Loss of parafibromin expression in a subset of parathyroid adenomas

in Endocrine-Related Cancer
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  • 1 1Departments of Molecular Medicine and Surgery,
  • 2 2Clinical Neuroscience and
  • 3 3Oncology and Pathology, Karolinska Institutet, Karolinska University Hospital, Solna, CMM L8: 01, SE-171 76 Stockholm, Sweden

Inactivation of the hyperparathyroidism–jaw tumour syndrome (HPT– JT) gene, HRPT2, was recently established as a genetic mechanism in the development of parathyroid tumours. Its encoded protein parafibromin has tumour-suppressor properties that play an important role in tumour development in the parathyroids, jaws and kidneys. Inactivating HRPT2 mutations are common in HPT– JT and parathyroid carcinomas, and have been described in a few cases of parathyroid adenomas with cystic features. In this study, 46 cases of cystic parathyroid adenomas previously investigated for HRPT2 mutations were characterized with regard to MEN1 gene mutations, cyclin D1 expression and parafibromin expression. In normal tissues and cell lines, parafibromin was ubiquitously expressed. Furthermore, parafibromin was detected as a dominating nuclear and a weaker cytoplasmic signal in transfected cell lines. In the three parathyroid tumours with inactivating HRPT2 mutations parafibromin expression was not detectable, and in one of two cases with aberrantly sized parafibromin the protein was delocalized. Both high and low cyclin D1 levels were found among HRPT2-mutated and -unmutated tumours, suggesting that these events are not mutually exclusive in parathyroid tumour development. The presented data suggest that in the majority of benign parathyroid tumours the expression of parafibromin remains unaltered, while the loss of parafibromin expression is strongly indicative of gene inactivation through mutation of the HRPT2 gene.

Abstract

Inactivation of the hyperparathyroidism–jaw tumour syndrome (HPT– JT) gene, HRPT2, was recently established as a genetic mechanism in the development of parathyroid tumours. Its encoded protein parafibromin has tumour-suppressor properties that play an important role in tumour development in the parathyroids, jaws and kidneys. Inactivating HRPT2 mutations are common in HPT– JT and parathyroid carcinomas, and have been described in a few cases of parathyroid adenomas with cystic features. In this study, 46 cases of cystic parathyroid adenomas previously investigated for HRPT2 mutations were characterized with regard to MEN1 gene mutations, cyclin D1 expression and parafibromin expression. In normal tissues and cell lines, parafibromin was ubiquitously expressed. Furthermore, parafibromin was detected as a dominating nuclear and a weaker cytoplasmic signal in transfected cell lines. In the three parathyroid tumours with inactivating HRPT2 mutations parafibromin expression was not detectable, and in one of two cases with aberrantly sized parafibromin the protein was delocalized. Both high and low cyclin D1 levels were found among HRPT2-mutated and -unmutated tumours, suggesting that these events are not mutually exclusive in parathyroid tumour development. The presented data suggest that in the majority of benign parathyroid tumours the expression of parafibromin remains unaltered, while the loss of parafibromin expression is strongly indicative of gene inactivation through mutation of the HRPT2 gene.

