Blockading RAS signalling controls tumour growth
Supplementary Information
MATERIALS AND METHODS
Expression vectors
A vector for expression of oncogenic mutant H-RASG12V (pcDNA3-HRASV12) was
made by sub-cloning full-length human H-RASG12V cDNA into the EcoRI site of
pcDNA3 (Invitrogen). The iDab#6-memb expression vector (pEF-iDabVH6) was made
by sub-cloning the anti-RAS iDab#6-memb (Tanaka et al., 2003) into the SfiI and NotI
sites of pEF-FLAG-Memb (Tanaka and Rabbitts, 2003).
Transgenic mice
CC10-Cre and CC10-iDab#6-memb mice: A CC10 genomic transgenic cassette was
constructed by sub-cloning the NotI and blunt-ended BamHI digested fragment
comprising human growth hormone polyA signal from pLck-hGH plasmid (Wildin et al.,
1995) into the NotI and blunt-ended XhoI sites of pMG2 (Forster et al., 2005) to give
pMG2-pA. The HindIII fragment of Rat Clara cell 10kDa promoter from pCC10CAT2300 (Stripp et al., 1992) was sub-cloned into the HindIII site of pMG2-pA to give
pCC10-pA. The final CC10-Cre transgene was made by sub-cloning the blunt-ended
XbaI/XhoI fragment containing the Cre cDNA from pPGK-Cre (a gift from Kurt
Fellenberg) into the EcoRV site of pCC10-pA. The CC10-iDab#6-memb transgene was
made by PCR amplification of the anti-RAS iDab#6-memb with an N-terminal FLAGtag and a C-terminal farnesylation signal peptide from pGCiG-anti-RAS VH#6 (Tanaka
et al., 2007), cleaving the PCR fragment with BamHI and cloning into the BamHI site of
pCC10-pA. Transgene fragments (prepared by excision with SceI) were injected into
pronuclei of fertilised eggs and transgenic strains were developed. The lines with high
copy number of the transgene were selected for further use and called CC10-Cre and
CC10-iDab#6-memb.
Conditional K-RasV12 mice: The mice bearing the Cre-dependent oncogenic K-Ras
transgene (Lox-GFP-polyA-Lox K-Ras driven by the -actin promoter) were kindly
provided by Prof. A. Berns (Meuwissen et al., 2001).
Genotyping of the adult mice was performed by Southern filter hybridization of genomic
DNA prepared from mouse tissues including tail biopsies by general phenol/chloroform
methods. Ten µg DNA were digested to completion with EcoRI, separated on 0.8%
agarose gels and transferred to positive-charged nylon membranes after denaturation. The
membranes were hybridized with anti-RAS iDab#6-memb cDNA (for genotyping of the
CC10-iDab#6-memb allele), Cre cDNA (for genotyping CC10-Cre) or EGFP cDNA (for
genotyping the conditional transgenic K-RasV12) using randomly 32P-dCTP labeled
probes.
To confirm Cre recombinase activation in lung Clara cells of CC10-Cre
transgenic mice, the mouse line was crossed with the lacZ reporter mouse line ROSA26
(Soriano, 1999). After genotyping by filter hybridization, lungs of the mice heterozygous
for the two alleles (CC10-Cre; ROSA26) were extracted and stained with -galactosidase
by perfusing X-gal staining solution (0.4 mg/ml X-gal, 5 mM MgCl2, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide in PBS) through the trachea and immersing
the whole lung in excess X-gal solution overnight at 37 ˚C.
Expression of anti-RAS iDab#6-memb in lungs of CC10-iDab#6-memb transgenic
mice was confirmed by Western blotting. Lungs were removed from the founder mice
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Blockading RAS signalling controls tumour growth
and snap-frozen on dry ice. They were homogenized in NP-40 lysis buffer and protein
samples were immunoprecipitated with agarose-conjugated anti-FLAG monoclonal
antibody (anti-FLAG M2 affinity gel, Sigma), fractionated on 15% SDS-PAGE and
stained with the same anti-FLAG antibody (M2, sigma).
Tumour group mice were observed for between 50 and 350 days (or until
appearance of any adverse symptoms) and humanely sacrificed, followed by dissection
and macroscopic plus microscopic examination. Sections were made after fixation in
10% neutral buffered formaldehyde, embedded in paraffin and 4mm sections stained with
haematoxylin and eosin.
