Selection and Analysis of a Mutant Cell Line Defective in the Hypoxia-inducible Factor-1 alpha -Subunit (HIF-1alpha )
CHARACTERIZATION OF HIF-1alpha -DEPENDENT AND -INDEPENDENT HYPOXIA-INDUCIBLE GENE EXPRESSION*

S. Morwenna WoodDagger , Michael S. Wiesener§, Kay M. Yeates, Noriko Okada, Christopher W. Pugh, Patrick H. Maxwell, and Peter J. Ratcliffepar

From the Erythropoietin Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the alpha -subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1alpha -defective, and HIF-1alpha -complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1alpha , whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1alpha . These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1alpha -dependent and HIF-1alpha -independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Naturally occurring genetic mutations have provided many insights into gene function. In an effort to extend this source of information, a large number of targeted or untargeted means of introducing mutations and selecting by genotype or phenotype have been devised. In somatic cells, one classical approach has been to subject a mutagenized culture to a substance whose toxicity is dependent on the activity of a set of genes involved in a signal transduction or metabolic pathway (1-3). However, the number of properties that are "intrinsically selectable" in this way is limited, leading some workers to explore the possibility of introducing selectable properties by transfection of recombinant plasmids (4, 5). For instance, stable transfection of a plasmid bearing an interferon-responsive promoter linked to a bacterial guanidine phosphoribosyl transferase gene has been used to select cells from the fibrosarcoma line HT1080 that are defective in the response to interferons (4). Similar promoters linked to the gene encoding the cell surface antigen CD2 have been used to select other lines defective in the interferon response using fluorescence-activated cell sorting (6).

The recent recognition of a widespread transcriptional response to hypoxia (see Refs. 7 and 8; for a review see Ref. 9), mediated by the activation of a heterodimeric basic-helix-loop-helix PAS1 protein complex termed hypoxia-inducible factor-1 (HIF-1) (10) presents an important challenge, in terms of both defining the responsive target genes and analyzing the underlying mechanism of oxygen sensing and signal transduction. Here we describe the use of a selection strategy based on the linking of HIF-1-binding hypoxia-responsive elements (HREs) with minimal promoters coupled to genes encoding cell surface antigens. We describe the selection, from a mutagenized culture of Chinese hamster ovary (CHO-K1) cells, of cells that are deficient in HRE-dependent transactivation and the identification of one line that is functionally defective in the alpha -subunit of HIF-1. We demonstrate the utility of this cell line in characterizing HIF-1alpha -dependent and HIF-1alpha -independent patterns of hypoxia-inducible gene expression.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines, Mutagenesis, and Transfections-- Cells were grown in Ham's F-12 (Sigma) supplemented with 10% fetal calf serum (Globepharm), L-glutamine (2 mM), penicillin (50 IU/ml), and streptomycin sulfate (50 µg/ml).

Transfections were performed by electroporation using a 1-millifarad capacitor array charged at 350 V. Plasmids bearing HREs linked to reporter genes or selectable markers are illustrated in Fig. 1. pHPGK-CD2 was as described previously (11). p(E1-25)5SV-ESel contained five copies of a 25-base pair HRE from the mouse 3'-erythropoietin enhancer coupled to the SV40 early promoter (from p(E1-25)5SV-GH (12)) ligated into pBluescript and linked to the complete E-selectin coding sequence and SV40 splice/poly(A) from pCDM8. pHTK-Luc contained two copies of a 24-base pair HRE from the mouse phosphoglycerate kinase-1 5'-enhancer lying 10 base pairs 5' to the TATA box of the herpes simplex virus thymidine kinase promoter cloned into pGL2 basic (Promega) bearing a firefly luciferase gene. pSV2Neo and pSV2Hyg contained neomycin and hygromycin phosphotransferase genes, respectively, linked to the SV40 early promoter. pCMVbeta Gal contained the beta -galactosidase gene from pSVbeta Gal (Promega) cloned into pcDNA3. A stable transfectant of CHO-K1 cells bearing two hypoxia-inducible cell surface markers was obtained by transfection with pHPGK-CD2 (20 µg) and pSV2Neo (1 µg) followed by selection in G418 (0.5 mg/ml) and then supertransfection of one clone with p(E1-25)5SV-ESel (20 µg) and pSV2Hyg (1 µg) and selection in hygromycin B (0.7 mg/ml).

Mutagenesis was performed by exposure of cells to 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine dihydrochloride (ICR191; Sigma) at 1 µg/ml for 5 h, using a protocol based on Ref. 13.

Transient transfection assays for HIF-1-dependent transcriptional responses were performed using the HRE-bearing plasmid, pHTK-Luc (10 µg) together with plasmid pCMVbeta Gal (6.25 µg) as a control for transfection efficiency. Cells from each transfection were split for parallel incubation under normoxic conditions (21% O2) or stimulated conditions; hypoxia (1%) or desferrioxamine (100 µM). The ability of the HIF-1alpha , ARNT, or EPAS-1 genes to reconstitute the hypoxia-inducible response was tested by transient co-transfection of cells with pHTK-Luc (10 µg), pCMVbeta Gal (6.25 µg), and the relevant human cDNA expression plasmid (pcDNA1/Neo/HIF-1alpha (containing the HIF-1alpha sequence from pBluescript/HIF-1alpha 3.2-2T7 (10) cloned into pcDNA1), pcDNA1/Neo/mARNT (14), or phEP-1 (15)). Transfected cells were incubated for 10 h at 21% oxygen (normoxia) and then grown in either normoxia or in hypoxia (1% O2) for a further 16 h before harvesting.

