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Supporting Information
for
A new oncolytic adenoviral vector carrying dual tumor suppressor genes
shows potent antitumor effect
Xin-Ran Liu1,2, Ying Cai1, Xin Cao1, Rui-Cheng Wei1, Hui-Ling Li1, Xiu-Mei Zhou3,
Kang-Jian Zhang1, Shuai Wu1, Qi-Jun Qian3,4, Biao Cheng2, Kun Huang2*, Xin-Yuan Liu1, 3*
1
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;
2
TongJi School of Pharmacy, Huazhong University of Science & Technology, Wuhan 430030,
China;
3
Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou
310018, China
4
Eastern Heptobiliary Hospital, Second Military Medical University, Shanghai 200438,
China.
Corresponding authors:
Xin-Yuan Liu
Address: 320 Yue Yang Road Shanghai, 200031 P.R.China
Tel: +86 21 54921126; Fax: +86 21 54921126
E-mail: [email protected]
Kun Huang
Address: 13 Hang Kong Road Wuhan, 430030 P.R.China
Tel: +86 27 83691499; Fax: +86 27 83691499
E-mail: [email protected]
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The supporting material provides detailed information on cell lines and culture conditions
used in this study; construction of the adenoviral plasmids; antibodies used for Western blot;
and the characterization results of ZD55SP/E1A-IL-24-shMPP1.
Cell lines and culture conditions
SW620 (human colorectal cancer cell line), HeLa (human cervical cancer cell line), A549
(human lung adenocarcinoma cell line), T24 (human urinary bladder carcinoma cell line),
NHLF-1 (human normal lung fibroblasts cell line), MRC-5 (human embryonic fibroblasts
cell line) and BEAS-2B (human normal bronchial epithelial cell line) cells were purchased
from ATCC (Rockville, MD); BEL-7404 (human hepatocarcinoma cell line), SCaBER
(human squamous cell carcinoma cell line) and L-02 (human normal hepatocytes cell line)
were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China). HEK293 (human embryonic kidney cell line) was obtained from
Microbix Biosystems Inc. (Toronto, Ontario, Canada). HEK293 and MRC-5 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY)
supplemented with 10% heat-inactivated FBS. L-02 and A549 were grown in RPMI 1640
(GIBCO BRL, Grand Island, NY) supplemented with 4% FBS (GIBCO BRL, Grand Island,
NY). Other cell lines were cultured in DMEM supplemented with 4% FBS. All cells were
incubated at 37ºC in a humidified atmosphere with 5% CO2.
Construction of the adenoviral plasmids
Plasmids pZXC2 and pZDΔ55 were digested with XbaI and BamHI, and we substituted the
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1170-bp fragment of pZXC2 for the homologous sequence of pZDΔ55 to construct
pXC2-Δ55. A 269-bp fragment of the 5’-UTR of the human survivin gene was amplified by
PCR from genomic DNA of HEK293 cells as the template, using primers as follows:
Forward: 5’-ATG AAT TCC GCG TTC TTT GAA AGC AG-3’
Reverse: 5’-AAT CTC GAG TGC CGC CGC CGC CAC CT-3’.
The PCR product was cloned into a pTG19-T vector to construct the plasmid pTG19-SP.
The plasmid pTG19-SP was digested with XhoI and SalI, and the 280-bp fragment of the
survivin promoter was cloned into the SalI site of pXC2-Δ55. After confirming that the
survivin promoter was in the right direction, the plasmid was named pSP-Δ55. Two sites on
plasmid pSP-Δ55 were used for carrying foreign DNA of expression cassettes.
To synthesize siRNAs in mammalian cells, we used a previously constructed RNAi system.
