aHIF: a Natural Antisense Transcript Overexpressed in Human Renal Cancer and During Hypoxia

Catherine A. Thrash-Bingham, Kenneth D. Tartof

Affiliations of authors: C. A. Thrash-Bingham, Fox Chase Cancer Center, Philadelphia, PA; K. D. Tartof, Urologic Oncology Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD.

Correspondence to: Kenneth D. Tartof, Ph.D., National Institutes of Health, Bldg. 10, Rm. 2B47, Bethesda, MD 20892-1501 (e-mail: kdtartof{at} helix.nih.gov).

K. D. Tartof would like to dedicate this work to Dr. Alfred Knudson on the occasion of the 25th anniversary of his two-hit hypothesis and for making this research possible. We thank Dr. Donald Chapman and C. Strobe for their expertise and equipment used in the hypoxia experiments and B. Bingham for comments on statistical analyses.


    ABSTRACT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Nonpapillary renal carcinoma is the predominant form of human kidney cancer and represents a distinct disease entity, morphologically and molecularly, from papillary renal carcinoma. We have discovered a natural antisense transcript that is complementary to the 3' untranslated region of hypoxia inducible factor {alpha} (HIF1{alpha}) messenger RNA (mRNA) and is strikingly overexpressed specifically in nonpapillary kidney cancer. HIF1{alpha} encodes a protein that is known to have two important functions: 1) to act as a transcription factor for hypoxia inducible genes and 2) to stabilize p53 protein during hypoxia. Because of the importance of HIF1{alpha}, we have characterized this natural antisense transcript, which we have named "aHIF." METHODS: Differential display, reverse transcription-polymerase chain reaction, ribonuclease protection, and DNA-sequencing methods were used in our analysis. RESULTS AND CONCLUSIONS: We show the following: 1) aHIF is a natural antisense transcript derived from HIF1{alpha} gene sequences encoding the 3' untranslated region of HIF1{alpha} mRNA; 2) aHIF is specifically overexpressed in all nonpapillary clear-cell renal carcinomas examined, but not in the papillary renal carcinomas examined; 3) aHIF is overexpressed in an established nonpapillary renal carcinoma cell line under both normoxic (i.e., normal aerobic) and hypoxic conditions; and 4) although aHIF is not further induced by hypoxia in nonpapillary disease, it can be induced in lymphocytes where there is a concomitant decrease in HIF1{alpha} mRNA. To our knowledge, this is the first case of overexpression of a natural antisense transcript exclusively associated with a specific human malignant disease.



    INTRODUCTION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When mammalian cells are exposed to reduced levels of oxygen, the expression of a large number of genes is increased (1). Among the first gene products to be overexpressed at the onset of hypoxia is pHIF1{alpha} (HIF1{alpha} [hypoxia inducible factor {alpha}] protein) (2). This increase occurs not at the level of transcription, but rather by a post-translational alteration that appears to alter the polypeptide's stability (3-6). The resulting protein, together with arylhydrocarbon receptor nuclear translocator (ARNT), forms a heterodimeric transcription factor that binds to an hypoxia response element located in the control region of many hypoxia inducible genes, such as erythropoietin (7,8), vascular endothelial growth factor (VEGF) (9), platelet-derived growth factor-ß (PDGFB) (10,11), glucose transporter (GLUT1) (12), nitrous oxide synthetase (13), and various glycolytic enzymes (14-16). Typically, the response to hypoxia, as measured by the increase in pHIF1{alpha} activity and the subsequent induction of downstream proteins that it regulates, is quite rapid, occurring over the course of several hours (2).

pHIF1{alpha} not only functions as a hypoxia transcription factor but also interacts with p53 during the hypoxic response. It has been known for some time that hypoxic cells accumulate p53 (17). Recently, it has been shown that, in response to hypoxia, pHIF1{alpha} binds to p53 and protects it from proteolytic degradation, thereby facilitating its accumulation under conditions of low oxygen (18). Moreover, it was further demonstrated that, in the absence of pHIF1{alpha}, p53 accumulates in hypoxic cells only when a proteosome inhibitor is present. This finding suggests that, by associating with pHIF1{alpha} during hypoxia, p53 is protected from proteolytic degradation via the proteosome pathway.

The rapid response to hypoxia reflects the fact that this condition is a potentially dangerous one that most cells experience only for brief periods of time. However, it has been estimated that perhaps half of all tumors are hypoxic or are composed of hypoxic regions within the malignant growth. As tumors become hypoxic, they may overexpress the genes discussed above in an attempt to influence tumor vascularization (19). From a practical point of view, hypoxic cells pose a particular problem for the management of malignant disease because they are especially resistant to virtually all forms of therapy.