Studies of inducible gene expression were performed on cells approaching confluence. Parallel incubations were performed on aliquots of cells in normoxia (humidified air with 5% CO2) and either hypoxia or desferrioxamine mesylate (100 µM; Sigma). Hypoxic conditions were generated in a Napco 7001 incubator (Precision Scientific, Chicago) with 1 or 0.1% O2, 5% CO2, and the remainder N2. For most genes, experiments were performed for a duration of 16 or 48 h in normal growth medium. For assays of the metalloproteinases and their inhibitors, the cells were grown in serum-free medium for 24 h before a further 24-h exposure in serum-free medium to the experimental conditions. To assay for gene expression in low glucose, cells were incubated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with either 25 mM or 0.5 mM glucose for 48 h.

Stable transfection of the mutant derivative Ka13 with a human HIF-1alpha gene was performed by electroporation using pcDNA3/Neo/HIF-1alpha (containing the HIF-1alpha sequence from pBluescript/HIF-1alpha 3.2-2T7 cloned into pcDNA3/Neo) (20 µg) and pPur (1 µg) (CLONTECH) followed by selection in puromycin (Sigma) 5 µg/ml.

Antibody Labeling of Cells and Immunoselection-- For fluorescence-activated cell scanning (FACS) analysis, cells were detached in 2 mM EDTA in HBSS, centrifuged at 1000 rpm at 4 °C, and washed twice in 3 ml of PFA (PBS with 0.1% sodium azide and 1% fetal calf serum). Antibody labeling was performed on ice for 30 min using a phycoerythrin-conjugated mouse IgG2b anti-human CD2 (Serotec) and fluorescein isothiocyanate-conjugated mouse IgG1 anti-human E-selectin (R & D Systems). Cells were then washed and fixed in 1% paraformaldehyde in PBS before FACS analysis (Becton Dickenson).

For immunological selection by panning (16), cells were harvested as above and then washed twice in panning buffer (PBS with 2 mM EDTA, 0.02% sodium azide, and 5% heat-inactivated fetal calf serum) at 4 °C. Antibody labeling (30 min, 4 °C) was performed either with mouse IgG2b anti-human CD2 (Serotec) at a 1:10 dilution or with mouse IgG1 anti-human E-Selectin (Serotec) at 1:100 dilution. The cells were then washed twice in panning buffer, resuspended in 5 ml of panning buffer, and poured onto prepared panning plates. Plates were left at room temperature for 15-60 min, after which the panning buffer was gently aspirated to harvest the nonadherent cells. These cells were washed in culture medium and plated out in selective medium. Panning plates were prepared as follows. Petri dishes were coated with 5 ml of a 10 µg/ml solution of affinity-conjugated goat anti-mouse IgG (Sigma) in 50 mM Tris, pH 9.5, for 1 h at room temperature. The dishes were then washed three times in PBS, blocked by overnight incubation with 5 ml of sterile blocking buffer (2 mg/ml bovine serum albumin in PBS), and stored at -20 °C.

Cell Fusions-- Parental cells were CHO-K1 cells and an hypoxanthine phosphoribosyl transferase-deficient (HPRT-) derivative of Ka13 selected in hypoxanthine-free medium containing 10 µM 6-thioguanine. For fusions, equal quantities of CHO-K1 and HPRT- Ka13 cells were mixed and plated such that the cells were overconfluent. After 4 h, the cells were washed with serum-free medium, and 0.5 ml of neutralized polyethylene glycol solution (Mr 1300-1600, 1 g/ml) was added for 1 min. The polyethylene glycol was diluted in 5 ml of serum-free medium over a further 2 min and then removed, after which the cells were washed gently in serum-free medium, incubated at 37 °C for 6 h in complete medium, and then trypsinized and replated. Selective medium (HAT containing G418) was added 24 h later.

Cell Extracts and Protein Analysis-- Nuclear extract was prepared using a modified Dignam protocol, and an electrophoretic mobility shift assay was performed as described in Ref. 17 using a double-stranded HIF-1 binding oligonucleotide 5'-GCCCTACGTGCTGCCTCGCATGGC-3' from the mouse erythropoietin enhancer (E24). For Western analysis, nuclear extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes. HIF-1alpha was detected using a polyclonal rabbit antiserum raised against a recombinant immunogen containing amino acids 530-652 of human HIF-1alpha , a peroxidase-conjugated goat anti-rabbit immunoglobulin (DAKO), and enhanced chemiluminescence.

RNA Analysis-- RNA was extracted by a modified acid/guanidinium thiocyanate/phenol/chloroform method (RNAzol B, Cinna/Biotec Laboratories, Houston), dissolved in hybridization buffer (80% formamide, 40 mM PIPES, 400 mM sodium chloride, and 1 mM EDTA, pH 8), and analyzed by an RNase protection assay or dissolved in diethylpyrocarbonate-treated distilled H2O for Northern blotting.

For Northern blotting, 30 µg of total RNA were fractionated on a denaturing agarose gel and transferred to a nylon membrane. The filters were hybridized with probes labeled with [32P]dCTP using random nonamer priming (Amersham Pharmacia Biotech) for ARNT and by polymerase chain reaction for HIF-1alpha .

