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Molecular Oncology, Markers, Clinical Correlates |
-Hydroxylase (CYP 27B1) in Human Malignant Glioma
Institut für Humangenetik, Theoretische Medizin [R. M. M., K. R., B. D., U. F., E. M.], Neurochirurgische Universitätsklinik [W-I. S.], and Abteilung für Neuropathologie [W. F.], Universität des Saarlandes, 66421 Homburg/Saar, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: To determine the amplification frequency, we performed comparative PCR studies and Southern blot hybridization experiments. To identify full-length clones of GAS89 we screened a HybriZAP library. Reverse transcription-PCR was performed to isolate splice variants and to determine expression levels.
Results: We identified for the gene GAS89 an amplification frequency of 25% in 28 examined glioblastoma multiforme samples. Screening a HybriZAP library, we isolated an incomplete gene sequence showing identity with the gene for 25-hydroxyvitamin D3 1,
-hydroxylase. Different full-length clones were then isolated using PCR primers chosen from the 3'- and 5'-untranslated regions. As determined by sequencing, the clones represent various splice variants of the 25-hydroxyvitamin D3 1,-hydroxylase gene. The clones encode truncated proteins but also one potentially functional enzyme variant. Reverse transcription-PCR studies revealed overexpression of several variants in glioblastoma samples with GAS89 amplification in comparison with normal brain RNA and glioblastoma without GAS89 amplification.
Conclusions: This is the first report of gene amplification for 25-hydroxyvitamin D3 1,-hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme. The endogenous expression of the 25-hydroxyvitamin D3 1,-hydroxylase gene and the appearance of alternative splice variants reveal a new feature of the molecular pathogenesis of glioblastoma and may represent a new target for glioma therapy.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of glioblastomas resulting in increased expression of the corresponding protein (3)
. Another, but less frequently amplified region, is chromosome 12q13–14 with an incidence of 15% of malignant gliomas (4)
. Recently, we were able to identify several cDNAs from a homogeneously staining region at chromosome 12q13–15 using microdissection-mediated cDNA capture (5)
. One of these genes, GAS41, could be identified as a potential transcription factor and was amplified in 23% of GBM (6)
.
In this study, we isolated and characterized the corresponding cDNA of another microdissected cDNA fragment (GAS89). Sequence comparison shows identity of the GAS89 cDNA with the gene encoding 25-hydroxyvitamin D3 1,-hydroxylase (CYP27B1), that catalyzes a key step in vitamin D3 metabolism. Precursors of vitamin D3 are metabolized in the skin under the influence of UV-light and are then hydroxylated in the liver to 25-hydroxyvitamin D3. The further hydroxylation to the active form of vitamin D3, calcitriol or 1,25(OH)2D3, is performed by the enzyme 25-hydroxyvitamin D3 1,-hydroxylase in the kidney. The cDNA for this enzyme was cloned from rat (7)
and human (8
, 9)
material. Using fluorescence in situ hybridization analysis, the gene was localized to chromosome 12q13–14, and the genomic DNA fragment was organized in 9 exons and 8 introns (10)
.
The biological active form of vitamin D3, calcitriol, is a secosteroid hormone best known for its role in calcium and bone metabolism. In addition, calcitriol provides antiproliferative and differentiating effects through binding to the vitamin D receptor belonging to the steroid/thyroid/retinoic acid receptor family that functions as a ligand-dependent transcription factor (11) .
Herein we report the endogenous expression of the 25-hydroxyvitamin D3 1,-hydroxylase in human malignant glioma that potentially could result in the production of biologically active calcitriol.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection of the cDNA library into Escherichia coli-XL1-blue MRF' was performed using standard protocols. The corresponding phage clones were transferred onto nylon membranes (Boehringer Mannheim, Mannheim, Germany). Plaque hybridization was done using the digoxygenin-labeled insert of plasmid pGAS89 according to the manufacturer’s protocol (DIG Nonradioactive Labeling and Detection kit, Boehringer Mannheim).
