Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185Neu protein (A. Maity et al., Cancer Res., 60: 5879–5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3′-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from −88 to +54 bp. Activity of the −88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.

VEGF3 is an important angiogenic factor (reviewed in Ref. 1) that is often overexpressed in tumors. In particular, VEGF is highly expressed in human glioblastomas, which are vascular tumors of the brain associated with a dismal prognosis. Numerous approaches to inhibit VEGF function, including VEGF antibodies, kinase inhibitors, and dominant negative VEGF receptors, efficiently inhibit tumor growth in animal models, indicating that expression of VEGF is important for tumor progression (reviewed in Ref. 2). Therefore, defining the mechanisms that regulate VEGF expression may have important implications for understanding tumor biology.

Previously we found that in U87MG human glioblastoma cells, VEGF expression is increased by EGFR activation (3). Furthermore, this increased VEGF expression was due to transcriptional activation of the VEGF promoter via a PI3K-dependent pathway. EGFR amplification occurs in 50–60% of cases of glioblastomas (4, 5). However, glioblastomas also frequently contain mutations in PTEN, a phosphatase that dephosphorylates the D-3 position of phosphoinositide phosphates such as phosphatidylinositol 3,4,5-trisphosphate to convert them to phosphatidylinositol 4,5-bisphosphate (for review, see Ref. 6). Homozygotic inactivation of PTEN occurs in at least 30% of primary glioblastomas and in 50–60% of glioblastoma cell lines (7, 8). Therefore, loss of PTEN function results in activation of pathways downstream of PI3K, leading us to hypothesize that PTEN inactivation may lead to increased VEGF expression in glioblastoma cells independent of EGFR activation. This led to the current investigation of the role of PTEN inactivation in VEGF regulation and the interplay with EGFR activation.

Tissue Culture and Reagents.

U87MG, U251MG, and SF188 cells were cultured in DMEM (4500 mg/liter glucose; Life Technologies, Inc.) containing 10% fetal bovine serum (Atlanta Biologicals) and grown in an incubator with 5% carbon dioxide and 21% oxygen or under hypoxic conditions as described below. U87/T691 cells were maintained in DMEM containing 10% fetal bovine serum and 0.4 mg/ml G418 (Life Technologies, Inc.). LY294002 and PD153035 were both dissolved in DMSO at a stock concentration of 10 mm.

Northern Blot Analysis.

RNA was isolated, and Northern blotting was performed as described previously (9). A 200-bp VEGF cDNA fragment excised with EcoRI from the pGEMh204 plasmid (a gift from B. Berse; Boston University School of Medicine) was used to make radioactive probes for hybridization. To verify equal loading between lanes, all gels were stained with ethidium bromide, and the membranes were probed with a DNA fragment of the 18S ribosomal RNA.

Protein Extraction, Western Blot Analysis, and ELISA.

Protein isolation and quantitation and Western blotting were performed as described previously (9). For detection of the phosphorylated form of Akt protein, we used an anti-phospho-Akt antibody directed against serine 473 (Cell Signaling catalogue number 9271S; 1:1000 dilution) followed by a goat antirabbit antibody (Bio-Rad; 1:2000 dilution). As a loading control, the blot was reprobed with an anti-pan-Akt antibody (Cell Signaling catalogue number 9272; 1:2000 dilution) followed by a goat antirabbit antibody (1:1000 dilution). Some blots were also reprobed with an anti-β-actin antibody (Sigma; 1:1000 dilution) followed by a goat antimouse antibody (1:2000 dilution). To detect PTEN, blots were probed with an anti-PTEN antibody (26H9; Cell Signaling; 1:1000 dilution) followed by goat antimouse antibody (1:2000 dilution).

VEGF ELISA assays were performed as described previously (3) using a commercial kit (R&D Systems).

Quantitation of Blots and Data Analysis.

Gels were scanned on an Agfa Arcus II photoscanner using Adobe Photoshop 4.0. Bands on the gels were quantitated using NIH Image 1.54 software. All results shown are representative of at least two independent reproducible experiments.

Plasmid Constructs and Transient Transfections.

