Article

Suppression of Rev3, the catalytic subunit of Pol?, sensitizes drug-resistant lung tumors to chemotherapy

The Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2010; 107(48):20786-91. DOI: 10.1073/pnas.1011409107
Source: PubMed

ABSTRACT

Platinum-based chemotherapeutic drugs are front-line therapies for the treatment of non-small cell lung cancer. However, intrinsic drug resistance limits the clinical efficacy of these agents. Recent evidence suggests that loss of the translesion polymerase, Polζ, can sensitize tumor cell lines to cisplatin, although the relevance of these findings to the treatment of chemoresistant tumors in vivo has remained unclear. Here, we describe a tumor transplantation approach that enables the rapid introduction of defined genetic lesions into a preclinical model of lung adenocarcinoma. Using this approach, we examined the effect of impaired translesion DNA synthesis on cisplatin response in aggressive late-stage lung cancers. In the presence of reduced levels of Rev3, an essential component of Polζ, tumors exhibited pronounced sensitivity to cisplatin, leading to a significant extension in overall survival of treated recipient mice. Additionally, treated Rev3-deficient cells exhibited reduced cisplatin-induced mutation, a process that has been implicated in the induction of secondary malignancies following chemotherapy. Taken together, our data illustrate the potential of Rev3 inhibition as an adjuvant therapy for the treatment of chemoresistant malignancies, and highlight the utility of rapid transplantation methodologies for evaluating mechanisms of chemotherapeutic resistance in preclinical settings.

Full-text

Available from: Trudy Gale Oliver
Suppression of Rev3, the catalytic subunit of Polζ,
sensitizes drug-resistant lung tumors to chemotherapy
Jason Doles
a,b
, Trudy G. Oliver
a
, Eleanor R. Cameron
a
, Gerald Hsu
a
, Tyler Jacks
a,b,c
, Graham C. Walker
b
,
and Michael T. Hemann
a,b,1
a
The Koch Institute for Integrative Cancer Research,
b
Department of Biology, and
c
The Howard Hughes Medical Institute, Massachusetts Institute of
Technology, Cambridge, MA 02139
Edited by Alan R. Lehmann, University of Sussex, Brighton, United Kingdom, and accepted by the Editorial Board October 15, 2010 (received for review
August 2, 2010)
Platinum-based chemotherapeutic drugs are front-line therapies
for the treatment of non-small cell lung cancer. However, intrinsic
drug resistance limits the clinical efcacy of these agents. Recent
evidence suggests that loss of the translesion polymerase, Polζ, can
sensitize tumor cell lines to cisplatin, although the relevance of
these ndings to the treatment of chemoresistant tumors in vivo
has remained unclear. Here, we describe a tumor transplantation
approach that enables the rapid introduction of dened genetic
lesions into a preclinical model of lung adenocarcinoma. Using this
approach, we examined the effect of impaired translesion DNA
synthesis on cisplatin response in aggressive late-stage lung can-
cers. In the presence of reduced levels of Rev3, an essential compo-
nent of Polζ, tumors exhibited pronounced sensitivity to cisplatin,
leading to a signicant extension in overall survival of treated re-
cipient mice. Additionally, treated Rev3-decient cells exhibited re-
duced cisplatin-induced mutation, a process that has been
implicated in the induction of secondary malignancies following
chemotherapy. Taken together, our data illustrate the potential
of Rev3 inhibition as an adjuvant therapy for the treatment of
chemoresistant malignancies, and highlight the utility of rapid
transplantation methodologies for evaluating mechanisms of che-
motherapeutic resistance in preclinical settings.
mouse models
|
error-prone synthesis
|
RNA interference
C
isplatin and related compounds are widely used in the treat-
ment of a variety of malignancies. Although these agents have
proven to be quite effective in treating certain tumor types, in
others, such as ovarian and lung cancer, clinical success has been
more variable. In particular, patients harboring advanced non-
small cell lung cancer (NSCLC) generally respond poorly to ag-
gressive chemotherapy, with median survival times commonly
falling short of a year (1). In light of studies showing that nearly
half of the patient population presents with advanced (stage IV)
disease, it is not surprising that the 5-y survival rate for all NSCLC
in the United States is less than 20%. Moreover, patients di-
agnosed with metastatic disease fare even worse (<4% 5-y sur-
vival) (2, 3). Therefore, a greater understanding of mechanisms of
cisplatin resistance are essential to improve treatment of patients
with advanced NSCLC and more broadly inform strategies to
target highly drug-resistant malignancies.
