Placenta Growth Factor Gene Expression Is Induced by Hypoxia in Fibroblasts: a Central Role for Metal Transcription Factor-11

[CANCER RESEARCH 61, 2696–2703, March 15, 2001] Placenta Growth Factor Gene Expression Is Induced by Hypoxia in Fibroblasts: A Central Role for Metal Transcription Factor-11

Christopher J. Green,2 Peter Lichtlen,2 Nhung T. Huynh, Marianna Yanovsky, Keith R. Laderoute, Walter Schaffner, and Brian J. Murphy3 The Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025 [C. J. G., N. T. H., M. Y., K. R. L., B. J. M.]; the Institut fur Molekularbiologie der Universitat Zurich, CH-8057 Zurich, Switzerland [W. S.]; and ESBATech AG, CH-8057 Zurich, Switzerland [P. L.]

ABSTRACT hPlGF isoforms in that it contains an additional 21 basic amino acid region at the COOH terminus that confers a heparin-binding ability Placenta growth factor (PlGF) is a mitogen for endothelial cells that can similar to that of VEGF (3, 5). However, whereas all VEGF potentiate the growth and permeabilizing effects on endothelium of vas- 165 isoforms bind to the tyrosine kinase receptors flt-1 and KDR/flk-1, cular endothelial growth factor. Here we report that hypoxia induces the PlGF homodimers are believed to bind only to flt-1 (see Ref. 6), expression of both PlGF mRNA and protein in immortalized/transformed mouse embryonic fibroblasts (mEFs) and in NIH 3T3 cells. Importantly, suggesting a role for the homodimers in endothelial cell-cell or the magnitude of the induction of PlGF expression by hypoxia is enhanced cell-matrix interactions (7). PlGF may also potentiate the angiogenic by the presence of oncogenic Ras. To investigate the transcriptional effects of VEGF (8) because the two proteins have the ability to form component of hypoxia-inducible PlGF expression, we cloned and se- heterodimers with each other (2, 9–11) that can activate the KDR/ quenced a 1350-bp fragment of the 5؅-flanking region of the mouse gene. flk-1 receptor (9). Like VEGF, PlGF-2 has also been shown to bind to Analysis of the promoter region indicated the presence of putative con- and activate neuropilin-1 (6, 12), a receptor found on many cell types sensus sequences for known hypoxia-responsive regulatory sites, including in addition to endothelial cells. This receptor can bind at least five metal response elements and Sp1-like sites. In the present study, we show different ligands including VEGF165 and has established roles in a that the induction of PlGF expression by hypoxia is dependent on the wide variety of cellular processes including axonal guidance (13). presence of the metal response element-binding transcription factor 1 These findings suggest that PlGF is a component of extensive com- (MTF-1). Thus, in mEFs with targeted deletions of both MTF-1 alleles, binatorial interactions involving the VEGF superfamily. hypoxia-induced increases of PlGF mRNA and protein levels were greatly In contrast to the widespread distribution of VEGF, PlGF expres- attenuated compared with those in wild-type mEFs. Moreover, transient transfection of a PlGF promoter reporter gene into NIH 3T3 cells resulted sion was originally considered to be restricted to cell types of placen- in hypoxia-responsive transcriptional activation of the reporter. Finally, tal origin such as primary cytotrophoblasts and in vitro differentiated ectopic expression of MTF-1 resulted in increased basal transcriptional syncytiotrophoblasts (e.g., see Ref. 14). However, accumulating evi- activity of a PlGF promoter reporter. Together, these findings demon- dence indicates that PlGF is synthesized in many nonplacental cells strate that the PlGF gene is responsive to hypoxia and that this response and other tissues including human thyroid, brain, lung, and skeletal is mediated by MTF-1. It remains to be determined whether this activa- muscle (reviewed in Ref. 15). PlGF is also highly expressed in dermal tion is the result of direct and/or indirect transcriptional activation by microvascular endothelial cells and retinal pericytes (16), whereas MTF-1. The stimulatory effect of oncogenic Ras on the induction of PlGF conditioned medium from cultured human keratinocytes was found to expression in hypoxic cells suggests that PlGF could be an important contain both PlGF homodimers and PlGF/VEGF heterodimers (11). It proangiogenic factor in the tumor microenvironment. has also been reported that migrating keratinocytes from healing wounds contain significant levels of PlGF from the third to the seventh day after injury (11). Other studies indicate that PlGF may be INTRODUCTION involved in the inflammatory process because PlGF homodimers and PlGF4 is a member of the VEGF family of proangiogenic factors PlGF/VEGF heterodimers were detected in the synovial fluid of (1). Both PlGF and VEGF form glycosylated homodimers that share patients with inflammatory arthopathies (17). PlGF is also expressed significant homology at the amino acid level within their platelet- in several types of solid tumors (18–21). Furthermore, PlGF is be- derived growth factor-like regions, including the conservation of eight lieved to be involved in the pathogenesis of PDR and other ischemic cysteine residues involved in intra- and interchain disulfide bond retinal diseases (22–24). formation (1, 2). hPlGF is also similar to VEGF (and platelet-derived Oxygen deprivation (hypoxia) appears to be a common feature of growth factor) in that alternative splicing of the mRNA from a single many of the pathophysiological conditions in which PLGF is ex- copy gene produces different isoforms of the protein [PlGF-1, PlGF-2, pressed. For example, hypoxia is a characteristic common to many solid tumors and is believed to contribute to increased expression of and PlGF-3 (3, 4)]. PlGF-2, or PlGF170, differs from the other two proangiogenic proteins (e.g., VEGF) and subsequent activation of angiogenesis (25, 26). However, the role of hypoxia in the regulation Received 9/7/00; accepted 1/12/01. The costs of publication of this article were defrayed in part by the payment of page of PlGF expression is currently unclear because the majority of cell charges. This article must therefore be hereby marked advertisement in accordance with lines used to examine its expression appear to be unresponsive to 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NCI