Clinical Cancer Research Meeting Calendar AACR Membership
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maas, R. M.
Right arrow Articles by Meese, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maas, R. M.
Right arrow Articles by Meese, E.
Clinical Cancer Research Vol. 7, 868-875, April 2001
© 2001 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Amplification and Expression of Splice Variants of the Gene Encoding the P450 Cytochrome 25-Hydroxyvitamin D3 1,{alpha}-Hydroxylase (CYP 27B1) in Human Malignant Glioma

Ruth Maria Maas, Katrin Reus, Britta Diesel, Wolf-Ingo Steudel, Wolfgang Feiden, Ulrike Fischer and Eckart Meese1

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Recently, we reported the isolation of six novel genes termed glioma-amplified sequences (GASs) from the glioblastoma cell line TX3868 using microdissected mediated cDNA capture (U. Fischer et al., Hum. Mol. Genet., 5: 595–600, 1996). The aim of this study was to further characterize the gene GAS89.

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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme. The endogenous expression of the 25-hydroxyvitamin D3 1,{alpha}-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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GBM2 is the most malignant and frequent intracranial tumor (1) . Despite intensive effort in surgery, radiotherapy, and chemotherapy, only minimal therapeutic advances toward a long-term survival for glioblastoma patients have been achieved in recent years. Many genetic alterations such as amplifications, deletions, and translocations are known to occur within the cells during tumor progression. Cytogenetic studies demonstrated that the most frequent aberrations in GBM are losses of chromosome 10, 19, and 22, as well as gains on chromosome 7 attributable to the amplification of the EGFR gene in the chromosomal region 7p11–12 (2) . EGFR is amplified in ~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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase in human malignant glioma that potentially could result in the production of biologically active calcitriol.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Library Construction and Screening.
cDNA was synthesized from poly(A) mRNA of the glioblastoma cell line TX3868 using oligo (dT) primers. Second-strand synthesis was performed according to the manufacturer’s instructions (Stratagene). After EcoRI adapter ligation and digestion with XhoI, the cDNA fragments were size fractionated with Sepharose CL-2B columns, and cDNAs >0.6 kb in length were ligated into the HybriZAP vector. Packaging was done using the Gigapack III Gold packaging extract from Stratagene. The library was amplified once prior to cDNA library screening.

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,{alpha}-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,{alpha}-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 [{alpha}-32P]dATP using the Random-primed DNA labeling kit (Boehringer Mannheim).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
pGAS89-b Represents a Splice Variant of the Human Gene for 25-Hydroxyvitamin D3 1,{alpha}-Hydroxylase.
Plasmid clone pGAS89 with an insert of 350 bp was previously isolated from a homogeneously staining region of the glioblastoma cell line TX3868 and subsequently localized on chromosome 12q13–15 (5) . To obtain the full-length cDNA, we screened a HybriZAP (Stratagene) cDNA library established from glioblastoma cell line TX3868 using the insert of plasmid pGAS89 as the probe. From 50,000 plaque-forming units that were examined, one positive phage clone was identified. The clone was reconfirmed by PCR with pGAS89 specific primers and then in vivo excised into a plasmid clone pGAS89-b with an insert length of 1.6 kb. Sequence analysis and homology search using the BLASTX and BLASTN algorithms revealed identity with parts of the sequence for the human cDNA for 25-hydroxyvitamin D3 1,{alpha}-hydroxylase, which was recently localized on chromosome 12q13–15 by somatic cell hybrid mapping (8 , 9 , 10) . The 25-hydroxyvitamin D3 1{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase in the TX3868 glioblastoma cell line.

Amplification Analysis for the 1,{alpha}-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,{alpha}-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. 1ACitation . 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. 1BCitation .



View larger version (66K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Amplification analysis of GAS89 in GBM. A, comparative PCR for tumors H549, H546, H485, H545, H556, H346, H412, H246, and H366. Three different concentrations of tumor and peripheral blood (pb) DNA (0.5, 1, and 2 ng) were amplified by PCR using primers specific for GAS89 (lower panel) and Muc2 or 9-1 plus as a single-copy control (upper panel). Arrows, PCR product sizes. GAS89 shows a strong amplification in tumor H549 and is weakly amplified in tumors H556, H346, and H246 in comparison with peripheral blood DNA. The "control" sample contained no template DNA. B, Southern blot hybridization using the insert of pGAS89-b as the probe and gel loading control. GAS89 was found to be amplified in tumors H91, H118, and H385 compared with peripheral blood DNA, whereas tumors H77 and H30 show no amplification.