Introduction

Primary hyperparathyroidism (PHPT) affects 1% of the general population (Lundgren et al. 1997), and commonly occurs as a sporadic disorder caused by a single parathyroid adenoma. The major known genes underlying PHPT are the oncogene cyclin D1, the calcium-sensing receptor (CASR), and the tumour-suppressor genes for multiple endocrine neoplasia type 1 (MEN1) in chromosome region 11q13 and hyperparathyroidism–jaw tumour syndrome (HPT–JT; HRPT2) in region 1q25. In HPT–JT (Jackson et al. 1990; OMIM 145001), the PHPT usually involves one or two adenoma(s), often with cystic features, and a significantly increased risk of parathyroid carcinoma. Inactivating germ-line HRPT2 mutations are reported in kindreds with HPT–JT and sometimes familial isolated PHPT (Carpten et al. 2002, Villablanca et al. 2004, Howell et al. 2003, Simonds et al. 2004, Cavaco et al. 2004, Cetani et al. 2004, Moon et al. 2004). The majority of sporadic parathyroid carcinomas also involve one or two mutations of germ-line and/or somatic type, as well as loss of parafibromin immunoreactivity (Shattuck et al. 2003, Howell et al. 2003, Cetani et al. 2004, Tan et al. 2004). In contrast, while MEN1 mutations are common in parathyroid adenomas, somatic HRPT2 mutations have only been reported in four out of 137 adenomas studied, notably all with cystic features (Carpten et al. 2002, Howell et al. 2003, Cetani et al. 2004, Krebs et al. 2005). These findings suggest that HRPT2 is a major gene involved in development of parathyroid malignancy (Weinstein & Simonds 2003), but of limited importance in benign parathyroid tumours. In agreement with these findings global gene expression profiling has identified distinct expression signatures in HRPT2-associated tumours compared with MEN1-linked tumours (Haven et al. 2004). However, the relative importance of HRPT2 and MEN1 in the adenoma group remains to be defined.

The HRPT2 gene is expressed in several human tissues, including – among others – parathyroid, bone and kidney, i.e. the principally affected organs in HPT–JT (Carpten et al. 2002). The encoded 531-amino acid protein parafibromin is a part of a human Paf1 complex and associates with proteins involved in cell proliferation, transcription regulation and histone modification (Rozenblatt-Rosen et al. 2005, Yart et al. 2005). In vitro studies have also demonstrated anti-proliferative effects and down-regulation of cyclin D1 expression by wild-type parafibromin, but not by mutated HRPT2 (Woodard et al. 2005, Yart et al. 2005). Here we have investigated parafibromin expression in vitro and in vivo, as well as in relation to HRPT2 and MEN1 gene status of cystic parathyroid adenomas.

Materials and methods

Cell lines

Six established human cell-lines were studied, including HEK-293 (embryonal kidney), HeLa (vulvar squamous cell carcinoma), U2020 (small cell lung carcinoma), HepG2 (hepatoblastoma), SH-SY5Y (neuroblastoma) and COS (simian virus 40-transformed monkey kidney cell lines COS-1 and COS-7). The cells were routinely maintained and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 supplemented with 10% fetal calf serum at 37 °C.

Tissue samples

All 46 cases studied have been previously published with respect to their clinical characteristics (Table 1), cystic features and the presence or absence of somatic HRPT2 mutations (Villablanca et al. 2002a, Carpten et al. 2002). In short, fresh-frozen tissue samples were obtained with informed consent and ethical approval at the Karolinska University Hospital, Solna, Sweden. All tumours were histopathologically diagnosed as parathyroid adenoma according to the World Health Organization classification (DeLellis et al. 2004), and presented cysts of larger and microscopic types. Representativity testing confirmed the diagnosis and a high proportion of tumour cells in all cases.

Histopathologically verified samples of secondary HPT glands, as well as normal parathyroid gland, pancreas and kidney, were similarly obtained as fresh-frozen tissue samples. Furthermore, paraffin-embedded, histopathologically verified normal parathyroid gland tissue was used as a positive control for immunohistochemistry.