Like many cytotoxic chemotherapeutic agents, cisplatin targets
DNA. Although only 5 to 10% of covalently bound cisplatin is
bound to DNA, it is this DNA damage that is largely responsible
for its cytotoxic properties (46). The predominant forms of
cisplatin-induced damage are intrastrand crosslinks: 1,2-(GpG)
(65%), 1,2 (ApG) (25%), and 1,3 (GpNpG) (510%), with in-
terstrand crosslinks and monoadducts accounting for 1 to 3%
(4). Binding of HMGB proteins to 1,2-intrastrand crosslinks can
contribute to cytotoxicity by shielding them from DNA repair (4,
5), although interstrand crosslinks are a particularly cytotoxic
form of DNA damage (7, 8). Numerous mechanisms of cisplatin
resistance have been identied, including decreasing drug uptake
(e.g., by down-regulation of the copper transporter CTR1), in-
creased efux, and increased glutathione-based detoxication (6,
9). In addition, resistance can also arise from changes that in-
crease a cells capacity to either repair or tolerate DNA damage
(1012). It is this latter group of DNA repair and tolerance-
based mechanisms that have come under recent scrutiny as po-
tential contributors to clinical cisplatin resistance.
REV3L, the catalytic subunit of the DNA Polζ, which plays
a key role in the DNA damage tolerance mechanism of trans-
lesion synthesis (TLS) (13, 14), is of unusual interest because of
its critical role in preventing cisplatin cytotoxicity. Notably, hu-
man cells expressing reduced levels of REV3L are more sensitive
to killing by cisplatin (14, 15). Additionally, in an siRNA-based
screen, a reduction in REV3L sensitized human cells to killing by
cisplatin to an extent equal or greater to a reduction of BRCA1
(16). Finally, chicken DT40 cells decient in Rev3 showed the
highest sensitivity to cisplatin of any of the DNA repair or check-
point mutants tested (17). In Saccharomyces cerevisiae, Polζ (Rev3
and its auxiliary subunit Rev7), functions together with Rev1 in the
mutagenic branch of TLS that is responsible for most mutations
induced by UV light and many chemical mutagens (18). The
mammalian Rev3 orthologs, human REV3L and mouse Rev3L,
are nearly twice the size of S. cerevisiae Rev3, mostly because of one
large intron (14).
In mammalian cells, as in yeast, REV1, REV3L, and REV7
are required for most of the mutagenesis induced by UV light and
chemical mutagens, such as benzo(a)pyrene diol epoxide (19, 20).
REV3L function has also been implicated in homologous re-
combination, somatic hypermutation, cell-cycle control, and ge-
nome stability (14, 21). Notably, in response to DNA damaging
agents, such as UV light and benzo(a)pyrene diol epoxide, loss of
Rev3 function has a greater effect on mutagenesis than cell sur-
vival (14). It seems likely that the striking sensitization to cisplatin
killing caused by a reduction in REV3L levels is a result of
REV3Ls roles in the repair of both cisplatin-induced intra- and
interstrand crosslinks (22). Consistent with this idea, inhibition of
REV7 (MAD2B) or REV1, which are also involved in the repair
of both intra- and interstrand crosslinks (22), similarly sensitizes
mammalian cells to cisplatin (22, 23).
Little is known, however, about the effects of REV3L sup-
pression on chemotherapeutic response in relevant preclinical
settings. In this study, we examined the impact of Rev3L de-
pletion on cisplatin response in a highly chemoresistant mouse
model of late-stage lung adenocarcinoma (24). Given the striking
similarities between lung tumors occurring in this Kras-driven
Author contributions: J.D., G.C.W., and M.T.H. designed research; J.D., T.G.O., E.R.C., and
G.H. performed research; J.D., T.J., G.C.W., and M.T.H. analyzed data; and J.D. and M.T.H.
wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission. A.R.L. is a guest editor invited by the Editorial
Board.
1
To whom correspondence should be addressed. E-mail: hemann@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1011409107/-/DCSupplemental.
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mouse model and human NSCLC, our data suggest a rationale
for targeting TLS as an adjuvant therapy in the treatment of
advanced lung cancer.
Results
Rev3 Deciency Sensitizes LSL-Kras
G12D
;p53
/
Lung Adenocarcinoma
Cells to Cisplatin.
To begin to examine the effects of Rev3L sup-
pression on cisplatin response in a clinically relevant mouse-
model system, we chose to use lung adenocarcinoma cell lines
derived from previously described LSL-Kras
G12D
;p53
/
mice.
Tumors generated in this context are thought to mirror human
NSCLC with respect to overt clinical phenotype, as well as to
core molecular mechanisms governing adenocarcinoma develop-
ment (24, 25). Interestingly, recent work has shown that au-
tochthonous LSL-Kras
G12D
;p53
/
lung tumors, like human
NSCLC, also show intrinsic resistance to front-line chemother-
apy (26). Most notably, these tumors proved to be refractory to
cisplatin therapy, providing a system in which we could evaluate
candidate drug-sensitizing genetic alterations. To this end, we
designed and retrovirally expressed three unique shRNAs tar-
geting REV3L, and subsequently veried suppression of REV3L
transcript by quantitative PCR (qPCR) in virally transduced
target cells (Fig. 1A). As impairment of TLS might have delete-
rious effects on cell growth or viability, we rst sought to de-
termine if our REV3L shRNAs impaired cell-cycle progression.