Grants CA57692 (to B. J. M.) and CA73807 and CA67166 (to oxygen deprivation (10, 16, 27, 28). The findings presented here K. R. L.). demonstrate that immortalized/transformed fibroblasts express PlGF 2 C. J. G. and P. L. contributed equally to these studies. and that exposure of these cells to physiologically relevant levels of 3 To whom requests for reprints should be addressed, at The Pharmaceutical Discovery Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025. Phone: hypoxia results in significant inductions of the steady-state levels of (650) 859-4213; Fax: (650) 859-5816; E-mail: [email protected]. both PlGF mRNA and protein. We also show that this activation is 4 The abbreviations used are: PlGF, placental growth factor; hPlGF, human PlGF; mPlGF, mouse PlGF; HRE, hypoxia response element; MRE, metal response element; dependent on the presence of the redox-sensitive transcription factor MT, metallothionein; MTF-1, metal response element-binding transcription factor 1; MTF-1 (29, 30) and that the hypoxia-inducible increase in PlGF PDR, proliferative diabetic retinopathy; RT-PCR, reverse transcription-PCR; VEGF, vascular endothelial growth factor; mEF, mouse embryonic fibroblast; Tag, T antigen; expression is due at least in part to increased transcription of the PlGF CMV, cytomegalovirus. gene, particularly in the presence of oncogenic ras. 2696

MATERIALS AND METHODS been cleaved with a variety of restriction enzymes and ligated with linkers containing primer sites for PCR amplification. Two nested PCR primers Cell Culture and Hypoxia. All cell cultures, including mEFs and NIH complementary to the 5Ј-end of the longest known mouse cDNA for PlGF 3T3, JAR (a human choriocarcinoma cell line), and HepG2 (a human hepa- (GenBank accession number X96793) were used. The two primers were toma cell line) cells, were maintained in DMEM containing 10% dialyzed fetal complementary to bases 268–291 and 295–318 of this cDNA sequence. Only bovine serum. Both SV40 large TAg and TAg/Ras-transformed wild-type one 500-bp fragment was obtained using this kit. Because only slightly more ϩ/ϩ Ϫ/Ϫ (MTF-1 ) and MTF-1 null (MTF-1 ) mEFs were used in these studies. To than 200 bp of this sequence were from the upstream region of the known obtain the mEFs, primary embryonic fibroblasts were isolated from 12.5-day- mPlGF cDNA, it was considered unlikely that this fragment would contain all old mouse embryos using standard techniques (31). PCR genotyping for of the necessary regulatory elements to function as a promoter in reporter gene MTF-1 null cells was performed using genomic DNA prepared from yolk sacs studies. Therefore, inverse PCR was used to clone a larger sequence of the and early passages of the primary cells. Wild-type and MTF-1 null primary mPlGF gene. Mouse genomic DNA (1 ␮g) was cleaved with BamHI, EcoRI, cells were then transfected with either 10 ␮g of plasmid CMV-TAg or 10 ␮g HindIII, KpnI, SstI, or XbaI. Each of the cleaved DNA preparations was of plasmid CMV-TAg and 10 ␮g of plasmid c-H-ras(A) (32) per 100-mm- dissolved in 0.5 ml of 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2,20mM DTT, diameter cell culture plate. The plasmid CMV-TAg directs expression of TAg, and1mM ATP, and then 2 units of T4 DNA ligase (Life Technologies, Inc., and c-H-ras(A) directs expression of oncogenic human H-ras. Cell foci were Rockville, MD) were added. The ligation reactions were then incubated at 5°C isolated, and immortalized cell lines were derived. All cell lines were geno- for 16 h. This low DNA concentration and low temperature ligation procedure typed again by PCR analysis for the presence of the MTF-1 wild-type and null favors the formation of circularized products. Each ligated DNA sample was alleles, as well as for genomic integration of TAg and oncogenic H-ras. precipitated with 3 volumes of ethanol, dried, and dissolved in 10 ␮l of water. For immunoblotting analyses, medium was removed, cells were washed The longest known sequence of the mouse cDNA for PlGF described above with PBS, and then fresh DMEM without fetal bovine serum was added was used to design the following two PCR primers: (a)5Ј-GAAGTCTGAA- immediately before hypoxia treatment. Our methods for exposing cells to GATGCGGTTTCCTTC-3Ј; and (b)5Ј-CACTCCTCACCAGTTTCCTCAG- Յ hypoxia (pO2 0.1% relative to air at pO2 of approximately 21%) have been CCA-3Ј. The first primer is complementary to positions 5–29 of the PlGF described in detail elsewhere (33). Other levels of hypoxia were achieved by cDNA sequence, and the second primer is the same as the sequence at positions continuous flow-though of specific levels of oxygen and 5% CO2 at 37°C, 101–125. Because these primers would amplify DNA in opposite directions, using a glass vacuum desiccator (34). After exposure to hypoxia, an analysis they are able to produce a fragment of the upstream PlGF promoter from the for cell viability using Alamar Blue indicated that the cells were not adversely circularized mouse genomic DNA. One-tenth (100 ng) of each of the five affected by these treatments. preparations of circularized mouse genomic DNA was used as the template for RT-PCR and Northern Analyses. Two DNA primers for RT-PCR were a PCR reaction using the Advantage 2 PCR Enzyme System (Clontech). In synthesized based on the mPlGF cDNA sequence (GenBank accession number addition to the DNA template, each PCR reaction contained 100 ␮lofAd- X80171; Ref. 35). The sequences of the sense and antisense primers were, vantage 2 PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 1 ␮lof respectively, 5Ј-TTTCTCAGGATGTGCTCTGTGAA-3Ј and 5Ј-CCTGGT- Advantage 2 polymerase mix, and 1 ␮M each of the two primers mentioned TACCTCCGGGAAATGAC-3Ј. These primers were designed to amplify a above. The reactions were amplified with a Perkin-Elmer GeneAmp PCR sequence spanning exons 4–7 (cDNA bases 473–619) of the PlGF gene, System 2400 using 35 PCR cycles (30 s at 94°C, 180 s at 72°C). After gel producing a 147-bp cDNA fragment by RT-PCR amplification of mPlGF electrophoresis, it was determined that only the HindIII- and SstI-cut circular- mRNA. This sequence was chosen to detect any mouse homologues of the ized genomic DNA fragments produced PCR products: a 3.0- and a 1.5-kb known human splice variants of PlGF mRNA, if such homologues were