Renal carcinomas are among those malignant diseases whose rate of occurrence is increasing and for which effective nonsurgical treatments are lacking, especially for late stage disease. In general, these tumors may be broadly assigned to one of two histologic patterns of growth, nonpapillary (85% of cases) or papillary (10% of cases). Nonpapillary disease may be further subdivided according to cell type, being either clear cell, granular cell, mixed clear and granular cell, and, more rarely, sarcomatoid. In both nonpapillary and papillary renal cancers, malignant cells arise from the epithelium of the proximal renal tubule.

Nonpapillary renal carcinoma is a distinct morphologic and molecular disease entity that is quite separate from papillary renal carcinoma. Nonpapillary tumors are characterized by dysfunction of the tumor suppressor gene VHL (von Hippel-Lindau), whereas papillary kidney cancers are not (20,21). Moreover, the hereditary form of basophilic papillary renal tumors possesses mutations in the MET proto-oncogene, while such alterations are not present in nonpapillary renal disease (22).

The Von Hipple-Lindau (VHL) locus, located at chromosome 3p25, has been demonstrated to be an early-acting tumor suppressor gene that is either mutated or silenced in most cases of sporadic nonpapillary clear-cell renal carcinomas (23,24). Normally, the VHL protein (pVHL) associates with several other proteins that regulate transcription (25) and control the cell cycle (26). In addition and for reasons unknown, cells in which pVHL is defective also overexpress VEGF, PDGFB, and GLUT1, genes that are controlled by several factors, including hypoxia. It has been shown that the increase in VEGF is due to stabilization of its messenger RNA (mRNA) (11,27), and its increased production by clear-cell renal carcinomas is probably responsible for the highly vascularized stroma characteristic of nonpapillary tumors. In fact, introduction of a functional VHL gene into clear-cell carcinomas corrects the misregulation of these loci under normal aerobic (normoxic) conditions and restores a typical hypoxia-inducible response (11). Therefore, these results suggest that pVHL may affect the post-transcriptional stability of mRNAs involved in the transduction of signals that detect oxygen levels.

In this article, we demonstrate the presence of a natural antisense transcript, referred to here as aHIF (antisense HIF1{alpha}), that is complementary to sequences in the 3' untranslated region (UTR) of HIF1{alpha} mRNA and is strikingly and specifically overexpressed in nonpapillary clear-cell renal carcinomas. Because HIF1{alpha} is of central importance for the hypoxic response, we have characterized aHIF in normal and malignant cells.


    MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue Specimens, Cell Culture, and RNA Isolation

Samples of normal and tumor renal tissues were obtained from patients, with no prior exposure to chemotherapy, who underwent surgery for kidney cancer at the Fox Chase Cancer Center. Loss-of-heterozygosity studies on most of these tumors have been described previously (28). Re-examination of the pathologic specimen from patient RCC34 indicates that it should be classified as a papillary renal carcinoma. Conditions for the establishment and maintenance of renal tumor cell lines and blood-derived lymphocytes immortalized with Epstein-Barr virus (EBV) are described elsewhere (29). Total RNA was extracted from 50 mg of tissue or from cultured cells with the use of an RNeasy Isolation Kit (Qiagen, Valencia, CA). After the first extraction, all samples were treated with deoxyribonuclease (DNase) and repurified on a second Qiagen column according to the manufacturer's instructions. This second purification was necessary to eliminate all traces of DNA contamination.

Lymphocytes (106/mL) obtained from patient RCC22 and immortalized with EBV were maintained in glass vessels at 37 °C; they were mixed by a magnetic stirrer and flushed with a water-saturated gas mixture (5% CO2/specified amount of O2/N2) at a rate of 1 L/minute. At the end of the incubation period, cells were immediately chilled on ice, and RNA was extracted as described above.

Oligonucleotides

The oligonucleotides used in reverse transcription-polymerase chain reaction (RT-PCR) experiments reported here are listed in Table 1.Go They (RT-PCR) either were obtained from commercial sources as indicated or were produced on a PE Applied Biosystems (Foster City, CA) DNA synthesizer.


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Table 1. Oligonucleotides used in the reported experiments*

 
Differential Display

Differential display reactions were carried out by use of the RNAimage Kit (GenHunter, Nashville, TN). Briefly, complementary DNAs (cDNAs) were synthesized from 200 ng of total RNA by use of Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo-deoxythymidylate (oligo-dT)-based primers H-T11A, H-T11G, or H-T11C in a 20-µL volume according to conditions recommended by the supplier. Two microliters of the reaction products was amplified by PCR by use of the same oligo-dT primer and one of the following short arbitrary sequence primers: H-AP9, H-AP10, H-AP11, H-AP12, and H-AP13. Samples were subjected to electrophoresis on 5% denaturing Long Ranger acrylamide gels (FMC BioProducts, Rockland, ME). After autoradiography, bands of interest were excised from the gels and DNA was eluted. DNA fragments were re-amplified by use of the same primer pair employed in the original differential display amplification and sequenced by use of the Sequenase PCR Sequencing Kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Semiquantitative RT-PCR