DNA content analysis did not reveal any change in population
doubling time, nor was there any cell-cycle defect, suggesting that
our level of REV3L inhibition was not grossly affecting normal
growth kinetics (Fig. 1 B and C). We then tested the effect of
REV3L-depletion on cisplatin response and found all three
shREV3L-expressing cell populations to be markedly sensitized
to drug relative to vector control-infected cells (Fig. 1D). Addi-
tionally, when treated with a high dose of cisplatin and evaluated
for long-term survival, cells lacking REV3L demonstrated a di-
minished capacity to recover from such an insult and conse-
ytilibaivevitaler
vector shRev3
#1 #2 #3
0 50 100 150 200 250
vector
shRev3-1
shRev3-2
shRev3-3
colony number
A
B
C
D
vector Rev3-1 Rev3-2 Rev3-3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
ANRm3veR%
*
p=0.004
p=0.001
p<0.001
p<0.001
p<0.001
p<0.001
no drug 5µ M 10µ M
0.5
0.6
0.7
0.8
0.9
1.0
1.1
vector
shRev3-1
shRev3-2
shRev3-3
Cisplatin dose
edidoimuid
ipo
r
p
stnuoc
vector
shRev3-1
shRev3-2
)
h
(em
itgn
i
lbuo
d
p=0.89
p=0.70
E
F
vector shRev3-1 shRev3-2
10
11
12
13
14
15
Fig. 1. Rev3-deciency sensitizes LSL-Kras
G12D
;p53
/
lung adenocarcinoma cells to cisplatin. (A) Quantitative RT-PCR (n 3) conrmation of Rev3 mRNA
suppression in transduced GFP sorted cell populations. Untreated control and Rev3 knock-down cells were counted and analyzed by ow cytometry to
determine (B) overall population doubling times, and (C) cell-cycle distribution proles (DNA content histogram). (D) Overall cell survival following cisplatin
treatment was compared for adenocarcinoma cells transduced with a Rev3 shRNA or a control. Cells were then treated with cisplatin and monitored for cell
survival (Cell-Titer-Glo) reagents relative to treated vector control cells (n = 3 independently treated samples for each construct, ± SD. *P < 0.05 for all three
shRev3 constructs at this dose). (E) A long-term (14 d) colony-outgrowth assay comparing shRev3 and vector control transduced lung adenocarcinoma cells
treated with 15 μM cisplatin. The images shown depict representative 10-cm plates stained with propidium iodide to visualize colonies. (F) Quantication of
images collected from three independently treated populations of cells. Data represent the mean colony number ± SD.
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quently formed fewer colonies compared with treated control
cells (Fig. 1 E and F).
REV3L deciency in human and mouse cell lines has been
associated with double-strand breaks and chromosome instability
(27, 28). Consistent with these prior observations, we saw a rel-
ative increase in the number and intensity of γ-H2AX foci,
a surrogate marker for DNA double-strand breaks, following
cisplatin treatment of REV3L knockdown cells (Fig. 2A). Ad-
ditionally, when examined over time by ow cytometry, REV3L-
decient cells failed to show a signicant decrease in either the
overall percentage of γ-H2AXpositive cells or in mean cellular
γ-H2AX immunouorescence intensity (Fig. 2B). Coincident
with the increase in cell death associated with this enhanced level
of unrepaired DNA damage, we observed a pronounced cell-cycle
arrest phenotype within the surviving cell population. Specically,
cisplatin treated REV3L-decient cells exhibited characteristics
of DNA-damage induced senescence, including the appearance
of a attened, vacuolized cell morphology, as well as the induction
of senescence-associated β-galactosidase activity (Fig. 2C and Fig.
S1). Thus, REV3L suppression impairs the repair of cisplatin-
induced DNA damage, leading to both cell death and irreversible
cell-cycle arrest.
Polζ is an error-prone DNA polymerase that is essential, not
only for much of the mutagenesis that is caused by agents such as
UV light, but for cisplatin-induced mutagenesis in human colon
carcinoma cells and immortal human broblasts as well (15, 29).