product, respectively. Because the known mouse cDNA sequence for PlGF has present in the mEF cells. One control without RNA and four RNA samples an SstI site in the 5Ј-untranslated region, most of this SstI inverse PCR containing 250 ng of total RNA each from aerobic and hypoxic wild-type fragment contained sequences upstream of the known cDNA. The 1.5-kb PCR mEFs (4 and8hofhypoxia) were used for RT-PCR analysis. A GeneAmp product from the SstI-cut circularized genomic DNA was cloned into the Srf I recombinant Thermus thermophilus Reverse Transcriptase kit (Perkin-Elmer site of pPCR-Script Amp SK(ϩ) (Stratagene, San Diego, CA), and this Applied Biosystems, Foster City, CA) was used according to the manufactur- template was sequenced from both strands using a PE Applied Biosystems er’s protocol. Thirty-five cycles each of 10 s at 95°C and 15 s at 60°C were Prism 310 Genetic Analyzer. Two separate clones were used for the sequenc- performed with a Perkin-Elmer GeneAmp System 2400, followed by a final ing, and they proved to be identical. The 3Ј-end of this sequence matched the incubation at 60°C for 7 min. The reaction products, along with DNA molec- 200-bp of promoter sequence found by using the Mouse GenomeWalker Kit ular weight markers of 517 to 75 bp, were resolved by nondenaturing 5% described above. The orientation of the pPCR-Script clones allowed the use of PAGE in Tris-borate EDTA buffer. The gel was stained with ethidium bro- the SstI site on the vector that was 3Ј to the insert and the SstI site on the 5Ј-end mide, and bands were visualized on an UV transilluminator. of the promoter fragment to cut out a 1.5-kb fragment. This SstI fragment was Our procedure for Northern analysis is described elsewhere (36); here, next cloned into the SstI site of the luciferase-containing pGL3-Basic reporter however, total RNA was isolated from cells by using a RNasy kit (Qiagen vector (Promega, Madison, WI), and the correct orientation was verified by Corp., Valencia, CA). Blots were probed with either an EcoRI/NotI (1.1 kb) restriction analysis. This construct was designated as pmPlGF(1.5kb)-Luc. The fragment of the mPlGF cDNA (Genome Systems, Mountain View, CA) or a promoter fragment in this clone corresponds to positions 1–1386 of the mPlGF PCR-generated PlGF cDNA fragment. To label the PlGF probe, we followed gene (Fig. 4A). the amplification reaction protocol described above, but with only the antisense Cell Transfections and Treatments. NIH 3T3 cells were plated at 5 ϫ 104 primer and [32P]dCTP and the other unlabeled deoxynucleotide triphosphates. cells/35-mm-diameter culture dish and grown to approximately 60–70% con- Signals were quantified by video densitometry using a Lynx 4000 image fluence before transfection using the Effectene Transfection Reagent (Qiagen) analyzer (Applied Imaging, Sunnyvale, CA). according to the manufacturer’s protocols. To determine the effect of hypoxia Immunoblotting Analysis. After treatment with hypoxia, conditioned me- on PlGF transcription, the cells were transfected with 0.4 ␮gof dia from the mEF cultures were concentrated approximately 14-fold by using pmPlGF(1.5kb)-Luc/35-mm-diameter plate. To investigate the effect of exog- Centraprep ultrafiltration units (Millipore Corp., Bedford, MA). Equivalent enous MTF-1 expression on basal PlGF transcription, the cells were cotrans- volumes (20 ␮l) of each concentrated sample in standard SDS loading buffer fected with pmPlGF(1.5kb)-Luc and varying amounts of a murine MTF-1 were analyzed by 12% SDS-PAGE. Blots were probed with a primary goat expression vector, CMV-MTF-1, which was created by inserting the mMTF-1 anti-mPlGF-2 antibody (R&D Systems, Minneapolis, MN), followed by an cDNA into the NotI site of a CMV expression vector (kindly provided by Dr. antigoat IgG horseradish peroxidase-conjugated secondary antibody (Dako, Glen Andrews; University of Kansas, Kansas City, MO). All transfections Carpenteria, CA). PlGF protein was detected using an ECL-PLUS Enhanced were normalized by cotransfection with a Renilla luciferase control vector Chemiluminescence detection system according to the manufacturer’s protocol (Dual-Luciferase Reporter Assay System; Promega). Where appropriate, a (Amersham Life Science, Arlington Heights, IL). Bluescript phagemid (Stratagene) was used to ensure equal administration of Inverse PCR of the PlGF Gene Promoter Region. Our initial attempt to total DNA. Cells were lysed, and luciferase activity was assayed by using a clone the promoter region of mPlGF used a Mouse GenomeWalker Kit Promega Biotec assay kit and a Turner Designs TD-20/20 luminometer (Turn- (Clontech, Palo Alto, CA). This kit supplies mouse genomic DNA that has er Designs, Inc., Sunnyvale, CA). 2697

RNA was extracted from both aerobic and hypoxic wild-type mEFs and subjected to RT-PCR analysis, as described in “Materials and Methods.” The primers were selected to amplify sequences coding for a stretch of basic amino acid residues in exon 6 that confer the heparin binding ability of mPlGF (35) and hPlGF-2 (5) proteins. The hPlGF-1 and hPlGF-3 transcripts are produced by alternative splicing that removes the region coding for the basic amino acid residues respon- sible for heparin binding. In the case of hPlGF-1, this change shortens the coding region by 63 bases compared to that of hPlGF-2. The PlGF-3 transcript lacks this region but contains an additional 216-bp exon not found in the other forms (3). The RT-PCR primers were designed to detect all three of these known PlGF isoforms. Specifi- cally, the primers were chosen to amplify a 147-bp fragment from the known mPlGF mRNA, a smaller 84-bp fragment corresponding to a homologue of hPlGF-1, and a 300-bp fragment corresponding to a homologue of hPlGF-3. This study showed that mouse fibroblasts express only one PlGF mRNA species, equivalent to hPlGF-2 (Fig. 1). The control lane (0; no RNA) in Fig. 1 shows no band, whereas the other lanes [Air and hypoxia (Hx)] contain a single band of the expected size (approximately 150 bp), corresponding to the size of the hPlGF-2 transcript. No bands corresponding to mouse homologues of hPlGF-1 or hPlGF-3 transcripts were detected. Hypoxia Causes Increased PlGF mRNA Levels through a Mechanism Involving MTF-1 and Ras. Fig. 2A (top panel) shows a representative Northern blot of PlGF mRNA from