These reactions were performed according to published procedures (30-32) as follows: Total RNA (200 ng) was reverse transcribed in 20-µL reactions with 1 mM each of the four standard deoxyribonucleotide triphosphates and 2.5 µM each of gene-specific reverse primers for aHIF, HIF1{alpha}, and ACTB (ß-actin; Table 2)Go by use of MMLV reverse transcriptase (GenHunter) for 1 hour at 37 °C. PCR amplification of each gene product was carried out in parallel 20-µL reactions to avoid depletion of substrates. Each tube contained 4 µL of the RT reaction, 1 µM of forward and reverse gene-specific primers, 30 µCi [{alpha}-32P]deoxyadenosine triphosphate (dATP), and 2.5 U Taq polymerase (Perkin-Elmer Applied Biosystems, Foster City, CA). Amplifications were carried out for 16 cycles for ACTB, 22 cycles for HIF1{alpha}, and 26 cycles for aHIF. These cycle numbers were chosen because they are within the exponentially increasing range with a reaction efficiency of 1.9 to 2.1 (31) and allow comparable amounts of [{alpha}-32P]dATP incorporation into each product. Equal volumes from each PCR amplification were loaded onto denaturing acrylamide gels and subjected to electrophoresis for 2 hours. After visualization by autoradiography, the DNA bands were excised from the gel, and radioactive incorporation was determined by use of a scintillation spectrometer. For the comparison of the amount of amplified aHIF and HIF1{alpha} fragments produced from different RNA samples, the amplified ACTB product of each sample was used as an internal standard. The radioactivity incorporated into aHIF and HIF1{alpha} fragments of each RNA sample was divided by the radioactivity incorporated into the ACTB fragment produced by that same sample, and a ratio was then calculated for each tumor/normal pair.


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Table 2. Relative amounts of natural antisense transcript (aHIF) and hypoxia inducible factor {alpha} (HIF1{alpha}) transcripts in nonpapillary and papillary renal cell carcinomas (RCCs)

 
cDNA Cloning

RNA from an EBV-transformed lymphocyte cell line obtained from patient RCC39 served as template for use with a 5' and 3' RACE (rapid amplification of cDNA ends) Systems Kit (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). Two types of 5' RACE experiments were performed with reverse transcriptase and gene-specific primer KT482, followed by either one or two nested PCR reactions. The first nested PCR used the Anchor primer and KT481. The second reaction utilized the Universal primer and KT477. For 3' RACE, an oligo-dT Adapter primer (Life Technologies. Inc.) was employed to synthesize cDNAs, and primers KT476 and KT480 were used in nested PCR amplification reactions with the oligo-dT primer. Amplifications were carried out as recommended by the supplier by use of aHIF gene-specific primer KT482 for cDNA synthesis and primers KT481 and KT477 for nested reactions in conjunction with the Anchor and Universal amplification primers. PCR products were cloned into the vector pCR2.1 (Invitrogen Corp., Carlsbad, CA). Cloned DNAs were cycle sequenced by use of an Applied Biosystems sequencer.

Ribonuclease Protection Assays

A 1049-base-pair (bp) aHIF fragment was PCR amplified from 5' RACE clone 3-7 by use of primers KT485 and KT489, designed to eliminate the oligo-C tail created by the RACE protocol. This fragment was cloned into pCR2.1 in both orientations. BamHI linearized plasmid served as substrate for riboprobe synthesis in a reaction containing 300-400 ng of DNA; 500 mM ATP, guanosine triphosphate, and cytosine triphosphate; 12 µM uridine triphosphate (UTP); 50 µCi [32P]UTP; 10 mM dithiothreitol; 40 U RNAsin inhibitor (Promega Corp., Madison, WI); 20 U T7 RNA polymerase; and 1x transcription buffer (Promega Corp.). Transcription products were separated on 5% denaturing Long Ranger gels. Gel slices containing full-length transcripts were excised and incubated overnight at 4 °C in gel elution buffer (Ambion, Austin, TX). Ribonuclease (RNase) protection assays were performed by use of a HybSpeed RPA Kit (Ambion). Typically, 40 µg of total RNA isolated from the RCC22 tumor cell line was mixed with 7 x 104 cpm of riboprobe labeled to a specific activity of about 109 cpm/µg. Conditions of probe hybridization and RNase T1 digestion were as recommended by the supplier (Ambion). The resulting protected fragments were separated on a 5% acrylamide gel and visualized by autoradiography. RNA size markers were synthesized by in vitro transcription of Century markers (Ambion) in the presence of [32P]UTP as recommended by the supplier.