Based on the four selected siRNA targeting sequences on the coding regions of the human
MPHOSPH1 gene (MPP1), eight single-strand oligonucleotides were designed and coded as
follows:
shMPP1-4873 Forward: 5’-GGT TGT ACC ACA CCA GTG ACA GTT AAG ATG AAG
ACA TCT TAA CTG TCA CTG GTG TGG TAC AAC CCT TTT TG-3’
shMPP1-4873 Reverse: 5’-AAT TCA AAA AGG GTT GTA CCA CAC CAG TGA CAG TTA
AGA TGT CTT CAT CTT AAC TGT CAC TGG TGT GGT ACA ACC-3’
shMPP1-4747 Forward: 5’-GGA TCT GTA GTC CTT GAC TCT TGT GAA GTG AAG
ACA CTT CAC AAG AGT CAA GGA CTA CAG ATC CCT TTT TG-3’
shMPP1-4747 Reverse: 5’-AAT TCA AAA AGG GAT CTG TAG TCC TTG ACT CTT GTG
AAG TGT CTT CAC TTC ACA AGA GTC AAG GAC TAC AGA TCC-3’
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shMPP1-4508 Forward: 5’-GAG AAG AAC GAG ATC AAC TGG TTG CAG CTG AAG
ACA GCT GCA ACC AGT TGA TCT CGT TCT TCT CCT TTT TG-3’
shMPP1-4508 Reverse: 5’-AAT TCA AAA AGG AGA AGA ACG AGA TCA ACT GGT
TGC AGC TGT CTT CAG CTG CAA CCA GTT GAT CTC GTT CTT CTC-3’
shMPP1-21nt Forward: 5’-GTG AAG AAG TGC GAC CGA AGA AGA CTT CGG TCG
CAC TTC TTC ACC TTT TTG-3’
shMPP1-21nt Reverse: 5’-AAT TCA AAA AGG TGA AGA AGT GCG ACC GAA GTC TTC
TTC GGT CGC ACT TCT TCA C-3’.
These eight single-strand oligonucleotides were annealed to generate 4 double-stranded
oligonucleotides and then cloned into the ApaI (blunted) and EcoRI sites of pBS/U6.
pBS/U6-shMPP1 was digested with BamHI, and the 0.4-kb fragment comprising the
U6-shMPP1 expression cassette was then cloned into the BglII site of pSP-Δ55. The 1.2-kb
fragment comprising the CMV/IL-24 expression cassette amplified by PCR was digested
with XhoI and then cloned into the XhoI site of pSP-shMPP1-Δ55 to generate the adenoviral
transfer plasmid pSP-IL-24-shMPP1-Δ55. Primers were as follows, with pCA13-IL-24 as the
template:
Forward: 5’-AGT CCT CGA GAA GAT CTA ATT CCC TGG-3’
Reverse: 5’-ACT GCT CGA GCC CAG ATC TTC GAT GCT-3’.
The same procedure was used for the construction of plasmids pSP-shMPP1-Δ55,
pSP-IL-24-Δ55 and pSP-EGFP-Δ55. The DNA sequences of all constructs were confirmed
by restriction enzyme digestion, PCR and DNA sequencing (data not shown).
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Generation, identification, production, purification and titration of the oncolytic
adenovirus
1 μg transfer plasmid together with 0.5 μg packaging plasmid were cotransfected into
the HEK293 cells by Effectene Transfection Reagent (Qiagen, Valencia, CA). When the
plaque appeared, each OV was isolated, and the viral genomic DNA was extracted by the
QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) and identified by PCR with primers as
follows:
Forward: 5’-GTG TTA CTC ATA GCG CGT AAT-3’
Reverse: 5’-CAC CGC CAA CAT TAC AGA GT-3’
Forward: 5’-AGA GCC CAT GGA ACC CGA GA-3’
Reverse: 5’-CAT CGT ACC TCA GCA CCT TCC A -3’.
After large scale of production in HEK293 cells, rec-Ads were concentrated and purified
by cesium chloride gradient ultracentrifugation at 20,000 rpm for 2 hours at 20°C. The
rec-Ads were collected and dialyzed overnight. The titration of Ads was determined by a
standard plaque assay (PFU/ml).
Western blot assay
Antibodies for actin, E1A, Bax, Bcl-xL, Caspase-8, Caspase-9, Caspase-3, poly
ADP-ribose polymerase (PARP), IL-24 and cyclin B1 were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA); antibody for MPHOSPH1 was obtained from Abnova
Corporation (Taipei, Taiwan); antibody for Mad2L1 was obtained from ProteinTech Group
Inc. (Chicago, IL); antibody for E1B55KD was obtained from Siemens Oncogene Science,
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Inc. (Cambridge, MA); antibody for p53 was obtained from Epitomics, Inc. (Burlingame,
CA); antibodies for PUMA and BTG2 were obtained from GeneTex Inc. (Irvine, CA); and
antibodies for p27 and p21 were obtained from Cell Signaling Technology, Inc. (Beverly,
MA). Secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP
biotin nick end labeling assays.
10-μm-thick sections were cut from the frozen pituitary blocks with a Leica CM 3050S
freezing microtome, thaw-mounted on gelatin-coated microscope slides and air-dried. The
frozen sections were washed with PBS twice, and endogenous peroxidase activity was
eliminated by 30-min incubation in 1% H2O2 and 0.4% Triton X-100 in PBS. Then, the
sections were blocked with blocking serum and incubated with mouse anti-MPP1 polyclonal
antibody (1:200 dilution; Abnova Corporation, Taipei, Taiwan) or goat anti-IL-24 polyclonal
antibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room
temperature. The mouse or goat ABC Staining System (Santa Cruz Biotechnology, Santa
Cruz, CA) were used with hematoxylin as counterstain. Apoptotic cells in tumor tissue
sections were assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL) staining with a TACS TdT Kit In Situ Apoptosis Detection Kit (R&D,
Minneapolis, MN). All sections were visualized with a Nikon Eclipse 80i microscope (Nikon,
Tokyo, Japan).