H-ras-transformed wild-type and MTF-1 null mEFs that were subjected to hypoxia Յ (pO2 0.1%; 2–16 h). The figure clearly shows that mPlGF mRNA Fig. 1. RT-PCR analysis of PlGF expression in mEFs. Total RNA from wild-type mEFs grown under aerobic and hypoxic conditions was used for RT-PCR analysis to levels were significantly increased in the wild-type mEFs compared determine whether more than one isoform of PlGF is expressed by these cells. Primers with the MTF-1 null mEFs by4hofhypoxia, with the greatest were designed to produce amplicons within exons 4–7 of the mouse gene, thus enabling accumulation occurring at 16 h of stress. Fold inductions of PlGF the detection of all potential mouse homologues of the known hPlGF isoforms. For details, see “Materials and Methods.” mRNA accumulations could not be reliably determined because the aerobic steady-state levels of the mRNA were barely detectable. We also observed increases in PlGF mRNA levels over a wide range of RESULTS Յ low pO2 levels ( 0.1% to 2.0%) in the primary mEFs and in NIH 3T3 Basal Expression of PlGF in Mouse Fibroblasts. During a cDNA fibroblasts (data not shown). An analysis of two human cell lines microarray analysis of hypoxia-responsive gene expression in wild- (HepG2 and JAR) in which hypoxia was reported to have no effect on type and MTF-1 null mEFs, we found that they express PlGF mRNA PlGF mRNA levels (e.g., see Ref. 10) was also included in these Ϸ and that the PlGF gene is responsive to hypoxia in a MTF-1-depend- studies. As predicted from this earlier work, neither mild (pO2 Յ ent manner. Three PlGF forms are expressed in humans (PlGF-1, 1–2%) nor severe (pO2 0.1% O2) hypoxic exposures affected PlGF PlGF-2, and PlGF-3) (3), but only one isoform has been reported for message accumulation in these experiments (data not shown). North- the mouse (35). Therefore, we used conventional RT-PCR analysis to ern analysis also showed that the induction of PlGF mRNA accumu- determine how many PlGF isoforms are expressed by mEFs. Total lation by hypoxia was strongly attenuated in the MTF-1 null mEFs

Fig. 2. Effects of hypoxia and oncogenic ras on PlGF mRNA accumulation in wild-type and MTF-1 null mEFs (MTF-1ϩ/ϩ and MTF-1Ϫ/Ϫ, respectively). A,H-ras-transformed Յ 32 mEFs were exposed to hypoxia (pO2 0.1%) for 0–16 h, and PlGF mRNA steady-state levels were detected by Northern blotting and probing with P-labeled cDNA probes for either mPlGF or VEGF (see “Materials and Methods”). The representative blot shown here was reprobed for ␤-actin mRNA. Lanes 1 and 6, aerobic controls; Lanes 2 and 7,2hofhypoxia; Յ Յ Lanes 3 and 8,4hofhypoxia; Lanes 4 and 9,8hofhypoxia; Lanes 5 and 10, 16 h of hypoxia. B, TAg-immortalized mEFs were exposed to hypoxia (pO2 0.1% or 0.01%) for Յ either 8 or 16 h and then assayed for PlGF mRNA levels as described above. Lanes 1 and 6, aerobic controls; Lanes 2 and 7,8hofhypoxia at 0.1% O2; Lanes 3 and 8,16hof Յ Յ Յ hypoxia at 0.1% O2; Lanes 4 and 9,8hofhypoxia at 0.01% O2; Lanes 5 and 10, 16 h of hypoxia at 0.01% O2. 2698

additional sequences extending to the translation start codon) and indicates some possible transcription factor binding sites. Putative sites in the 5Ј-region flanking region of the gene include four sequences corresponding to a MTF-1 binding site (MRE) and at least five Sp1-like binding sites. Consensus sequences corresponding to AP-2 and tandem retinoid X receptor/peroxisome proliferator activated receptor-␦ binding sites are also indicated in the figure. A 203-kb region of human chromosome 14 has recently been sequenced that contains the gene for PlGF (GenBank accession number AC006530). The region containing the promoter of the hPlGF Fig. 3. Hypoxia regulates PlGF protein expression in a MTF-1-dependent manner. gene is shown in Fig. 4B. A comparison of the human and mPlGF After hypoxia (Hx) for 8 and 16 h, conditioned media from wild-type and MTF-1 null promoter regions shows both similarities and differences. For exam- mEF cultures were concentrated, and protein levels were determined by standard immu- ple, both promoters have two closely spaced Sp1-like sequences that noblotting and chemiluminescence antibody detection. Arrows, MTF-1 protein and non- specific (N.S.) bands. are inverted relative to each other at about 400 bp upstream from the translation start codon. The hPlGF promoter also has four 9-bp repeats (CTGCGGGCT) within a 100-bp-region approximately 500 (Fig. 2A, top panel). However, there was a residual induction of PlGF bp upstream from the translation initiation site. Two of these repeats mRNA accumulation in these cells, indicating other, less stimulatory are part of a 14-bp sequence (GGCGCTGCGGGCTG). It is not levels of control of the PlGF gene in hypoxic mEFs. Finally, the known whether these sequences are relevant for the transcriptional accumulation of VEGF mRNA in both MTF-1 wild-type and knock- control of hPlGF, but they do have similarity to the core consensus out mEFs (Fig. 2A, middle panel) was not significantly different, sequence for the MRE, TGCRCNC (37, 38). This fragment of the indicating that MTF-1 does not regulate the hypoxic response of this human promoter contains five other possible MREs, of which two functionally related gene. tandem copies (positions 500–521) display perfect MRE core con- Fig. 2B shows a representative Northern blot from a similar study sensus sequences in addition to G/C-rich 3Ј-flanking regions. Another using TAg-immortalized wild-type and MTF-1 mEFs. In this study, putative MRE overlaps with a possible HRE at positions 279–290 (no Յ cells were subjected to two different levels of hypoxia (pO2 0.1% consensus HRE was obvious in the mPlGF proximal promoter). Յ and pO2 0.01%) for 8- and 16-h exposures. Although significant MTF-1 Is Involved in the Transcriptional Regulation of the inductions of PlGF mRNA levels were observed in the wild-type TAg PlGF Gene. To define the molecular basis of the hypoxia-associated mEFs, the detectable levels were much lower and sustained over a expression of the PlGF gene, we examined whether the increase in shorter period than those found in the corresponding wild-type mEFs steady-state levels of PlGF mRNA in hypoxic mEFs is dependent on transformed with a ras oncogene (see Fig. 2, A and B, top panels). transcriptional activation. We cloned the