Suppression of Rev3, the catalytic subunit of Polζ,
sensitizes drug-resistant lung tumors to chemotherapy
Jason Dolesa,b, Trudy G. Olivera, Eleanor R. Camerona, Gerald Hsua, Tyler Jacksa,b,c, Graham C. Walkerb,
and Michael T. Hemanna,b,1
aThe Koch Institute for Integrative Cancer Research,bDepartment of Biology, andcThe 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 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 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 significant extension in overall survival of treated re-
cipient mice. Additionally, treated Rev3-deficient 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
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 ofmechanisms 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 (4–6). The predominant forms of
cisplatin-induced damage are intrastrand crosslinks: 1,2-(GpG)
(65%), 1,2 (ApG) (25%), and 1,3 (GpNpG) (5–10%), 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 identified, including decreasing drug uptake
(e.g., by down-regulation of the copper transporter CTR1), in-
isplatin and related compounds are widely used in the treat-
ment of a variety of malignancies. Although these agents have
creased efflux, and increased glutathione-based detoxification (6,
9). In addition, resistance can also arise from changes that in-
crease a cell’s capacity to either repair or tolerate DNA damage
(10–12). 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 deficient 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
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,
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
REV3L’s 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 conflict of interest.
This article is a PNAS Direct Submission. A.R.L. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1011409107PNAS Early Edition
| 1 of 6
mouse model and human NSCLC, our data suggest a rationale
for targeting TLS as an adjuvant therapy in the treatment of
advanced lung cancer.
Rev3 Deficiency Sensitizes LSL-KrasG12D;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-KrasG12D;p53fl/flmice.
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-KrasG12D;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 verified 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 first 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-
y t i l i b
a i v
v i t a l e
#1 #2 #3
050 100150 200250
vector Rev3-1Rev3-2 Rev3-3
no drug 5µ M 10µ M
d i d
u i d i p
n i l b
suppression in transduced GFP sorted cell populations. Untreated control and Rev3 knock-down cells were counted and analyzed by flow cytometry to
determine (B) overall population doubling times, and (C) cell-cycle distribution profiles (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) Quantification of
images collected from three independently treated populations of cells. Data represent the mean colony number ± SD.
Rev3-deficiency sensitizes LSL-KrasG12D;p53−/−lung adenocarcinoma cells to cisplatin. (A) Quantitative RT-PCR (n ≥ 3) confirmation of Rev3 mRNA
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| www.pnas.org/cgi/doi/10.1073/pnas.1011409107Doles et al.
quently formed fewer colonies compared with treated control
cells (Fig. 1 E and F).
REV3L deficiency 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 flow cytometry, REV3L-
deficient cells failed to show a significant decrease in either the
overall percentage of γ-H2AX–positive cells or in mean cellular
γ-H2AX immunofluorescence intensity (Fig. 2B). Coincident
with the increase in cell death associated with this enhanced level
ofunrepairedDNAdamage, weobserved apronouncedcell-cycle
arrest phenotype within the surviving cell population. Specifically,
cisplatin treated REV3L-deficient cells exhibited characteristics
of DNA-damage induced senescence, including the appearance
ofsenescence-associated β-galactosidase activity (Fig.2C andFig.
S1). Thus, REV3L suppression impairs the repair of cisplatin-
induced DNA damage, leading to both cell death and irreversible
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 fibroblasts as well (15, 29).
mutagenesis in our LSL-KrasG12D;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-TG–mediated toxicity, this assay allows for
the quantitation of cisplatin-induced hprt mutation. Cells ex-
reduction in 6-TG resistant colonies (4.7- to 6.6-fold) relative to
control cells (Fig. 2D). Although shREV3L-3–transduced cells
also showed a decrease in colony number, this decrease was not
statistically significant. Notably, this shRNA also produces less
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 difficult
(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 × 104)
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 specific 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
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
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 (∼3–4 wk).
