1 Institute of Pathology, Technical University Munich, Munich,
2 Institute of Pathology, GSF-National Research Center of Environment and Health, Neuherberg,
3 Department of Dermatology, University of Essen, Essen and
4 Department of Dermatology, Technical University of Munich, Munich, Federal Republic of Germany
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Abstract |
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Abbreviations: BCC, basal cell carcinoma; LOH, loss of heterozygosity; NBCCS, nevoid basal cell carcinoma syndrome; RMS, rhabdomyosarcoma.
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Introduction |
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PTCH is predicted to contain 12 transmembrane-spanning domains and two large extracellular loops and thus does not resemble any known tumor suppressor gene (for review see ref. 9). Within the plasma membrane PTCH forms a receptor complex with its signaling partner Smoothened (SMO) that transduces hedgehog (HH) signaling (10,11). A main impact of this signaling pathway is on the control of cell differentiation and proliferation. A physiological activation of this pathway occurs during embryogenesis and is induced by HH. Binding of HH to PTCH suspends the inhibition of SMO, which leads to signal transduction and induction of target genes. Mutational inactivation of PTCH results in a pathological activation of this signaling pathway and is characterized by increased levels of GLI 1 and PTCH mRNA (for review see ref. 12).
We have recently established a murine model of Ptch hemizygosity by replacement of exons 6 and 7 of the Ptch gene by a neomycin resistance cassette (13). Heterozygous Ptchneo67/+ mice develop medulloblastoma and RMS, two frequent childhood tumors (13,14). As in human tumors associated with PTCH mutations, mutational inactivation of murine Ptch resulted in a pathological activation of its signaling pathway with consecutive expression of high levels of Gli1 and Ptch mRNA (12,13).
To determine if deletion of both copies of Ptch is a prerequisite in RMS development in heterozygous Ptchneo67/+ mice, we examined whether the normal Ptch allele was deleted or inactivated by a mutation in these tumors. Furthermore, we tried to elucidate if both alleles contributed to the high Ptch mRNA expression previously found in RMS of these mice. Finally we examined the status of PTCH in human BCCs, allowing us to compare the role of PTCH in human PTCH-associated tumors with its role in murine Ptch-associated tumors.
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Materials and methods |
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Samples
RMS and non-cancerous skeletal muscle (SM) taken as a reference were excised from heterozygous Ptchneo67/+ mice maintained on a CD-1 background. Human nodular, solid BCCs were obtained at surgery. All tissues were snap-frozen and stored in liquid nitrogen for later extraction of RNA and DNA. The identity of the tumors as murine RMS or human BCCs was confirmed on haematoxylin and eosin (H&E) stained sections by a trained pathologist.
Tissue preparation and microdissection
Murine RMS were examined prior to extraction of DNA or RNA on H&E stained sections and only tumor tissue that consisted of a pure population of 9095% of tumor cell was used to obtain tumor DNA or RNA samples.
BCCs were sectioned at 10 µm in a cryostat, mounted on glass slides and non-neoplastic epidermis and neoplastic tissue was microdissected using the Palm Laser-MicroBeam System (P.A.L.M., Bernried, Germany). After selecting the cells of interest, adjacent cells were photolysed by the microbeam. At least 1000 selected cells were picked from the slides using conventional sterile needles and transferred into reaction tubes containing 200 µl STE buffer (20 mM TrisHCl pH 8.0, 10 mM NaCl, 10 mM EDTA, 0.5% w/v sodium dodecyl sulfate and 1 mg/ml proteinase K) for isolation of DNA or 200 µl guanidine isothiocyanate (GITC) Solution (Life Technologies, Rockville, MD, USA) and 1.6 µl ß-mercaptoethanol for isolation of total RNA.
Isolation of DNA and total RNA
DNA from fresh frozen microdissected human BCCs, from macrodissected murine RMS and from murine tails was isolated from STE buffer by phenolchloroform extraction according to standard procedures.
Total RNA of fresh frozen murine RMS was isolated using Trizol (Life Technologies) and digested with DNase (Roche Diagnostics, Mannhein, Germany) according to the manufacturer's instructions.
Total RNA from microdissected BCC tissue was lysed in GITC buffer and isolated by addition of 0.1 volume 2 M sodium acetate (pH 4.0), 1 volume water-saturated phenol and 0.3 volumes chloroform followed by precipitation with an equal volume of isopropanol in the presence of 10 µg carrier glycogen. The RNA pellet was washed once in 70% ethanol, air-dried and resuspended in RNase-free water.