To evaluate the effect of REV3L knockdown on cisplatin-induced
mutagenesis in our LSL-Kras
G12D
;p53
/
cells, we performed
a hypoxanthine phosphoribosyl-transferase (hprt) mutation assay
where control and REV3L knockdown cells were treated with
cisplatin, allowed to recover, and then selected in the presence of
the toxic nucleoside analog 6-thioguanine (6-TG). As hprt func-
tion is required for 6-TGmediated toxicity, this assay allows for
the quantitation of cisplatin-induced hprt mutation. Cells ex-
pressing shREV3L-1 and shREV3L-2 shRNAs showed a dramatic
reduction in 6-TG resistant colonies (4.7- to 6.6-fold) relative to
control cells (Fig. 2D). Although shREV3L-3transduced cells
also showed a decrease in colony number, this decrease was not
statistically signicant. Notably, this shRNA also produces less
cisplatin sensitization than shREV3L-1 and shREV3L-2 in colony
outgrowth assays. Taken together, these cell-based assays suggest
that reducing the level of REV3L not only sensitizes highly re-
sistant lung cancer cells to cisplatin, but also prevents cisplatin-
induced mutation in surviving cells.
Development of a Genetically Tractable Lung Adenocarcinoma
Transplant System.
Although cell-based treatment studies may
yield important insight into potential tumor responses to therapy,
achieving durable therapeutic responses in malignancies in their
native microenvironment has proven considerably more difcult
(30). Thus, we decided to evaluate the potential of REV3L in-
hibition as a strategy to improve upon existing cisplatin-based
chemotherapeutic regimens in an established preclinical model of
NSCLC. To this end, we adapted methodologies previously used
for tumor transplantation in hematopoietic malignancies for use
in our lung adenocarcinoma cell line (31). Such an approach
allows for rapid manipulation of in vivo tumor cell genetics,
without the requirement for generating stable genetically engi-
neered mouse models. Lung adenocarcinoma cells (5 × 10
4
)
were intravenously injected via tail vein into syngeneic immuno-
competent recipient mice. As early as 20 d posttransplantation,
highly proliferative tumor foci were detectable in the lung (Fig.
3A), with nearly every recipient mouse exhibiting a disseminated
disease within 30 to 35 d (Fig. 3B). Notably, tumor presentation
was specic to the lung, suggesting that either the route of cell
delivery or the lung microenvironment restricts the development
of transplanted malignancies to the appropriate target organ.
To assess the ability of these transplanted cells to recapitulate
the original disease, we examined overall tumor histology. Trans-
planted tumors exhibited features reminiscent of their epithelial
origin in the lung: namely, alveolar and sheet-like structures (Fig.
3B). Interestingly, we found that the transplants displayed a
markedly aggressive morphology, consistent with hallmark fea-
tures of late-stage carcinomas. In some cases, the transplanted
tumors breached the visceral pleura, penetrating the pleural space
proximal to the chest cavity (Fig. 3B, Lower, Right). Immunohis-
tochemical staining of tumor specimens with antibodies targeting
Nkx2.1 and HMGA2, two markers of lung adenocarcinoma pro-
gression, further suggested that these transplants represent
a highly aggressive version of the disease suitable for modeling the
treatment of late-stage lung cancer (Fig. 3C).
REV3L-Depletion Sensitizes Lung Adenocarcinoma Transplants to
Cisplatin in Vivo.
Using the most potent shRNA targeting
REV3L, we transplanted pure populations of retrovirally infected
control and REV3L knockdown lung adenocarcinoma cells into
syngeneic recipient mice and allowed tumors to form (34 wk).
Mice were subsequently killed 48 h following cisplatin treatment
A
B
CD
% -gal+ cells/HPF
vector shRev3-1
0.00
0.25
0.50
0.75
p=0.003
0
10000
20000
30000
40000
50000
0h
2h 6h
24h 48h
H2AX index
*
*
vector
shRev3-1
untreated 12h cisplatin 12h cisplatin
p=0.009
p=0.007
0.0
0.2
0.4
0.6
0.8
1.0
1.2
shRev3-1 shRev3-2vector
relative survival in 6-TG
vector
shRev3-1
Fig. 2. Rev3 depletion promotes cisplatin-induced DNA damage but limits
associated mutagenesis. (A) Immunouorescence images of control and
shRev3 expressing lung adenocarcinoma cells treated with 10 μM cisplatin
and incubated with an antiγ-H2AX antibody. Color images are stained with
DAPI (blue) for DNA. (B) Flow-cytometric analysis of γ-H2AX immunouo-
rescence in cisplatin-treated cells. The γ-H2AX index was calculated by mul-
tiplying the number of γ-H2AXpositive cells by the mean intensity of the
γ-H2AX population (n = 3 independently treated samples at each time point).
*P < 0.05 at the indicated time points. (C) Senescent cells were identied
using a standard X-gal staining protocol and manually quantied from rep-
resentative microscope images. Data represent the mean of six 40× high-
power elds from two independent samples for each experimental condi-
tion. (D) A cisplatin in vitro mutagenesis assay. Shown is the relative colony
forming ability of control and Rev3-decient cells treated with 15 μM cisplatin
and then selected for 6-thioguanine resistance. Each datapoint represents an
independently treated and selected experimental replicate.