RNase protection assays were performed as described previously (18). Quantitation of the protected species was performed using a PhosphorImager (Molecular Dynamics) and was related to an internal control assay for a constitutively expressed U6 small nuclear RNA. Fully homologous riboprobe templates for Chinese hamster genes were generated by polymerase chain reaction using oligonucleotides based on published sequences for other species and cloned into pSP72 or pGemTeasy. Details of riboprobe templates for Chinese hamster or mouse genes are given in Table I. For endogenous genes, the quantity of RNA analyzed was as follows: U6, small nuclear 5 ng; GAPDH, 2 µg; GRP78, 10 µg; Glut-1 and HO-1, 20 µg each; ornithine decarboxylase and tissue inhibitor of metalloproteinases-2, 30 µg each; gelatinase 92 and transferrin receptor, 40 µg each; AK3 and HIF-1alpha , 50 µg each; and EPAS-1 and VEGF, 100 µg each.

Reporter Gene Assays-- Luciferase activity was determined in cell lysates using a commercial assay system (Promega) and a TD-20e luminometer (Turner Designs). beta -Galactosidase activity in cell lysates was measured using o-nitrophenyl-beta -D-galactopyranoside as substrate in a 0.1 M phosphate buffer pH 7.0 containing 10 mM KCl, 1 mM MgSO4, and 30 mM beta -mercaptoethanol.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Characterization of a Cell Line Bearing Surface Markers Controlled by Hypoxia-responsive Elements-- A central difficulty in using selection against exogenous genetic markers introduced by transfection to derive mutants that are functionally defective in components of an interacting transcriptional mechanism is that the transfected gene is generally much less stable than the endogenous genome. As a result, selection against an inducible transfected marker gene will generate a large excess of cells that have simply lost or suppressed expression of the marker over those bearing defects in the endogenous mechanism of gene regulation. We employed two strategies to limit this problem. First, two separately integrated, hypoxically inducible immunoselectable markers were used. This enabled us to limit selection of cells that had simply lost one or the other integration (by alternate selection against each of the two hypoxically inducible antigens) and also allowed us to detect such cells rapidly by FACS analysis. Second, positive selection for co-transfected drug resistance markers was continued throughout the negative immunoselection procedure.

First, to assess the efficiency of various HIF-1-dependent HREs in supporting hypoxia-inducible gene expression in CHO-K1 cells, transient transfections were performed with plasmids bearing different HRE/promoter combinations coupled to cDNAs for cell surface markers. Inducible marker gene expression was measured by mRNA and FACS analysis (data not shown). The two plasmids that showed the highest induction of marker expression were chosen for stable transfection and are illustrated in Fig. 1. CHO-K1 cells were first transfected with pHPGK-CD2 and pSV2Neo, and clones were selected in G418. One such clone was further transfected with p(E1-25)5SV-ESel and pSV2Hyg, and clones were selected in hygromycin B. Several resistant clones were assayed for hypoxia-inducible expression of the transfected cell surface markers, and one (C4.5) was selected on the basis of showing the greatest inducible expression of both markers. Fig. 2A shows FACS analysis of normoxic and hypoxic cultures of C4.5 cells after labeling cells with monoclonal antibodies against human CD2 and E-selectin. Both markers are clearly inducible by hypoxia. Untransfected CHO-K1 cells did not express either marker gene as assessed by RNase protection and FACS.


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Fig. 1.   Schematic illustration of plasmids expressing hypoxia-inducible marker genes. pHPGK-CD2 contained three copies of a 24-base pair HRE from the mouse phosphoglycerate kinase (PGK) gene reiterated within the surrounding phosphoglycerate kinase-1 promoter/enhancer sequence and coupled to cDNA sequences encoding human CD2. p(E1-25)5SV-ESel contained five copies of a 25-base pair HRE from the mouse erythropoietin gene linked to the SV40 virus early promoter and coupled to the cDNA sequences encoding human E-selectin. pHTK-Luc contained two copies of the 24-base pair mouse phosphoglycerate kinase-1 HRE linked to a herpes simplex virus thymidine kinase (TK) promoter and coupled to a firefly luciferase gene.


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Fig. 2.   Cell surface expression of CD2 and E-selectin in stably transfected CHO cells. FACS analyses of cell surface expression of CD2 (left panels) and E-selectin (right panels) are shown for wild type C4.5 cells (A), a pool of C4.5 cells that had undergone mutagenesis and negative selection by panning (B), a clone, Ka13, which was isolated from the above pool (C), and hybrid CHO-K1/Ka13 cells (D). Each panel shows surface marker expression in normoxic (N, thin line) and hypoxic (H, thick line) cells. C4.5 cells show hypoxia-inducible marker expression of both markers, which is reduced in a significant proportion of the cells after mutagenesis and selection. The inducible response is absent in Ka13 cells but restored in the hybrid cells by fusion with CHO-K1.

