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`. . Proceedings of the National Academy of Sciences
`\...:~""'·"'"'
`of the United States of America
`.
`DOES NOT LEA VE
`THE LIBRARY
`
`April 10, 2001 I vol. 98 I no. 8 I pp. 4277-4816 I www.pnas.org
`
`univ. of Minn.
`Bio~Medical
`Library
`
`Where the poly(A)-binding protein binds translation factors
`
`Ancient carbon cycle tells of past biological diversity
`
`New World patterns of human migration
`
`Retrovirus linked with schizophrenia
`
`Pharmaceutical intervention for weight control
`
`Breckenridge Exhibit 1096
`Wen
`Page 001
`
`

`

`4581 Selective ablation of retinoid X receptor a in
`~ hepatocytes impairs their lifespan and
`regenerative capacity
`Takeshi Imai, Ming Jiang, Philippe· Kastner, Pierre Cbambon,
`and Daniel Metzger
`
`IMMUNOLOGY
`
`4587 Localization of co4+ T cell epitope hotspots to exposed
`~ strands of HIV envelope glycoprotein suggests
`structural influences on antigen processing
`Sherri Surman, Timothy D. Lockey, Kar en S. Slobod,
`Bart Jones, Janice M. Riberdy, Stephe n W. While,
`Peter C. Doherty, and Julia L. Hurw itz
`4593 lmmucillin H, a powerful transition-state analog
`~ inhibitor of purine nucleoside phosphorylase,
`selectively inhibits human T lymphocytes
`Greg A. Kicska, Li Long, H eidi Ha rig, Craig Fairchild,
`Peter C. Tyler, Richard H. Furneaux, Vern L. Schramm
`and Howard L. Kaufman
`
`4599 Role of MEKK2-MEK5 in the regulation of TNF-a gene
`~ expression and MEKK2-MKK7 in the activation of
`c-Jun N-terminal kinase in mast cells
`Kosuke Chayama, Philip J. Papst, Timothy P. Garrington,
`Joanne C. Pratt, Tamotsu Ishizuka, Saipbone Webb,
`Soula Ganiatsas, Leonard I. Zon, Weiyong Sun,
`Gary L. Johnson, and E rwin W. Gelfand
`
`MEDICAL SCIENCES
`
`4605 Comparative evaluation of the antitumor activity of
`~ antiangiogenic proteins delivered by gene transfer
`Calvin J. Kuo, Filip Farnebo, Evan Y. Yu,
`Rolf Christofferson, Rebecca A. ·swearingen, Robert Carter,
`Horst A. von Recum, Jenny Yuan, Junne Kamihara,
`Evelyn Flynn, Robert D'Amato, Judah Folkman,
`and Richard C. Mulligan
`4611 An important function of Nrf2 in combating oxidative
`~ stress: Detoxification of acetaminophen
`Kaimin Chan, Xiao-Dong Han, and Yuet Wai Kan
`4617 Correlation of breath ammonia with blood urea
`nitrogen and creatinine during hemodialysis
`L. R. Narasimhan, William Goodman, and C. Kumar N. Patel
`
`4622 PTEN controls tumor-induced angiogenesis
`~ Shenghua Wen, Javor Stolarov, Michael P. Myers,
`Jing Dong Su, Michael H. Wigler, Nicholas K. Tonks,
`and Donald L. Durden
`4628 Role of tumor-host interactions in interstitial diffusion
`~ of macromolecules: Cranial vs. subcutaneous tumors
`A lain Pluen, Yves Boucher, Saroja Ra manujan,
`Trevor D. McKee, Takeshi Gohongi, Emmanuelle di Tomaso,
`Edward B. Brown, Yotaro Izumi, Robert B. Campbell,
`David A. Berk, and R akesh K. Jain
`4634 Retroviral RNA identified in the cerebrospinal fluids and
`brains of individuals with schizophrenia
`Hllkan Karlsson, Silke Bachmann, Johannes Schroder,
`Justin McArthur, E. Fuller Torrey, and Robert H. Yolken
`-t See commentary on page 4293
`4640 A phosphatidylinositol 3-kinase/ Akt/ mTOR pathway
`~ mediates and PTEN antagonizes tumor necrosis factor
`inhibition of insulin signaling through insulin
`receptor substrate-1
`Osman Nidai Ozes, H akan Akca, Lindsey D. Mayo,
`Jason A. Gustin, Tomohiko Maehama,
`Jack E. Dixon, and David B. Donner
`
`vi
`
`I www.pnas.org
`
`4646 Antisense-mediated depletion of p300 in human cells
`leads to premature G, exit and up-regulation of c-MYC
`Sivanagarani Kolli, Ann Marie Buchmann, Justin Williams,
`Sigmund Weitzman, and Bayar Thimmapaya
`
`4652 Ciliary neurotrophic factor activates leptin-like
`·~ pathways and reduces body fat, without cachexia or
`rebound weight gain, even in leptin-resistant obesity
`P. D. Lambert, K. D. A nderson, M. W. Sleeman, V. Wong,
`J. Tan, A. Hijarunguru, T. L. Corcoran, J. D. Murray,
`K. E. T habet, G.D. Yancopoulos, and S. J. Wiegand
`-t See commentary 011 page 4279
`
`MICROBIOLOGY
`
`4658 Complete genome sequence of an M1 strain of
`Streptococcus pyogenes
`Joseph J. Ferretti, William M. McShan, Dragana Ajdic,