Doles et al. PNAS Early Edition
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to analyze the effects of cisplatin on tumor-cell proliferation rate
and survival. Histopathological evaluation of harvested tumors
corroborated our in vitro observations, as we noted both a de-
crease in mitotic index (Fig. 4A) and an increase in apoptosis (Fig.
4B) in treated REV3L-decient transplants.
To more carefully examine the effect of REV3L suppression
on lung adenocarcinoma response to cisplatin, we took an in vivo
imaging-based approach that allowed us to study individual tu-
mor dynamics over a course of therapy. Using a microcomputed
tomography (microCT) imaging platform, we were able to image
the lung and surrounding tissues at regular intervals to identify
and subsequently track disease progression in individual tumor-
bearing mice over the course of cisplatin treatment. As illus-
trated in reconstructed 3D isosurface and 2D axial images taken
10 d following the initiation of therapy (10 mg/kg cisplatin on day
0), Rev3-decient transplants exhibited an enhanced response to
cisplatin relative to controls (Fig. 5). This nding was evidenced
at the level of individual tumors (Fig. 5 AC), as well as in the
broader context of the entire lung (Fig. 5 D and E). Indeed,
quantitative evidence of overall tumor regression or at a mini-
mum, growth stasis, was observed in cisplatin-treated REV3L-
knockdown transplants, whereas most control transplants con-
A
C
Nkx2.1 Hmga2
H&E
s
uonohtcotuA
tnalpsnarT
suonohtcotuA
tnalpsnarT
10x 20x
3
H
pE
&
H
B
Fig. 3. Histological analysis of LSL-Kras
G12D
;p53
/
adenocarcinoma trans-
plants. (A) H&E (Uppe r) and antiphospho-histone-H3 (pH3) immunohisto-
chemical (Lower) staining of lung adenocarcinoma transplants harvested at
18 d postinjection. Arrowheads demarcate pH3-positive cells. (B)H&E
staining of tumor transplants harvested at 30 d postinjection, as well as
representative images of an autochthonous LSL-Kras
G12D
;p53
/
lung ade-
nocarcinoma. The dotted line represents the visceral pleural boundary, with
an arrowhead highlighting the tumor mass extending into the pleural space.
(C) Anti-Nkx2.1 and anti-HMGA2 immunostaining of early- and late-stage
lung adenocarcinomas, respectively. The arrowheads indicate a region in the
autochthonous tumor with high expression of the late-stage marker HMGA2
and corresponding down-regulation of the early-stage marker Nkx2.1.
A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
# CC3+ cells/HPF
0
2
4
6
8
10
12
14
# pH3+ cells/HPF
vec shRev3 vec shRev3
cisplatin
vec shRev3 vec shRev3
cisplatin
untreated
48hr cisplatin
vector
shRev3
p=0.002
p=0.015
p=0.002
p=0.67
p=0.32
p=0.013
untreated
48hr cisplatin
vector
shRev3
B
Fig. 4. Rev3 depletion sensitizes transplanted lung adenocarcinoma to cis-
platin. (A) Anti-phosphoH3 and (B) cleaved caspase 3 staining of control and
shRev3-transduced tumor transplants 48 h following treatment with 10 mg/
kg cisplatin. Tumors were treated upon detection of tumo r mass by microCT.
P values were determined using two-tailed Students t tests.
A
C
D
vector
Day 0
Day 10 cisplatin
shRev3
B
before cisp 10d cisp
0
10
20
30
D
E
F
control shRev3
0.0
0.5
1.0
1.5
Day 0
Day 10 cisplatin
vectorshRev3
Day 0
Day 10 cisplatin
vectorshRev3
tumor volume (mm^3)
% change in lung volume
% survival
0 5 10 15 20 25 30
0
20
40
60
80
100
control
shRev3
day
p=0.011
p<0.0001
vector
shRev3
Fig. 5. Rev3 depletion promotes cisplatin efcacy in vivo. (A) Representa-
tive axial images of mouse lungs harboring transplanted lung adenocarci-
noma cells. The darker areas represent healthy, air-lled lung space, whereas
the lighter shades highlight denser tissues, including areas populated by
tumor cells. Red arrowheads demarcate individual tumors in treated control
mice that respond poorly to cisplatin treatment. (B) Three-dimensional iso-
surface projections of selected lung regions. Green staining indicates lung
adenocarcinoma mass. (C) Individual tumor volume calculations for several
control and shRev3 transplants. (D) Inverse 3D isosurface projections of
healthy lung volume before and after cisplatin treatment. White/gray sur-
faces indicate disease-free, healthy lung space, whereas hollowed-out voids
indicate the presence of tumor material. (E) Quantication of healthy lung
volumes from D. P values were determined using a Students t test. (F)A
Kaplan-Meier curve comparing survival of mice bearing shRev3-infected
transplants versus mice bearing control tumors following treatment with
cisplatin (vector, n = 11; shRev3, n = 12; median survival time = 11 and 22.5 d,
respectively). P values were determined using a log-rank test.