The construction of the plasmid pGL3-1.5kbVEGFprom has been described previously (3). The pcDNA/PTEN construct was obtained from Charles Sawyer (University of California Los Angeles School of Medicine). pCMV6/Akt K179M and pCMV6/myr Akt were obtained from Phil Tschilis (Thomas Jefferson Medical School). Transfections were performed using Fugene (Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, cells were split into 60-mm dishes so that 24 h later they were approximately 50% confluent. At this time, each dish was transfected using 6 μl of Fugene and 2 μg of the reporter plasmid and 1 μg of pSV-β-galactosidase to control for transfection efficiency (Promega). Cells were harvested by removing the media, washing twice with PBS, and directly adding 100 μl of lysis buffer/dish. Of this lysate, 80 μl were used for luciferase determinations, and 10 μl were used for β-galactosidase assays. These determinations were performed using the LucLite kit (Packard Instrument Co.) and the β-galactosidase Enzyme Assay System (Promega). Luciferase readings were performed on a TopCount Microplate Scintillation and Luminescence Counter (Packard Instrument Co.).

An upstream primer (5′-TAGCTCGAGCCGGGCGGCCGGGGC-3′) engineered with a XhoI restriction site (underlined) and a downstream primer (5′-TACAAGCTTCTAGCCCCAGCGCCACGA-3′) engineered with a HindIII restriction site (underlined) were used to PCR amplify the fragment of the VEGF promoter from −88 to +54. This fragment was excised with XhoI and HindIII and subcloned into the plasmid pGL3-Basic to make pGL3(−88/+54)VEGFprom. To make the construct pGL3(−70/+54)VEGFprom, the same downstream primer was used, but the upstream primer was 5′-TAGCTCGAGGGGTCCCGGCGGGGCGGA-3′ engineered to contain a XhoI site. After PCR amplification, the fragment was excised with XhoI and HindIII and subcloned into these sites in pGL3-Basic.

PTEN Adenovirus.

Adenovirus expressing PTEN or phosphatase dead PTEN and capable of replicating in the “packaging” 293 cell line was made using the pAdEasy protocol. The virus was stored in single-use aliquots at −80°C. U87MG cells were harvested 48 h after adenoviral infection.

Phosphorylated Akt Levels Correlate with VEGF mRNA Expression in Human Glioblastoma Cells.

Previous results indicated that U87/T691 cells, in which EGFR kinase activation and signaling are inhibited by introduction of a truncated Erb-B2 receptor, contain less VEGF mRNA than the parental U87MG cells (3). EGFR activation increases PI3K activity, and phosphorylation of Akt occurs downstream of PI3K activation (reviewed in Ref. 10). Akt phosphorylation was examined in U87MG and U87/T691, and, as expected, the level of phospho-Akt was lower in the latter cell line than in the former cell line by 40% (Fig. 1,A). Of note, however, the level of phospho-Akt was still higher in U87/T691 cells than in SF188 cells that express wild-type PTEN and exhibit low levels of 3′ phospholipids phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (Ref. 11; Fig. 1,A). U251MG, another glioblastoma cell line containing mutant PTEN, expresses a similar level of phospho-Akt as U87MG (Fig. 1,A). Relative levels of VEGF mRNA in the four glioblastoma cell lines mirrored the levels of phospho-Akt, with U87MG and U251MG cells expressing the most VEGF mRNA, SF188 cells expressing the least VEGF mRNA, and U87/T691 cells expressing an intermediate amount of VEGF mRNA (Fig. 1 B).

Wild-type PTEN Decreases VEGF Expression in U87MG and U251MG Glioblastoma Cells.