Mutagenesis and Selection of Clones with Impaired Responses to Hypoxia-- For mutagenesis, the CHO line C4.5 was exposed to ICR191. In pilot experiments, one exposure to a dose of 1 µg/ml of mutagen for 5 h resulted in nearly 1 in 103 mutants at the HPRT locus per clonable cell (data not shown). Since we anticipated that disabling mutations of hypoxia-inducible transcription would be much less frequent, approximately 7 × 107 C4.5 cells, in 10 separate aliquots, were exposed to ICR191 at 1 µg/ml for 5 h. Previous reports had described a higher frequency of mutation following multiple exposures to ICR191 (19, 20), so cells were allowed to recover and then subjected to a further two exposures of the same dose and duration. The cells were maintained in the 10 separate aliquots throughout these further exposures to ICR191 and the subsequent selection procedure. Cell mortality after one exposure was approximately 55%.

The isolation of cells that were unable to support HRE-dependent induction of gene expression was made by antibody labeling of the HRE-linked cell surface markers and selection by panning. After a 16-h hypoxic exposure, cells were harvested, labeled with primary antibody to CD2 or E-selectin, and then placed on a prepared dish coated with secondary antibody. Cells that failed to adhere to the panning plates were collected, washed, and replated in tissue culture dishes. Once the cells had multiplied to about 5 × 106/pool, they were again made hypoxic, and another round of panning was undertaken. To measure the extent of selection obtained in this way, preliminary experiments were performed in which a marker CHO cell (bearing a constitutively expressed intercellular adhesion molecule-3 gene) was mixed with C4.5 cells in a ratio of 1:1000. After three rounds of panning of this mixed cell pool for nonexpression of E-selectin, the pool was tested for intercellular adhesion molecule-3 expression by FACS. These cells had now reached a prevalence of approximately 10% in the pool (and remained undetectable by FACS in a parallel pool that had simply been passaged without selection). From this, it was estimated that for a mutation frequency likely to be less than 1 in 106, approximately 10 rounds of panning would be required to enrich sufficiently for such a mutant population. Panning of the mutagenized C4.5 cells was therefore performed alternately with anti-CD2 and anti-E-selectin antibodies, to a total of 10 rounds. Throughout the procedure, selection was continued in neomycin and hygromycin B. Each pool was then tested for inducibility of both CD2 and E-selectin and compared with C4.5 cells. Results for one of the pools are shown in Fig. 2B. A clear reduction in the hypoxia inducible expression of both markers was observed in a significant proportion of cells.

Isolation of a Clone That Is Functionally Defective for the alpha -Subunit of HIF-1-- Clonal populations from each pool were obtained by limiting dilution and then examined for noninducibility of both surface markers by FACS analysis. 44 clones that showed impaired regulation of these markers were tested further for defects in HRE activation by transient transfection with pHTK-Luc, a plasmid bearing an HRE-dependent luciferase reporter gene. In this assay, 32 of these clones showed levels of HRE activity that were the same or similar to wild type C4.5. However, 11 showed a clear but incomplete reduction in the hypoxia-inducible response ranging from 20 to 65% of that observed in wild type C4.5 cells. For nine of these clones, this change was stable over 12 weeks of continuous passage. One clone, Ka13, showed an absent response. Fig. 2C illustrates expression of CD2 and E-selectin in Ka13. Ka13 was stable with respect to this phenotype and has been subject to detailed further analysis.

First, the behavior of Ka13 was assessed in cell fusion studies. An HPRT- variant of Ka13 was selected in 6-thioguanine (a toxic analogue of the substrate hypoxanthine), and this phenotype was confirmed by sensitivity to HAT medium (the HPRT gene is required for survival in HAT). Equal quantities of the HPRT- Ka13 cells and the parental CHO-K1 cells were fused using polyethylene glycol, and hybrids (acquiring a neomycin phosphotransferase gene from Ka13 and an HPRT gene from the parental K1 cells) were selected in HAT medium containing G418. Each of several pools from two independent fusions showed restoration of the hypoxic response (Fig. 2D).

Since cDNAs encoding the heterodimeric HRE binding complex HIF-1 had been identified at an earlier stage in this work (10), we next tested if the defect in Ka13 could be corrected by transfection with cDNAs encoding HIF-1alpha or ARNT. Ka13 cells were co-transfected with pHTK-Luc and expression plasmids encoding HIF-1alpha , ARNT, or no cDNA. Results are shown in Fig. 3A. Co-expression of HIF-1alpha , but not ARNT, supported a high level of reporter gene expression. Increasing levels of expression in normoxic cells were observed with increasing concentrations of HIF-1alpha expression plasmid and could be further enhanced by exposure of transfected cells to hypoxia. For comparison, the activity of pHTK-Luc in the parental C4.5 cells is shown. These results suggested that Ka13 cells were defective in some aspect of HIF-1alpha expression or function but retained the ability to respond to hypoxia and regulate the activity of a heterologous HIF-1alpha gene.


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Fig. 3.   Transient transfection analysis of Ka13 cells. A, restoration of the hypoxia-inducible response by transfection with HIF-1alpha but not ARNT. Ka13 cells were cotransfected with the reporter plasmid pHTK-Luc and the indicated amounts of pcDNA1/Neo/HIF-1alpha , pcDNA1/Neo/mARNT, or an empty expression vector (pcDNA1). Transfection with HIF-1alpha restored hypoxia-inducible reporter gene activity in Ka13 cells in a dose-dependent manner. B, activity of a Gal4-HIF-1alpha chimeric gene in C4.5 and Ka13 cells. Cells were cotransfected with pUASTKLuc reporter and pGAL (containing no HIF-1alpha sequence) or pGAL/alpha 530-826 (containing amino acids 530-826 from HIF-1alpha ). pGAL/alpha 530-826 showed hypoxia-inducible activity in both cell types. All data are corrected luciferase activity, expressed in arbitrary units, for cells incubated in normoxia (N) and hypoxia (1% oxygen) (H). C, comparison of the hypoxia-inducible response after transfection with HIF-1alpha and EPAS-1. Ka13 cells were cotransfected with the reporter plasmid pHTK-Luc and 1 µg of pcDNA1/Neo/HIF-1alpha and/or phEP-1 or with an empty expression vector (pcDNA1). Both HIF-1alpha and EPAS-1 restore hypoxic induction; when transfected together in these amounts, an enhanced response was observed.