Transfection assays and live cell imaging

Three constructs were used for transfection studies. Plasmid pCMV.SPORT6.HRPT2 was the IMAGE clone 6170851 that contained the full-length HRPT2 cDNA (GenBank accession no. BC065037) in pCMV-SPORT6 (Invitrogen). To create the plasmid pcDNA3.HRPT2 ~mGFP, a ClaI site was introduced in pCMV.SPORT6.HRPT2 by site-directed mutagenesis, thereby changing the codons for the last amino acids and the stop codon in the HRPT2 cDNA from TTCTGA to ATCGAT. Next, the obtained HRPT2 Asp-718/ClaI fragment was inserted into Asp-718/ClaI-opened pcDNA3.Cav2 ~mGFP (Uhles et al. 2003), replacing the caveolin 2 cDNA with the HRPT2 cDNA. To generate the third plasmid, pCMV.SPORT6.HRPT2, the HRPT2 SfoI/NotI fragment was inserted into pRcCMVi.mGFP ~HRPT2 following cleavage with the respective restriction enzymes, in-frame into XmaI (filled in)/NotI-opened pRcCMVi.mGFP0. The accuracy of the constructs was verified by restriction analysis and/or DNA sequencing. As a negative control, a plasmid without the HRPT2 sequence (pcDNA3 ~mGFP) was used. The COS and HeLa cell lines were grown in DMEM with glutamine and supplemented with 10% fetal calf serum. Cells were plated in triplicate in six-well plates and transfected at 50% confluence. Transfection was performed with 2 μg plasmid DNA from each construct and Fugene or Lipofectamine reagent in DMEM without serum. Twenty-eight and 36 h after transfection the cells were harvested by washing in PBS and trypsinized.

The distribution of monomeric green fluorescent protein (mGFP)-tagged HRPT2 variants (mGFP ~HRPT2 and HRPT2 ~mGFP) in transfected COS cells was analyzed by laser-scanning confocal microscopy in a Leica TCS SP2 microscope (Leica Microsystems, Heidelberg, Germany) equipped with a Leica HCX P1 Apo ×63/1.20/0.17 UV objective lens as described by Uhles et al. (2003). The following settings were used: excitation wavelength 488 nm (Ar laser) and 543 nm (HeNe laser), a 488/543 double dicroic mirror, and detection at 505–525 nm.

Antibodies

Rabbit polyclonal antibodies were raised against the C-terminal fragment of human parafibromin, amino acids 509–531. Two rabbits (coded APVF and UPVF) were immunized repeatedly according to a standard protocol with 200 μg peptide in Freund’s complete and incomplete adjuvant (Polak & Van Noorden 1986). Pre-immune sera were collected before immunization and used as a negative control in western blot analysis and immunohistochemical analyses. A3nity purification was performed with antisera from rabbit APVF (511U) by binding the peptide used for immunization to a column (UltraLink, Pierce, Rockford, IL, USA). The antibodies were commercially raised by Agri-Sera Co. (Umeå, Sweden). Mouse monoclonal antibodies against Ki-67 (Mib-1) and β-tubulin were purchased from Dako Cytomation (Glostrup, Denmark) and Abcam (Cambridge, UK), respectively. Rabbit monoclonal antibody against cyclin D1 (SP4) was from Lab Vision, and antibodies against green fluorescent protein (GFP) were from Abcam. Secondary goat-anti rabbit and goat anti-mouse were from BioRad Laboratories.

Western blot analyses

Proteins were extracted from tissues and cell pellets using standard methods (Dignam et al. 1983), and measured by a dye-binding assay (Bradford 1976). From each sample 30–75 μg protein was electrophoresed in 10% Tricine gels or 8% Tris/Gly gels, blotted onto 0.2 μm nitrocellulose filters (Schleicher and Schuell, Dassel/Relliehausen, Germay) and incubated overnight at 4 °C with anti-parafibromin (511U; dilution 1:500), anti-cyclin D1 (1:400), anti-β tubulin (1:1000), or anti-GFP (1:500). Anti-β tubulin served as an internal control for protein quality and loading, and the filters were stained with Ponceau Red (Sigma–Aldrich) as a control for protein presence. This was followed by washing, incubation with the secondary antibodies (goat anti-rabbit at a dilution of 1:12 500 and goat anti-mouse at a dilution of 1:25 000), detection with enhanced chemiluminiscence (ECL; Amersham Pharmacia Biotech) for 1 min, and exposure to Hyperfilm (Amersham Pharmacia Biotech). In control experiments, anti-parafibromin was blocked by pre-incubation with peptide antigen (i.e. blocking peptide, 1 h at room temperature and 3 h at 4–8 °C). Dot-blots with immobilized peptide antigen were incubated with anti-parafibromin from both rabbits (dilutions 1:300, 1:1000 and 1:3000).

