greater sensitivity of
knocked down cells to ionizing radiations compared to
wild type cells. DNA-PKcs single deficient cell lines were
more radiosensitive than RAD54 deficient cells support-
ing the hypothesis that the NHEJ mechanism plays a pre-
dominant role in the repair of radio-induced DSBs. Such
radiosensitivity was enhanced in double KD RAD54/
DNA-PKcs cells, revealing that the correct function of
both repair mechanisms is essential for cell survival.
These results extend those previously obtained by differ-
ent authors showing that HR and NHEJ factors cooperate
in the maintenance of genome stability in mammalian
cells [32-34].
To study gene amplification ability of the cells KD in
the different functions, we analyzed MTX resistance,
which is frequently due to amplification of the DHFR
gene. In the different cell lines, we determined the fre-
quency of MTX resistant clones, the proportion of ampli-
fied clones among the resistant ones and the organization
of the amplified DNA on mitotic chromosomes. The
RAD54 deficient cell lines showed a frequency of MTX
clones slightly higher than proficient cells, but lower than
that observed in DNA-PKcs knocked down cells, while
the proportion of amplified clones in the two deficient
cell lines was similar (67%), and greater than that
observed in HeLa cells (36%). These results suggest that,
when the HR repair mechanism is impaired, the fraction
of DSBs undergoing an improper repair and being
engaged in gene amplification is lower than in cells defi-
cient in NHEJ, but still higher than in proficient cells.
This confirms again that the two repair mechanisms col-
laborate to prevent genome instability. In the cell lines
deficient for both RAD54 and DNA-PKcs, the frequency
of MTX resistant clones was lower than in proficient
cells. Although we could still find amplified resistant
clones, it is likely that when both the repair mechanisms
are impaired, fewer cells can survive to the events invol-
ving DNA breakage that leads to gene amplification. The
lack of increase in the frequency of amplified mutants in
the double-deficient cells may derive from a balance
between the increased generation of amplifications, due
to the persistence of unrepaired DSBs, and the death of
the cells that cannot survive to the breakage events
required to trigger gene amplification, because of the
severe DNA repair mechanism impairment.
In the amplified RAD54 deficient cells we found three
major structures bearing the extra-copies of the DHFR
gene: DMs, HSRs and isochromosomes of the long arm
of chromosome 5, where the DHFR gene is located.
Analysis of the organization of HSRs by double colour
FISH with probes for the selected gene and for an adja-
cent region revealed that the amplicons were organized
as inverted repeats, indicating that BBF cycles were the
most likely mechanism of origin. It has been postulated
that DMs can originate from breakages within HSRs
[35,36]. In one clone, we could clearly demonstrate the
transition from HSRs to DMs; in fact, at early stages
after selection, both DMs and HSRs were detected in
the MTX resistant clonal population, while after further
propagation in culture, DMs remained the only struc-
tures bearing amplification.
Isochromosomes of the long arm of chromosome 5
were a peculiarity of the cells deficient for RAD54,
either single KD or double KD for RAD54 and DNA-
PKcs; in fact, they were present in about 40% of the
MTX resistant clones isolated from these cells lines,
while they were detected only in a minority of the
mitoses of a single clone derived from DNA-PKcs defi-
cient cells.
Although studies dealing with the molecular mechan-
isms responsible for the formation of isochromosomes
are scarce, pericentromeric or centromeric breakage, fol-
lowed by unequal recombination among highly repeti-
tive sequences, have been hypothesized as possible
causes of this cytogenetic abnormality [37-39]. Human
centromeres contain extensive copies of repetitive ele-
ments called a-satellite [40] and it is well known that
centromeres, jointly with telomeres, are regions of the
chromosomes where recombination occurs very fre-
quently [41,42]. Recently, Nakamura and co-workers
[39] showed in the yeast Schizoccharomyces pombe that
a failure in the HR mechanism, because of a defect in
the RAD51 recombinase, increases the frequency of iso-
chromosome formation. In yeast, DNA breaks within
the centromere can be repaired through gene conver-
sion, which depends on RAD51, or through break-
induced replication (BIR), which is RAD51 independent.
In the absence of a proficient recombination-mediated
repair, DSB repair can preferentially occurs through
BIR, which can lead to isochromosome formation when
DNA synthesis is primed by an inverted repeated
sequence that snaps back to align with its complemen-
tary sequence. Interestingly, it was shown [39] that
RAD51 is associated with centromeres during the S
phase, supporting the hypothesis that this protein can
suppress the rearrangements of centromeric repeats that
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result in isochromosome formation. Tinline-Purvis and
collaborators [43] found that defects in other HR func-
tions increases the frequency of isochromosome forma-
tion. In addition, they demonstrated that also breaks far
from the centromeres can lead to these chromosome
anomalies, because of the occurrence of an extensive
processing of DNA ends, when the efficiency of gene
conversion is reduced.