`Dragutin J. Savic, Gorana Savic, Kevin Lyon,
`Charles Primeaux, Steven Sezate, Alexander N. Suvorov,
`Steve Kenton, Hong Shing Lai, Shao Ping Lin, Yudong Qian,
`Hong Gui J ia, Fares Z. Najar, Q un Ren, Hua Zhu, Lin Song,
`Jim White, Xiling Yuan, Sandra W. Clifton, Bruce A. Roe,
`and Robert McLaughlin
`
`4664 Purification and characterization of an autoregulatory
`~ substance capable of regulating the morphological
`transition in Candida albicans
`Ki-Bong Oh, Hiroshi Miyazawa, Tosbimichi Naito,
`and Hideaki Matsuoka
`4669 Polymerization of a single protein of the pathogen
`~ Yersinia enterocofitica into needles punctures
`eukaryotic cells
`Egbert Hoiczyk and Gunter Blobel
`4675 Epstein-Barr virus latent-infection membrane proteins
`are palmitoylated and raft-associated: Protein 1
`binds to the cytoskeleton through TNF receptor
`cytoplasmic factors
`Masaya Higuchi, Kenneth M. Izumi, and Elliott Kieff
`4681 Proteomic analysis of the bacterial cell cycle
`~ Bjorn Griinenfelder, Gabriele Rummel, J iri Vohradsky,
`Daniel Roder, Hanno Langen, and Urs Jena!
`
`NEUROBlOLOGY
`
`4687 Mechanisms of migraine aura revealed by functional
`~ MRI in human visual cortex
`Nouchine Hadjikhani, Margarita Sanchez de! Rio, Ona Wu,
`Denis Schwartz, Dick Bakker, Bruce Fischl,
`Kenneth K. Kwong, F. Michael Cutrer, Bruce R. Rosen,
`Roger B. H. Toolell, A. Gregory Sorensen,
`and Michael A. Moskowitz
`4693 Rethinking the role of phosducin: Light-regulated
`~ binding of phosducin to 14-3-3 in rod inner segments
`Koichi Nakano, Jing Chen, George E. Tarr, Tatsuro Yoshida,
`Julia M. F lynn, and Mark W. Bitensky
`4699 Allosteric modulation of Ca2+ channels by G proteins,
`voltage-dependent facilitation, protein kinase C, and
`CavfJ subunits
`Stefan Herlitze, Huijun Zhong, Todd Scheuer,
`and William A. Catterall
`4705 Control of gating mode by a single amino acid residue
`in transmembrane segment 153 of the N-type
`Ca2 + channel
`Huijun Zhong, Bin Li, Todd Scheuer,
`and William A. Catterall
`
`4710 Net
`sut
`the
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`So1
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`471 6 Lac
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`
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`Wen
`Page 002
`
`

`

`PTEN controls tumor-induced angiogenesis
`
`Shenghua Wen*, Javor Stolarov†, Michael P. Myers†, Jing Dong Su*, Michael H. Wigler†, Nicholas K. Tonks†,
`and Donald L. Durden*‡
`
`*Section of HematologyyOncology, Department of Pediatrics, Herman B Wells Center for Pediatric Research, Department of Biochemistry and Molecular
`Biology, Indiana School of Medicine, Indianapolis, IN 46202; and †Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724
`
`Contributed by Michael H. Wigler, February 7, 2001
`
`Mutations of the tumor suppressor PTEN, a phosphatase with
`specificity for 3-phosphorylated inositol phospholipids, accom-
`pany progression of brain tumors from benign to the most malig-
`nant forms. Tumor progression, particularly in aggressive and
`malignant tumors, is associated with the induction of angiogene-
`sis, a process termed the angiogenic switch. Therefore, we tested
`whether PTEN regulates tumor progression by modulating angio-
`genesis. U87MG glioma cells stably reconstituted with PTEN cDNA
`were tested for growth in a nude mouse orthotopic brain tumor
`model. We observed that the reconstitution of wild-type PTEN had
`no effect on in vitro proliferation but dramatically decreased tumor
`growth in vivo and prolonged survival in mice implanted intracra-
`nially with these tumor cells. PTEN reconstitution diminished
`phosphorylation of AKT within the PTEN-reconstituted tumor,
`induced thrombospondin 1 expression, and suppressed angiogenic
`activity. These effects were not observed in tumors reconstituted
`with a lipid phosphatase inactive G129E mutant of PTEN, a result
`that provides evidence that the lipid phosphatase activity of PTEN
`regulates the angiogenic response in vivo. These data provide
`evidence that PTEN regulates tumor-induced angiogenesis and the
`progression of gliomas to a malignant phenotype via the regula-
`tion of phosphoinositide-dependent signals.