Tetracycline-inducible cell lines.
The iDab#6-memb conditional expression clone pTet0-iDab#6-memb-ires-GFP was
generated by sub-cloning the fragment containing anti-RAS iDab#6-memb with an inframe FLAG-tag at the N-terminus and membrane localization signal peptide at Cterminus, an internal ribosomal entry site (IRES) and GFP from pGC-IRES-iDab#6memb (Tanaka et al., 2007) into the SacII and BamHI sites of the plasmid pUHD10-3
(Gossen et al., 1995).
The human cancer cell lines HT1080, HCT116 and SW480 and their derivatives were
grown in DMEM with 10 % FCS and penicillin and streptomycin (P/S), the cell line
DLD1 grown in RPMI with 10% FCS and P/S. To establish cells with the tetracyclineinducible (TET-ON) system in which tetracycline/doxycycline can induce gene
expression in the cells, the parental cell lines were transfected with pUHD172-1neo
(Gossen et al., 1995) and selected with 1 mg/ml of G418 (Geneticin, Invitrogen).
Colonies obtained after selection were individually tested by transient transfection with
pTet0-iDab#6-memb-ires-GFP that allows GFP expression in a reverse tetracycline
transactivator (rtTA)-dependent fashion after treatment of cells with 1 g/ml doxycycline
24 hours after transfection. Analysis of fluorescence was by flow cytometry after an
additional 24 hrs.
One clone from each parental cell line (viz. HT1080-tetON, DLD1-tetON,
HCT116-tetON and SW480-tetON) were chosen to generate inducible cell lines for
further analysis. The pTet0-iDab#6-memb-ires-GFP construct were linearized with ScaI
and co-transfected into each of the TET-ON clones with linearized pPGK-hygro (te Riele
et al., 1990) at a 20 to 1 molar ratio. Single colonies were obtained by selection with 1
mg/ml G418 and 0.3 mg/ml hygromycin B (Calbiochem) for 1 week. The clones were
tested by growth with and without 1 g/ml doxycycline and flow cytometric detection of
green fluorescence after 48 hrs. Clones showing low background GFP expression in the
absence of doxycycline and the highest percentage of GFP-positivity in the presence of
doxycycline were selected. Doxycycline-dependent expression of iDab#6-memb in the
chosen clones (HT1080-tetON-iDabVH, DLD1-tetON-iDabVH, HCT116-tetONiDabVH and SW480-tetON-iDabVH) was confirmed by Western blot analysis with antiFLAG antibody (M2, Sigma).
To establish these lines with ubiquitous Firefly luciferase expression for
bioluminescence imaging, pEF-FLuc was generated by sub-cloning the fragment of
Firefly luciferase cDNA with a polyA signal into the NcoI and XbaI sites of
pEF/myc/cyto (Invitrogen). The plasmid were linearized with XmnI and co-transfected
into each TET-ON-iDab#6-memb cell together with pPGK-puro (Tucker et al., 1996) at a
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Blockading RAS signalling controls tumour growth
20 to 1 molar ratio. Single colonies were obtained by selection with 1 mg/ml G418, 0.3
mg/ml hygromycin B (Calbiochem) and 1-5 g/ml puromycin for about 1 week. The
luciferase expression in each clone was assayed with the Dual-luciferase assay (Promega)
according to Manufacture’s instructions and the clones HCT116-tetON-iDab#6-membluc and SW480-tetON-iDab#6-memb-luc were used for the mouse transplantation assays.
Animal studies
All animal studies including the transgenic mouse model, the xeno-transplant
tumourigenecity assay in immunodeficient mice and mouse imaging were performed
under a licence complying with Home Office Animals (Scientific Procedures) Act 1986
(UK) and approved by the University of Leeds, Leeds Institute of Molecular Medicine
Institutional Ethical Committee.
CC10-Cre and CC10-iDab#6-memb mice were generated and validated as described.
Conditional K-RasV12 mice were kindly provided by Prof. A. Berns (Meuwissen et al.,
2001). These mice were crossed with CC10-Cre transgenic mice to generate mice
carrying both alleles (conditional KRasV12; CC10-Cre). Mice with both transgenes were
also crossed to CC10-iDab#6-memb transgenic mice to generate mice heterozygous for
the three alleles (conditional KRasV12; CC10-Cre; CC10-iDab#6-memb).