Statistics

For the comparison of differences in expression of aHIF and HIF1{alpha} transcripts between tumor types, radioactive counts from excised bands were normalized against counts for ACTB (ß-actin) amplified in the same experiment. RT-PCR reactions for all samples obtained from the patients were performed in duplicate. Data were expressed as ratios of normalized tumor cpm divided by adjusted normal cpm from the same patient. The data were analyzed by two-sided nested analysis of variance (Nested ANOVA) (33,34).


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
aHIF: Transcript Antisense to the 3' UTR of HIF1{alpha} mRNA

To search for genes whose expression might be specifically associated with nonpapillary clear-cell renal carcinoma, we examined RNAs from five normal and nonpapillary clear-cell tumor pairs as well as from one renal oncocytoma normal/tumor pair in duplicate by differential display (35). As a result of these experiments, we discovered a distinct transcript that is strikingly overexpressed only in clear-cell tumors relative to normal kidney tissue and not expressed in either normal or tumor tissue from the renal oncocytoma (Fig. 1).Go No other transcript detected in this experiment was similarly overexpressed.



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Fig. 1. Portion of a differential display gel that shows elevated expression of a reverse transcription-polymerase chain reaction (RT-PCR) product in nonpapillary clear-cell renal carcinomas RCC9, RCC35, RCC22, RCC33, RCC13 but not in a renal oncocytoma (ONCO) from patient RCC16. Total RNA from normal (N) and tumor (T) kidney tissue was assayed in duplicate. Reamplification and sequencing of the overexpressed bands yielded a 187 nucleotide product that was found to be identical to an HIF1{alpha} (hypoxia inducible factor {alpha}) gene sequence.

 
Overexpressed bands were extracted from the acrylamide gel, amplified, and sequenced. The resulting 187 nucleotide (nt) product was compared with nucleic acid database entries and found to be identical with a portion of the 3' UTR of HIF1{alpha}. Despite this apparent identity, it was not possible to find complementarity between the HIF1{alpha} mRNA sequence and the RT and PCR primers used to generate the observed fragment. However, the same RT and PCR primers could form duplexes sufficient to account for the 187 nt differential display PCR fragment on a template that is the antisense to the 3' UTR of HIF1{alpha} mRNA. We refer to this antisense transcript as "aHIF." Three additional lines of evidence demonstrate that aHIF is a natural antisense transcript that is complementary to a large portion of the 3' UTR of HIF1{alpha}.

First, primer-specific RT-PCR amplification demonstrates overexpression of an aHIF transcript in clear-cell tumors. Forward and reverse oligonucleotide primers capable of amplifying a 92 nt fragment contained within the 187 nt UTR were synthesized (Table 1Go). DNase-treated RNA from clear-cell renal tumor RCC33 and corresponding normal kidney tissue were reverse transcribed in separate reactions by use of either the reverse or the forward primer. The resulting cDNAs were then PCR amplified in the presence of both primers and [32P]dATP, and the products were separated by acrylamide gel electrophoresis. As illustrated in Fig. 2,Go RT reactions with the reverse primer yielded similar amounts of HIF1{alpha}-derived product in both normal and tumor tissues. In contrast, RT with the forward primers produced a conspicuous aHIF-derived fragment only with tumor RNA. These HIF1{alpha} and aHIF products were not due to spurious DNA contamination because PCR amplification of RT reactions lacking reverse transcriptase (RT- lanes) failed to produce HIF1{alpha} or aHIF fragments.



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Fig. 2. Reverse transcription-polymerase chain reaction (RT-PCR) evidence that natural antisense transcript (aHIF) is an antisense transcript derived from the 3' untranslated region (UTR) of hypoxia inducible factor {alpha} (HIF1{alpha}). Forward and reverse primers located within the 3' UTR of HIF1{alpha} were used in the RT and PCR reactions to produce a 92-base-pair segment. For HIF1{alpha}, the RT reaction was initiated with the reverse primer (KT476) followed by PCR amplification with both primers (KT476 and KT478). For aHIF, the RT reaction was initiated with the forward primer (KT478) and then PCR amplified with both primers. Equal amounts of total RNA from normal (N) and tumor (T) tissues from patient RCC33 (33) were used. Aliquots were withdrawn at the indicated PCR cycle number, and the RT-PCR products were separated on an acrylamide gel. Samples for the RT- (no reverse transcriptase) lanes were PCR amplified for 24 or 26 cycles for HIF1{alpha} or aHIF, respectively. See text for additional details.