Sequence Analysis.
Sequencing was performed according to the manufacturer’s instructions using the Amersham Thermo Sequenase-labeled primer cycle sequencing kit with 7-deaza-dGTP. Plasmid inserts were sequenced with an automated sequencer (Licor 4000 L; MWG Biotech, Ebersberg, Germany). The obtained sequences were edited using the SEQUENCHER 3.0 program (GeneCodes; Ann Arbor, MI). Homology search was done with the BLASTN and BLASTX algorithms (12)
.
Tumors and Tissues.
Tumor samples were kindly provided by the Department of Neurosurgery, Homburg, Universität des Saarlandes (Homburg, Germany). The samples had been snap-frozen in liquid nitrogen immediately after surgical excision and stored at -75°C until usage. Cell line TX3868 was established and cultured as described previously (5)
. All of the tumor samples originated from grade IV glioblastoma, as determined by the Department of Neuropathology. Normal brain RNA was obtained from Clontech, Palo Alto, California. Normal kidney RNA was a gift from Dr. H-P. Sattler, Department of Human Genetics.
DNA and RNA Isolation.
RNA isolation was carried out according to the manufacturer’s instructions (Stratagene). In brief, frozen tissue was homogenized, proteins and DNA were extracted using phenol/chloroform (pH 5.3–5.7), and the remaining RNA was precipitated with isopropanol. The RNA was quantified spectrophotometrically, and its integrity was controlled by agarose gel electrophoresis in 3-(N-morpholino) propane sulfonic acid buffer.
Genomic DNA from frozen tissue samples and blood lymphocytes was isolated according to standard protocols (13) . To screen E. coli transformants, plasmid DNA from bacterial cultures was isolated using alkaline lysis without a further purification step (14) . A column purification was added for plasmids that were sequenced (Plasmid Mini kit; QIAgen, Hilden, Germany).
Comparative PCR.
Comparative PCR was performed as described previously (15)
. The aim of this method is to determine the number of copies of a specific gene in tumor DNA samples versus normal blood DNA. Concentration of DNA samples were determined by absorbance measurement and adjusted to 5 ng/µl. Three dilutions from each test sample (0.5, 1.0, and 2.0 ng/µl) were used as templates in different reactions to obtain the same concentrations in normal and tumor DNA. Primers for the gene Mucin 2 (MUC2A: 5'-CATTCTCAACGACAACCCCT-3'; MUC2Z: 5'-GCAAGAGATGTTACTGCC-3'), which maps at 11p15.5 and for clone 9-1 plus (9-1 forward: 5'-GCTTTATTGCCCTTGTCTATG-3'; 9-1 reverse: 5'-TCCTATCTGCTTCTACCATCC-3') located at 14q were used for calibration purposes. Both genes represent single copy genes. When each dilution of the tumor and blood DNA was adjusted to the same concentration, a final PCR with the GAS89-specific primers H89 forward and H89 reverse was performed to detect amplification of the GAS89 gene. The PCR reactions were carried out in a thermal cycler (PTC100; MJ Research) with the following cycling conditions for the MUC primers: 26 cycles of 1 min denaturation at 94°C, 45 s annealing at 60°C, 45 s extension at 72°C. The PCR for the 9-1 primers was run for 26 cycles at 94°C for 45 s, 58°C for 45 s, and 72°C for 45 s. The GAS89 specific primers H89 forward (5'-TGCAGCATCAATGAACACTAT-3') and H89 reverse (5'-GGCCCTTCTGATCATGTATGC-3') were deduced from the originally isolated 350-bp fragment of pGAS89 that was localized in this study to the 3'-untranslated region of the gene by sequence comparison. PCR conditions for the H89 primer were: 27 cycles of 1 min at 94°C, 45 s at 56°C, and 45 s at 72°C.
RT-PCR.