Tumourigenecity assays using human cancer cell xeno-grafts in mice.
Cell lines carrying a tetracycline-responsive anti-RAS iDab#6-memb conditional gene
(described above) were used for xeno-grafts. 106 HCT116-tetON-iDab#6-memb-luc cells
were injected subcutaneously into each flank of 6 to 8 week-old CB17/Lcr-Prkdcscid/Crl
SCID mice (Charles River) or 106 SW480-tetON-iDab#6-memb-luc or parental SW480tetON cells were injected into each flank of 6 to 8 week-old CD-1 nude mice. The mice
were fed with normal diet and water until their subcutaneous tumour reached palpable
size (> 4 mm diameter, approximately 5 days after injection). The mice were then divided
two groups and one of which was supplied with Doxycyclin via drinking water (2 mg
Dox/ml in 10% black-currant juice) and the Dox Diet (200 mg/kg, BIO-serve).
Subcutaneous tumour growth was monitored by measuring tumour size twice a week
with callipers and by bioluminescence as described below. The assay was terminated
when the tumour sizes reached a maximum of 17 mm. After humane sacrifice, the mice
were dissected and pathological and biological examinations were performed.
Mouse tumour bioluminescence imaging (BLI).
Bioluminescence was measured in transplanted subcutaneous tumours non-invasively
using the IVIS imaging system (Xenogen Corp) following injection of the luciferase
substrate luciferin as described (Tanaka and Rabbitts, 2008). All of the images were
taken after intraperitoneal injection of luciferin (140 mg/kg body weight; Synchem).
During image acquisition, mice were sedated continuously by inhalation of 3%
isofluorane. Image analysis and bioluminescence quantification was performed using
Living Image software (Xenogen).
Protein analysis and kinase assays
NIH3T3 cells were grown in DMEM medium with 10% foetal calf serum (FCS). 24
hours before transfection, the cells were washed with PBS and DMEM without FCS was
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Blockading RAS signalling controls tumour growth
added for serum starvation. pcDNA3-HRASGV12 and pEF-iDab#6-memb were
transiently co-transfected into these serum-starved cells using Lipofectamine 2000
(Invitrogen) according to Manufacture’s instructions. Forty-eight hours after transfection,
the cells were harvested and Western blots were performed to detect specific proteins
described below.
Western blotting
For culture cells, the cells were harvested and lysed in 1 x SDS sample buffer (62.5mM
Tris-Cl pH 6.8 at 25 ˚C, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue).
For xeno-transplanted tumours, the tissues were removed and snap-frozen on dry ice,
before homogenization in 0.5ml NP-40 lysis buffer (50 mM Hepes (pH 7.5), 150 mM
NaCl, 1 mM EDTA, 10 mM MgCl2, 1% NP-40 and 10 % glycerol), followed by addition
of equal volume of 2x SDS-PAGE loading buffer. The prepared protein samples were
boiled and fractionated on 12 or 15% SDS-PAGE, transferred to PVDF membranes
(Millipore) and incubated with the appropriate antibodies including anti-ERK1/2, antiphospho ERK1/2 (Thr202/Tyr204), anti-Akt, anti-phospho Akt (Ser473) (all polyclonal
antibodies were from Cell Signaling Technology), anti-FLAG (M2, Sigma) or anti-pan
Ras (Ab-3, Oncogene Research). Antibodies were detected with the appropriate
Horseradish Peroxidase-linked secondary antibody and visualized with a
chemiluminescence (ECL) kit (Amersham).
Histology and Immunohistochemistry
Mouse organs were fixed in 10 % neutral buffered formaldehyde, embedded in paraffin
and 4 m sections stained with haematoxylin and eosin. For immunohistochemistry, 4
m thick slides were stained with anti-CC10 monoclonal antibody as follows. The tissue
sections were de-paraffinized and hydrated and antigen retrieval by microwave heating in
sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After
incubating with 3% hydrogen peroxide in PBS and blocking with normal rabbit serum,
the sections were stained with goat anti-CC10 monoclonal antibody (1: 100; T-18, SantaCruz) and biotinylated rabbit anti-goat (1:1000; DAKO) were used as the secondary
antibody. Visualization was achieved using streptavidin-coupled horse radish peroxidase
(HRP) (Vector) and diaminobenzidine as chromogen.
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