 
Second, RACE primers derived from the 187 nt amplified product were used to obtain cDNA clones extending in the 5' and 3' directions along the aHIF transcript (Fig. 3,Go A). Sequencing of six independent RACE clones and one RT-PCR clone revealed that aHIF is at least 1577 nt in length and contains no predicted open reading frame (Fig. 3Go, B). The 5' end of the aHIF transcript consists of 695 nt of unique sequence that is contiguous with genomic sequences and is not found in any nucleic acid database. This is followed by 882 nt that exactly complements the terminal portion of the 3' UTR of HIF1{alpha} mRNA. Since a differential display clone and a 3' RACE clone isolated with different primers terminate at the same sequence and the 5' RACE clones continue to be extended by use of additional primers, it is likely that we are at or very near the 3' terminus of aHIF but have not reached the 5' end of the transcript.



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Fig. 3. A) Organization of the hypoxia inducible factor {alpha} (HIF{alpha}) messenger RNA (mRNA) and the natural antisense RNA aHIF. B) Partial sequence of the aHIF transcript. 5' and 3' rapid amplification of complementary DNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR) primers (seeTable 2Go and "Materials and Methods" section) together with RNAs from a lymphocyte cell line overexpressing aHIF RNA yielded the partial sequence shown here. Nucleotides (nt) 1-695 are unique to aHIF. Bold, underlined T at nt 696 is the beginning of that portion of the transcript that is antisense to sequences in HIF1{alpha} mRNA. The Genbank accession number for the aHIF sequence is U85044. ORF = open reading frame; kb = kilobase.

 
With respect to the 3' end of aHIF, two additional observations suggest that it is not polyadenylated. First, the reverse primer used in the differential display experiment that is designed to form a duplex with terminal polyadenylated stretches (see "Materials and Methods" section) hybridized instead to an internal region of the aHIF transcript and not with a long terminal polyA tract. Second, when RNA from clear-cell renal carcinoma cell line RCC22 was fractionated on an oligo-dT column (data not shown), it was possible to enrich considerably for HIF1{alpha} mRNA but not for aHIF. Attempts to obtain a satisfactory northern blot of aHIF have been hampered for two reasons: 1) It is a very AU (i.e., adenine-uridine)-rich sequence and 2) because it is not polyadenylated, it is not possible to isolate it from the vast amount of ribosomal RNA that is otherwise present.

Finally, an RNase protection experiment demonstrates that aHIF is an antisense transcript derived from the opposite strand of the 3' UTR of HIF1{alpha}. As illustrated in Fig. 4Go, a cDNA obtained from the RACE experiments containing the last 1049 bp of the aHIF transcript (see "Materials and Methods" section) was cloned, in both orientations, adjacent to a T7 promoter. The full-length in vitro synthesized riboprobe is 1163 nt, owing to an additional 114 nt derived from plasmid sequences flanking the insert. HIF1{alpha} and aHIF transcripts are expected to overlap with 737 nt and 1049 nt of their complementary riboprobes, respectively. Full-length 32P-labeled riboprobes complementary to HIF1{alpha} and aHIF were synthesized in vitro, purified on an acrylamide gel, and then hybridized to total RNA isolated from RCC22, a cell line established from a clear-cell renal carcinoma (28). Following RNase T1 digestion, the RNA duplexes were denatured and subjected to electrophoresis on an acrylamide gel.



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Fig. 4. Ribonuclease (RNase) protection experiment to detect sense/antisense hypoxia inducible factor {alpha} (HIF1{alpha}) and aHIF natural antisense transcripts. Upper part of the figure contains a diagram indicating the cloned transcription template from which HIF1{alpha} and aHIF complementary transcripts are synthesized. Dark blocks indicate plasmid-derived sequences. HIF1{alpha} and aHIF overlap the template partially and completely, respectively. For lower part of the figure, total RNA from kidney tumor cell line RCC22 was hybridized to 32P-labeled riboprobes specific for HIF1{alpha} or aHIF transcripts and then digested with RNase T1. Lanes 1 and 4, 32P-labeled HIF1{alpha} and aHIF riboprobes, respectively; lanes 2 and 5, hybridization of total RCC22 RNA with HIF1{alpha} and aHIF riboprobes followed by RNase T1 digestion, respectively; lanes 3 and 6, HIF1{alpha} and aHIF riboprobes annealed in the absence of RCC22 RNA and treated with RNase T1, respectively.

 
As shown in Fig. 4,Go the 1100 nt 32P-labeled riboprobes (lanes 1 and 4) were reduced to protected fragments of about 750 and 1000 nt (lanes 2 and 5), respectively, following hybridization and nuclease digestion. These bands were not detected in lanes 3 and 6. In the case of the aHIF sense riboprobe complementary to HIF1{alpha}, two bands of about 750 nt and differing from each other by about 15 nt were consistently observed (lane 2). This may have been due to breathing of the AU-rich duplex that can occur during RNase T1 digestion.

aHIF Overexpression in Clear-Cell Renal Tumors

Semiquantitative RT-PCR was used to assess the relative abundance of aHIF and HIF1{alpha} transcripts with respect to ACTB in various renal tumors. The efficiency of RT-PCR amplification of aHIF, HIF1{alpha}, and ACTB transcripts was found to be 2.09 ± 0.02 (mean ± standard deviation), 2.06 ± 0.03, and 1.97 ± 0.05 per cycle, respectively, over the cycle numbers used here. The PCR cycle numbers chosen for the assay of these products in the experiments described below are within the exponentially increasing range of the reactions and permit comparable amounts of [{alpha}-32P]dATP incorporation without exhausting the substrate.