Despite EGFR inhibition, U87/T691 cells still have significant phospho-Akt expression compared with SF188 cells. U87/T691 cells contain mutant PTEN, whereas SF188 cells express wild-type PTEN, suggesting that the differences in VEGF expression might be due to the difference in PTEN status. To determine the effect of wild-type PTEN in U87MG cells, we infected these cells with adenovirus expressing wild-type PTEN. This led to a decrease in phospho-Akt levels compared with control samples not treated with virus or infected with phosphatase dead PTEN adenovirus (Fig. 2,A). Expression of PTEN protein by wild-type PTEN adenovirus was confirmed by Western blotting (Fig. 2,B). Infection of cells with virus expressing wild-type PTEN also led to a significant decrease in VEGF mRNA expression (Fig. 2,C) and secreted VEGF protein compared with controls (Fig. 2,D). Therefore, introduction of wild-type PTEN into U87MG cells decreased levels of phospho-Akt protein, VEGF mRNA, and VEGF protein. Wild-type PTEN had a similar effect on decreasing phospho-Akt and VEGF mRNA expression in another glioblastoma line, U251MG (Fig. 3, A and B).

Antagonism of PI3K Activity in U87/T691 Cells Decreases VEGF Expression.

We sought to determine whether PTEN could decrease VEGF mRNA expression in cells in which EGFR signaling was already inhibited. To this end, U87/T691 cells were infected with adenovirus expressing wild-type PTEN. Even though phospho-Akt expression was already decreased in U87/T691 compared with U87MG cells (Fig. 1,A), introduction of wild-type PTEN adenovirus into U87/T691 cells further decreased the level of phospho-Akt (Fig. 4,A). Likewise, wild-type PTEN decreased VEGF mRNA expression in U87/T691 cells (Fig. 4,C), despite the already lower VEGF mRNA levels evident in U87/T691 compared with U87MG (Fig. 1 B).

We corroborated the effects of PTEN by using a pharmacological inhibitor of PI3K, LY294002. As expected, treatment of U87/T691 cells with this agent further decreased the level of phospho-Akt protein and VEGF mRNA (Fig. 4, B and D, respectively). Therefore, inhibition of signaling downstream of PI3K by either introduction of wild-type PTEN or treatment with a pharmacological inhibitor further decreases VEGF expression in U87/T691 cells, which already have inhibition of EGFR signaling due to expression of a mutant Erb-B2 receptor.

EGFR Inhibition and PTEN Have a Combined Effect to Decrease VEGF Expression in U87MG Cells.

Fig. 4, A–D, showed that inhibition of signaling downstream of PI3K led to a further decrease in VEGF expression, beyond the effects of EGFR inhibition. To confirm that expression of PTEN and EGFR activation both contribute to VEGF mRNA expression, U87MG cells were exposed to adenovirus expressing wild-type PTEN, the EGFR inhibitor PD153035, or both. The combination of wild-type PTEN and PD153035 resulted in a lower level of VEGF mRNA than either treatment by itself (Fig. 4 E). Therefore, in U87MG cells, loss of PTEN and EGFR activation both contribute to increased VEGF expression in an additive manner.

PI3K Inhibition Decreases Activity of −88/+54 VEGF Promoter in U87MG Cells.

Previously, we (3) found that a luciferase reporter construct containing 1.5 kb of the VEGF promoter could be negatively regulated by the PI3K inhibitor LY294002 and by Δp85, a dominant negative mutant of the p85 subunit of PI3K (12). The VEGF promoter region contained in this construct includes the HIF-1 binding site known to be critical for induction of transcription by hypoxia (Fig. 5,A). A region of the VEGF promoter encompassing −88 to +54 was chosen for further study because it had previously been found to be regulated by transforming growth factor α (13) and mitogen-activated protein kinase (Ref. 14; Fig. 5,A). Activity of this promoter region was down-regulated by Δp85, a dominant negative mutant of the p85 subunit of PI3K (data not shown), as well as by the pharmacological inhibitor LY294002 (Fig. 5,B). In contrast, the −70/+54 VEGF promoter construct exhibited extremely low activity in U87MG cells (Fig. 5,B), suggesting that elements residing between −88 and −70 were responsible for basal activity in these glioblastoma cells. Activity of the −88/+54 VEGF promoter construct could also be down-regulated in EGFR-inhibited U87/T691 cells by treatment with LY294002 or by cotransfection with a PTEN-expressing construct (Fig. 5, C and D).

EGFR Inhibition and Wild-type PTEN Have an Additive Effect to Decrease VEGF Promoter Activity in U87MG Cells.