 
Identification and Cloning of Splice Variants of the 1,{alpha}-Hydroxylase Gene.
To obtain the entire cDNA from the glioblastoma cell line, we used primers deduced from the 5'-coding region and the 3'-noncoding region of the known cDNA for 1,{alpha}-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,{alpha}-hydroxylase indicated that these clones represented additional splice variants and were named hydroxylase-variant 2, 3, and 4 (Hyd-V2, -V3, and -V4; Fig. 2Citation Citation ). 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,{alpha}-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.



View larger version (70K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. A, sequence analysis of variants Hyd-V2, -V3, and -V4 and comparison with the known sequence of 1{alpha}-hydroxylase. Arrows, the exons and intron 2. B, structures of the normal and the alternatively spliced 1,{alpha}-hydroxylase mRNAs. Variant Hyd-V1 corresponds to the insert of pGAS89-b and is incomplete at the 5'-end. Heavy bars (in the normal 1,{alpha}-hydroxylase mRNA), the coding regions for the ferredoxin-binding site in exon 6 and for the haem-binding site in exon 8. Arrows, premature stop codons.

 


View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2A. Continued

 
The second variant, Hyd-V3, contains only a part of exon 8. Exon 8 contains an additional alternative 5'-splice site, AG, at the appropriate position obviously recognized in variant Hyd-V3. The partial deletion leads to a frameshift in the remaining part of exon 8, which results in a premature termination signal. The encoded protein consists of 414 amino acids and contains the ferredoxin but lacks the haem-binding site of the 1,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase gene as determined by comparative PCR. Normal tissues were normal brain and kidney tissues in which the 1{alpha}-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. 2BCitation 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,{alpha}-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)Citation . 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,{alpha}-hydroxylase cDNA or variant Hyd-V4, respectively. Similar expression patterns were detected in tumor samples without amplification of GAS89. In tumors carrying 1,{alpha}-hydroxylase gene amplifications and in TX3868, the glioblastoma cell line from which GAS89 was isolated, the expression level of the normal 1,{alpha}-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,{alpha}-hydroxylase gene in GBM and the expression level of several splice variants and the normal cDNA of the 1,{alpha}-hydroxylase gene.



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Expression analysis of the 1,{alpha}-hydroxylase gene in tumor samples and normal tissues (kidney and normal brain). A, RT-PCR standardization of DNaseI-treated tumor and normal tissue RNA with glyceraldehyde-3-phosphate dehydrogenase specific primers. B, Southern blot hybridization of RT-PCR with the insert of pGAS89-b as the probe. Primers were chosen from exon 3 and exon 8 of the 1,{alpha}-hydroxylase gene. H385 and H91 were glioblastomas with strong GAS89 amplification, TX3868 is the cell line from which GAS89 was primarily isolated; H282 and H322 were glioblastomas without GAS89 amplification. Arrows, variants of the 1,{alpha}-hydroxylase cDNA.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we describe the isolation of the 25-hydroxyvitamin D3 1,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase expression in keratinocytes, brain, and testis (8) . There are also reports concerning extrarenal sites of 1,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-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,{alpha}-hydroxylase gene and their tissue-specific preference. The effect of the expression of alternative transcripts on the 1,{alpha}-hydroxylase activity level in glioma has to be investigated in future experiments.

The overexpression of the 1,{alpha}-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) .


   ACKNOWLEDGMENTS
 
We thank Doris Hemmer for contribution to the amplification analysis and Daniela Scherer for technical assistance. We also thank Brenda Glass for critically reading the manuscript.


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

1 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 Back

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,{alpha}-hydroxylase, 25-hydroxyvitamin D3 1,{alpha}-hydroxylase. Back