Immunohistochemistry

Immunohistochemical staining with anti-parafibromin (511U) was performed on 41 cystic parathyroid adenomas, and normal parathyroid controls. Ki-67 expression was determined in 15 cystic parathyroid adenomas with aberration in HRPT2, parafibromin or MEN1, as well as in four additional parathyroid adenomas with published MEN1 mutations (Farnebo et al. 1998). Paraffin sections cut at 4 μm were deparaffinized, rehydrated, and microwave-heated in citrate buffer (pH 6) for 20 min. Sections were incubated with 0.3% hydrogen peroxide in water for 30 min, blocked in 1% BSA for 20 min and incubated with primary antibody overnight (511U at dilution 1:300; Ki-67 at dilution 1:100). Antigen–antibody binding was visualized using the avidin–biotin–peroxidase complex (ABC) method (Vectastain, Elite kit; Vector Laboratories, Burlingame, CA, USA) for 30 min, diaminobenzedine tetrahydrochloride for 6 min, and counterstained in haematoxylin. In control experiments, anti-parafibromin was blocked by preincubation with peptide antigen at 4 °C overnight. Parafibromin expression was scored by two of the authors whereby the subcellular localization (nucleus/cytoplasm) and the level of expression were evaluated. Proliferation index was determined by scoring at least 1500 cells from multiple areas of the slide in a randomized fashion as having either positive or negative Ki-67 expression.

Multiple-tissue mRNA expression analyses

A panel of human multiple-tissue cDNAs was purchased from Clontech (MTC Panel 1, catalogue no. K1420-1), and screened for HRPT2 expression by reverse transcriptase PCR (RT-PCR) using the primers HRPT2-F (5′-AGATGGCGGACGTGCTTAGCG-3′) and HRPT2-R (5′-TTGGATCCAGACGAACTTCA-3′), and the Advantage 2 PCR kit (Clontech). For each reaction, a parallel amplification with G3PDH control primers supplied in the kit was performed as a positive control. The experiments were performed on two separate occasions with consistent results.

RNA isolation, cDNA synthesis and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from 41 of the 46 adenomas and from two normal parathyroid samples using TRIzol Reagent (Invitrogen). After purification with the RNeasy Kit (Qiagen), the 260/280 nm ratio (interval 1.9–2.1) was determined by spectrophotometry. The integrity of the RNA was confirmed by denaturing gel electrophoresis, whereby sharp 28 S and 18 S bands were demonstrated. cDNA was synthesized from 2 μg each purified RNA sample using MultiScribe reverse transcriptase primed with random hexamers (High Capacity cDNA Archive Kit; Applied Biosystems, Foster City, CA, USA), according to the protocol of the manufacturer.

The mRNA expression levels HRPT2 were determined using the TaqMan technology and an ABI Prism 7700 sequence detection system (PE Applied Biosystems). HRPT2 sequence-specific primers and probe (Hs00363810_m1, Assay-on-Demand; Applied Biosystems) with specificity towards HRPT2 exons 2 and 3 were used for 41 tumours (Table 3). In addition, 10 of the 15 tumour samples with detected aberrations in MEN1, HRPT2 or parafibromin were analyzed using an additional assay with specificity towards HRPT2 exons 10 and 11 (Hs00225998_m1). The primers and probe for the housekeeping gene 36B4, used as a reference, have been published previously (Forsberg et al. 2005). To establish a standard curve for relative quantification, cDNA derived from HeLa cells transfected with an HRPT2-containing plasmid was used. The qRT-PCR was performed in 25 μl reactions containing 1 ×TaqMan Universal Master Mix, 1 ×Target Assay Mix and 5 μl cDNA from each sample as a template, using 96-well optical reaction plates covered with optical caps (Applied Biosystems). The thermocycling conditions constituted 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All qRT-PCR experiments were performed in quintets. Analysis of the raw data was performed using the Sequence Detection System (SDS) version 1.9.1 software (Applied Biosystems). Following quantification in relation to the generated standard curves, HRPT2 expression values were normalized to the respective 36B4 value, and subsequently normalized to 1 for the normal parathyroid reference. To ensure the sensitivity of the qRT-PCR method, an additional experiment with tumour samples 1:1 and diluted 1:2 was carried out. The 1:2-diluted tumour cDNAs exhibited a 2-fold reduction in HRPT2 mRNA expression compared with the original samples (data not shown), demonstrating the sensitivity of the method.