mPlGF promoter fragment This important finding suggests that hypoxia and oncogenic Ras can into a pGL3 reporter vector [pmPlGF(1.5 kb)-Luc; see “Materials and cooperate to induce PlGF expression mediated by MTF-1. A similar Methods”] and determined its responsiveness to hypoxia. The PlGF dependence involving oncogenic Ras was observed in the hypoxia- promoter reporter construct was transiently transfected into NIH 3T3 Յ associated activation of the mouse metallothionein-I gene (data not cells, and the cells were exposed to hypoxia (pO2 0.01% for 16 h) shown). We have demonstrated elsewhere that two isoforms of MT before lysis to determine luciferase activity. Fig. 5 shows that this are transcriptionally activated by hypoxia through a MTF-1-depend- PlGF promoter reporter construct was significantly responsive to ent mechanism (30). hypoxia (enhancements in luciferase activities relative to the aerobic Hypoxia Induces PlGF Protein Expression in a MTF-1-depend- controls were 4.5 Ϯ 2.3; n ϭ 4), indicating the presence of one or ent Manner. To determine whether the hypoxia-associated increases more HREs. in PlGF mRNA correspond to PlGF protein inductions, conditioned Finally, the involvement of MTF-1 in the regulation of PlGF media from H-ras-transformed wild-type and MTF-1 null mEFs sub- expression was confirmed in studies using MTF-1 null mEFs that Յ jected to 8 and 16 h of hypoxia (pO2 0.1% O2) were analyzed for were cotransfected with pmPlGF(1.5 kb)-Luc and varying amounts of PlGF protein levels by standard immunoblotting procedures. Fig. 3 a mMTF-1 cDNA expression vector (see “Materials and Methods”). shows a representative immunoblot of PlGF protein secreted from The basal transcriptional activities of the PlGF promoter reporter these aerobic and hypoxic cultures. Secreted PlGF protein was unde- construct were significantly enhanced by ectopic expression of tectable in aerobic wild-type cells but was found in the hypoxic cell mMFT-1, with maximum increases of approximately 13-fold at 0.025 media with a strong induction by 16 h of hypoxia. In agreement with ␮g of pmMTF-1 (Fig. 6). the Northern analyses, the relative induction of secreted PlGF protein by hypoxia was attenuated in the MTF-1 null mEFs but was not DISCUSSION completely blocked. Again, a similar result was found for mouse MT-1 protein (30). Hypoxia is established as an important environmental stimulus of Isolation and Characterization of the mPlGF Promoter Region. angiogenesis in various pathological conditions, such as solid tumor We successfully cloned and sequenced a genomic fragment of the development, some inflammatory states, and PDR. The induction of 5Ј-flanking region of the mPlGF gene (GenBank accession number VEGF in response to hypoxia is an important mediator of angiogen- AF2855629) by using inverse PCR amplification, as described above. esis in these pathologies (39, 40). The expression of other components This fragment contains 1357 bp upstream of the largest known mPlGF of the proangiogenic response, such as VEGF receptors (flt-1 and cDNA sequence. The exact site(s) of transcriptional initiation is KDR/flk-1) and angiopoietin-2, is also induced by hypoxia in various presently under investigation. A structural analysis of the promoter cells or tissues (7, 41–43). The present study extends these findings to region was performed in an effort to detect putative MRE consensus show that the PlGF gene, a member of the VEGF family of proan- sequences or other regulatory sites that may explain the gene’s re- giogenic factors, can also be activated by hypoxia. Specifically, we sponsiveness to hypoxia in wild-type but not MTF-1 null mEFs. Fig. have demonstrated for the first time that immortalized/transformed 4A shows the primary structure of the 5Ј-region of the gene (including fibroblasts (mEFs) express PlGF and that hypoxia causes increased 2699

Fig. 4. Nucleotide sequence of the 5Ј-flanking region of the mouse (A) and human (B) PlGF genes. Nucleotides are numbered relative to the SstI site of the mPlGF gene. The ATG translation start codon and the longest reported mPlGF cDNA are indicated. Putative MRE, Sp1, Sp2, and HRE sequences are underlined. In addition, MRE-like repeats are indicated in the sequence of hPlGF.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. PlGF INDUCTION BY HYPOXIA IS MTF-1 DEPENDENT expression of the PlGF gene in these cells by either a direct or indirect mechanism involving the transcription factor MTF-1. We have also shown that the induction of VEGF by hypoxia is not dependent on the presence of MTF-1 (Fig. 2). As discussed below, this induction of PlGF by hypoxia is potentiated by a ras oncogene. Although PlGF expression has been considered to occur primarily in tissues composed of normal rather than transformed cells, several reports describe its expression in solid tumors, including hypervascu- lar renal cell carcinomas (21), cervical squamous cell carcinomas (19), and some brain tumors (18, 20). PlGF expression has also been detected in a several established human tumor cell lines, including trophoblast choriocarcinoma cells (e.g., BeWo, JAR, and JE-3; see Refs. 10 and 27), HepG2 hepatoma cells (10), and U-25/MG glioma cells (20) and in melanoma cell cultures (44). Our study demonstrates Fig. 6. Ectopic expression of MTF-1 increases basal transcriptional activity of that H-ras-transformed mEFs express at least one isoform of PlGF pPlGF(1.5 kb)-Luc. To determine the effect of ectopic expression of the metal transcription factor MTF-1 on the proximal promoter region of the mPlGF gene, varying amounts (PlGF-2). Studies of the effect of hypoxia on PlGF expression have of a CMV-mMTF-1 vector (0–0.1 ␮g) were cotransfected with pPlGF(1.5 kb)-Luc (0.35 Ϸ ␮ yielded different results. For example, moderate hypoxia (pO2 g) in MTF-1 null mEFs. After a 24–36-h recovery period, cells were lysed and assayed 1–2%) was found to have no stimulatory effect on PlGF mRNA levels for luciferase activity. The data represent the means of three independent experiments. Error bars, SD of the mean. in a variety of cell types, including choriocarcinoma cells, HepG2 cells, dermal microvascular endothelial cells, retinal pericytes (9, 16, 27, 28), and cells in human placental tissue (28). In contrast, PlGF-1 anism of MTF-1 involvement in the activation of