To determine the effects of wild-type PTEN on VEGF promoter activity, U87MG cells were cotransfected with a vector expressing PTEN along with the 1.5-kb VEGF promoter reporter construct. PTEN decreased VEGF reporter activity in a dose-dependent manner (Fig. 6,A). PTEN also decreased activity of the −88/+54 VEGF promoter luciferase construct (Fig. 6 B). Therefore, inhibition of signaling downstream of PI3K by PTEN down-regulates VEGF promoter activity through the region located from −88 to +54.

To determine the combined effect of EGFR inhibition and introduction of wild-type PTEN on VEGF promoter regulation, U87MG cells were transfected with the 1.5-kb VEGF promoter reporter and, in addition, cotransfected with PTEN, treated with the EGFR inhibitor PD153035, or both. Fig. 6,C shows that EGFR inhibition decreased VEGF promoter activity, as did expression of wild-type PTEN, but that the two treatments combined decreased VEGF promoter activity more than either treatment alone. Therefore, PTEN and inhibition of EGFR signaling have an additive effect on regulating the VEGF promoter. Similar results were obtained when the −88/+54 VEGF promoter reporter was used instead of the 1.5-kb reporter (Fig. 6 D).

Akt Regulates VEGF Promoter Activity in U87MG and SF188 Cells.

Because Akt is a known downstream effector in the PI3K pathway and is negatively regulated by PTEN (11, 15), we tested the ability of Akt to modulate VEGF promoter activity. Cotransfection of a plasmid expressing dominant negative Akt decreased activity of the −88/+54 VEGF promoter reporter in U87MG cells (Fig. 6,E). Conversely, cotransfection of a plasmid expressing a constitutively active myristoylated Akt led to increased activity of the promoter in SF188 cells (Fig. 6 F).

Oncogenic changes such as Ras mutations that occur during cancer progression contribute to deregulated VEGF expression (16). Although Ras mutations rarely occur in glioblastomas (17), EGFR amplification, which can activate similar downstream pathways as Ras, is common. The link between EGFR activation and VEGF expression has been established by us (3) and others in both glioblastoma cells (18) and other cell types (19, 20). The finding that the PI3K pathway is involved in VEGF regulation led us to investigate the role of PTEN mutation, which commonly occurs in glioblastomas (7, 8) and leads to unopposed PI3K activity and activation of downstream effectors such as Akt (11, 15).

In the current study, we found that introduction of wild-type PTEN into U87MG cells decreased VEGF mRNA levels. Inhibition of EGFR using a pharmacological inhibitor also decreased VEGF expression in these cells; however, introduction of wild-type PTEN along with EGFR inhibition led to a greater decrease in VEGF expression than either treatment alone. Therefore, EGFR activation and loss of PTEN have an additive effect to increase VEGF mRNA levels and VEGF promoter activity under normoxic conditions.

We have shown that two genetic alterations commonly seen in glioblastomas can have a combined effect to increase VEGF levels. EGFR amplification occurs at similar frequencies in glioblastomas with or without PTEN mutations, suggesting that the two genetic changes are not redundant (21). Based on our results, one could speculate that a possible reason why some glioblastomas contain both EGFR amplification and PTEN mutation is that there is an additive effect on increasing the expression of certain genes that contribute to tumor growth, one of them being VEGF. It is possible that EGFR amplification augments the effect of PTEN loss by increasing signaling downstream of PI3K. Alternatively, EGFR amplification may activate another pathway that cooperates with the PI3K pathway to increase VEGF expression.

The PI3K pathway has been shown to regulate VEGF levels in many cell types including chick allantoic cells (22), human umbilical endothelial cells (23), and some cell types transformed with ras including fibroblasts (24), intestinal epithelial cells (25), and endothelial cells (26). At least two groups have suggested that the PI3K pathway regulates VEGF through HIF-1 (27, 28). In contrast to these reports, our results indicate that there is an element located in the −88/+54 region of the promoter, far removed from the HIF-1 binding site, that is responsive to the PI3K/Akt pathway. Binding sites for AP2 and Sp1 are located in this region, and future studies will examine whether these sites are critical for activation by PI3K.