To address this defect further, Ka13 cells and wild type C4.5 cells were co-transfected with a Gal4-HIF-1alpha fusion gene (pGAL/alpha 530-826) (21) and a galactosidase-responsive reporter (pUASTKLuc) (Fig. 3B). Similar inducible activity was observed in both cell types, confirming that the underlying regulatory mechanism was functioning normally.

Expression of HIF-1alpha mRNA and protein was next measured. Using RNase protection and Northern analysis, the total level of mRNA in Ka13 cells was shown to be greatly reduced when compared with wild type (Fig. 4B). No abnormal transcripts were detected, and although the level of HIF-1alpha mRNA in Ka13 cells was at the limit of detection on the Northern blot, prolonged exposure of the autoradiograph revealed a faint signal corresponding to the mobility of wild type HIF-1alpha mRNA. In contrast, mRNA levels for ARNT were normal.


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Fig. 4.   A, electrophoretic mobility shift assay for HIF-1 binding activity in C4.5 and Ka13 cells. Binding to radiolabeled oligonucleotide E24 in nuclear extracts prepared from cells incubated in normoxia (N) or hypoxia (1% oxygen) (H) for 12 h. The inducible (I) and constitutive (C) species are indicated. FP, free probe. No inducible species was demonstrable in Ka13 cells. B, Northern analysis of HIF-1alpha and ARNT mRNA in wild type C4.5 and mutant Ka13 cells in normoxic (N) and hypoxic (H) conditions. HIF-1alpha expression in C4.5 cells was detected at the expected length, but HIF-1alpha mRNA in Ka13 was at the limit of detection with Northern analysis; prolonged exposure of the autoradiograph revealed a faint signal corresponding to the mobility of wild type HIF-1alpha mRNA. ARNT mRNA expression was equivalent in both cell lines. C, Western analysis for HIF-1alpha protein on nuclear extracts from HeLa, C4.5, and Ka13 cells grown in conditions of normoxia (N), hypoxia (1% oxygen) (H), or 100 µM desferrioxamine (D). Immunodetection was with a rabbit polyclonal antibody to human HIF-1alpha . Extracts from HeLa cells were run in parallel for comparison. HeLa and wild type C4.5 cells show a similar nuclear accumulation of HIF-1alpha in stimulating conditions (hypoxia or desferrioxamine). There was no detectable HIF-1alpha protein in Ka13 cells. D, RNase protection assay for EPAS-1 mRNA from Chinese hamster liver homogenates and CHO cells. EPAS-1 was not detectable in CHO cells (100 µg of RNA).

Western analysis for HIF-1alpha protein was performed on nuclear extracts from wild type and mutant CHO cells (Fig. 4C). For comparison, nuclear extracts from HeLa cells were run in parallel. HeLa and wild type C4.5 cells show similar nuclear accumulation of HIF-1alpha after stimulation, whereas there was no detectable HIF-1alpha immunoactivity in Ka13 cells.

Electrophoretic mobility shift assays using a HIF-1 binding oligonucleotide from the mouse erythropoietin 3'-enhancer demonstrated loss of the HIF-1 complex in nuclear extracts from Ka13 cells (Fig. 4A). Species showing the normal HIF-1 mobility shift were undetectable in Ka13, although the constitutive species was observed as in wild type C4.5 cells.

Recently, another basic-helix-loop-helix PAS protein, termed EPAS-1, has been described. Since it possesses substantial sequence similarly to HIF-1alpha , dimerizes with ARNT, and binds similar DNA sequences (15, 22, 23), we examined EPAS-1 mRNA levels in wild type and mutant cells by RNase protection. No signal was detected in either cell type in assays of up to 100 µg of RNA, although EPAS-1 mRNA was clearly detectable by this assay in RNA prepared from Chinese hamster liver (Fig. 4D).

We also tested the ability of a human EPAS-1 expression plasmid to correct the defect in HRE activity in transient transfection assays. As shown in Fig. 3C, results were similar to those obtained with HIF-1alpha expression. Expression of EPAS-1 led to an increase in both normoxic and hypoxic HRE activity and restoration of hypoxia-inducible reporter gene expression.