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tinued to grow and displace healthy lung volume (Fig. 5E). Im-
portantly, Kaplan-Meier analysis of overall survival supported
these imaging-based observations, with mice harboring Rev3-
decient transplants surviving nearly twice as long compared
with the control cohort (Fig. 5F ).
Discussion
Our experiments provide a striking illustration of how reducing
the activity of a key translesion DNA polymerase can make an
intractable lung cancer model of NSCLC susceptible to cisplatin-
based chemotherapy. Thus, REV3L represents a bona de lung-
cancer drug target. Inhibiting REV3L activity or expression may
be particularly effective in this context, because cisplatin treat-
ment, itself, increases REV3L mRNA levels (15) and elevated
REV3L has been shown to promote cisplatin resistance (12).
Notably, therapies that exploit DNA repair deciencies, including
the use of poly(ADP ribose) polymerase inhibitors in BRCA1/2-
decient tumor cells (32, 33), have emerged as a promising ap-
proach to target chemoresistant malignancies. Although it is un-
clear whether advanced malignancies acquire a similar dependence
upon REV3L function, cell-based studies examining gliomas
a highly chemoresistant malignancyhave documented elevated
levels of REV3L in this setting and shown that down-regulation of
REV3L sensitizes these cells to cisplatin (12). Interestingly, mis-
match repair-decient, p53-decient tumor cells also show
increases up to 20 times in REV3L levels (34), suggesting that
malignancies driven by mutagenesis in the absence of mismatch
repair may be particularly reliant upon REV3L function.
The striking cisplatin sensitization seen in the absence of
REV3L may result from the dual requirement for REV3L
function in the repair of both intrastrand crosslinks, which con-
stitute the majority of the lesions, and highly toxic interstrand
crosslinks, which are much less frequent. The functions of Polζ
(REV3L/REV7), REV1, Polη, and RAD18 are all required for
replicative bypass of cisplatin intrastrand crosslink (22). Polζ has
been shown to cooperate with Polη and Polκ in error-free and
error-prone TLS, respectively over a 1,2-GpG cisplatin adduct
(35). In addition, Polζ and REV1 have been hypothesized to
facilitate repair of interstrand crosslinks independently of pro-
liferating cell nuclear antigen monoubiquitination (22). Bio-
chemical analyses of replication-dependent interstrand crosslink
repair using Xenopus extracts have implicated Polζ in the TLS
across from the crosslink during the repair process (36).
A potential benet to a chemotherapeutic strategy that relies on
combining a reduction in REV3L activity/expression with a DNA
damaging chemotherapeutic agent is that cisplatin-induced mu-
tagenesis might also be reduced. In vitro studies of immortal hu-
man broblasts have shown that reduced levels of REV3L lowers
cisplatin-induced mutation, including mutation that leads to cis-
platin resistance (15). In the companion article (37), we use an-
other clinically relevant mouse model to illustrate how interfering
with REV1/REV3L/REV7 pathway of mutagenic TLS can reduce
the frequency of acquired drug resistance following tumor relapse.
Although specic TLS inhibitors have not yet been developed,
improvements to in vivo RNAi delivery methodologies suggest
that adjuvant siRNA therapies may be achievable in accessible
tumor sites (38). Additionally, the development of specic in-
hibitors targeting critical protein interactions in TLS polymerase
complexes may hold signicant therapeutic promise. Although it
is reasonable to believe that rapidly growing tumor cells have
a greater requirement for TLS function, future work will be re-
quired to determine whether TLS inhibition can be achieved in
tumors without enhancing cisplatin-related normal cell toxicity.
Genetically engineered mouse models of cancer provide the
opportunity to validate candidate drug targets in relevant patho-
physiologic settings (30, 39). Although autochthonous tumor
models represent the ideal context for such studies, efcient
mechanisms to introduce diverse genetic alterations into such
models are currently lacking. This lack is particularly true for drug
sensitization experiments, where all tumor cells may need to be
modied to see a therapeutic effect. Here, we have developed
a tumor transplant approach that allows for rapid ex vivo modi
-
cation of lung adenocarcinoma cells. Importantly, transplanted
tumors develop in the appropriate organ system, in the presence of
an adaptive immune system, and are pathologically similar to ag-
gressive autochthonous tumors. Although this approach does not
supplant the subsequent value of autochthonous tumor models, it
may inform their development. Additionally, we have recently
shown that large-scale RNAi-based screening approaches can be
performed in vivo (40). Thus, robust transplantation of cell lines
derived from Kras-driven lung tumor models may not only serve as
an attractive setting in which to evaluate putative mediators of
chemotherapeutic response, but may also function as a platform
from which to screen for and identify novel genetic factors capable
of sensitizing aggressive NSCLC to existing chemotherapies.