Northern blot analysis
For analysis of expression of wild-type and mutant Ptch transcripts of Ptchneo67/+ heterozygous mice 10 µg of total RNA isolated from RMS and other non-neoplastic tissue of Ptchneo67/+ heterozygous CD-1 mice were electrophoresed on a 1% agarose-formaldehyde gel, transferred to a nylon membrane and hybridized with 32P-labeled probes according to standard protocols. The cDNA probes used for detection of mutant or wild-type Ptch transcripts corresponded to nucleotides 7051011 and 35094279 of the murine Ptch cDNA, respectively (amplified with primer pairs 13/14 and 21/9, Figure 2; Table I
). Filters were washed and exposed to a film at 80°C.
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RNA isolated from microdissected BCC samples was reverse-transcribed in a final volume of 20 µl using SuperScriptII reverse transcriptase (Life Technologies) in the manufacturer's buffer containing 1 mM dNTPs, 10 U of RNase inhibitor (Life Technologies), 250 ng of random hexamers (Roche Diagnostics) and 500 ng of oligo(dT)15 primers (Roche Diagnostics). After digestion with DNase, the cDNAs were diluted with DNase free water to a total volume of 90 µl and stored at 20°C until use.
Real-time quantitative RTPCR
Real-time quantitative RTPCR analysis for human PTCH in BCC was carried out using an ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems, Foster City, CA, USA). The PTCH-specific primers spanning exon 7 and 8 of the human PTCH-cDNA were Ex7F and Ex8R and the fluorogenic intron-spanning probe was Ex7/Ex8 (Table II). PCR amplifications were carried out with the TaqMan Universal PCR Master Mix (Applied Biosystems) using 10 µl of the diluted BCC cDNA (see reverse transcription), 100 nmol/l of the probe and 300 nmol/l forward primer and reverse primer in a 30 µl final reaction mixture. After 2 min incubation at 50°C, AmpliTaq Gold was activated by incubation for 10 min at 95°C. Each of the 40 PCR cycles consisted of 15 s denaturation at 95°C and hybridization of probe and primers for 1 min at 60°C. Amplification of TBP was performed as an endogenous control. The TBP primers, the TBP probe and amplification conditions are described elsewhere (15). For each experimental sample, the amount of target and endogenous reference was determined from standard curves constructed by fivefold serial dilution of plasmids (1000 pg to 0.32 pg) containing fragments of either human PTCH cDNA (target) or human TBP DNA (endogenous reference). All data shown are the average of at least two independent experiments.
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Four highly polymorphic microsatellite markers (D9S1820, D9S196, D9S287 and D9S1786) on human chromosome 9 in the vicinity of the PTCH gene were used to confirm the obtained data. The primer sequences of the microsatellite markers were obtained from the Genome Database (http://www.genome.wi.mit.edu). PCR amplification of genomic DNA was performed under standard conditions in a 15 µl reaction mixture with 1.5 mM MgCl2 PCR Buffer, 1.25 mM dNTPs, 20 pmol of each primer (forward primers were fluorescently labeled) and 1.5 U Taq Polymerase. Amplification efficiency was determined by resolving 2 µl of the reaction products on an agarose gel. Equal amounts of the PCR products from normal and tumor DNA were subsequently analyzed using an automated ABI 377 sequencer and the ABI Prism Gene Scan software (Applied Biosystems).
Nucleotide sequencing
All sequencing reactions were performed using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Sequences were analyzed using the Sequencher software (Gene Codes Corp., Ann Arbor, MI).
Sequence analysis of Ptch in murine tumor samples
Primer pairs 4/5 and 6/7 were used to amplify 87% of the wild-type Ptch transcript from cDNA isolated from 5 RMS of heterozygous Ptchneo67/+ mice (Figure 2a, Table I
). For analysis of 11% of the remaining 3'-end of the wild-type transcript, genomic DNA was amplified from the same tumors using the primer pair 8/9 (Figure 2a
, Table I
). The Ptch transcript derived from the targeted allele in tumor tissue was amplified with primer pair 7/10 (Figure 2b
, Table I
).
Nucleotide polymorphisms detected in the wild-type and the mutant transcripts were confirmed on tail DNA isolated from 5 wild-type CD-1 mice (purchased from Charles River) and 1 wild-type 129Sv mouse using the primer pairs 11/12, 15/16, 17/18, 19/20, 21/22 and 8/9 (Table I) for amplification and sequencing.
To quantify the expression of the Ptch transcripts in tumor tissue a fragment amplified with primer pair 11/12 from cDNA of RMS was subcloned into the pCRII cloning vector (Invitrogen BV, Groningen, Netherlands). We have excluded by sequencing the existence of polymorphisms between CD1 and 129 mouse strains within regions binding PCR primers to avoid biased amplification of one allele. Ninety subclones were sequenced from both sides using the T7 and Sp6 primers. Inserts originating from either the wild-type or the mutant transcript were distinguished by nucleotide polymorphism at positions 3438 and 3498 of the murine Ptch gene.