Prior to utilization, the RNA that served as a template for RT-PCR was DNaseI treated. The existence of genomic DNA contaminants was then excluded by Alu-PCR. First-strand cDNA was synthezised with the PROSTAR First strand RT-PCR kit (Stratagene) using 5 µg of total RNA, oligo (dT)-Primer and Moloney murine leukemia virus-reverse transcriptase in a volume of 50 µl and stored at -20°C. Five µl of the first-strand cDNA were used in the following PCR. To obtain the entire cDNA fragments of the 1,-hydroxylase from TX3868 cDNA, the primer 5'-end (5'-TATGATGCTCAGGAGAAGCG-3') was chosen from the 5'-untranslated region of the known cDNA. Primer H89 forward binds to the antisense strand in the 3'-untranslated region and was, therefore, used as the 3'-primer for this purpose. PCR was carried out with the Expand High Fidelity PCR system (Boehringer Mannheim) for 33 cycles as follows: 94°C for 1 min, 56°C for 45 s, and 72°C for 1 min and 30 s. PCR products were excised from the gel and extracted with the QIAquick Gel Extraction kit (QIAgen). Cloning into the pGEM-T Easy Vector System (Promega, Mannheim, Germany) was performed according to the manufacturer’s instructions. To analyze the 1,-hydroxylase splice variants, primers 5'-gap (5'-TGAACAACGTAGTCTGCGACCTT-3') and 3'-gap (5'-TGACACAGAGTGACCAGCGTATTT-3') were chosen from exon 3 and exon 8. After 34 cycles (1 min at 94°C, 1 min at 60°C, 1 min 72°C) of amplification with Taq-DNA Polymerase (Pharmacia, Freiburg, Germany), the resulting bands were cloned using the pMOSBlue blunt-ended cloning kit (Amersham, Braunschweig, Germany).
Southern Blot Analysis.
PCR products were separated on 1%-agarose gels and then transferred to a Gene-Screen Nylon membrane in 20x SSC. Labeling and detection of the probe was performed with the DIG Non-Radioactive Labeling and Detection kit (Boehringer Mannheim) as recommended by the manufacturer. The membrane was hybridized with the 1.6-kb insert from clone pGAS89-b in 5x SSC, 1% blocking reagent, 0.1% lauroylsarcosin, and 0.02% SDS overnight at 42°C. Washing was done twice for 5 min in 2x SSC/0.1% SDS at room temperature and twice for 15 min in 0.1x SSC/0.1% SDS at 55°C.
DNA (5 µg) from peripheral blood lymphocytes and from tumor samples was digested with EcoRI, separated on a 0.8% agarose gel and blotted onto a nylon membrane (GeneScreen). Southern hybridization was carried out in a 500-mM phosphate buffer (pH 7.2) as described previously (6)
. Insert DNA (50 ng) was labeled with [-32P]dATP using the Random-primed DNA labeling kit (Boehringer Mannheim).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Hydroxylase.-hydroxylase, which was recently localized on chromosome 12q13–15 by somatic cell hybrid mapping (8
, 9
, 10)
. The 25-hydroxyvitamin D3 1-hydroxylase, a mitochondrial cytochrome P450 enzyme, catalyzes the hydroxylation of 25-hydroxyvitamin D3 to 1,25(OH)2D3, the active form of vitamin D3. The 1,-hydroxylase gene is located in the amplification unit 12q13–14 between MDM2 and CDK4/SAS according to the National Center for Biotechnology Information Map-View program.
The insert of the primary isolated clone pGAS89 was identical with a sequence in the 3'-untranslated region of the 1,-hydroxylase gene. The 5'-region of the isolated clone pGAS89-b was incomplete probably resulting from an incomplete reverse transcription reaction during the cDNA library construction. In contrast to the known sequence for the 1,-hydroxylase cDNA, pGAS89-b exhibited deletions of exons 4 and 5, and insertion of intron 2 at the 5'-end and was, therefore, termed hydroxylase-variant 1 (Hyd-V1). Isolation of this clone was the first evidence for aberrantly spliced variants of the gene for 1,-hydroxylase in the TX3868 glioblastoma cell line.