`
`The reversible phosphorylation of proteins and lipids is critical
`
`to the control of signal transduction in mammalian cells and
`is regulated by kinases and phosphatases (1). The product of the
`tumor suppressor gene PTENyMMAC (hereafter termed
`PTEN) was identified as a dual specificity phosphatase and has
`been shown to dephosphorylate inositol phospholipids (2–9).
`The PTEN gene is mutated in 40–50% of high-grade gliomas as
`well as prostate, endometrial, breast, lung, and other tumors (2,
`3, 10). In addition, PTEN is mutated in several rare autosomal
`dominant cancer predisposition syndromes, including Cowden
`disease, Lhermitte-Duclos disease, and Bannayan-Zonana syn-
`drome (11–14). The phenotype of PTEN-knockout mice reveal
`a requirement for this phosphatase in normal development and
`confirm its role as a tumor suppressor (15–17).
`PTEN is a 55-kDa protein comprising an N-terminal catalytic
`domain, identified as a segment with homology to the cytoskel-
`etal protein tensin and containing the sequence HC(X)5R, which
`is the signature motif of members of the protein tyrosine
`phosphatase family, and a C-terminal C2 domain with lipid-
`binding and membrane-targeting functions (18). The sequence
`of the extreme C terminus of PTEN suggests a function in
`binding PDZ domain-containing proteins. PTEN is a dual
`specificity phosphatase that displays a pronounced preference
`for acidic substrates (5). Importantly, PTEN possesses lipid
`phosphatase activity, preferentially dephosphorylating phos-
`phoinositides at the D3 position of the inositol ring. It is the only
`enzyme known to dephosphorylate the D3 position in inositol
`phospholipids, suggesting that PTEN may function as a direct
`antagonist of phosphatidylinositol 3-kinase (PI3-kinase) and
`phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]-
`dependent signaling (7, 13). Reconstitution of PTEN in tumor
`cells that carry mutations in the PTEN gene have established
`that this phosphatase regulates the PI3-kinase-dependent acti-
`vation of AKT, a major player in cell survival (6, 14). However,
`despite progress in understanding the biochemistry of PTEN,
`
`the role of this phosphatase in tumor progression, as it relates to
`its diverse effects on cell growth, angiogenesis, andyor survival,
`remains unclear.
`Mutations in PTEN accompany progression of brain tumors
`from grade IyII to malignant grade III and IV in vivo (19, 20).
`Tumor progression is associated with angiogenesis, the formation
`of new blood vessels from existing vascular structures, with in-
`creases in microvessel density (MVD) and increased invasion of
`tumor cells into brain parenchyma (21–23). For tumor growth to
`occur, tumor dormancy must be broken, an event termed the
`angiogenic switch. During angiogenesis endothelial cells are in-
`duced to degrade the basement membrane of existing vessels, break
`away, and migrate to the site of the tumor, where they proliferate
`to form linear structures that differentiate to form blood vessels.