Analysis of PTCH in human tumor samples
Genomic DNA isolated from microdissected non-neoplastic and neoplastic tissue derived from 10 BCCs was amplified and sequenced with primer pair 12F/12R and 23F/23.R2 (Table II). To determine allelic expression of PTCH in tumor tissue, cDNA isolated from neoplastic tissue was amplified and sequenced with the intron-spanning primer pairs Ex12F/Ex12R or Ex23F/Ex23R (Table II
) that detect the polymorphic sites in exon 12 or exon 23, respectively. To quantify the level of expression of each PTCH allele, the resulting PCR fragments were cloned into the pCRII cloning vector (Invitrogen BV) and the subclones were sequenced using the T7 primer.
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Results |
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No evidence for mutations in the wild-type Ptch allele in RMS of heterozygous Ptchneo67/+ mice
To determine whether the wild-type Ptch allele was mutationally inactivated in the 5 tumors of Ptchneo67/+ mice, we sequenced 87% of Ptch wild-type cDNA amplified from the RMS using primer pairs 4/5 and 6/7. A further 11% was amplified from genomic DNA of these tumors using primers 8 and 9 (Figure 2a). No mutations have been detected in the 98% of the wild-type Ptch coding region in any of the five tumors examined. Although 26 bp of the C-terminal end of the wild-type Ptch allele has not been sequenced in RMS the data suggest that the wild-type allele does not harbor mutations in the protein-coding region.
Expression of the mutant allele in tissues of heterozygous Ptchneo67/+ mice
Amplification of cDNA from RMS of the same 5 heterozygous Ptchneo67/+ mice with primer pair 7/10 (Figure 2) uniformly revealed an aberrant Ptch transcript, in which the neo-cassette of the targeted 129Sv-derived Ptchneo67/+ allele was excised and exon 5 was spliced together with exon 8 of the murine Ptch gene (Figure 2b
).
Overxpression of the mutant Ptch allele in RMS of heterozygous Ptchneo67/+ mice
In heterozygous Ptchneo67/+ mice the mutant Ptchneo67/+ allele is derived from 129Sv ES cells. Heterozygous Ptchneo67/+ mice crossed onto the CD-1 background contain the targeted 129Sv-derived Ptchneo67/+ allele as well as a wild-type CD-1-derived Ptch allele. When comparing the nucleotide sequence of the Ptch open reading frame derived from the mutant and the wild-type Ptch cDNA, several sequence discrepancies between the two transcripts were detected. Since the mutant transcript was derived from the 129Sv mouse strain and the wild-type transcript was derived from the CD-1 mouse strain, tail DNA isolated from 129Sv and CD-1 mice was examined. Our data showed that the variations represented 7 strain-specific polymorphisms. Six of these polymorphisms have been described previously for mouse strains C57BL/6 and 129Sv (16). Four of the polymorphisms showed allelic variance within the CD-1 genome and two of them resulted in amino acid exchanges (Table III). The polymorphisms were used to investigate expression of the Ptch alleles in normal and RMS tissue.
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To quantify the expression level of the mutant transcript in RMS, one of the fragments derived from amplification of Ptch cDNA isolated from RMS using the intron-spanning primer pair 11/12 was subcloned in the pCRII vector and 89 subclones were sequenced from both sides using the T7 and Sp6 primers. Inserts originating from wild-type or mutant transcripts were distinguished by the nucleotide polymorphisms at position 3438 and 3498 as described above. Eighty-seven subclones were derived from the mutant 129Sv allele whereas two subclones were derived from the wild-type CD-1 Ptch allele. Thus, the mutant transcript was 48-fold overexpressed in RMS of Ptchneo67/+ mice.
Frequency of RMS of Ptchneo67/+ mice is modified by the genetic background
At 225 days of age, 15% of heterozygous Ptchneo67/+ mice on CD-1 background had developed soft tissue tumors histologically confirmed as RMS (Figure 4). Within this period no RMS were detected in animals maintained on the C57BL/6 background. This indicates that the frequency of RMS in heterozygous Ptchneo67/+ mice is strongly affected by genetic background.
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Altogether these results indicate that unlike in mice, most human tumors exhibit heterozygosity at the PTCH locus and express PTCH biallelically.