Received 11/27/00; revised 1/23/01; accepted 1/23/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kleihues, P., Burger, P. C., Collins, V. P., Newcomb, E. W., Ohgaki, H., and Cavenee, W.K. Glioblastoma. In: P. Kleihues and W. K. Cavenee (eds.), Tumors of the nervous system, pp. 29–39. Lyon: IARC Press, 2000.
  2. Van de Kelft E. Molecular pathogenesis of astrocytoma and glioblastoma multiforme. Acta. Neurochir. (Wien), 139: 589-599, 1997.[CrossRef][Medline]
  3. Eley G., Frederick L., Wang X. Y., Smith D. I., James C. D. 3' end structure and rearrangements of EGFR in glioblastomas. Genes Chromosomes Cancer, 23: 248-254, 1998.[CrossRef][Medline]
  4. Reifenberger G., Reifenberger J., Ichimura K., Meltzer P. S., Collins V. P. Amplification of multiple genes from chromosomal region 12q13–14 in human malignant gliomas: preliminary mapping of the amplicons shows preferential involvement of CDK4, SAS, and MDM2. Cancer Res., 54: 4299-4303, 1994.[Abstract/Free Full Text]
  5. Fischer U., Gracia U., Elkahloun A., Trent J. M., Meese E., Meltzer P. S. Isolation of genes amplified in human cancers by microdissection mediated cDNA capture. Hum. Mol. Genet., 5: 595-600, 1996.[Abstract/Free Full Text]
  6. Fischer U., Heckel D., Michel A., Janka M., Hulsebos T., Meese E. Cloning of a novel transcription factor-like gene amplified in human glioma including astrocytoma grade I. Hum. Mol. Genet., 6: 1817-1822, 1997.[Abstract/Free Full Text]
  7. Shinki T., Shimada H., Wakino S., Anazawa H., Hayashi M., Saruta T., DeLuca H. F., Suda T. Cloning and expression of rat 25-hydroxyvitamin D3–1{alpha}-hydroxylase cDNA. Proc. Natl. Acad. Sci. USA, 94: 12920-12925, 1997.[Abstract/Free Full Text]
  8. Fu G. K., Lin D., Zhang M. Y., Bikle D. D., Shackleton C. H., Miller W. L., Portale A. A. Cloning of human 25-hydroxyvitamin D-1 {alpha}-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol. Endocrinol., 11: 1961-1970, 1997.[Abstract/Free Full Text]
  9. Monkawa T., Yoshida T., Wakino S., Shinki T., Anazawa H., Deluca H. F., Suda T., Hayashi M., Saruta T. Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 {alpha}-hydroxylase. Biochem. Biophys. Res. Commun., 239: 527-533, 1997.[CrossRef][Medline]
  10. Fu G. K., Portale A. A., Miller W. L. Complete structure of the human gene for the vitamin D 1{alpha}-hydroxylase, P450c1{alpha}. DNA Cell. Biol., 16: 1499-1507, 1997.[Medline]
  11. Christakos S., Raval-Pandya M., Wernyj R. P., Yang W. Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3. Biochem. J., 316: 361-371, 1996.
  12. Altschul S. F., Gish W., Miller W., Myers E.W., Lipman D. J. Basic local alignment search tool. J. Mol. Biol., 215: 403-410, 1990.[CrossRef][Medline]
  13. Muellenbach R., Lagoda P. J., Welter C. An efficient salt-chloroform extraction of DNA from blood and tissues. Trends Genet., 5: 391 1989.[Medline]
  14. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7: 1513-1523, 1979.[Abstract/Free Full Text]
  15. Brass N., Heckel D., Meese E. Comparative PCR: an improved method to detect gene amplification. Biotechniques, 24: 22-24, 1998.[Medline]
  16. Elkahloun A. G., Bittner M., Hoskins K., Gemmill R., Meltzer P. S. Molecular cytogenetic characterization and physical mapping of 12q13–15 amplification in human cancers. Genes Chromosomes Cancer, 17: 205-214, 1996.[CrossRef][Medline]
  17. Reifenberger G., Ichimura K., Reifenberger J., Elkahloun A. G., Meltzer P. S., Collins V. P. Refined mapping of 12q13–q15 amplicons in human malignant gliomas suggests CDK4/SAS and MDM2 as independent amplification targets. Cancer Res., 56: 5141-5145, 1996.[Abstract/Free Full Text]
  18. Albertson D. G., Ylstra B., Segraves R., Collins C., Dairkee S. H., Kowbel D., Kuo W. L., Gray J. W., Pinkel D. Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene. Nat. Genet., 25: 144-146, 2000.[CrossRef][Medline]
  19. van Leeuwen J. P. T. M., Pols H. A. P. Vitamin D: anticancer and differentiation Feldmann D. Glorieux F. H. Pike J. W. eds. . Vitamin D, : 1089-1099, Academic Press London 1997.