Fig. 1.

Phospho-Akt protein and VEGF mRNA levels in glioblastoma cells. A, Western blot with protein lysates collected from SF188, U87MG, U87/T691, and U251MG cells growing in media supplemented with 10% serum. Membranes were probed with an anti-phosphorylated Akt antibody and then reprobed with a pan-Akt antibody (loading control). Numbers at the bottom of A represent ratios of intensity of the phospho-Akt (P-Akt) band to the pan-Akt band. B, Northern blot of RNA from the same cell lines as described in A. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Membrane was probed for both VEGF and 18S ribosomal RNA (loading control). Numbers at the bottom of B represent the ratios of intensity of the upper VEGF band to the 18S band.

Fig. 1.

Phospho-Akt protein and VEGF mRNA levels in glioblastoma cells. A, Western blot with protein lysates collected from SF188, U87MG, U87/T691, and U251MG cells growing in media supplemented with 10% serum. Membranes were probed with an anti-phosphorylated Akt antibody and then reprobed with a pan-Akt antibody (loading control). Numbers at the bottom of A represent ratios of intensity of the phospho-Akt (P-Akt) band to the pan-Akt band. B, Northern blot of RNA from the same cell lines as described in A. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Membrane was probed for both VEGF and 18S ribosomal RNA (loading control). Numbers at the bottom of B represent the ratios of intensity of the upper VEGF band to the 18S band.

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Fig. 2.

Effect of adenovirus expressing PTEN on phospho-Akt protein and VEGF mRNA levels in U87MG cells. U87MG cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. For A–C, cells were harvested 48 h later. Aliquots were obtained for Western and Northern blotting. A, Western blot was initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. B, Western blot was initially probed for PTEN and then reprobed for β-actin. C, Northern blot was initially probed for VEGF and then reprobed for 18S. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of C represent ratios of intensity of the upper VEGF band to the 18S band. D, aliquots were collected from media 24 h after infection of U87MG cells with no virus or adenovirus expressing wild-type or phosphatase dead PTEN. VEGF protein levels were determined by ELISA and normalized to the number of cells in each dish.

Fig. 2.

Effect of adenovirus expressing PTEN on phospho-Akt protein and VEGF mRNA levels in U87MG cells. U87MG cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. For A–C, cells were harvested 48 h later. Aliquots were obtained for Western and Northern blotting. A, Western blot was initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. B, Western blot was initially probed for PTEN and then reprobed for β-actin. C, Northern blot was initially probed for VEGF and then reprobed for 18S. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of C represent ratios of intensity of the upper VEGF band to the 18S band. D, aliquots were collected from media 24 h after infection of U87MG cells with no virus or adenovirus expressing wild-type or phosphatase dead PTEN. VEGF protein levels were determined by ELISA and normalized to the number of cells in each dish.

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Fig. 3.

Effect of adenovirus expressing PTEN on phospho-Akt protein and VEGF mRNA levels in U251MG cells. U251MG cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. Aliquots were obtained 48 h later for Western and Northern blotting. A, Western blot was initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. B, Northern blot was initially probed for VEGF and then reprobed for 18S. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of B represent ratios of the intensity of the upper VEGF band to the 18S band.

Fig. 3.

Effect of adenovirus expressing PTEN on phospho-Akt protein and VEGF mRNA levels in U251MG cells. U251MG cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. Aliquots were obtained 48 h later for Western and Northern blotting. A, Western blot was initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. B, Northern blot was initially probed for VEGF and then reprobed for 18S. Ethidium bromide staining of the gel confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of B represent ratios of the intensity of the upper VEGF band to the 18S band.

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Fig. 4.