Amplification Analysis for the 1,-Hydroxylase Gene in Glioblastoma Samples.
To determine the frequency of gene amplification, tumor samples were analyzed using comparative PCR with pGAS89-specific primers. In 7 of 28 tested GBM samples (25%), an amplification of the 1,-hydroxylase gene was detected compared with peripheral blood DNA. One representative example of a strong amplification (H549) and three samples with weak GAS89 amplifications (H556, H346, and H246) are shown in Fig. 1A
. Additionally to comparative PCR, amplification analysis was performed by Southern blot hybridization using the insert of pGAS89-b as the probe. Only tumors with high amplification levels (H91, H118, and H385) revealed by comparative PCR show strong signals (7.65 kb) in the hybridization experiments as shown in Fig. 1B
.
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-Hydroxylase Gene.-hydroxylase. RT-PCR resulted in two major products that were excised from the gel and subcloned into pGEM-Teasy. Restriction analysis of the resulting transformants revealed three different inserts. Sequence analysis of the subcloned cDNAs and comparison with the known sequence for 1,-hydroxylase indicated that these clones represented additional splice variants and were named hydroxylase-variant 2, 3, and 4 (Hyd-V2, -V3, and -V4; Fig. 2
). Variant Hyd-V2 lacks exons 4 and 5 resulting in a truncated protein of 207 amino acids in contrast to 508 amino acids encoded by the full-length 1,-hydroxylase gene. This is attributable to a termination codon that occurs after the first 29 bp in Exon 6 and is caused by a frameshift. The resulting protein contains neither the ferredoxin nor the haem-binding site of the P450 protein and, therefore, probably represents an inactive variant of the enzyme.
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-hydroxylase. It is unlikely that this variant could produce an active enzyme.
Variant Hyd-V4 is complete with regard to the exons of the known cDNA but additionally contains the 3'-region of intron 2 using an alternative 5'-splice signal-AG in intron 2. Translation of the Hyd-V4 cDNA would result in an in-frame insertion of 27 amino acids to generate a protein product of 535 amino acids containing both the ferredoxin- and the haem-binding site. This protein product may represent an active variant with regard to the 1,-hydroxylase specific properties.
Expression of Splice Variants in Several Normal Tissues and Glioblastoma.
To examine the correlation between amplification and the appearance of splice variants of the 1,-hydroxylase gene, we performed RT-PCR with RNA from normal tissues and tumor samples. We used five glioblastoma samples with amplification and nine glioblastoma samples without amplification of the 1,-hydroxylase gene as determined by comparative PCR. Normal tissues were normal brain and kidney tissues in which the 1-hydroxylase is primarily active. Because attempts to amplify the entire cDNA only result in weak bands, we used primers chosen from exons 3 and 8 restricting our contemplation at the lack of exon 4 and 5. The variants summarized in Fig. 2B
should generate two products (763 bp and 389 bp) that cannot be ascribed to single templates. Both Hyd-V1 and Hyd-V2 can produce the signal of 389 bp, whereas the product of 763 bp can be generated by both Hyd-4 and the normal cDNA of the1,-hydroxylase gene. Variant Hyd-V3 cannot be amplified with the primers used in this study because of the deletion of the binding site of the reverse primer at the beginning of exon 8.