Plasmids expressing shMPP1.
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We constructed four plasmids encoding U6 promoter-driven shRNAs against different
target sites within the MPHOSPH1 transcript, and transfected these vectors into HEK293
cells. As shown in Fig. S1, we chose the pBS/U6-M21 plasmid vector, which exhibits the
strongest interfering effect, to construct the Ad-mediated RNA silencing system.
Figure S1
Figure S1. RNA interference effects of four constructed plasmids expressing shMPP1
were valued by RT-PCR. The knockdown effect of four constructed plasmids encoding the
U6 promoter-driven shMPP1 on MPHOSPH1 in HEK293 cells was examined by qPCR at 48
hours after transfection. GAPDH served as an internal control. Data are presented as means ±
SD (bars) (n=3, **P < 0.01, ***P < 0.001).
Identification of the rec-Ads
To investigate the expression of E1A under the control of the survivin promoter, human
normal and cancer cell lines were infected with ZD55SP/E1A, ZD55, or Ad.WT. Results
from western blots indicated that the expression levels of E1A in the tumor cells (SW620,
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A549, BEL-7404) infected with ZD55SP/E1A were much higher than those in normal cells
(MRC-5 and BEAS-2B); in SW620, ZD55SP/E1A showed much stronger expression of E1A
than ZD55 did, similar to Ad.WT (Fig. S2A). These results confirm that the expression of
E1A of the OV ZD55SP/E1A was strictly controlled by the survivin promoter.
The expression level of E1B55KD was also examined in SW620 cells infected with
different adenovirus, and we observed no expression of E1B55KD in the cells infected with
ZD55SP/E1A,
ZD55SP/E1A-IL-24,
ZD55SP/E1A-shMPP1
and
ZD55SP/E1A-IL-24-shMPP1 against Ad.WT (Fig. S2B), showing no Ad.WT contamination
in the rec-Ads. We further investigated whether the two expression cassettes on the Ad
function correctly. The western blot analysis detected a strong expression of foreign IL-24 as
well as a marked downregulation of MPHOSPH1 in SW620 cells infected with
ZD55SP/E1A-IL-24-shMPP1 (Fig. S2C). The RT-PCR analysis also demonstrated that
infection of ZD55SP/E1A-IL-24-shMPP1 and ZD55SP/E1A-shMPP1 resulted in a
significant downregulation of MPHOSPH1 both in A549 and SW620 cells (Fig. S2D). These
results suggest that ZD55SP/E1A-IL-24-shMPP1 successfully express IL-24 and deliver
shRNAs in vitro.
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Figure S2
Figure S2. Identification of the rec-Ads. (A) E1A expression level was examined by
western blot. Left: Normal and tumor cells were infected with ZD55SP/E1A; middle: SW620
cells were infected with ZD55 and ZD55SP/E1A; right: HeLa cells (left 3 lanes) and SW620
cells (right 3 lanes) were infected with ZD55SP/E1A and Ad.WT. (B) E1B55KD expression
levels in SW620 cells treated with different oncolytic Ads were examined by western blot.
Lane 1, mock; lane 2, Ad.WT; lane 3, ZD55SP/E1A; lane 4, ZD55SP/E1A-IL-24; lane 5,
ZD55SP/E1A-shMPP1; lane 6, ZD55SP/E1A-IL-24-shMPP1. (C) IL-24 and MPHOSPH1
expression levels in SW620 cells treated with different oncolytic Ads were examined by
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western blot. Lane 1, mock; lane 2, ZD55SP/E1A; lane 3, ZD55SP/E1A-IL-24; lane 4,
ZD55SP/E1A-IL-24-shMPP1. (B, C) Western blots results carried out at 48 hours after
infection at MOI of 1. (D) Quantification of the MPHOSPH1 transcript level by RT-PCR in
A549 cells (left) and SW620 cells (right) 48 hours after infection with ZD55SP/E1A,
ZD55SP/E1A-shMPP1 and ZD55SP/E1A-IL-24-shMPP1 at MOI=1. GAPDH was used as an
internal control. Data are presented as means ± SD (bars) (n=3, **P < 0.01, ***P < 0.001)
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