Effect of PTEN and/or EGFR inhibition on phospho-Akt protein and VEGF mRNA expression in glioblastoma cells. For A and C, U87/T691 cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. Cells were harvested 48 h later for protein and RNA. For B and D, U87/T691 cells were treated with LY294002 (40 μm) or DMSO (control). Sixteen h later, aliquots were harvested for RNA and protein. E, U87MG cells were treated in one of four ways: (a) infected with wild-type PTEN expressing adenovirus at t = 0 h; (b) infected with wild-type PTEN expressing adenovirus at t = 0 h and then exposed to PD153035 (5 μm) at t = 24 h; (c) exposed to PD153035 at t = 24 h; or (d) untreated. Cells were harvested at t = 48 h for RNA and analyzed by Northern blotting. A and B, Western blots were initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. C and D, Northern blots were probed for VEGF and then reprobed for 18S RNase (control). Ethidium bromide staining of the gels confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of C–E represent ratios of the intensity of the upper VEGF band to the 18S band.

Fig. 4.

Effect of PTEN and/or EGFR inhibition on phospho-Akt protein and VEGF mRNA expression in glioblastoma cells. For A and C, U87/T691 cells were infected with adenovirus expressing either wild-type (wt) or phosphatase dead PTEN. Cells were harvested 48 h later for protein and RNA. For B and D, U87/T691 cells were treated with LY294002 (40 μm) or DMSO (control). Sixteen h later, aliquots were harvested for RNA and protein. E, U87MG cells were treated in one of four ways: (a) infected with wild-type PTEN expressing adenovirus at t = 0 h; (b) infected with wild-type PTEN expressing adenovirus at t = 0 h and then exposed to PD153035 (5 μm) at t = 24 h; (c) exposed to PD153035 at t = 24 h; or (d) untreated. Cells were harvested at t = 48 h for RNA and analyzed by Northern blotting. A and B, Western blots were initially probed for phospho-Akt, then reprobed for pan-Akt, and then reprobed for β-actin. C and D, Northern blots were probed for VEGF and then reprobed for 18S RNase (control). Ethidium bromide staining of the gels confirmed equal loading of the lanes by visual inspection (data not shown). Numbers at the bottom of C–E represent ratios of the intensity of the upper VEGF band to the 18S band.

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Fig. 5.

Effect of LY294002 or PTEN on VEGF promoter activity in glioblastoma cells. A, schematic of VEGF promoter constructs. The 1.5-kb VEGF promoter contains a 47-bp hypoxia responsive element (HRE, shaded box) located between −985 and −939. The −88/+54 VEGF promoter contains two Sp1 binding sites and one AP-2 binding site. These sites are deleted in the −70/+54 VEGF promoter. B, U87MG cells were cotransfected with pSV-β-galactosidase and −88/+54 or −70/+54 promoter constructs. Total amount of DNA was 0.5 μg for all transfections (pcDNA3 was added as filler DNA as needed). Twenty-four h later, PI3K inhibitor LY294002 (40 μm) was added. Twenty-four h after the addition of LY294002 (48 h after transfection), samples were harvested for luciferase and β-galactosidase assays. The ratio of luciferase:β-galactosidase activity is plotted on the Y axis. C, same as in B except that U87/T691 cells were transfected with the −88/+54 VEGF promoter construct. D, U87/T691 cells were transfected with the −88/+54 VEGF promoter reporter along with pcDNA-PTEN. For B–D, values represent the means of three independent transfections, and error bars represent 1 SD of the mean.

Fig. 5.

Effect of LY294002 or PTEN on VEGF promoter activity in glioblastoma cells. A, schematic of VEGF promoter constructs. The 1.5-kb VEGF promoter contains a 47-bp hypoxia responsive element (HRE, shaded box) located between −985 and −939. The −88/+54 VEGF promoter contains two Sp1 binding sites and one AP-2 binding site. These sites are deleted in the −70/+54 VEGF promoter. B, U87MG cells were cotransfected with pSV-β-galactosidase and −88/+54 or −70/+54 promoter constructs. Total amount of DNA was 0.5 μg for all transfections (pcDNA3 was added as filler DNA as needed). Twenty-four h later, PI3K inhibitor LY294002 (40 μm) was added. Twenty-four h after the addition of LY294002 (48 h after transfection), samples were harvested for luciferase and β-galactosidase assays. The ratio of luciferase:β-galactosidase activity is plotted on the Y axis. C, same as in B except that U87/T691 cells were transfected with the −88/+54 VEGF promoter construct. D, U87/T691 cells were transfected with the −88/+54 VEGF promoter reporter along with pcDNA-PTEN. For B–D, values represent the means of three independent transfections, and error bars represent 1 SD of the mean.