Four representative tumor samples, cell line TX3868, and normal tissues were blotted and hybridized with the insert of pGAS89-b as the probe to confirm specificity (Fig. 3)
. The strongest signal in kidney was that of 763 attributed to the normal cDNA containing both exons 4 and 5, as expected. Variants Hyd-V2 and Hyd-V1 that probably encode truncated proteins were also expressed but at a lower level. This proportion was reverted in normal brain in which the expression level of variants Hyd-V2/V1 was stronger than the expression of the normal 1,-hydroxylase cDNA or variant Hyd-V4, respectively. Similar expression patterns were detected in tumor samples without amplification of GAS89. In tumors carrying 1,-hydroxylase gene amplifications and in TX3868, the glioblastoma cell line from which GAS89 was isolated, the expression level of the normal 1,-hydroxylase cDNA/Hyd-V4 was higher than in normal brain. The cDNA fragment of 763 bp was barely visible in the normal brain sample. An additional product of about 1000 bp appeared in most tumors with amplification and in TX3868. Cloning and sequencing of this fragment revealed a new variant Hyd-V5 containing intron 5. This caused a frameshift resulting in a premature stop codon soon within exon 6. Summarizing the RT-PCR results, we assume there is a correlation between the amplification of the 1,-hydroxylase gene in GBM and the expression level of several splice variants and the normal cDNA of the 1,-hydroxylase gene.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase cDNA from an amplified region of chromosome 12q13–15 derived from the glioblastoma cell line TX3868. Chromosome 12q13–15 is frequently amplified in human sarcomas and brain tumors and several genes of potential significance for neoplastic growth are located in this genomic segment such as Gli, SAS, CDK4, and MDM2 (16)
. Amplification mapping of chromosome 12q13–15 in malignant glioma reveals the highest amplification level (about 15%) for the two loci CDK4/SAS and MDM2 suggesting these three genes as likely targets of the amplification unit (4)
. A refined mapping of this region in 24 primary gliomas using seven known loci located between CDK4 and MDM2 shows no continuity in this region of the amplicon (17)
. However, it is still possible that all amplicons share another amplified region between MDM2 and CDK4/SAS which might contain one or more as yet unidentified genes. One of the candidate target genes on chromosome 12q13–15 is GAS41, probably encoding a transcription factor. GAS41 is amplified in 23% of glioblastoma, in 50% of astrocytoma grade II and grade III, and even in 80% of analyzed pilocytic astrocytoma (6)
. The relatively high amplification frequency (25%) detected in this study for the 1,-hydroxylase gene suggests that this gene may be a further likely target gene of the amplification unit 12q13–15. To our knowledge, this is the first report concerning the chromosomal alteration of the 1,-hydroxylase gene in malignant tissue. Another gene from vitamin D metabolism, the 24-hydroxylase, could recently be detected as a likely target oncogene of the amplification unit 20q13.2 in breast cancer tumors and cell lines using array CGH (18) . The 24-hydroxylase inactivates the biologically active 1,25(OH)2D3 by a further hydroxylation step so that overexpression attributable to amplification may abrogate vitamin D3-mediated growth control. This known function supports its candidacy for being an oncogene although 24-hydroxylase has not been previously implicated in breast cancer.
It is more difficult to define the role of the 1,-hydroxylase gene in the pathogenesis of malignant glioma because this enzyme catalyzes the synthesis of biologically active vitamin D3. Amplification and overexpression of this gene should exert antiproliferative and prodifferentiating effects according to the in vitro studies. Analysis of numerous normal and cancer cell lines demonstrates that at high concentrations (10-9 to 10-7 M) 1,25(OH)2D3 inhibits the growth of tumor cells in vitro, and it has also been shown that 1,25(OH)2D3 has beneficial effects in several in vivo models of various types of cancer (19
, 20)
. If the overexpression of the 1,-hydroxylase gene results in an overproduction of 1,25(OH)2D3, the accumulation of this metabolite should have beneficial effects as shown by the in vitro and in vivo studies. The question about the cellular and systemic consequences of the endogenous expression of the 1,-hydroxylase in malignant tissues remains to be clarified.