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Discussion |
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We have investigated the mutational status and the expression of Ptch in RMS in murine Ptch mutants. Here we show that the wild-type Ptch allele is retained and appears to be intact in RMS of heterozygous Ptchneo67/+ animals. This conclusion is based on several lines of evidence. First of all, there is no evidence for the loss of the chromosomal segment harboring the wild-type allele of Ptch, as determined by LOH analysis. Secondly, mRNA transcripts derived from the wild-type allele can be detected in RMS and they carry no inactivating mutations within the protein-coding region. Interestingly, using an independent Ptch mutant mouse strain (14), Wetmore et al. and Zurawel et al. also found no evidence for the mutational inactivation of the wild-type allele in the other frequent tumor in murine Ptch mutants, medulloblastoma (16,22).
Surprisingly, the overexpression of Ptch in RMS appears to be caused by an overabundance of transcripts derived almost exclusively from the mutant allele. This observation is specific for the tumor and it is unlikely caused by different stabilities of the wild-type and mutant Ptch alleles. Indeed, both transcripts are expressed at comparable levels in non-tumor tissues. An overexpression could be caused either by a silencing of the wild-type allele or by an increased transcription of the mutant one. In respect of the first possibility, the wild-type allele of Ptch could be switched off by a mechanism different from mutational inactivation. There is growing evidence that epigenetic silencing rather than mutation is a common mechanism for loss of function of one or sometimes both alleles of tumor suppressor genes. Examples include VHL, MLH1, p16 and possibly BRCA1 and the underlying mechanism involves allele-specific methylation of CpG islands in the promoter regions of these genes (19,23).
The strong under-representation of transcripts derived from the wild-type allele in the Ptch transcript pool in RMS is in agreement with such a mechanism. In addition, a selective silencing of the wild-type allele would explain the apparent disagreement between the Ptch mRNA expression status in RMS and the current model of its regulation. According to this model, Ptch represses its own transcription via a negative feedback loop. Conversely, reduced Ptch activity, such as that expected in the case of a mutational inactivation increases Ptch transcription (for review see ref. 24). This model is in agreement with the overexpression of Ptch transcripts observed in a variety of tumors associated with Ptch mutations. However, it does not explain the dramatic difference between the relative expression levels of wild-type and the mutated Ptch alleles. To explain this difference, one would have to postulate an additional mechanism, such as a selective silencing of the wild-type allele. To confirm or reject this hypothesis it will be necessary to provide evidence for functionally relevant and tumor-specific differences in the methylation status of both Ptch alleles in RMS. Furthermore, an analysis of the protein expression from either allele will have to be conducted. This should become possible following the development of the appropriate exon-specific antibodies. Alternatively, it is possible that the wild-type allele is expressed at `normal' levels in RMS and that haploinsufficiency for Ptch is sufficient for the formation of this tumor. To verify this hypothesis it will be necessary to first identify the cell subpopulation from which RMS develop and to determine its Ptch expression level. If the expression of wild-type allele is indeed not repressed in RMS, this would mean that RMS formation requires an event at a gene locus different from Ptch. The strong dependency of the RMS prevalence on the genetic background is in agreement with a significant contribution of other gene loci to RMS formation. The experimental verification could be provided by a time-point defined and skeletal muscle-specific inactivation of the wild-type allele. This should be achievable by means of conditional knockout technology. Furthermore, it should be possible to identify loci responsible for the differences in the Ptch phenotypes on different genetic backgrounds by means of QTL analysis.
In either case these data suggest that Ptch may not act as a classic tumor suppressor gene in mice. In humans, the mode of action of PTCH may be different. In accordance with previous studies (7,25) and similarly to murine tumors, all BCCs examined showed high levels of PTCH expression. However, in contrast to RMS in mice some human BCCs exhibited LOH at the PTCH locus. Obviously, all PTCH transcripts in these BCCs were derived from the remaining PTCH allele. Approximately every second BCC showed retention of heterozygosity at the PTCH locus and most of these tumors expressed PTCH biallelically. Thus, unbalanced expression of PTCH appears not to be strictly maintained in all PTCH-associated tumors. Altogether, these results raise the possibility that tumorigenesis associated with PTCH mutations may vary depending on tissue or species context. Similar observations have been made with other tumor suppressor genes. For instance, loss of function of the cyclin-dependent kinase inhibitor p16 may occur through deletion, point mutation, or promoter hypermethylation and interestingly, the frequency of each mechanism differs between tumor types (23). A proof of tissue- or species-specific differences in the inactivation (e.g. through silencing) of PTCH will have to include an analysis of human BCCs with mutations in only one allele of this gene. This task should become possible following identification of all cis-acting genetic elements, which control PTCH expression.
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Notes |
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6 To whom correspondence should be addressed at: Institute of Human Genetics, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Federal Republic of Germany Email: hhahn{at}gwdg.de
* These authors contributed equally to this work.
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Acknowledgments |
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References |
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