`Factors that control angiogenesis include growth factors, matrix
`metalloproteinases, plasminogen activators, thrombospondins, in-
`tegrins avb3, avb5, and a5b1, etc. (23–25). The angiogenic switch
`involves a shift in the balance of angiogenic stimulators and
`angiogenic inhibitors. Stimulators include the growth factors, vas-
`cular endothelial growth factor and basic fibroblast growth factor,
`and the induction of matrix remodeling via matrix metal-
`loproteinases (26). Inhibitors include thrombospondin 1 (TSP-1),
`angiostatin, endostatin, tissue inhibitors of metalloproteinases, and
`others (24, 27). It has been observed that neovascularization and
`PTEN mutations are associated with high-grade gliomas and are
`not observed in low-grade glial tumors, leading to the hypothesis
`that these two events may be causally linked.
`Regulation of PI3-kinase-dependent signals, including activa-
`tion of AKT by vascular endothelial growth factor and its
`receptors, the protein tyrosine kinases Flt-1 and KDR, have been
`implicated in brain tumor angiogenesis (28). Data generated in
`the chicken chorioallantoic membrane model suggests that PI3-
`kinase-dependent pathways may regulate angiogenesis and vas-
`cular endothelial growth factor expression in endothelial cells
`(29). Furthermore, correlative studies in prostate tumor speci-
`mens have demonstrated that tumors containing PTEN muta-
`tions have higher microvessel counts than tumors expressing
`wild-type (WT) PTEN (30). However, whether PTEN is causally
`linked to induction of angiogenesis by the tumor cell remains
`unproven. These and other observations led us to hypothesize
`that PTEN may control tumor-induced angiogenesis and con-
`tribute to the high mortality associated with malignant brain
`tumors.
`
`Materials and Methods
`Cell Culture, Constructs, and Reagents. WT PTEN or mutant PTEN
`(G129E, R130 M) cDNAs were subcloned into the pBabe-puro
`retroviral expression vector. Stable clones of U87MG cells
`expressing WT PTEN (WT.E1, WT.C7) or mutant PTEN
`(G129E, R130 M) were established under puromycin selection
`
`Abbreviations: PI3-kinase, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylino-
`sitol 3,4,5-trisphosphate; TSP-1, thrombospondin 1; WT, wild type; MVD, microvessel
`density.
`‡To whom reprint requests should be addressed. E-mail: ddurden@iupui.edu.
`
`The publication costs of this article were defrayed in part by page charge payment. This
`article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
`§1734 solely to indicate this fact.
`
`4622– 4627 u PNAS u April 10, 2001 u vol. 98 u no. 8
`
`www.pnas.orgycgiydoiy10.1073ypnas.081063798
`
`Breckenridge Exhibit 1096
`Wen
`Page 003
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`

`

`MEDICALSCIENCES
`
`Stable expression of PTEN and PTEN mutants in U87MG cells regulates
`Fig. 1.
`AKT. (A) Cell lysates from the U87MG (U87) cell line and U87 cells infected with
`a retroviral vector encoding PTEN (pBabe-Puro-PTEN) or mutants of PTEN
`(pBabe-Puro-PTEN-G129E or R130 M) were resolved by SDSyPAGE, and equal
`amounts of proteins were loaded per lane, immunoblotted with antisera to
`PTEN, phospho-AKT, and total AKT, and visualized by enhanced chemilumi-
`nescence. The basal levels of PTEN (Top), phosphorylated AKT (Ser-473) (Mid-
`dle), and total AKT (Bottom) are shown. The status of the PTEN gene in each
`stable cell line was designated as: WT.E1 and WT.C7, two separate clones
`expressing WT PTEN; R130 M and G129E are mutated PTEN, R130 M is inert as
`both a protein and a lipid phosphatase. The G129E PTEN can dephosphorylate
`acidic phosphopeptides, but cannot dephosphorylate lipid substrate,
`PtdIns(3,4,5)P3. The U87MG (U87) cell line is the parental cell line isolated from
`a human glioblastoma multiforme patient. (B) Comparison of in vitro growth
`of U87MG cells transduced with mutants of PTEN. Equal number of cells (1 3
`105) were incubated in RPMI 1 10% FBS for different times, and cell numbers
`were quantitated by direct cell counting.
`
`determined for five tumors per experimental group and analyzed
`by Student’s t test.
`
`Results
`Effect of PTEN Reconstitution on in Vitro Growth of Tumor. To
`determine whether PTEN exerts control over angiogenesis and
`the growth of glial tumors, we developed an orthotopic brain
`tumor model in which PTEN-deficient tumor cells were genet-
`ically manipulated in vitro and then stereotactically injected into
`the skin or frontal cerebral cortex of nude mice. The U87MG cell
`line is derived from a patient diagnosed with glioblastoma
`multiforme, a highly malignant and uniformly fatal brain tumor.