RT-PCR experiments using primers within the unique 5' domain of aHIF demonstrated that this antisense transcript was specifically overexpressed in all nonpapillary clear-cell renal cell carcinomas examined. RNA from each of 10 different primary clear-cell tumors and the corresponding normal tissues was reverse transcribed in a reaction containing the reverse primers for aHIF, HIF1{alpha}, and ACTB transcripts. The resulting cDNAs were PCR amplified in three separate reactions using primers specific for each transcript, and the synthesized fragments were examined by acrylamide gel electrophoresis. As illustrated by the four representative examples presented in Fig. 5,Go A, aHIF was strikingly overexpressed in all nonpapillary carcinomas in comparison with normal kidney tissue, whereas the abundance of HIF1{alpha} and ACTB transcripts in these same specimens was relatively constant. In contrast, papillary renal carcinomas (Fig. 5Go, B) displayed no consistent differential elevation of either aHIF, HIF1{alpha}, or ACTB products.



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Fig. 5. The natural antisense transcript aHIF is overexpressed in nonpapillary clear-cell renal carcinomas (RCCs). A) Normal and tumor tissue RNAs from four nonpapillary clear-cell RCCs (13, 14, 33, 35, and 33) were assayed for aHIF, HIF1{alpha} (hypoxia inducible factor {alpha}), and ACTB (ß-actin) transcripts. Normal tissue from RCC33 was used as a control in a reaction in which reverse transcriptase was omitted (RT-). B) Normal and tumor tissue RNAs from four papillary RCCs (6, 36, 63, and 17) were assayed as in A. In all cases, aHIF, HIF1{alpha}, and ACTB reverse transcribed complementary DNAs were polymerase chain reaction (PCR) amplified for 26, 22, and 16 cycles, respectively. HIF1{alpha} reverse and forward specific primers KT467 and 466 are located at base pairs (bp) 2472 and 2013, respectively, in the open reading frame (ORF) depicted in Fig. 3Go, A. aHIF reverse and forward specific primers KT492 and KT491 are located at bp 417 and 675 in Fig. 3Go, B, respectively. Reverse and forward primers for ACTB are located at bp 60 and 331, respectively. See text for additional details.

 
The RT-PCR-amplified aHIF, HIF1{alpha}, and ACTB fragments from 10 nonpapillary clear-cell and seven papillary tumor and normal tissue pairs were excised from acrylamide gels, and their radioactivity was quantitated. For all tissue samples, aHIF- and HIF1{alpha}-amplified fragments were normalized to ACTB amplified from the same RT reaction. As demonstrated by the data presented in Table 2Go, nonpapillary clear-cell renal tumors exhibited a 10- to 100-fold increase in aHIF relative to normal kidney tissue. There was a statistically significant increased expression of aHIF in nonpapillary tumors in comparison with papillary disease (P = .0005 by Nested ANOVA). Although aHIF was markedly overexpressed in nonpapillary clear-cell tumors, it was only a modestly abundant transcript. On the basis of the difference in PCR cycle number, there was about 250-fold more ACTB transcript than aHIF.

Overexpression and Hypoxic Induction of aHIF in Renal Carcinoma Cells and Lymphocytes

Because aHIF is associated with the HIF1{alpha} locus, we wanted to determine if the aHIF transcript could be induced by hypoxia. Therefore, RCC22, one of our established nonpapillarly renal carcinoma cell lines, was exposed to varying oxygen conditions. Cells were equilibrated in a normal oxygen atmosphere (21%) for 1 hour and then in 21%, 0.3%, 0.1%, and 0% oxygen for 2 hours. After RNA purification, the samples were assayed by RT-PCR for the presence of aHIF and ACTB transcripts. As shown in Fig. 6Go, A, the expression of aHIF remained elevated and at about the same levels, whether in a normoxic or in a hypoxic atmosphere. The amount of ACTB remained relatively constant under these conditions as well.