PlGF by hypoxia are and PlGF-2 mRNA levels were found to be induced in hypoxic ongoing. In the context of oncogenesis, our findings suggest a role for U-25/MG brain meningioma cells (20). Induced PlGF protein expres- activated ras in the transmission of hypoxic signals to MTF-1 with sion has also been observed in ischemic retinal diseases [PDR, retinal subsequent activation of PlGF expression. Interestingly, oncogenic vein occlusion, and acute retinal necrosis (22, 24)] and at sites of ras can also cooperate with hypoxia to induce VEGF expression (45, wound healing (11) and inflammation (17), both of which may in- 46), apparently through a mechanism that does not involve down- volve hypoxic stress. Taken with our finding of strong hypoxia- stream activation of MTF-1 (see “Results”). inducible PlGF expression in immortalized/transformed mEFs, these To our knowledge, this report is the first to show the sequence of other reports suggest that the response of the PlGF gene to hypoxia is the mPlGF proximal promoter region and the first sequence exami- not only cell- or tissue-type specific but is also susceptible to onco- nation of the hPlGF promoter. The cloned fragment of the mPlGF genic stimuli. promoter region contains 5Ј-sequences from the longest known mouse Using mEFs with targeted deletions of both MTF-1 alleles, we cDNA (GenBank accession number X96793), although the transcrip- observed that the hypoxia-inducible expression of PlGF is dependent tion start site remains undefined. An analysis of this longest cDNA on the presence of the MTF-1 transcription factor. Consistent with this fragment upstream from the translation start site shows that the GC finding, the PlGF promoter reporter studies described here indicate content (57.8%) is only slightly enriched, and the CpG/GpC dinucle- that hypoxia-inducible PlGF expression is controlled at least in part at otide ratio (0.35) is well below what would be expected for a random the transcriptional level. The residual hypoxic response of the PlGF sequence. These findings are compatible with the idea that the PlGF gene detected by both the Northern and immunoblotting analysis may gene is not a housekeeping gene but is probably highly regulated be attributed to the contribution of other transcription factors and/or because both high GC content and the lack of CpG dinucleotide posttranscriptional regulation. Studies to determine the precise mech- suppression are considered common features of constitutively active promoters (47). A computer search for transcription factor binding sites identified four putative MRE consensus sequences, three of which are clustered within a 200-bp region from positions 1000 to approximately 1200 (Fig. 4A). Two of these sites are within 15 bp of each other and just downstream from a putative Sp1 sequence. MREs are highly conserved 13–15-bp sequences that contain the heptanucle- otide core consensus TGCRCNC and a partially overlapping, less conserved, GC-rich 3Ј-flanking region (37, 38). All four sites have almost perfect consensus core sequences with varying degrees of similarity in the flanking GC-rich regions. The hPlGF promoter contains at least five putative MREs (Fig. 4B). Two perfect matches for the Sp1 core consensus site (GGGCGG) were found within 25 bp of each other in the proximal region of the mouse promoter. These two elements may be functionally important because they are also conserved in the human gene at approximately the same distance from the translation start site. Three other possible Sp1-like motifs (GGGC/T/ AGG) are also present. Members of the Sp family of transcription factors are regulated by hypoxic stress though a mechanism involving relief of Sp3 repression of Sp1 (48). It is worth noting that both the Fig. 5. PlGF is transcriptionally activated by hypoxia exposure. The pPlGF(1.5 human MT-IIA and mouse MT-I promoters also harbor Sp1 elements kb)-Luc construct was transiently transfected into NIH 3T3 cells that were subsequently near their MREs (see Ref. 30). The possible regulatory roles of both Յ incubated in 5% CO2:air or hypoxia (pO2 0.1%) for up to 16 h. The data represent the average mean value from three independent experiments of the arbitrary luciferase activity the putative MRE and Sp1 sequences in the transcriptional activation levels in extracts of aerobic and hypoxic cells. Error bars, SD of the mean. of PlGF by hypoxia are currently under investigation. It is possible 2701

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. PlGF INDUCTION BY HYPOXIA IS MTF-1 DEPENDENT that MTF-1 activates PlGF by an indirect mechanism involving other 9. Cao, Y., Chen, H., Zhou, L., Chiang, M. K., Anand-Apte, B., Weatherbee, J. A., downstream targets of MTF-1. For example, we have recently ob- Wang, Y., Fang, F., Flanagan, J. G., and Tsang, M. L. Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell served that MTF-1 regulates the expression of the transcription factor expression, and high affinity binding to Flk-1/KDR. J. Biol. Chem., 271: 3154–3162, CCAAT enhancer-binding protein ␣.5 1996. 10. Cao, Y., Linden, P., Shima, D., Browne, F., and Folkman, J. In vivo angiogenic Sequence analysis also indicated that the cloned mPlGF proximal activity and hypoxia induction of heterodimers of placenta growth factor/vascular promoter fragment does not contain consensus sequences for the endothelial growth factor. J. Clin. Investig., 98: 2507–2511, 1996. HIF-1 transcription factor (RCGTG). The hypoxia inducible factor-1 11. Failla, C. M., Odorisio, T., Cianfarani, F., Schietroma, C., Puddu, P., and Zambruno, G. Placenta growth factor is induced in human keratinocytes during wound healing. heterodimer is considered the major transcription factor affecting a J. Investig. Dermatol., 115: 388–395, 2000. wide range of hypoxia-responsive genes, including VEGF, erythopoi- 12. Gluzman-Poltorak, Z., Cohen, T., Herzog, Y., and Neufeld, G. Neuropilin-2 and etin, heme oxygenase-1, tyrosine hydroxylase, the urokinase receptor, neuropilin-1 are receptors for the 165-amino acid form of vascular endothelial growth factor (VEGF) and of placenta growth factor-2, but only neuropilin-2 functions as a nitric oxide synthase, and numerous glycolytic enzymes (e.g., see Ref. receptor for the 145-amino acid form of VEGF. J. Biol. Chem., 275: 18040–18045, 49). However, other transcription factors, including AP-1, CCAAT 2000. ␤ ␬ 13. