NVP-AEW541 does not affect the interaction between JCV T-antigen, IRS-1, pRb
and p53. Since the NVP-AEW541 treatment of JCV T-antigen positive BsB8 cells
resulted in growth retardation and the inhibition of IGF-I-mediated phosphorylation
events, we have asked whether NVP-AEW541 could affect the binding between JCV Tantigen and its major cellular targets, pRb, p53 and IRS-1. Results in Figure 4B clearly
demonstrate that in BsB8 cells these anticipated protein–protein interactions were not
affected by the NVP-AEW541 treatment or by the presence or absence of IGF-I
stimulation. This suggests that although JCV T-antigen –mediated inactivation of pRb,
p53 (Bollag et al., 2000; Wang et al., 2004) and nuclear translocation of IRS-1
contributed to the transformation of BsB8 cells (Lassak et al., 2002), these molecular
events seem to be independent from NVP-AEW541–mediated growth retardation
observed in this JCV T-antigen dependent experimental model of medulloblastoma.
Figure 4B. Lack of an effect of NVP-AEW541 treatment on interactions between JCV
T-antigen and its binding proteins, IRS-1, pRb and p53. Cell culture conditions are the
same as described for Fig.3. Total protein extracts were isolated from the quiescent [cells
exposed to serum-free medium (SFM) for 48 hrs] or IGF-I stimulated cells in the
presence (+) or absence (-) of 1M NVP-AEW541. The aliquots of corresponding protein
extracts (500g) were immunoprecipitated (IP) with anti-IRS-1, anti-pRb and anti-p53
antibodies and Western blots (W) were developed with anti-T-antigen antibody.
Supplemental Methods
Cell Culture: BsB8 and Daoy were maintained as monolayer cultures in Dulbecco’s
Modified Eagle Medium (DMEM) (GIBCO-BRL, Grand Island, NY) containing 10%
fetal bovine serum (FBS), at 37oC in a 9.6% CO2 atmosphere. D384 were cultured in
suspension, in the Eagle’s MEM supplemented with non-essential amino acids (GIBCOBRL, Grand Island, NY), 2 mM L-glutamine, 1mM sodium pyruvate, and 20% FBS. The
cells were made quiescent by 48 hours incubation in serum-free medium (SFM) (DMEM
supplemented with 0.1% bovine serum albumin). Cellular growth and signaling responses
were tested by stimulating quiescent cells with 50ng/ml of recombinant IGF-I (GIBCOBRL, Grand Island, NY). Cell growth and survival after NVP-AWE-541 (Novartis
Pharma AG, Basel, Switzerland) treatment were tested in serum containing growth
media. In the experiments in which IGF-I –mediated phosphorylation events were tested,
quiescent cells were pre-incubated with 1M NVP-AWE-541 for 0, 20 min, 6 hrs, 24 hrs
and 48 hrs, and the IGF-1 stimulation was applied for 30 minutes.
Cell Growth and Survival in Suspension: Quiescent BsB8 cells were detached from a
culture dish with 0.02% EDTA (disodium ethylenediamine tetraacetate), and seeded on
dishes coated with poly(2-hydroxyethyle methacrylate) [poly(HEMA)] (Aldrich,
Milwaukee, WI), prepared according to the methodology previously described (Reiss et
al. 1998a). Cells were seeded in 10% FBS at concentration of 1 x 105 cells/35mm dish,
and were treated either with NVP-AWE-541, or were left untreated. In some experiments
cell cultures were additionally treated with 20mM lithium chloride (J.T. Baker Inc,
Phillipsburg, NJ); or 0.1–4.0mM sodium nitroprusside dihydrate (Lausen, Switserland).
At 48 and 72 hors time points the cells were collected, dissociated with 0.25% trypsin,
and counted in a Bright-line Hemocytometer in the presence of Trypan blue. Results
represent an average of at least three experiments, and are expressed as percent increase
or decrease in cell number over T0, which represents an initial cell number after plating,
and normalized by plating efficiency.
Western blot and Immunoprecipitation: To evaluate phosphorylation levels of the
selected IGF-IR signaling molecules, monolayer cultures were lysed for 5 minutes on ice
with 400 l of lysis buffer A [50 mM HEPES; pH 7.5; 150 mM NaCl; 1.5 mM MgCl 2; 1
mM EGTA; 10% glycerol; 1% Triton X-100; 1mM phenylmethylsulfonyl fluoride
(PMSF); 0.2 mM Na-orthovanadate and 10g/ml aprotinin]. Total protein extract (50g)
was separated on a 4-15% gradient SDS-PAGE (BioRad, Hercules, CA). The following
primary antibodies were utilized: anti-pY1316 IGF-IR rabbit polyclonal (K2895, kindly
provided by Dr. Olaf Mundigl, Roche Diagnostics, Penzberg, Germany); anti-pY612
IRS-1 rabbit polyclonal (BioSource, Camarillo, CA); anti- pS473 Akt, anti-pT202/Y204
-pS21/9 GSK3/
Beverly, MA. To monitor loading conditions we utilized anti- IGF-IR (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-IRS-1 (Upstate USA Inc., Charlottesville, VA),
anti-GSK3, anti-Akt and anti-Erks antibodies (Cell Signaling, Danvers, MA).
IRS-1, p53 and pRb were immunoprecipitated from 500 g of pre-cleared protein
extract with rabbit anti-IRS-1 antibody (Upstate Biotechnology), rabbit anti-p53 antibody
(Chemicon International Inc., Temecula, CA), and rabbit anti-pRb antibody (EMD
Biosciences Inc. San Deigo CA) in the presence of agarose conjugated protein A
(Calbiochem, San Diego, CA). Corresponding blots were developed with anti-T-antigen
antibody, which recognizes both SV40 and JCV T-antigen (Lassak et al., 2002).
Immunocytofluorescence: For immunolabeling, monolayer cultures of BsB8 cells were
fixed and permeabilized with the buffer containing 0.02% Triton X-100 and 4%
formaldehyde in PBS. Fixed cells were washed 3X in PBS and blocked in 1% BSA for 30
min at 370C. Immunocyrofluorescent detection of tyrosine phosphorylated IGF-IR in
quiescent Bsb8 cells (48 hrs in SFM), and in BsB8 cells stimulated with IGF-I in the
presence or absence of NVP-AEW541. Preincubation was performed in the presence of
anti-pY1316 IGF-IR rabbit polyclonal antibody (K2895, kindly provided by Roche) and
FITC conjugated anti-rabbit secondary antibody. The images were visualized with an
inverted Nikon Eclipse TE300 fluorescence microscope equipped with a Retiga 1300
camera (QImaging). Series of three-dimensional images of each individual picture were
deconvoluted to one two-dimensional picture and resolved by adjusting the signal cut-off
to near maximal intensity to increase resolution.