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Fig. 6. Hypoxia induction of the natural antisense transcript aHIF. A) Effect of hypoxia on renal cell carcinoma 22 (RCC22). Upper panel shows the levels of aHIF and ACTB (ß-actin) transcripts as assayed by reverse transcription-polymerase chain reaction (RT-PCR) techniques, at the indicated oxygen atmospheres by use of the same number of amplification cycles as that used in Fig. 5Go. RNA from the 0.1% oxygen sample served as the RT- (no reverse transcriptase) control. Lower panel plots the ratio of 32P-labeled aHIF/ACTB transcripts at each oxygen concentration. B) Effect of hypoxia on lymphocytes. Upper panel shows the levels of aHIF and ACTB transcripts at the indicated oxygen atmospheres as assayed by RT-PCR. RNA from the 0.1% oxygen sample served as the RT- control. Lower panel plots the ratio of 32P-labeled aHIF/ACTB transcripts at each oxygen concentration. C) Time course for the hypoxic induction of aHIF in lymphocytes. Upper panel shows levels of aHIF, HIF1{alpha} (hypoxia inducible factor {alpha}), and ACTB transcripts as assayed by RT-PCR by use of the same number of amplification cycles as that used in Fig. 5Go. RNA from the 6.5-hour sample served as the RT- control. Lower panel plots the ratios of 32P-labeled aHIF/ACTB and HIF1{alpha}/ACTB transcripts as a function of time. This experiment was carried out in duplicate.

 
However, when an EBV-transformed lymphocyte cell line derived from a normal individual was equilibrated in a normal oxygen atmosphere (21%) for 1 hour and then exposed to 21%, 1%, 0.3%, 0.1%, and 0% oxygen for 2 hours, a different result was obtained. As shown in Fig. 6,GoB, there was a striking induction of aHIF at oxygen concentrations of 0.3% or less, while the amount of ACTB remained relatively constant. In this case and in the RCC22 experiment (Fig. 6Go, A and B), the RT-control demonstrated that this induction was not due to contaminating DNA. Quantitation of the amount of radioactivity in each band indicated that, in lymphocytes, there was about an eightfold increase in aHIF expression relative to ACTB under conditions of 0.3% oxygen or less (Fig. 6Go, B).

Because the amount of aHIF was measured relative to ACTB in the cell being assayed, it was not possible by this method to compare the absolute levels of aHIF in RCC22 and lymphocytes. The difference in aHIF/ACTB ratios in RCC22 and lymphocytes might be due to differences in the amounts of aHIF, ACTB, or both. Nevertheless, it is clear that RCC22 cells express aHIF at high levels in normoxic and hypoxic conditions, whereas lymphocytes express high levels of aHIF only in a hypoxic environment. It is possible that aHIF is not further induced by hypoxia in RCC22 because it is already maximally expressed in normoxic conditions.

Two other cell lines were also assayed for possible inducibility of aHIF by hypoxia. aHIF remains very low and not induced by hypoxia in Hep3B (human hepatocarcinoma) cells and HeLa (human ovarian carcinoma) cells (data not shown). The reason for this is not known. It may be an intrinsic property of these cell types, or it may the reflect changes that such cell lines have undergone over the very long time they have been maintained in culture that have impaired aHIF expression. The pattern of expression of aHIF in a variety of mammalian cells and tissues is being investigated.

For the determination of the effect of hypoxia on aHIF, HIF1{alpha}, and ACTB over time, lymphocytes were equilibrated in an atmosphere of 21% for 1 hour and then shifted to 0.1% oxygen. At appropriate intervals, aliquots of cells were removed, RNA was extracted, and the presence of aHIF, HIF1{alpha}, and ACTB RNA was assayed by RT-PCR. As shown in Fig. 6Go, C, induction of aHIF began at about 2 hours and increased over the next 6.5 hours. During that period, the amount of ACTB remained virtually constant. Quantitation of the radioactivity incorporated in each band is graphically displayed in the lower portion of Fig. 6Go, C. The results indicate that, by 6.5 hours of hypoxia, aHIF increased about eightfold while HIF1{alpha} appeared to be reduced about twofold. This inverse relationship suggests that aHIF expression may negatively affect the abundance of HIF1{alpha} mRNA.


    DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our results demonstrate the following: 1) aHIF is a natural antisense transcript derived from the 3' UTR of HIF1{alpha}; 2) aHIF is strikingly overexpressed in all of the nonpapillary clear-cell renal carcinomas that we have examined, but not in papillary renal carcinomas; 3) aHIF is overexpressed in an established nonpapillary renal carcinoma cell line under both normoxic and hypoxic conditions; and 4) while aHIF is not further induced by hypoxia in nonpapillary disease, it can be induced in lymphocytes and there is a decrease in HIF1{alpha}. To our knowledge, this is the first case of overexpression of a natural antisense transcript exclusively associated with a specific human malignant disease. As discussed below, these results may have important implications for understanding the sequence of events beginning with dysfunction of the VHL gene that affects aHIF expression and the downstream consequences that this may have for pHIF1{alpha} and p53.