Mice were subsequently killed 48 h following cisplatin treatment
% -gal+ cells/HPF
untreated12h cisplatin12h cisplatin
relative survival in 6-TG
associated mutagenesis. (A) Immunofluorescence images of control and
shRev3 expressing lung adenocarcinoma cells treated with 10 μM cisplatin
and incubated with an anti–γ-H2AX antibody. Color images are stained with
DAPI (blue) for DNA. (B) Flow-cytometric analysis of γ-H2AX immunofluo-
rescence in cisplatin-treated cells. The γ-H2AX index was calculated by mul-
tiplying the number of γ-H2AX–positive 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 identified
using a standard X-gal staining protocol and manually quantified from rep-
resentative microscope images. Data represent the mean of six 40× high-
power fields from two independent samples for each experimental condi-
tion. (D) A cisplatin in vitro mutagenesis assay. Shown is the relative colony
and then selected for 6-thioguanine resistance. Each datapoint represents an
independently treated and selected experimental replicate.
Rev3 depletion promotes cisplatin-induced DNA damage but limits
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-
creasein mitotic index (Fig.4A) andanincrease in apoptosis (Fig.
4B) in treated REV3L-deficient 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-deficient transplants exhibited an enhanced response to
cisplatin relative to controls (Fig. 5). This finding was evidenced
at the level of individual tumors (Fig. 5 A–C), 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 l p
a l p
plants. (A) H&E (Upper) 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-KrasG12D;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.
Histological analysis of LSL-KrasG12D;p53−/−adenocarcinoma trans-
# CC3+ cells/HPF
# pH3+ cells/HPF
vec shRev3 vec shRev3
vec shRev3 vec shRev3
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 tumor mass by microCT.
P values were determined using two-tailed Student’s t tests.
Rev3 depletion sensitizes transplanted lung adenocarcinoma to cis-
Day 10 cisplatin
before cisp10d cisp
Day 10 cisplatin
Day 10 cisplatin
tumor volume (mm^3)
% change in lung volume
tive axial images of mouse lungs harboring transplanted lung adenocarci-
noma cells. The darker areas represent healthy, air-filled 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) Quantification of healthy lung
volumes from D. P values were determined using a Student’s 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.
Rev3 depletion promotes cisplatin efficacy in vivo. (A) Representa-
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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-
deficient transplants surviving nearly twice as long compared
with the control cohort (Fig. 5F).
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 fide 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 deficiencies, including
the use of poly(ADP ribose) polymerase inhibitors in BRCA1/2-
deficient tumor cells (32, 33), have emerged as a promising ap-
proach to target chemoresistant malignancies. Although it is un-
upon REV3L function, cell-based studies examining gliomas—
a highly chemoresistant malignancy—have 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-deficient, p53-deficient 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).
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 fibroblasts 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
Although specific 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 specific in-
hibitors targeting critical protein interactions in TLS polymerase
complexes may hold significant 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, efficient
mechanisms to introduce diverse genetic alterations into such
modelsare currently lacking.This lack isparticularly true fordrug
sensitization experiments, where all tumor cells may need to be
modified to see a therapeutic effect. Here, we have developed
a tumor transplant approach that allows for rapid ex vivo modifi-
cation of lung adenocarcinoma cells. Importantly, transplanted
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
an attractive setting in which to evaluate putative mediators of
chemotherapeutic response, but may also function as a platform
of sensitizing aggressive NSCLC to existing chemotherapies.
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/LTRmiR30-SV40-GFP (LMS) vector. Sequences
(5′–3′) 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 (0–15 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 identifica-
tion was purchased from USB Corporation.
RT-qPCR, Immunohistochemistry, and Immunofluorescence. 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 CO2asphyxiation and lungs were fixed overnight in 10% neutral-buff-
ered formalin. Lung lobes were separated and embedded in paraffin
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% H2O2for 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 immunofluorescence were grown and treated on
poly-L-lysine coated coverslips, fixed 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 instruments and deconvolution software.
In Vitro Viability Assays and FACS. For short-term viability assays, cells were
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 × 105lung
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 flow cytometry was performed using Becton-Dickinson FACScan
or MoFlo flow 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 fixed 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 incorporation buffer before FACS analysis and sub-
sequently analyzed using FloJo software.
Doles et al.PNAS Early Edition
| 5 of 6
Mutagenesis (hprt) Assay. Retrovirally transduced cells were initially cultured Download full-text
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
variantswithimpaired hprtfunction.Resultantcolonies were visualizedusing
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(∼5×104)were
intravenously injected into the tail vein of syngeneic C57BL6/Jx129-JAE male
recipient mice and monitored weekly using a GE Healthcare microCT imaging
injection. Images were acquired and processed usingGE 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
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