`This tumor and other human glioblastomas and glioblastoma cell
`lines contain a mutation in both PTEN alleles, resulting in a null
`genotype. In light of these observations, we reconstituted the
`PTEN gene in the parental U87MG (U87) cells.
`Stable derivatives of the parental U87 cells were generated
`after transduction with retroviruses encoding cDNA for WT
`PTEN or specific mutants of this phosphatase. In particular, we
`used missense mutations in the PTP signature motif to ascertain
`the importance of the enzymatic activity of PTEN to its tumor
`suppressor function. This included R130 M, in which all phos-
`phatase activity is abrogated (5, 33), and G129E, which has been
`identified in Cowden disease and endometrial cancer and in
`which the activity toward inositol phospholipids is severely
`attenuated but protein phosphatase activity is essentially intact.
`Tumor cells were characterized biochemically for levels of
`activated AKT (phospho-S473-AKT), growth in vitro, and PTEN
`expression (Fig. 1). Anti-PTEN blots confirmed that parental
`
`(2 mgyml) (6). Muristirone-induced expression of PTEN in
`U87MG cells was performed as described by J. Stolarov (31).
`Antibodies were obtained specific for PTEN (6), AKT and
`phospho-S473-AKT (New England Biolabs, #9270), TSP-1 (Cal-
`biochem, #605230), and anti-BrdUrd mAb clone BU33 (Sigma,
`#B9285).
`
`Tumor Implantation. Cells were cultured in fresh medium for 24 h
`and harvested, adjusting the cell concentration to 1 3 106 in 10
`ml of RPMI medium. Mice, under general anesthesia were placed
`into the stereotactic device (model 963, Kopf Instruments,
`Tujunga, CA). Stereotactically controlled drill assembly was
`used to provide a hole 0.3 mm deep and of 0.8 mm diameter in
`cranium at a position 0.5 mm anterior and 1.2 mm lateral to the
`landmark. Tumor cells (1 3 106) were
`bregmal anatomical
`introduced slowly through a 10-ml Hamilton syringe at a depth
`of 2.5 mm at a rate of 2 mlymin. We then slowly removed the
`needle at a rate of 0.5 mmymin. After needle removal we sealed
`the hole with bone wax and closed the incision with a wound clip.
`In some of the mice, 5 3 106 tumor cells were implanted s.c. into
`the right flank to monitor tumor volume and to perform
`biochemical and immunohistochemical analysis of tumor tissue.
`All animal experiments performed were approved by the Animal
`Care Committee at Indiana University School of Medicine.
`
`Biochemical Analysis. Immunoblots were performed on cell lysates
`obtained from U87 cells grown in tissue culture or from multiple
`cryostat sections of s.c. tumor tissues. A Bradford assay was
`performed to determine protein concentration of each lysate.
`Equivalent amounts of protein were resolved by SDSyPAGE and
`transferred to nitrocellulose. Membranes were probed with
`antisera specific for PTEN, AKT, phospho-S473-AKT, or TSP-1.
`The RNase protection assay was performed by using a RPA III
`kit from (Ambion) according to the manufacturer’s specifica-
`tions. Briefly, 20 mg of total RNA was precipitated and resus-
`pended in 10 ml of hybridization buffer containing the radioac-
`tive probe. The RNA then was heated to 95°C for 10 min and
`hybridized for 16 h at 42°C. A total of 150 ml of this mixture was
`treated with 1:100 dilution of RNase in RNase buffer for 30 min.
`RNase was inactivated, and RNA was reprecipitated and re-
`solved on 5% acrylamide gel. RNA probes were synthesized by
`using MAXI SCRIPT using PCR templates and T7 polymerase. The
`glyceraldehyde-3-phosphate dehydrogenase probe was provided
`in the kit, and TSP-1 probe represents a 590-nt sequence in the
`39 untranslated region of TSP-1 sequence. All probes were
`sequenced.
`
`Immunohistochemical Analysis. MVD was determined for each s.c.