Hypoxia-inducible Gene Expression in Ka13 Cells-- The above analysis indicates that Ka13 cells manifest a profound defect in HIF-1alpha expression. To explore the effect of this on hypoxia-inducible gene expression, the regulation of particular groups of genes was examined first in wild type C4.5 cells and Ka13 cells. Table I lists all of the genes tested. Several of these genes (gelatinase 92, tissue inhibitor of metalloproteinases 2, transferrin receptor, and ornithine decarboxylase) showed only a low level or inconsistent regulation by hypoxia in wild type C4.5 cells and were not analyzed further. Fig. 5A shows results for six genes selected from different functional groups that showed inducible responses or were otherwise of particular interest: the VEGF, Glut-1, GAPDH, AK3, HO-1, and GRP78 genes. Striking differences were observed. For some genes, a high level of hypoxia-inducible expression was observed in wild type C4.5 cells that was very much reduced or completely abolished in Ka13. Surprisingly, little or no induction of VEGF mRNA by hypoxia was observed in the wild type C4.5 cells, and VEGF expression was similar in Ka13 cells incubated in hypoxic conditions, although a small induction was seen in Ka13 cells treated with desferrioxamine. The largest induction by hypoxia was observed for Glut-1 mRNA. When cells were exposed to an atmosphere of 1% oxygen, Glut-1 mRNA expression was induced 5-fold in C4.5 cells but was not induced in Ka13 cells. For GAPDH and AK3, a similar abrogation of hypoxia-inducible expression was observed in Ka13 cells. In contrast, HO-1 and GRP78 showed inducible responses to hypoxia that were not obviously reduced in Ka13 cells. This suggested that HIF-1alpha was critical for the induction of certain genes at 1% oxygen but that independent pathways of hypoxia-inducible gene expression might also operate. To examine this further, the expression pattern of four genes was examined after exposure to more severe hypoxia, 0.1% oxygen for 48 h (Fig. 5B). For all four genes, the level of induction was greater than that observed with 1% oxygen for 16 h. In Ka13 cells, the response to hypoxia was again abolished for Glut-1 and GAPDH gene expression. The behavior of HO-1 and GRP78 genes differed from Glut-1 in that the responses were well preserved in Ka13 cells; for instance, HO-1 induction was 17- and 13-fold with 0.1% oxygen in C4.5 and Ka13 cells, respectively. This suggested that different pathways of hypoxia-inducible expression were operating on these genes in CHO cells. Since (like HIF-1), HO-1 and Glut-1 genes have both been demonstrated to be inducible by cobaltous ions (24-26), we examined the response of these two contrasting genes to cobaltous ions and other stimuli known to induce HIF-1. Results are shown in Fig. 6. Glut-1 mRNA was induced to a similar extent by hypoxia, cobaltous ions, and desferrioxamine in C4.5 cells. All of these responses were abolished in Ka13 cells. In contrast, HO-1 was induced to a much greater extent by cobaltous ions but was not inducible by desferrioxamine in C4.5 cells, and these responses were similar in Ka13 cells. When responses to desferrioxamine were compared across the set of genes, two distinct patterns were observed (Fig. 5A). For Glut-1, AK3, and GAPDH genes, the response to hypoxia was mimicked closely by desferrioxamine, whereas for HO-1 this was not the case.

                              
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Table I
Details of riboprobes used in RNase protection assays
For some genes (indicated with an asterisk) a fully homologous Chinese hamster probe was generated by polymerase chain reaction using oligonucleotides designed from a non-hamster sequence deposition as indicated.


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Fig. 5.   Induction of endogenous gene expression in C4.5 and Ka13 cells. mRNA levels were assayed by RNase protection. The data for each gene have been normalized to the level of normoxic expression in C4.5 cells. Each bar represents the mean of at least three experiments (except for GAPDH at 48 h, where n = 2). A, cells were incubated in parallel in normoxic conditions (N), hypoxia (1% oxygen) (H), or 100 µM desferrioxamine (D) for 16 h. B, cells were incubated in parallel in either normoxic conditions (N) or hypoxia 0.1% oxygen (H 0.1%) for 48 h.


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Fig. 6.   Induction of glucose transporter-1 and heme oxygenase-1 in different conditions in C4.5 and Ka13 cells. An RNase protection assay of Glut-1 and HO-1 mRNA is shown. Aliquots of cells were incubated in normoxia (N), hypoxia (1% oxygen) (H 1%), hypoxia (0.1% oxygen) (H 0.1%), 100 µM desferrioxamine (D), or 100 µM cobalt (C) for 16 h.

Since low glucose has also been reported to induce several of these genes (27, 28), mRNA levels for VEGF, Glut-1, HO-1, and GRP78 were measured after incubation of cells in low glucose (0.5 mM) medium. Results are summarized in Fig. 7. Only HO-1 showed induction by low glucose; as with induction by hypoxia, this response was preserved in Ka13 cells.


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Fig. 7.   Expression of Glut-1, VEGF, HO-1, and GRP78 in C4.5 and Ka13 cells incubated in conditions of high glucose (25 mM) (HG) or low glucose (0.5 mM) (LG) for 16 h. Data are from RNase protection assays. All values given have been normalized to the normoxic expression in C4.5 cells. Induction by low glucose conditions was only seen for HO-1 and was equivalent in C4.5 and Ka13 cells.