It is known that, in at least 80% of all sporadic nonpapillary clear-cell renal carcinomas, there is loss of VHL function as a result of mutation or gene silencing (21,24). This situation indicates that loss of VHL function is a very early event in the development of nonpapillary renal cancer. Moreover, considerable molecular data demonstrate that pVHL mediates a post-transcriptionally controlled increase in the expression of a number of loci associated with oxygen stress, including VEGF, GLUT1, and PDGFB (11,27). The overexpression of aHIF is consistent with this pattern. Hence, it will be of interest to determine if VHL acts through a transcriptional or post-transcriptional mechanism to cause the increased expression of aHIF RNA.

In the nonpapillary renal carcinoma cell line RCC22, hypoxia fails to increase further the already elevated expression of aHIF. This result suggests that the aHIF transcript may be maximally expressed in these cells. However, in lymphocytes, aHIF is present at low levels under normoxic conditions but is readily inducible by hypoxia. This inducibility implies that aHIF may play a regulatory role in the hypoxic response. In this regard, it is of interest to note that the increase in aHIF and the decline in HIF1{alpha} occur at a time (about 4 hours) when pHIF1{alpha} begins to decrease (2). Thus, aHIF may facilitate the reduced expression of pHIF1{alpha} during periods of prolonged hypoxia. It is also worth noting that the reciprocal regulation of the sense transcript by expression of its counter transcript has been reported for several genes (36-40). Assay of pHIF1{alpha} in conjunction with the expression of aHIF and HIF1{alpha} transcripts will help to decide this issue.

The potential for aHIF to reduce HIF1{alpha} abundance could have important implications for the regulation of p53. Increased aHIF expression could lead to a decrease in the amount of pHIF1{alpha} and, therefore, a lower abundance of p53. Because p53 acts as a tumor suppressor, loss of p53 function results in deregulated cell proliferation (41-43). An interesting example of how the level of p53 can be altered to achieve oncogenic transformation involves the MDM2 locus. MDM2 binds p53 and accelerates its degradation through the ubiquitin pathway (44,45). Of particular interest is the fact that MDM2 is amplified in more than one third of 47 human sarcomas examined (46). It has been proposed that, in this subgroup of cancers, loss of p53 function occurs not by mutation of p53 but rather by enhancing its degradation. Experiments to determine the effect of aHIF expression on p53 and pHIF1{alpha} are under way.

Much remains to be discovered about the aHIF transcript itself. Because the aHIF sequence reported here is incomplete, it is not known if it is linked to a protein-coding region or possesses other regulatory information. Also, we do not know the sequences regulating aHIF expression, although a hypoxia response element would appear to be implicated. If aHIF forms a duplex structure with the 3' UTR of HIF1{alpha} mRNA in vivo, this might affect the abundance of the HIF1{alpha} transcript or the protein it encodes. In addition, such a putative duplex might interact with proteins such as double-stranded RNA adenosine deaminase-editing enzymes (47-49), double-stranded RNA-activated protein kinase (50), interferon-induced RNase L (51), or inosine-specific RNase (52). Experiments are under way to address these points.

The overlap between aHIF and HIF1{alpha} transcripts is substantial, corresponding to 882 bp of the 1169 bp in the 3' UTR of HIF1{alpha} mRNA. It is of interest to note that the HIF1{alpha} mouse and human 3' UTRs are about 89% identical over their entire length (53). This high degree of noncoding sequence conservation is relatively common. Recent comparisons of vertebrate genes included in the Genbank database reveal that about 5%-30% have highly conserved 3' UTRs (54,55). Such extensive homology throughout a noncoding sequence implies that there is strong selective pressure acting on the entire region that has an important or essential role in the cell (54).

An intriguing hypothesis to explain the strong conservation in 3' UTRs such as this proposes that long, nearly perfect RNA duplexes are formed between sense and antisense transcripts and function as a signal for the stabilization or destruction of the target mRNA (56). Although there may be some small nt sequences that bind regulatory proteins within or near these duplexed regions, it is the long length of the duplex per se that is the signaling element. Sequence conservation over the entire length of 3' UTR is maintained because the presence of mutations would, in the heterozygote, disrupt the duplex.

The overexpression of a natural antisense transcript in clear-cell renal carcinoma suggests a novel regulatory mechanism that may be of importance for understanding the control of gene expression in normal cells as well as in several diseases, including cancer. The system reported here provides a means to explore this phenomenon further.

Supported by grants from the Lucille P. Markey Charitable Trust; Public Health Service grant CA06927 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; a gift from the Tuttleman Family Foundation in tribute to Dr. Michael Kriegler; and an appropriation from the Commonwealth of Pennsylvania.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received June 18, 1998; revised October 21, 1998; accepted November 10, 1998.



             
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