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., and Klagsbrun, M. Neuropilin-1 enhancer-binding protein , nuclear factor B, and early growth is expressed by endothelial and tumor cells as an isoform-specific receptor for response factor-1, have also been implicated in the cellular response vascular endothelial growth factor. Cell, 92: 735–745, 1998. to hypoxia (see Ref. 33). Taken with our previous work, the present 14. Shore, V. H., Wang, T. H., Wang, C. L., Torry, R. J., Caudle, M. R., and Torry, D. S. Vascular endothelial growth factor, placenta growth factor, and their receptors in study confirms that MTF-1 is another hypoxia-responsive transcrip- isolated human trophoblast. Placenta, 18: 657–665, 1997. tion factor. Furthermore, we hypothesize that MTF-1 regulates a 15. Persico, M. G., Vincenti, V., and DiPalma, T. Structure, expression, and receptor- subset of hypoxic stress proteins that could be important for the binding properties of placenta growth factor (PlGF). Curr. Top. Microbiol. Immunol., 237: 31–40, 1999. adaptive response of transformed as well as normal cells to this stress. 16. Yonekura, H., Sakurai, S., Liu, X., Migita, H., Wang, H., Yamagishi, S., Nomura, M., For example, we demonstrated that MTF-1 is required for the hy- Abedin, M. J., Unoki, H., Yamamoto, Y., and Yamamoto, H. Placenta growth factor poxia-inducible expression of the human MT-IIA and mouse MT-I and vascular endothelial growth factor B and C expression in microvascular endothelial cells and pericytes. Implication in autocrine and paracrine regulation of 2ϩ genes, critical determinants of Zn homeostasis in mammalian cells angiogenesis. J. Biol. Chem., 274: 35172–35178, 1999. (30). We postulated that MTF-1 is a redox-sensitive regulatory protein 17. Bottomley, M. J., Webb, N. J., Watson, C. J., Holt, L., Bukhari, M., Denton, J., Freemont, A. J., and Brenchley, P. E. Placenta growth factor (PlGF) induces vascular that influences malignant progression through control of MT expres- endothelial growth factor (VEGF) secretion from mononuclear cells and is co- sion and that of other known MTF-1 targets such as the hypoxia- expressed with VEGF in synovial fluid. Clin. Exp. Immunol., 119: 182–188, 2000. responsive gene ␥-glutamylcysteine synthase in the tumor microenvi- 18. Donnini, S., Machein, M. R., Plate, K. H., and Weich, H. A. Expression and localization of placenta growth factor and PlGF receptors in human meningiomas. ronment (30, 31). The findings presented here suggest that MTF-1 J. Pathol., 189: 66–71, 1999. may also be an important regulator of tumor angiogenesis through its 19. Kodama, J., Seki, N., Tokumo, K., Nakanishi, Y., Yoshinouchi, M., and Kudo, T. control of hypoxia-inducible PlGF expression. Placenta growth factor is abundantly expressed in human cervical squamous cell carcinoma. Eur. J. Gynaecol. Oncol., 18: 508–510, 1997. In summary, we have shown that immortalized/transformed mEFs 20. Nomura, M., Yamagishi, S., Harada, S., Yamashima, T., Yamashita, J., and express PlGF and that exposure of these cells to either mild or severe Yamamoto, H. Placenta growth factor (PlGF) mRNA expression in brain tumors. J. Neurooncol., 40: 123–130, 1998. hypoxia results in transcriptional activation of the PlGF gene. These 21. Takahashi, A., Sasaki, H., Kim, S. J., Tobisu, K., Kakizoe, T., Tsukamoto, T., findings imply that PlGF, a member of the VEGF family of proan- Kumamoto, Y., Sugimura, T., and Terada, M. Markedly increased amounts of giogenic factors, participates in the stimulation of tumor angiogenesis. messenger RNAs for vascular endothelial growth factor and placenta growth factor in renal cell carcinoma associated with angiogenesis. Cancer Res., 54: 4233–4237, Furthermore, we demonstrate that the hypoxia-inducible expression of 1994. PlGF is dependent on the presence of the MTF-1 transcription factor 22. Khaliq, A., Foreman, D., Ahmed, A., Weich, H., Gregor, Z., McLeod, D., and and is induced strongly in cooperation with oncogenic H-Ras. Boulton, M. Increased expression of placenta growth factor in proliferative diabetic retinopathy. Lab. Investig., 78: 109–116, 1998. 23. Simpson, D. A., Murphy, G. M., Bhaduri, T., Gardiner, T. A., Archer, D. B., and Stitt, A. W. Expression of the VEGF gene family during retinal vaso-obliteration and REFERENCES hypoxia. Biochem. Biophys. Res. Commun., 262: 333–340, 1999. 1. Maglione, D., Guerriero, V., Viglietto, G., Delli-Bovi, P., and Persico, M. G. Isolation 24. Yamashita, H., Eguchi, S., Watanabe, K., Takeuchi, S., Yamashita, T., and Miura, M. of a human placenta cDNA coding for a protein related to the vascular permeability Re: “Expression of placenta growth factor (PIGF) in ischaemic retinal diseases.” Eye, factor. Proc. Natl. Acad. Sci. USA, 88: 9267–9271, 1991. 13: 372–374, 1999. 2. DiSalvo, J., Bayne, M. L., Conn, G., Kwok, P. W., Trivedi, P. G., Soderman, D. D., 25. Laderoute, K. R., Alarcon, R. M., Brody, M. D., Calaoagan, J. M., Chen, E. Y., Palisi, T. M., Sullivan, K. A., and Thomas, K. A. Purification and characterization of Knapp, A. M., Yun, Z., Denko, N. C., and Giaccia, A. J. Opposing effects of hypoxia a naturally occurring vascular endothelial growth factor placenta growth factor on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic heterodimer. J. Biol. Chem., 270: 7717–7723, 1995. inducer vascular endothelial growth factor. Clin. Cancer Res., 6: 2941–2950, 2000. 3. Cao, Y., Ji, W. R., Qi, P., and Rosin, A. Placenta growth factor: identification and 26. Sutherland, R. M., Ausserer, W. A., Murphy, B. J., and Laderoute, K. R. Tumor characterization of a novel isoform generated by RNA alternative splicing. Biochem. hypoxia and heterogenity: challenges and opportunities for the future. Semin. Radiat. Biophys. Res. Commun., 235: 493–498, 1997. Oncol., 6: 59–70, 1996. 4. Maglione, D., Guerriero, V., Viglietto, G., Ferraro, M. G., Aprelikova, O., Alitalo, K., 27. Gleadle, J. M., Ebert, B. L., and Ratcliffe, P. J. Diphenylene iodonium inhibits the Del Vecchio, S., Lei, K. J., Chou, J. Y., and Persico, M. G. Two alternative mRNAs induction of erythropoietin and other mammalian genes by hypoxia. Implications for coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from the mechanism of oxygen sensing. Eur. J. Biochem., 234: 92–99, 1995. a single gene of chromosome 14. Oncogene, 8: 925–931, 1993. 