Furthermore, we were able to demonstrate a correlation between amplification of the gene and its overexpression in human malignant glioma. The main site of expression of the 1,-hydroxylase gene is in the kidney, and the major part of circulating 1,25(OH)2D3 is made by the proximal tubular cells of the renal cortex (21)
. By using RT-PCR, it was possible to detect 1,-hydroxylase expression in keratinocytes, brain, and testis (8)
. There are also reports concerning extrarenal sites of 1,-hydroxylase activity, which could be the cause of hypercalcemia associated with lymphoma and certain solid tumors (22
, 23)
. Recently it was found that the extrarenal 1,-hydroxylase activity in a human non-small cell lung carcinoma is attributable to the same gene as in the kidney (24)
.
We reported the cloning and expression of several alternatively spliced 1,-hydroxylase cDNAs from the glioblastoma cell line TX3868, including transcripts encoding truncated proteins. Differences in the expression levels of variants Hyd-V1, Hyd-V2, Hyd-V4, and Hyd-V5 and the normal cDNA were found in kidney, normal brain, and glioblastoma with and without amplification of the 1,-hydroxylase gene. Variant Hyd-V3 could not be detected in this RT-PCR study because of the primer location. However it was obvious that the expression pattern in glioma with amplification is different from that in normal brain, where the variant without exons 4 and 5 encoding a truncated protein is dominant. Alternative splicing is a frequent feature in the expression of many P450 genes, is considered as an important factor in regulating the enzyme level, and may be the cause for tissue-specific variation (25)
. Until now, there has been no indication of the existence of alternatively spliced mRNAs of the 1,-hydroxylase gene and their tissue-specific preference. The effect of the expression of alternative transcripts on the 1,-hydroxylase activity level in glioma has to be investigated in future experiments.
The overexpression of the 1,-hydroxylase gene in glioma could result in a higher level of 1,25(OH)2D3. A prerequisite for the antiproliferative action of 1,25(OH)2D3 is the availability of the vitamin D receptor. Previous works demonstrated the presence of the vitamin D receptor in normal brain, in which its physiological function remains unknown, and in human anaplastic astrocytoma and glioblastoma (26)
. Furthermore the known antiproliferative and prodifferentiating effects of 1,25(OH)2D3 have been observed on rat and human glioblastoma cell lines (27
, 28)
. These results suggest that 1,25(OH)2D3 may be a potentially useful agent in the therapy of human malignant glioma (27)
. However, the clinical use of 1,25(OH)2D3 is impaired by its potent hypercalcemic effects. One possibility of reducing hypercalcemia is the use of less calcemic synthetic vitamin D3 analogues which have also been shown to induce cell death of rat glioma cells (29)
. Pending further mechanistic studies, our findings raise the possibility that the supplementation of patients’ treatment with 25(OH)D3 may promote the local synthesis of 1,25(OH)2D3 by glioma cells without the systemic side effects of hypercalcemia as discussed for the chemopreventive therapy of invasive prostate cancer (30)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Human Genetics, Building. 60, Medical School, University of Saarland, 66421 Homburg/Saar, Germany. Phone: 49-6841-166038; Fax: 49-6841-166186; E-mail: hgemee{at}med-rz.uni-sb.de 
2 The abbreviations used are: GBM, glioblastoma multiforme; GAS, glioma-amplified sequence; RT-PCR, reverse transcription-PCR; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 1,-hydroxylase, 25-hydroxyvitamin D3 1,-hydroxylase.
Received 11/27/00; revised 1/23/01; accepted 1/23/01.
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-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA, 94: 12920-12925, 1997.-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol. Endocrinol., 11: 1961-1970, 1997.-hydroxylase. Biochem. Biophys. Res. Commun., 239: 527-533, 1997.[CrossRef][Medline]
-hydroxylase, P450c1. DNA Cell. Biol., 16: 1499-1507, 1997.[Medline]
and CYP24) in human non-small cell lung carcinomas. Endocrinology, 140: 3303-3310, 1999.,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line. Cancer Lett., 100: 3-10, 1996.[CrossRef][Medline]
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