Methods
Cell Culture, Retroviral Vectors, and Chemicals. Mouse lung adenocarcinoma
cells were cultured in standard DMEM/10% FBS media. Short hairpin RNA
constructs were designed and cloned as previously described (41). The vector
used coexpressed GFP under the control of the SV40 promotor and is iden-
tical to the published MSCV/LTRmiR3 0-SV40-GFP (LMS) vector. Sequences
(53) targeted by shRNAs are as follows: shRev3-1: TTTACTACAGATAC-
CATGCTG; shRev3-2: TATCTTTATAAGCTGCTCCTG; shRev3-3: TACAGTTATA-
CAAATATCCTA. Retrovirally infected cells were then selected with puro-
mycin. Cisplatin was purchased from Calbiochem and used at the indicated
concentrations (015 μM). For in vivo studies, cisplatin was dissolved in
a 0.9% NaCl solution, protected from light, and immediately injected in-
traperitoneally into tumor-bearing mice. X-gal for senescent cell identica-
tion was purchased from USB Corporation.
RT-qPCR, Immunohistochemistry, and Immunouorescence. For real-time
quantitative PCR, total RNA was isolated after retroviral infection and GFP
sorting for GFP-high expressing cells. RT-qPCR was performed using SYBR
green on a BioRad thermal cycler. GAPDH and Rev3 primer sequences are
available upon request. For immunohistochemistry assays, mice were killed
by CO
2
asphyxiation and lungs were xed overnight in 10% neutral-buff-
ered formalin. Lung lobes were separated and embedded in parafn
according to standard procedures. Lungs were sectioned at 4 μm and stained
with H&E for tumor pathology. For detection of cleaved caspase 3 (1:500;
Cell Signaling) and phospho-histone-H3 (1:200; Cell Signaling), TTF-1
(Nkx2.1, 1:200; Epitomics), HMGA2 (1:500; Biocheck), tissue sections were
subjected to antigen retrieval in citrate buffer, blocked in 3% H
2
O
2
for 10
min, blocked for 1 h in 5% serum/PBS-T, and stained overnight at 4 °C.
Secondary antibodies were used according to Vectastain ABC kits (Vector
Laboratories). Cells for immunouorescence were grown and treated on
poly-
L-lysine coated coverslips, xed with 100% methanol for 5 min at
20 °C, and stored for later use. Anti γ-H2AX (1:500; Upstate) was used
along with an Alexa secondary (568) antibody (Molecular Probes) to visual-
ize γ-H2AX foci. Stained coverslips were imaged and analyzed using Applied
Precision DeltaVision instrument s and deconvolution software.
In Vitro Viability Assays and FACS. For short-term viability assays, cells were
seeded in triplicate (6 × 10
3
per well) in 96-well plates and treated as indicated
with cisplatin. After 48-h treatment, cell viability was measured using Cell-
Titer-Glo (Promega) on an Applied Biosystems microplate luminometer.
Long-term viability assays were performed by initially treating 4 × 10
5
lung
adenocarcinoma cells with 15 μM cisplatin for 24 h. Four days following
treatment, cells were split 1:20 onto a fresh 10-cm plate and allowed to form
colonies for 10 d. To visualize colonies, plates were washed with 0.05%
ethidium bromide (in 50% EtOH) for 10 to 15 s and imaged using a UV-gel
box/camera. Images were processed and colonies counted using ImageJ
software. All ow cytometry was performed using Becton-Dickinson FACScan
or MoFlo ow cytometers. Cell death was detected by propidium iodide in-
corporation (0.05 mg/mL), and dead cells were excluded from GFP analysis.
Live cell sorting was performed using GFP coexpression as a marker of cell
transduction. For γ-H2AX assays, cells were xed in 70% EtOH, then stained
using an anti-γ-H2AX antibody (1:2,500; Upstate) followed by an Alexa (488)
secondary antibody. Stained cells were then costained in a sodium citrate/
propidium iodide incorpo ration buffer before FACS analysis and sub-
sequently analyzed using FloJo software.
Doles et al. PNAS Early Edition
|
5of6
MEDICAL SCIENCES
Page 5
Mutagenesis (hprt) Assay. Retrovirally transduced cells were initially cultured
for a minimum of 2 wk in media containing hypoxanthine, aminopterin, and
thymidine (HAT) to remove any preexisting hprt- variants from the pop-
ulation. Cells were then split into fresh media (without HAT) 24 h before
treatment with cisplatin. Target cells were then mutagenized with 15 μM
cisplatin for 1 h, allowed to recover, and passaged for an additional 10 d (in
the absence of HAT) to stabilize any induced mutations. Mutagenized cells
were then split onto fresh 10-cm plates in media containing 6-TG to select for
variants with impaired hprt function. Resultant colonies were visualized using
0.05% ethidium bromide (see above) and imaged using a UV-gel box/camera.