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Fig. 6.

Effect of PTEN and EGFR inhibition on VEGF promoter activity in U87MG cells. In A–D, U87MG cells were transfected with a VEGF promoter reporter construct. In A and C, the VEGF reporter was the 1.5-kb promoter construct; in B and D, it was the −88/+54 bp promoter construct. In A and B, cells were cotransfected with pSV-β-galactosidase and pcDNA-PTEN along with the VEGF reporter. Total amount of DNA was 0.5 μg for all transfections (pcDNA3 was added as filler DNA as needed). In C and D, in addition to being transfected with the VEGF promoter reporter, cells were treated in one of four ways: (a) transfected with control pcDNA3; (b) transfected with pcDNA-PTEN; (c) transfected with pcDNA3 and then exposed to PD15153035 for 24 h; or (d) transfected with pcDNA-PTEN and then exposed to PD153035 for 24 h. In E, U87MG cells were cotransfected with pSV-β-galactosidase, the −88/+54 VEGF promoter reporter, and pCMV6/HA-AktK179M (0.2 μg). In F, SF188 cells were cotransfected with pSV-β-galactosidase, the −88/+54 VEGF promoter reporter, and pCMV6/myr-Akt (0.2 μg). In A–F, lysates were harvested 48 h after transfection for luciferase and β-galactosidase assays. The ratio of luciferase:β-galactosidase activity is plotted on the Y axis.

Fig. 6.

Effect of PTEN and EGFR inhibition on VEGF promoter activity in U87MG cells. In A–D, U87MG cells were transfected with a VEGF promoter reporter construct. In A and C, the VEGF reporter was the 1.5-kb promoter construct; in B and D, it was the −88/+54 bp promoter construct. In A and B, cells were cotransfected with pSV-β-galactosidase and pcDNA-PTEN along with the VEGF reporter. Total amount of DNA was 0.5 μg for all transfections (pcDNA3 was added as filler DNA as needed). In C and D, in addition to being transfected with the VEGF promoter reporter, cells were treated in one of four ways: (a) transfected with control pcDNA3; (b) transfected with pcDNA-PTEN; (c) transfected with pcDNA3 and then exposed to PD15153035 for 24 h; or (d) transfected with pcDNA-PTEN and then exposed to PD153035 for 24 h. In E, U87MG cells were cotransfected with pSV-β-galactosidase, the −88/+54 VEGF promoter reporter, and pCMV6/HA-AktK179M (0.2 μg). In F, SF188 cells were cotransfected with pSV-β-galactosidase, the −88/+54 VEGF promoter reporter, and pCMV6/myr-Akt (0.2 μg). In A–F, lysates were harvested 48 h after transfection for luciferase and β-galactosidase assays. The ratio of luciferase:β-galactosidase activity is plotted on the Y axis.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grant 1R01 CA093638-01 (to A. M.) and grants from the Veterans Administration Review Program, Brain Tumor Society, and NIH Grant 1R01 CA90586-01 (to D. M. O.).

3

The abbreviations used are: VEGF, vascular endothelial growth factor; PI3K, phosphatidylinositol 3′-kinase; EGFR, epidermal growth factor receptor; HIF-1, hypoxia-inducible factor-1.

We thank Amita Sehgal for use of the TopCount Microplate Scintillation and Luminescence Counter.

Note Added in Proof

While this manuscript was being reviewed, Takahashi et al. published that Akt can regulate the proximal VEGF promoter independently of the hypoxia-inducible element in cultured skeletal muscle cells (Takahashi, A., Kureishi, Y., Yang, J., Luo, Z., Guo, K., Mukhopadhyay, D., Ivashchenko, Y., Branellec, D., Walsh, K. Mol. Cell Biol. 22:4803–4814, 2002).

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