`and brain tumors by CD31 staining, and a proliferative index was
`determined by using anti-BrdUrd mAb staining performed on
`cryostat sections (7 mm), fixed in acetone, blocked in 1% goat
`serum, and stained with anti-CD31 antibody (PharMingen,
`#01951D). Antibody staining was visualized with peroxidase-
`conjugated anti-mouse and counterstained with hematoxylin. A
`negative control was performed on each tumor tissue stained
`with mouse IgG. Two sections from each tumor were scanned
`under low-power magnification (340) to identify areas of highest
`CD31-positive vessel density (32), followed by digitization of
`three fields from this area. For in vivo BrdUrd labeling, mice
`received 100 ml of BrdUrd in PBS (10 mM) injected into the tail
`vein 1 h before tumor harvesting. The digitized images repre-
`senting one 320 field were counted for the number of CD31-
`positive vascular elements and number of BrdUrd-positive cellsy
`field. The number of cells positive for BrdUrd staining ranged
`between 5% and 8% of total number of tumor cells. Data were
`collected from two independent observers without knowledge of
`which tumors were viewed. The average number of microvessels
`or cells positive for BrdUrd staining per digitized field was
`
`Wen et al.
`
`PNAS u April 10, 2001 u vol. 98 u no. 8 u 4623
`
`Breckenridge Exhibit 1096
`Wen
`Page 004
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`

`

`U87 cells do not express PTEN and that after reconstitution of
`mutant or WT PTEN expression, U87 cells express amounts
`comparable to WT physiological
`levels. Expression of WT
`PTEN, to levels similar to those observed in a mouse brain lysate
`and primary human astrocytes, suppressed the activated state of
`AKT observed in PTEN-deficient U87 cells (Fig. 1 A, lanes 3 and
`5). After expression of the R130 M and G129E mutant forms of
`PTEN, the levels of phospho-AKT were similar to those ob-
`served in the parental U87 cells (Fig. 1 A, lanes 1, 2, and 4),
`suggesting that the lipid phosphatase activity of PTEN was
`essential for the effects on the PtdIns(3,4,5)P3-dependent acti-
`vation of AKT. Importantly, the growth of the different PTEN-
`expressing U87 cell lines in vitro was similar in 2%, 5%, and 10%
`FBS (data not shown and Fig. 1B). Therefore, we compared
`these cell lines further in our in vivo models.
`
`Effect of PTEN Reconstitution on inVivoGrowth and Proliferation. We
`implanted athymic nude mice s.c. and by intracranial injection.
`Production of s.c. tumors allowed us to monitor the size of the
`tumor and perform direct biochemical analysis of tumor tissue
`for PTEN expression and levels of AKT activation without
`significant contamination from other tissues. Tumor tissue
`blocks were processed for hematoxylinyeosin staining. Greater
`than 95% of tissue analyzed was tumor. We compared the levels
`of PTEN in tumor tissue and numerous normal tissues within the
`athymic nude mouse. Using anti-PTEN antisera, we detected the
`expression of PTEN in all tissues, with the exception of skeletal
`and heart muscle (data not shown), but no PTEN was detected
`in parental U87-derived tumor tissue (Fig. 2C, lane 4). These
`results indicate that the tumor tissue sampled represents pre-
`dominantly tumor cell-derived proteins. As observed in the cell
`lines grown in vitro, s.c. tumors, derived from U87 cells recon-
`stituted with mutant or WT PTEN, displayed similar levels of
`PTEN expression (Fig. 2C, lanes 1–3 and 5). Phospho-AKT
`activity was higher in PTEN-null U87 cells and U87 cells
`reconstituted with R130 M and to a lesser extent in U87 cells
`expressing the G129E mutant (Fig. 2C, lane 1) as compared with
`the WT PTEN-transduced cells (Fig. 2C, compare lanes 1, 2, and
`4 to lanes 3 and 5). The pattern of phosphorylated AKT was
`similar when the different U87 mutant expressing cell lines were
`assayed in vitro or in vivo (compare Fig. 1 A to 2C).
`Despite the similar in vitro growth rate, there was a dramatic
`difference in the growth of tumors derived from parental U87 cells
`compared with cells reconstituted with WT PTEN (Fig. 2 A and B).
`The average volume of U87-derived tumors on day 25 after
`implantation was 848 6 203 mm3, compared with 91 6 27 mm3 for
`tumors derived from WT PTEN-reconstituted cells (n 5 5, P ,
`0.0001). Interestingly, the reconstitution of U87 cells with catalyt-
`ically impaired PTEN (G129E or R130 M mutants) (Fig. 2A) shows
`an intermediate level of growth suppression, a result that suggests
`some residual function of these mutants in vivo.