28. Khaliq, A., Dunk, C., Jiang, J., Shams, M., Li, X. F., Acevedo, C., Weich, H., Whittle, 5. Hauser, S., and Weich, H. A. A heparin-binding form of placenta growth factor M., and Ahmed, A. Hypoxia down-regulates placenta growth factor, whereas fetal (PlGF-2) is expressed in human umbilical vein endothelial cells and in placenta. growth restriction up-regulates placenta growth factor expression: molecular evidence Growth Factors, 9: 259–268, 1993. for “placental hyperoxia” in intrauterine growth restriction. Lab. Investig., 79: 151– 6. Migdal, M., Huppertz, B., Tessler, S., Comforti, A., Shibuya, M., Reich, R., Bau- 170, 1999. mann, H., and Neufeld, G. Neuropilin-1 is a placenta growth factor-2 receptor. J. Biol. 29. Dalton, T., Qingwen, L., Bittel, D., Liang, L., and Andrews, G. K. Oxidative stress Chem., 273: 22272–22278, 1998. activates metal-responsive transcription factor-1 binding activity. J. Biol. Chem., 271: 7. Fong, G. H., Rossant, J., Gertsenstein, M., and Breitman, M. L. Role of the Flt-1 26233–26241, 1996. receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 30. Murphy, B. J., Andrews, G. K., Bittel, D., Discher, D. J., McCue, J., Green, C. J., (Lond.), 376: 66–70, 1995. Yanovsky, M., Giaccia, A., Sutherland, R. M., Laderoute, K. R., and Webster, K. A. 8. Park, J. E., Chen, H. H., Winer, J., Houck, K. A., and Ferrara, N. Placenta growth Activation of metallothionein gene expression by hypoxia involves metal response factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in elements and metal transcription factor-1. Cancer Res., 59: 1315–1322, 1999. vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J. Biol. Chem., 269: 31. Gunes, C., Heuchel, R., Georgiev, O., Muller, K. H., Lichtlen, P., Bluthmann, H., 25646–25654, 1994. Marino, S., Aguzzi, A., and Schaffner, W. Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J., 17: 2846–2854, 1998. 5 P. Lichtlen, Y. Wang, T. Belser, O. Georgiev, U. Certa, and W. Schaffner. Target 32. Schonthal, A., Herrlich, P., Rahmsdorf, H. J., and Ponta, H. Requirement for fos gene gene search for the metal-responsive transcription factor MTF-1, submitted for publica- expression in the transcriptional activation of collagenase by other oncogenes and tion. phorbol esters. Cell, 54: 325–334, 1988. 2702

33. Laderoute, K. R., Mendonca, H. L., Calaoagan, J. M., Knapp, A. M., Giaccia, A. J., 41. Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., and Stork, P. J. Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression and Yamamoto, H. Possible participation of autocrine and paracrine vascular endo- is induced by low oxygen conditions found in solid tumor microenvironments. A thelial growth factors in hypoxia-induced proliferation of endothelial cells and peri- candidate MKP for the inactivation of hypoxia-inducible stress-activated protein cytes. J. Biol. Chem., 270: 28316–28324, 1995. kinase/c-Jun N-terminal protein kinase activity. J. Biol. Chem., 274: 12890–12897, 42. Oh, H., Takagi, H., Suzuma, K., Otani, A., Matsumura, M., and Honda, Y. Hypoxia 1999. and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in 34. Murphy, B. J., Robin, E. D., Tapper, D. P., Wong, R. J., and Clayton, D. A. Hypoxic bovine microvascular endothelial cells. J. Biol. Chem., 274: 15732–15739, 1999. coordinate regulation of mitochondrial enzymes in mammalian cells. Science (Wash- 43. Takagi, H., King, G. L., and Aiello, L. P. Identification and characterization of ington DC), 223: 707–709, 1984. vascular endothelial growth factor receptor (Flt) in bovine retinal pericytes. Diabetes, 35. DiPalma, T., Tucci, M., Russo, G., Maglione, D., Lago, C. T., Romano, A., Saccone, 45: 1016–1023, 1996. S., Della Valle, G., De Gregorio, L., Dragani, T. A., Viglietto, G., and Persico, M. G. 44. Graeven, U., Rodeck, U., Karpinski, S., Jost, M., Andre, N., and Schmiegel, W. The placenta growth factor gene of the mouse. Mamm. Genome, 7: 6–12, 1996. Expression patterns of placenta growth factor in human melanocytic cell lines. 36. Murphy, B. J., Laderoute, K. R., Chin, R. J., and Sutherland, R. M. Methallothionein J. Investig. Dermatol., 115: 118–123, 2000. IIA is up-regulated by hypoxia in human A431 squamous carcinoma cells. Cancer 45. Mazure, N. M., Chen, E. Y., Yeh, P., Laderoute, K. R., and Giaccia, A. J. Oncogenic Res., 54: 5808–5810, 1994. 37. Skroch, P., Buchman, C., and Karin, M. Regulation of human and yeast metallothionein transformation and hypoxia synergistically act to modulate vascular endothelial gene transcription by heavy metal ions. Prog. Clin. Biol. Res., 380: 113–128, 1993. growth factor expression. Cancer Res., 56: 3436–3440, 1996. 38. Stuart, G. W., Searle, P. F., Chen, H. Y., Brinster, R. L., and Palmiter, R. D. A 46. Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirsawa, S., Sasazuki, T., and Kerbel, 12-base-pair DNA motif that is repeated several times in metallothionein gene R. S. Mutant ras oncogenes up-regulate VEGF/VGF expression: implications for promoters confers metal regulation to a heterologous gene. Proc. Natl. Acad. Sci. induction and inhibition of tumor angiogenesis. Cancer Res., 55: 4575–4580, 1995. USA, 81: 7318–7322, 1984. 47. Bird, A. P. CpG-rich islands and the function of DNA methylation. Nature (Lond.), 39. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. Vascular endothelial growth factor 321: 209–213, 1986. induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature (Lond.), 48. Discher, D. J., Bishopric, N. H., Wu, X., Peterson, C. A., and Webster, K. A. Hypoxia 359: 843–845, 1992. regulates ␤-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 bind- 40. Shweiki, D., Neeman, M., Itin, A., and Keshet, E. Induction of vascular endothelial ing to a conserved GC element. J. Biol. Chem., 273: 26087–26093, 1998. growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: 49. Semenza, G. L. Expression of hypoxia-inducible factor 1: mechanisms and conse- implications for tumor angiogenesis. Proc. Natl. Acad. Sci. USA, 92: 768–772, 1995. quences. Biochem. Pharmacol., 59: 47–53, 2000.

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Christopher J. Green, Peter Lichtlen, Nhung T. Huynh, et al.

Cancer Res 2001;61:2696-2703.

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