Images were processed and colonies counted using ImageJ software.
In Vivo Transplantation and Imaging. Lungadenocarcinomacells(5× 10
4
)were
intravenously injected into the tail vein of syngeneic C57BL6/Jx129-JAE male
recipient mice and monitored weekly using a GE Healthcare microCT imaging
device (45-μm resolution, 80 kV, with 450-μA current) beginning 3 wk following
injection. Images were acquired and processed using GE eXplore software. The
Massachusetts Institute of Technology Committee on Animal Care reviewed
and approved all mouse experiments described in this study.
ACKNOWLEDGMENTS. We thank members of the G.C.W. and M.T.H.
laboratories for helpful advice and discussions. This study is supported in
part by National Institutes of Health Grant RO1 CA128803 (to M.T.H.); Koch
Institute Support (core) Grant P30-CA14051 from the National Cancer
Institute; National Institute on Environmental Health Sciences Grant
ES015818 and Grant P30 ES002109 from the Center of Environmental Health
Sciences, Massachusetts Institute of Technology (to G.C.W.); and a Massachu-
setts Institute of Technology Department of Biology training grant and
a Ludwig Center Graduate Fellowship (to J.D.). M.T.H. is a Rita Allen Fellow
and the Latham Family Career Development Assistant Professor of Biology,
T.J. is a Howard Hughes Medical Institute Investigator and a Daniel K.
Ludwig Scholar, and G.C.W. is an American Cancer Society Research
Professor.
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  • Source
    • "Our results with the Rev3l knock-in polymerase mutant mouse are relevant to development of REV3L as a target for chemotherapy. Suppression of REV3L sensitizes cancer cells to cisplatin in mouse model systems, and can limit chemo-resistance [12, 53] because loss of pol z diminishes point mutagenesis2345 . These studies used siRNA knockdown of REV3L to demonstrate this effect, but future use of small molecule DNA polymerase inhibitors may be more clinically feasible. "
    [Show abstract] [Hide abstract] ABSTRACT: DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.
    Full-text · Article · Jan 2016 · PLoS Genetics
  • Source
    • "The activities of REV3 and REV1 have been linked to the drug resistance to cisplatin and cyclophosphamide in murine models of both B-cell lymphoma and lung cancer[23,25]. Using short hairpin RNA to inhibit REV1 or REV3 deficient in tumor cells significantly sensitized these tumors to treatment[23,25]. In some in vitro studies, cultured human cell lines showed that suppressing either Rev1 or REV3L reduced the rate of emergence of cisplatin resistance[19,50,51]. "
    [Show abstract] [Hide abstract] ABSTRACT: REV3L, the catalytic subunit of DNA Polymerase ζ (Polζ), plays a significant role in the DNA damage tolerance mechanism of translesion synthesis (TLS). The role of REV3L in chemosensitivity of cervical cancer needs exploration. In the present study, we evaluated the expression of the Polζ protein in paraffin-embedded tissues using immunohistochemistry and found that the expression of Polζ in cervical cancer tissues was higher than that in normal tissues. We then established some cervical cancer cell lines with REV3L suppression or overexpression. Depletion of REV3L suppresses cell proliferation and colony formation of cervical cancer cells through G1 arrest, and REV3L promotes cell proliferation and colony formation of cervical cancer cells by promoting G1 phase to S phase transition. The suppression of REV3L expression enhanced the sensitivity of cervical cancer cells to cisplatin, and the overexpression of REV3L conferred resistance to cisplatin as evidenced by the alteration of apoptosis rates, and significantly expression level changes of anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia sequence 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl) and proapoptotic Bcl-2-associated x protein (Bax). Our data suggest that REV3L plays an important role in regulating cervical cancer cellular response to cisplatin, and thus targeting REV3L may be a promising way to alter chemosensitivity in cervical cancer patients.
    Full-text · Article · Mar 2015 · PLoS ONE
  • Source
    • "The long stretches of error-prone synthesis are also likely relevant to other situations where mutagenic processes promote adaptation, evolution or human disease and where the role of clustered mutations is yet to be established. For example, Polz/ Rev1-dependent TLS is believed to be responsible for the acquired drug resistance and the development of secondary tumors in patients undergoing chemotherapy with DNA-damaging agents62636465. The ability of TLS enzymes to generate multiple mutations in extended stretches of DNA likely accelerates the emergence of chemoresistance. Future molecular characterization of therapy-resistant tumors could help clarify the role of the TLS-associated localized hypermutability in tumor evolution. "
    [Show abstract] [Hide abstract] ABSTRACT: Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.
    Full-text · Article · Mar 2015 · PLoS Genetics
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