Supplementary Text
Cell lines These cell lines were stably reconstituted with a retroviral vector encoding the wildtype (WT) pten cDNA or empty vector to prepare U87MG-PTEN and U373MG-PTEN cell
lines. Briefly, WT pten cDNA was subcloned into a retroviral expression vector containing a
muristirone-inducible promoter or into the pBABEpuro vector for expression in U87MG or
U373MG cells [1]. In addition, U87MG clones were engineered to contain VgEcR and retinoid
X receptor nuclear receptors/transcription factors that confer muristirone-responsive expression
of PTEN when cloned into a retroviral vector, which contains a muristirone responsive promoter,
as described previously [2, 3]. Stable clones of U87MG cells, which would allow for expression
of WT PTEN or mutants of PTEN (G129E, lipid phosphatase dead or G129R, catalytically dead)
were established by subcloning the PTEN cDNA into the muristirone-inducible retroviral vector,
which contains a puromycin selectable marker as described elsewhere.
Generation of GFAPV12Ras cell line. GFAPV12Ras Tg mice develop high grade malignant
gliomas in vivo [4]. In this animal model described by Ding et al, 100% of mice develop glial
tumors. The tumors are evident from abnormal neurological function of the mice necessitating
euthanasia or by routine histopathological analysis of neurologically normal mice. Glial tumors
ranging from low grade to high grade gliomas were described with significant mouse strain
effects noted. We bred these mice with PTENfl/fl animals obtained from Jackson laboratory. We
isolated normal astrocytes and tumor derived glioma cells from PTEN deficient mice. The
GFAPV12Ras Tg mice have been successfully bred with PTENfl/fl mice to generate or PTENfl/fl
x GFAP V12 Ras glioma cell line. The GFAP V12 Ras PTENfl/fl glioma cells are then transduced
with a retroviral vector, pMIEG-cre which contains cre recombinase and an IRES directed EGFP
or empty pMIEG control vector. This allowed us to delete one or both alleles of PTEN and
develop isogenic PTEN -/-, PTEN+/- or PTEN+/+ GFAPV12 Tg positive glioma cell lines,
termed GFAP V12 Ras PTENfl/fl cre+, GFAP V12 Ras PTENfl/wtcre+ or GFAP V12 Ras
PTENfl/fl, respectively.
Proliferation assays.
U373MG-Null and U373MG-PTEN reconstituted, U87MG-Null and
U87MG-PTEN cells were incubated for 48 hours with 2ME2 in medium containing 10% FBS
(37o C, 5% CO2). This was followed by the addition of 10 µl of Cell Proliferation Reagent WST1 (Roche Applied Science, Indianapolis, IN) and incubation for one hour. Cell viability was
expressed as a mean percentage + the standard deviation of absorbance. All experiments were
performed in triplicate. For combined dose effect analysis and analysis of synergism,
isobologram method was used. This method defined the doses of 2ME2 and LY294002 used to
produce a single agent effect, the IC50 , and these doses were plotted on the x and y axes. The
line connecting these two points is the line of additivity, and the response of the two drugs used
in combination at their IC50 levels were added to this plot. This combined effect is defined as
synergistic, additive or antagonistic when the point lies below, on or above the line of additivity,
respectively.
CD31 immunohistochemistry. At the end of the efficacy experiments, tumors were harvested
and placed in optimum cutting temperature blocks for frozen section analysis or fixed in 10%
buffered formalin and/or processed into paraffin. Sections of tumor tissue at 4-μm thickness were
stained with rat anti-mouse CD31 antibody for detection of the murine tumor
microvasculature.Ten fields for each tumor were counted in a blinded manner for CD31-positive
vascular staining at 20x. The average number of microvessels was determined for five tumors
per experimental group and analyzed by the Student’s t test.
Orthotopic GBM xenograft survival studies: U373MG-Null and U373MG-PTEN Human
GBM cells were stereotaxically implanted (Model 963,David Kopf Instruments,Tujunga, CA)
into the right frontal lobe of 6-8 week old nude (nu/nu) mice (1 x 106 tumor cells in .6 µl PBS).
Three days later animals were randomized and treated with 200 mg/kg daily dose of 2ME2
intraperitonelly. Survival analysis was done using Kaplan-Meir graph. The experiments were
repeated three times with n=15 mice in each experimental group.
1.
2.
3.
4.
Wen, S., et al., PTEN controls tumor-induced angiogenesis. PNAS, 2001. 98(8): p. 46224627.
Su, J.D., et al., PTEN and phosphatidylinositol 3'-kinase inhibitors up-regulate p53 and
block tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial
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Wen, S., et al., PTEN controls tumor-induced angiogenesis. Proc Natl Acad Sci U S A,
2001. 98(8): p. 4622-7.
Ding, H., et al., Astrocyte-specific expression of activated p21-ras results in malignant
astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res,
2001. 61(9): p. 3826-36.
Legends to Supplementary Figures:
Fig. S1. Effect of 2ME2 on proliferation. Parental U373MG-Null and PTEN reconstituted
U373MG-PTEN cell lines (A) as well as U87MG-Null and U87MG-PTEN cell lines (B) were
treated with different concentrations of 2 ME2 and used for WST assay.
Fig. S2. 2ME2 induced apoptosis is not altered by PTEN status. U87MG-Null and U87MGPTEN cell lysates were probed with a cleaved caspase 3 specific antibody that detects
endogenous levels of the large fragment (17/19 kD) of activated caspase3. β-actin was used as a
loading control. 2ME2 was shown to markedly induce apoptosis after 48 hours in both PTENnull and PTEN-positive U87 tumor lines. No major differences were observed between the two
cell lines in terms of apoptosis induction by 2ME2 in vitro.