Concluding remarks
Our results indicate that a failure of HR moderately
increases the frequency of gene amplification in human
cells, suggesting that NHEJ is not sufficient to correctly
repair all DNA breaks when HR is impaired. In addition,
our observation of an increased frequency of isochromo-
some formation in HeLa cells deficient for RAD54 sug-
gests that, similarly to what observed in fission yeast,
also in human cells homologous recombination proteins,
such as RAD54, can play a role in preventing rearrange-
ments of the centromeres.
Methods
Cell culture and transfection
HeLa cells were routinely cultured at 37°C in 5% CO2,
in DMEM supplemented with 10% fetal calf serum, glu-
tamine and non-essential amino acids. During transfec-
tion and selection experiments 0.1 mg/ml penicillin and
100 U/ml streptomycin were added.
To inhibit RAD54 expression four plasmids containing
the neomycin (G418) resistance gene with four different
inserts for shRNA production under the control of the U1
promoter were used (SureSilencing™ kit, Cat. number
KH01719N). The insert sequence of plasmids shRNAp-1,
shRNAp-2, shRNAp-3 and shRNAp-4 were TCTCGtcac-
cagcattgtgaatagatCTTCCTGTCAatctattcacaatgctggtgaCT,
TCTCGaaggttgtagaacgcttcaatCTTCCTGTCAattgaagcgttc-
tacaaccttCT, TCTCGtgtggttgttgaccctattctCTTCCTGT-
CAagaatagggtcaacaaccacaCT and TCTCGcgagttgaaggagc
tgtttatCTTCCTGTCAataaacagctccttcaactcgCT, respec-
tively. An additional plasmid (p-scrambled) containing a
scrambled sequence, which is not homologous to any
human gene (TCTCggaatctcattcgatgcatacCTTCCTGT-
CAgtatgcatcgaatgagattccCT) was used as control. To inhi-
bit DNA-PKcs expression a plasmid previously described
[14], containing the puromycin resistance gene and an oli-
gonucleotide for the expression of an shRNA against
DNA-PKcs, was used; the plasmid pSSP, not containing
the oligonucleotide insert, was used to produce the control
cell line pSSP1.Transfection experiments were carried out
as previously described [44]. Briefly, 105cells were seeded
in complete medium in 10 cm dishes; after 24 h the med-
ium was replaced with serum free medium and 1-5 μg of
plasmid DNA in a 1 mM PEI (Polyethylenimine, Sigma)
solution was added. After 7 h at 37°C, the medium was
replaced with complete medium. After 24 h, selective
medium containing 1 μg/ml puromycin or 800 μg/ml
G418 was added. Puromycin and/or G418 resistant colo-
nies were isolated after 4-5 weeks.
Immunological detection of RAD54 and DNA-PKcs
For indirect immunofluorescence analysis of RAD54 and
DNA-PKcs expression, cells were spread on slides with a
cytospin centrifuge and permeabilized at 37°C for
15 minutes in KCM buffer (KCl 120 mM, NaCl 20 mM,
Tris-HCl 10 mM, Na-EDTA 0.5 mM and Triton X-100
0.1% v/v). The slides were then incubated with either
the anti-RAD54 (Abcam) or the anti-DNA-PKcs (Ab-4,
Neomarkers) antibody diluted 1:200 in KCM containing
1% BSA at 37°C for 1 h. After washing with KB-buffer
(Tris-HCl 10 mM, NaCl 150 mM and BSA 1%) the
slides were treated with a goat anti-mouse texas-red
conjugated secondary antibody (Jackson ImmunoRe-
search) at 37°C for 30 min. The cells were washed again
with KB-buffer, stained with DAPI (4, 6-diamidino-2-
phenylindole) and visualized with a ZEISS Axioplan
fluorescence microscope. Images were captured with a
Photometric CCD camera and processed with the IP-
Lab software.
To obtain total cell extracts for Western blots the cells
were washed twice with cold PBS, resuspended in
Laemmli buffer and boiled for 10 min. Proteins were
separated by SDS-PAGE gel electrophoresis and electro-
blotted to nitrocellulose membranes (Amersham). The
membranes were then incubated with the antibody
against DNA-PKcs diluted 1:3000 or with the antibody
against RAD54 diluted 1:100; and with the antibody
against tubulin (NeoMarkers), diluted 1:3000. A goat
antimouse monoclonal (Pierce), diluted 1:5000 was used
as secondary antibody. The pre-incubation of the mem-
branes and the dilution of all antibodies were performed
in 1xPBS containing 0,05% Tween20 and 7.5% non-fat
dry milk. The signals detection was performed by che-
miluminescence (Immun-Star WesternC Kit, BioRad).