  20. Studzinski G. P., Moore D. C. Sunlight—can it prevent as well as cause cancer?. Cancer Res., 55: 4014-4022, 1995.[Abstract/Free Full Text]
  21. Brunette M. G., Chan M., Ferriere C., Roberts K. D. Site of 1,25(OH)2 vitamin D3 synthesis in the kidney. Nature (Lond.), 276: 287-289, 1978.[CrossRef][Medline]
  22. Davies M., Hayes M. E., Yin J. A., Berry J. L., Mawer E. B. Abnormal synthesis of 1,25-dihydroxyvitamin D in patients with malignant lymphoma. J. Clin. Endocrinol. Metab., 78: 1202-1207, 1994.[Abstract]
  23. Mawer E. B., Hayes M. E., Heys S. E., Davies M., White A., Stewart M. F., Smith G. N. Constitutive synthesis of 1,25-dihydroxyvitamin D3 by a human small cell lung cancer cell line. J. Clin. Endocrinol. Metab., 79: 1994, 1994554-560, [Abstract]
  24. Jones G., Ramshaw H., Zhang A., Cook R., Byford V., White J., Petkovich M. Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1{alpha} and CYP24) in human non-small cell lung carcinomas. Endocrinology, 140: 3303-3310, 1999.[Abstract/Free Full Text]
  25. Ding S., Lake B. G., Friedberg T., Wolf C. R. Expression and alternative splicing of the cytochrome P-450 CYP2A7. Biochem. J., 306: 161-166, 1995.
  26. Magrassi L., Bono F., Milanesi G., Butti G. Vitamin D receptor expression in human brain tumors. J. Neurosurg. Sci., 36: 27-30, 1992.[Medline]
  27. Magrassi L., Butti G., Pezzotta S., Infuso L., Milanesi G. Effects of vitamin D and retinoic acid on human glioblastoma cell lines. Acta. Neurochir. (Wien), 133: 184-190, 1995.[CrossRef][Medline]
  28. Naveilhan P., Berger F., Haddad K., Barbot N., Benabid A. L., Brachet P., Wion D. Induction of glioma cell death by 1,25(OH)2 vitamin D3: towards an endocrine therapy of brain tumors?. J. Neurosci. Res., 37: 271-277, 1994.[CrossRef][Medline]
  29. Baudet C., Chevalier G., Naveilhan P., Binderup L., Brachet P., Wion D. Cytotoxic effects of 1 {alpha},25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line. Cancer Lett., 100: 3-10, 1996.[CrossRef][Medline]
  30. Schwartz G. G., Whitlatch L. W., Chen T. C., Lokeshwar B. L., Holick M. F. Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Cancer Epidemiol. Biomark. Prev., 7: 391-395, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Neuro OncolHome page
A. Idbaih, E. Criniere, Y. Marie, A. Rousseau, K. Mokhtari, M. Kujas, Y. El Houfi, C. Carpentier, S. Paris, B. Boisselier, et al.
Gene amplification is a poor prognostic factor in anaplastic oligodendrogliomas
Neuro-oncol, August 1, 2008; 10(4): 540 - 547.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
U. Fischer, A. Keller, P. Leidinger, S. Deutscher, S. Heisel, S. Urbschat, H.-P. Lenhof, and E. Meese
A Different View on DNA Amplifications Indicates Frequent, Highly Complex, and Stable Amplicons on 12q13-21 in Glioma
Mol. Cancer Res., April 1, 2008; 6(4): 576 - 584.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Wu, S. Ren, L. Nguyen, J. S. Adams, and M. Hewison
Splice Variants of the CYP27b1 Gene and the Regulation of 1,25-Dihydroxyvitamin D3 Production
Endocrinology, July 1, 2007; 148(7): 3410 - 3418.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
M. F. Holick
The Vitamin D Epidemic and its Health Consequences
J. Nutr., November 1, 2005; 135(11): 2739S - 2748S.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
B. Diesel, J. Radermacher, M. Bureik, R. Bernhardt, M. Seifert, J. Reichrath, U. Fischer, and E. Meese
Vitamin D3 Metabolism in Human Glioblastoma Multiforme: Functionality of CYP27B1 Splice Variants, Metabolism of Calcidiol, and Effect of Calcitriol
Clin. Cancer Res., August 1, 2005; 11(15): 5370 - 5380.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Ren, L. Nguyen, S. Wu, C. Encinas, J. S. Adams, and M. Hewison
Alternative Splicing of Vitamin D-24-Hydroxylase: A NOVEL MECHANISM FOR THE REGULATION OF EXTRARENAL 1,25-DIHYDROXYVITAMIN D SYNTHESIS
J. Biol. Chem., May 27, 2005; 280(21): 20604 - 20611.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maas, R. M.
Right arrow Articles by Meese, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maas, R. M.
Right arrow Articles by Meese, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online