Analysis of Gene Expression in Ka13 Cells Bearing a Stably Integrated Human HIF-1alpha Gene-- The above data demonstrate that the induction of some but not all genes in response to hypoxia is abrogated in HIF-1alpha -deficient Ka13 cells, suggesting a critical role for this transcription factor in these responses. To test this further, Ka13 cells were transfected with pcDNA3/Neo/HIF-1alpha and pPur, with selection in puromycin. Surviving colonies were pooled and checked for expression of the marker genes. Since expression of the cell surface markers was only increased in a small proportion of the pool, immunoselection by panning for high hypoxic expression of E-selectin was undertaken to augment this population. After four rounds of panning, clones were obtained by limiting dilution. RNase protection analysis indicated that these clones expressed varying levels of human but not Chinese hamster HIF-1alpha RNA. Several clones were analyzed for Glut-1, GAPDH, and AK3 mRNA expression, and in all clones increased levels were observed. Results are shown for one clone expressing a moderate level of human HIF-1alpha RNA in Fig. 8; restoration of induction by both hypoxia and desferrioxamine can be seen. In some clones expressing higher levels of HIF-1alpha mRNA (as assessed by RNase protection assays of mRNA), an enhanced level of Glut-1 mRNA was observed in normoxic cells, suggesting that overexpression of HIF-1alpha can increase expression of certain endogenous target genes in normoxic cells.


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Fig. 8.   RNase protection assay for glucose transporter-1 expression in C4.5 cells, Ka13 cells, and Ka13 cells bearing a stably integrated human HIF-1alpha gene (KH3-H10 cells). Cells were incubated in normoxia (N), hypoxia (1% oxygen) (H), or 100 µg of desferrioxamine (D) for 16 h. The inducible response was restored in KH3-H10 cells.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The purpose of these experiments was to select mutant cells with a defective transcriptional response to hypoxia. Since we were unaware of hypoxia-inducible genes with intrinsically selectable properties, we used stably integrated recombinant plasmids to provide selectable markers consisting of HREs linked to cell surface antigens. This strategy was successful in enabling the isolation of several clones that showed reduced HRE activity as assessed by transient transfection assays and one with absent HRE activity, which proved to be defective in the alpha -subunit of the transcriptional complex HIF-1. Nevertheless, there were some difficulties to be overcome that merit comment.

First, despite high levels of hypoxia-inducible activity observed for some of the HRE/promoter combinations in transient transfection assays, inducibility of the most responsive stably integrated marker was only 5-fold. This suggested that the use of cytotoxic selection markers, where more stringent control of the level of gene expression is required, might present difficulties. Hence, we adopted an immunoselection strategy similar to that which has been described for the selection of interferon unresponsive mutants (6). The technique of panning has been widely used for expression cloning of genes encoding cell surface antigens (16). In these experiments, we measured its efficacy in selecting low expressing cells and demonstrated that with multiple rounds it provided a relatively simple and inexpensive method of achieving the necessary level of selection even with relatively low levels of marker gene induction.

In pilot experiments, the measured mutation frequency of approximately 1 in 103 at the (haploid) HPRT locus was similar to that previously reported using ICR191, indicating that adequate exposure to the mutagen was achieved (29). It is clearly not possible to comment precisely on the frequency of ablative mutations at (presumably diploid) loci encoding genes involved in the response to hypoxia from our isolation of one HIF-1alpha -deficient line. Nevertheless, this result is not inconsistent with the relatively low frequency of ablative mutants reported for other loci using similar methodology (4, 20). The very much higher rate of loss or suppressed expression of the transfected marker gene that is generally observed when tissue culture cells are selected in this way will therefore potentially obscure the selection of such rare events (4, 30). As has been reported by others, we found that continued positive selection with a co-transfected drug resistance gene (4) and the use of two separately integrated markers (5, 6) limited this problem. Nevertheless, a substantial number of clones (32 out of 44) that showed reduced expression of both the stably integrated HRE-linked markers used for selection were indistinguishable from wild type in the hypoxia-inducible response assayed by a subsequent transient transfection with a third HRE-linked reporter, suggesting that such effects still occurred quite frequently. Eleven clones were identified that showed reduced HRE activity when assessed by transient transfection. Nine of these clones showed a partial reduction in HRE activity, which remained stable in tissue culture. Although eight came from one pool and might represent the same mutation, the other must represent an independent mutation. The defect(s) in these cells have not yet been defined, and hypoxia-inducible expression of the endogenous Glut-1 gene was normal in all. Presumably, they represent defects that impair the the full response of the HRE in transfected cells but are not rate-limiting as far as expression of Glut-1 mRNA is concerned. Since we were concerned to identify mutations that were critical for the response to hypoxia, we concentrated our analysis on one line that possessed a much more striking defect in HRE activity.

Our analysis of this line (Ka13) demonstrated a defect in HIF-1alpha function that could be complemented by transient or stable transfection with a human HIF-1alpha gene. Normal inducibility of a transfected Gal4/HIF-1alpha fusion gene indicated that Ka13 cells were not defective in the oxygen sensing process that activates HIF-1alpha (21). Southern blotting showed no genomic rearrangement or deletion at the HIF-1alpha locus. Based on the known action of ICR191 (31), the defect is most likely to be a frame-shifting mutation of the HIF-1alpha gene that prevents correct translation and leads to an unstable mRNA or a defect in a transcription factor that is itself essential for HIF-1alpha expression.

At least two proteins, HIF-1alpha and EPAS-1, have been shown to dimerize with ARNT and bind similar DNA sequences (15, 22, 23). Our finding of absent HRE activity in the HIF-1alpha -deficient line is not inconsistent with this, since neither wild type nor mutant cells contained detectable EPAS-1 mRNA.