Sensitivity to irradiation
Irradiation was carried out using a60Co-ray source at a
dose rate of 1.3 Gy/min. Exponentially growing cells
were tripsinized, resuspended in complete medium and
irradiated. After irradiation, the cells were diluted and
seeded in 10 cm plates (500 cells/plate). After 10 days,
colonies were fixed with methanol, stained with Coo-
massie Blue (1% Page Blue in 50% methanol and 7.5%
acetic acid) and the number of colonies with more than
50 cells was counted. The g-ray dose reducing survival
to 50% (LD50) was then calculated after plotting survival
against g-ray doses.
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Sensitivity to methotrexate and analysis of gene
amplification frequency
Sensitivity to methotrexate (MTX) was determined by
measuring the inhibition of growth in massive cell cul-
tures. Samples of 2 × 105cells were plated in 6 cm
dishes in complete medium containing different MTX
concentrations. After 96 h at 37°C, the cells were tripsi-
nized, centrifuged, washed in PBS and centrifuged again.
The pellets were dissolved in 0.1 M NaOH for 30 min
at 50°C; absorbance of cellular lysates was measured at
260 nm. The drug concentration inhibiting cell growth
by 50% (Dose Response 50 or DR50) was determined
from a plot of absorbance against drug concentration.
To measure gene amplification frequency, samples of
105cells were seeded in selective medium containing
two different MTX concentrations, 45 nM and 55 nM
MTX (4.5 and 5.5 times the LD50). In addition to two
single RAD54 defective and two double RAD54/DNA-
PKcs defective cell lines, we included in the experiments
the two previously isolated DNA-PKcs defective cell
lines CRPK1.1 and CRPK1.4 [14] and their control cell
line (a clone from HeLa cells transfected with the empty
plasmid pSSP, named pSSP-1). Five samples were plated
per each drug concentration. After 2 weeks, the surviv-
ing MTX resistant colonies were fixed, stained and
counted. The mutation frequency was calculated from
the mean number of colonies among the cultures [26].
For each cell line, the mean was corrected for the plat-
ing efficiency, determined by counting the number of
colonies recovered after seeding 500 cells in 10 cm
dishes.
Cytogenetic analysis
To detect the presence of the DHFR gene, fluorescence
in situ hybridizations (FISH) was performed using the
BAC clone CTC-325J23 (CalTech human BAC library,
AC008434). The chromosome 5 specific BAC probe
used (RP11- 297G19) was obtained from the RPCI-11
human bacterial artificial chromosome (BAC) library.
The whole-chromosome 5 painting probe derives from
flow sorted human chromosomes. Chromosome
spreads and FISH were performed as previously
described [45]. Briefly, probes were labeled by nick
translation with Cy3-dUTP and Alexa-dUTP and
chromosomes were stained with DAPI (4, 6-diamidino-
2-phenylindole). Digital images were captured as
indicated above.
Additional material
Additional file 1: Number of methotrexate (MTX) resistant colonies
in RAD54 and DNA-PKcs defective cell lines. Mean number of MTX
resistant colonies per plate in cell lines with different repair functions
impaired for each experiment, including standard deviations.
Acknowledgements
This work was supported by grants from the European Commission Euratom
and Integrated Project RISC-RAD. Aurora Ruiz-Herrera was supported by an Intra-
European Fellowship Euratom and Alexandra Smirnova by Regione Lombardia,
Progetto Biomedicina. We are grateful to Antonio Faucitano and Armando
Buttafava (University of Pavia) for rendering available to us the irradiation source,
to Mariano Rocchi (University of Bari) for the BAC clones from the RPCI-11 library
and to Monserrat Garcia-Caldes for the chromosome 5 painting probe.
Author details
1Dipartimento di Genetica e Microbiologia “Adriano Buzzati-Traverso”,
Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy.2Istituto di Genetica
Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy.3Departament de
Biologia Cel.lular, Fisiologia i Immunologia and Institut de Biotecnologia i
Biomedicina (IBB), Universitat Autònoma de Barcelona, 08193, Campus
Bellaterra, Barcelona, Spain.
Authors’ contributions
ARH: research design, performed experiments, data analysis, manuscript
writing. AS: performed experiments, data analysis. LK: performed
experiments, data analysis. SGN: performed experiments, data analysis. CM:
data analysis, manuscript writing. EG: study conception, research design, data
analysis, manuscript writing. All authors have read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 12 November 2010 Accepted: 18 March 2011
Published: 18 March 2011
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doi:10.1186/2041-9414-2-5
Cite this article as: Ruiz-Herrera et al.: Gene amplification in human cells
knocked down for RAD54. Genome Integrity 2011 2:5.
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