`Despite dramatic differences in the size of these tumors, in vivo
`BrdUrd labeling of tumor cells revealed no significant difference
`in number of cells in S phase: 72 6 6 BrdUrd-positive cells per
`field in parental U87MG tumor mass versus 68.5 6 3 in WT
`PTEN reconstituted tumors (data not shown). The percentage
`of TUNEL-positive nuclei within the U87MG and WT PTEN
`reconstituted tumors was similar. These results suggested that
`something other than the proliferative or apoptotic rate of the
`U87MG versus U87MG reconstituted with WT PTEN ac-
`counted for the difference in growth potential. We observed that
`the loss of inositol phospholipid phosphatase activity results in
`deregulated tumor growth comparable to the total ablation of
`catalytic activity and that the despite differences in overall
`growth in vivo, the proliferative rate of these tumors is similar.
`These findings are consistent with a role for PTEN in the
`regulation of another aspect of tumor biology, such as angio-
`
`Effects of PTEN on growth of U87MG cells in vivo. (A) Cell growth in vivo.
`Fig. 2.
`To determine the rate of cell growth in vivo, equal amounts of cells (5 3 106) from
`each cell line were implanted at the right ventral flank by s.c. injection. The
`formation and growth of the s.c. tumor was monitored, and the volume of the
`tumor was determined by a three-dimensional measurement at the times indi-
`cated (day 0, the date of implantation, no tumor is detected). Data were analyzed
`by Student’s t test, and differences were significant comparing the PTEN deficient
`(U87MG, R130 M, G129E) to the WT PTEN (WT.E1, WT.C7), n 5 5, number of mice;
`P , 0.0001. (B) Stereophotography of s.c. tumor sites in mice implanted with the
`parental U87 tumor, PTEN minus (Left) versus WT PTEN reconstituted tumor cells
`(Right). These tumors represent 25 and 42 days after implantation for PTEN minus
`versus WT PTEN reconstituted tumors, respectively. (Magnification, 340.) (C)
`Immunoblot of cryostat tissue sections from s.c. tumor for the expression pattern
`of PTEN, AKT, and phosphorylated AKT. Frozen tissue sections were solubilized in
`Laemmli sample buffer, total protein was quantitated, and equal protein was
`loaded on SDSyPAGE. The data shown are representative of tissue analysis from
`five animals per experimental group.
`
`genesis. This led us to assess the effect of PTEN reconstitution
`on the induction of angiogenesis in this model.
`
`PTEN Suppresses Tumor-Induced Angiogenesis. To examine the
`effect of PTEN on angiogenesis, we compared parental U87 cells
`to cells reconstituted with WT or mutant PTEN. We stained
`cryostat sections from s.c. tumors for CD31 (PECAM). CD31 is
`an endothelial marker used to measure the MVD of these
`tumors. MVD was assessed from multiple digitized images of
`CD31-stained tumor tissue at 3100 magnification (three fields
`were evaluated per tumor) and counted blindly for the number
`of CD31-positive microvessels per unit surface area as described
`(32). Reconstitution of PTEN expression in U87 cells dramat-
`ically suppressed the angiogenic response in vivo (Fig. 3 A and
`B). Quantitation of MVD in tumors derived from parental U87
`cells (77 6 13) and U87 cells expressing WT PTEN (38 6 7)
`revealed an ’50% suppression of angiogenesis (Fig. 3C) (n 5 5,
`P , 0.001). The MVD of tumors derived from U87 cells
`reconstituted with catalytically impaired PTEN (R130 M, 84 6
`15 or G129E, 69 6 16) were not significantly different (P . 0.05)
`from the parental U87 cell line (Fig. 3C). The levels of phospho-
`AKT detected within the tumor mass in vivo provide a correla-
`tion between the loss of the inositol lipid phosphatase function
`of PTEN, the phosphorylation status AKT, and the angiogenic
`phenotype within the tumor.
`
`PTEN Induction of Thrombospondin. Recent in vitro data suggest a
`link between PTEN and downstream targets including AKT,
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`4624 u www.pnas.orgycgiydoiy10.1073ypnas.081063798
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`Wen et al.
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`A
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`B
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`C
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`D
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`E
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`PTEN suppresses angiogenesis. Immunohistochemical analysis of staining with CD31 antibody to evaluate the angiogenesis response within the parental
`Fig. 3.