Transient co-transfection experiments in several types of cells with normal endogenous HIF-1alpha expression have shown that overexpression of the HIF-1alpha will drive the transcription of an HRE-linked reporter even in normoxia (32). Ka13 cells provided an opportunity to test this against a background of absent endogenous HIF-1alpha expression. The results show a progressive increase in normoxic HRE activity as more HIF-1alpha was transfected into Ka13 cells. Hypoxia greatly increased activity, so that with high plasmid doses the HRE activity was much greater than in untransfected wild type cells. A similar result was obtained with transfection of EPAS-1, indicating that both the HIF-1alpha and EPAS-1 genes can independently support hypoxia-inducible gene expression and that sufficient expression of either will drive substantial HRE activity even in normoxic cells.

Studies of hypoxia-inducible responses among endogenous genes in these cells showed marked differences from wild type cells. For some genes, hypoxia-inducible expression was completely abrogated, indicating a critical dependence on HIF-1alpha . Such a pattern was observed for the glucose transporter Glut-1, the glycolytic GAPDH gene, and AK3, an isoenzyme of adenylate kinase (NTP:AMP phosphotransferase), suggesting an important function for HIF-1alpha in adapting energy metabolism to hypoxia. Complete loss of the hypoxia-inducible response for Glut-1 contrasts with the partial loss of inducibility observed in the ARNT-deficient mutant Hepa-1 line, c4, which also fails to form the HIF-1 complex (33) and is of interest in relation to the dual regulation by hypoxia-induced transcription and mRNA stabilization, which has been observed for Glut-1 in several cell lines (27, 34). Either the response in CHO cells is entirely transcriptional or both responses must in some way be dependent on HIF-1alpha expression.

Stable transfection of Ka13 cells with human HIF-1alpha restored hypoxia-inducible expression of this group of genes. Interestingly, some clones with stable overexpression of the human HIF-1alpha gene also showed increased expression of Glut-1 mRNA in normoxic cells, indicating that the level of HIF-1alpha gene expression could have substantial effects on the expression of certain target genes even under basal conditions.

In contrast with the striking dependence on HIF-1alpha gene expression observed for Glut-1 and GAPDH, hypoxia-inducible expression of other genes appeared to be largely or entirely independent of HIF-1alpha expression. This was observed most strikingly for heme oxygenase-1, where marked induction in 1% oxygen, 0.1% oxygen, and after exposure to cobaltous ions was observed in both wild type and mutant cells. This independence from HIF-1alpha was also observed, in least in part, for GRP78. Both GRP78 and heme oxygenase-1 have been well recognized as oxygen-regulated proteins in CHO cells (35, 36). The data on HO-1 are particularly interesting in that a recent analysis of HO-1 expression defined a functional HIF-1 site in the HO-1 promoter and showed abrogation of hypoxia-inducible HO-1 expression in the ARNT-deficient mutant Hepa-1 cell line, c4 (25). Either hypoxia-inducible expression of HO-1 is differently controlled in CHO cells or there are other partners for ARNT that confer hypoxia inducibility and operate specifically on the HO-1 promoter.

Analysis of gene expression in embryonic stem cells bearing a targeted mutation of the ARNT gene has recently suggested the existence of an ARNT-dependent transcriptional response to low glucose (28). We therefore tested responses to low glucose in the CHO cells. Surprisingly, only HO-1 was induced by low glucose. This response was preserved in Ka13 cells, indicating independence from HIF-1alpha .

Overall, our experiments have demonstrated the feasibility of selecting mutant CHO cells that are defective in HRE activation and demonstrated the utility of such cells in dissecting pathways of oxygen-regulated gene expression in mammalian cells. In a number of other systems, several different complementation groups of mutants have been defined, corresponding to critical individual components of the response. It seems likely that HIF-1 activation is a complex process involving more than one mechanism of activation (21, 37-39). Further attempts to isolate mutant cells with specific defects in these pathway(s) should prove very useful in understanding oxygen-regulated gene expression in mammalian cells.

    ACKNOWLEDGEMENTS

Thanks for kind gifts go to O. Hankinson for pcDNA1/Neo/mARNT; to S. McKnight for phEP-1; to G. Semenza for pBluescript/HIF-1alpha 3.2-3T7; D. Simmons for the cDNA encoding E-selectin; and G. Stark for plasmids pDW9-27CD2 (from which sequences encoding CD2 were derived), pSV2Neo, and pSV2Hyg.

    FOOTNOTES

* This work was supported by grants from the Wellcome Trust and the Medical Research Council (MRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A Wellcome Clinical Training Fellow.

§ Supported by a grant from the German Research Foundation.

An MRC Senior Fellow.

par To whom correspondence should be addressed: Room 420, Inst. of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Tel.: 44 1865 222382; Fax: 44 1865 222500; E-mail: peter.ratcliffe{at}imm.ox.ac.uk.

1 The abbreviations used are: PAS, Per-aryl hydrocarbon receptor-ARNT-Sim; AK3, adenylate kinase-3; ARNT, aryl hydrocarbon receptor nuclear translocator desferrioxamine; EPAS-1, endothelial PAS protein-1; FACS, fluorescence-activated cell scanning; Glut-1, glucose transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein 78; HO-1, heme oxygenase-1; HAT, hypoxanthine-aminopterin-thymidine; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-responsive element; HPRT, hypoxanthine phosphoribosyl transferase; ICR191, 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine dihydrochloride; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); VEGF, vascular endothelial growth factor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.

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