`U87MG tumor (A) and PTEN reconstituted tumors (B), implanted into the s.c. tissue. In PTEN minus and tumors expressing mutants of PTEN, there are more new
`vessels formed (angiogenesis) (Upper, arrow indicated) than in WT PTEN reconstituted tumor (Lower), indicating the PTEN has direct influence on angiogenesis
`during tumor growth. (C) MVD counts were performed on tumor tissue stained with anti-CD31 antibody as described (32) to determine the effect of expression
`of PTEN and specific PTEN mutants (G129E or R130 M) on tumor-induced angiogenesis. Bars represent SD, five animals per group. Statistical analysis by Student’s
`t test demonstrateed significant difference between MVD of PTEN null and PTEN catalytic mutants as compared with WT PTEN reconstituted tumors, n 5 5,
`number of mice; P , 0.001. (D) PTEN regulates the expression of TSP-1 in U87MG cells. RNase protection assay was used to measure levels of TSP-1 mRNA in WT
`PTEN expressing U87 cells or cells transduced with a mutant catalytically dead PTEN (G129R). U87MG cells were infected with retrovirus encoding WT PTEN (WT),
`the catalytically dead, G129R mutant (GR), or empty vector retrovirus (2) and selected for 10 days in puromycin. RNA was harvested and RNase protection assays
`were carried out by using probes for TSP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A probe for GAPDH was used as a normalization control.
`(E) Thrombospondin immunoblot analysis. U87MG transduced with WT PTEN (WT) or a catalytic mutant PTEN (G129R) in an ecdysone inducible expression system
`(36) were induced (48 h) with 0.5 mM muristirone or assayed without induction to determine the effect of PTEN expression on the induction of TSP-1 by Western
`blotting. Supernates from cells were prepared and proteins were resolved on SDSyPAGE and probed with anti-TSP-1 antibody. There is clear up-regulation of
`TSP-1 in WT PTEN-transduced U87 cultures compared with U87 cells expressing the lipid phosphatase-deficient G129R mutant PTEN.
`
`MEDICALSCIENCES
`
`HIF1a, and vascular endothelial growth factor in the potential
`control of angiogenesis (34, 35). We used the RNase protection
`assay to confirm an observation we initially made during a cDNA
`microarray analysis comparing the effect of PTEN reconstitu-
`tion on U87MG TSP-1 expression. RNase protection assay was
`performed with a TSP-1 specific probe in the same U87MG cells
`constitutively expressing WT PTEN or G129R PTEN (Fig. 3D).
`The data demonstrate that WT but not mutant PTEN expression
`induces TSP-1 in U87 cells. To confirm these results, we per-
`formed Western blot analysis for TSP-1 expression with a
`retroviral-based ecdysone-inducible PTEN expression system
`(36). Inducible and dose-dependent expression of PTEN was
`confirmed in U87 cells, and we noted that the induced expression
`of WT PTEN and not G129R PTEN (not shown) resulted in
`augmented TSP-1 expression (Fig. 3E), and WT PTEN sup-
`pressed the activation of phospho-AKT without affecting total
`AKT (data not shown). The induced expression of mutant
`G129R had no effect on phospho-AKT. Therefore, our data
`demonstrate that PTEN positively modulates the expression of
`TSP-1, a negative regulator of angiogenesis (Fig. 3 D and E) (37,
`38). These data identify one potential mechanism by which
`PTEN may regulate angiogenesis through the control of TSP-1.
`
`PTEN Is a Determinant of Survival in Orthotopic Brain Tumor Model.
`Brain tumor-induced angiogenic responses occur in the context
`of brain-specific stromal and extracellular matrix interactions.
`This fact led us to determine whether the expression of the
`PTEN affects the survival of mice in an orthotopic brain tumor
`model. For these experiments, U87 cells expressing either WT or
`mutant forms of PTEN were implanted under stereotactic
`control into the right frontal lobe of nude mice (Fig. 4B, see
`
`arrow for site of implantation). The results indicate that recon-
`stitution of WT PTEN in U87 cells suppressed the malignant
`potential of these cells in an orthotopic animal model. Thus,
`there was 90% survival at 40 days in animals implanted with the
`WT PTEN-reconstituted U87 cells compared with 100% mor-
`tality of mice implanted with the parental cells at 27 days (Fig.
`4C) (n 5 15, P , 0.0001). PTEN reconstituted tumor cells grew
`more slowly when implanted in the frontal lobe (Fig. 4, compare
`A and B) and remained circumscribed to that area of brain (data
`not shown). U87 cells reconstituted with mutants of PTEN,
`either ablated in inositol lipid phosphatase activity (G129E) or
`catalytically inactive (R130 M), displayed a phenotype similar to
`the PTEN-negative, parental U87 cells (Fig. 4C). Animals with
`tumors derived from U87 cells rec