Mutation analysis of the MEN1 gene

The coding region of MEN1 (exons 2–10) was sequenced according to published conditions (Villablanca et al. 2002b), using primers derived from the intronic regions to allow the detection of splice mutations (Table 2), and an ABI 377 automated sequencer (PE Applied Biosystems). Detected mutations were precisely determined by sequencing at least 10 positive clones after one-step cloning according to a previously published protocol (Aldred et al. 2006), and the mutated exons were sequenced in constitutional DNA to confirm the somatic status of the mutation.

Loss of heterozygosity (LOH) status in 1q25 (HRPT2) and 11q13 (MEN1)

The 46 matched blood and tumour DNA samples were previously genotyped for microsatellite markers located close to and flanking HRPT2 in 1q25 and MEN1 in 11q13 (Carpten et al. 2002, Villablanca et al. 2002a; Table 3).

HRPT2 mutations

All 46 tumours were previously screened for HRPT2 mutations by sequencing of the entire coding region, as reported in Carpten et al. (2002). Tumours T20, T41 and T42 harboured a somatic inactivating HRPT2 mutation (Table 3). In addition, case T3 exhibited a nucleotide alteration (AAT → AGT) in exon 8 in tumour and constitutional DNA, which was predicted to lead to a missense alteration of asparagine to serine at position 272 (N272S). The patient is an isolated case of PHPT and was surgically treated at the age of 83, whereby one adenoma with cystic features (0.5 g) was removed. She had no personal or family history of familial isolated PHPT or HPT–JT and at present time she has no clinical or biochemical signs of PHPT.

Results

In vitro expression of parafibromin by transfection to COS and HeLa cells

Western analysis of HRPT2-transfected cells revealed a ~60 kDa parafibromin-specific band in both COS and HeLa cells (Fig. 1C), corresponding to predicted wild-type parafibromin. In cells transfected with GFP-tagged constructs (mGFP~HRPT2 and HRPT2 ~mGFP) a ~90 kDa band was detected using antibodies against parafibromin as well as against GFP (Fig. 1D), in agreement with the size expected. However, the non-transfected cells revealed no or only weak expression of the ~60 kDa product. Live-cell imaging of mGFP ~HRPT2 and HRPT2 ~ mGFP resulted in strong nuclear signal and a weaker cytoplasmic signal using both constructs (Fig. 1A and B).

Expression of HRPT2 and parafibromin in normal tissues and established cell lines

Analysis of a multiple-tissue cDNA panel revealed a single transcript of ~1.5 kb in size, in agreement with the primer design (data not shown). This transcript was seen in all tissues analyzed, including brain, heart, kidney, liver, lung, pancreas, placenta and skeletal muscle, suggesting that the HRPT2 gene is ubiquitously expressed in normal human tissues.

By western blot analysis, expression of a ~60 kDa band corresponding to parafibromin was detected in the cell lines studied, including SH-SY5Y, HepG2, U2020 and HEK-293 cells (Fig. 2A). The band detected by western blot analysis was successfully blocked by preincubation with the antigen, supporting the specificity of the finding (Fig. 2A). In addition to the ~60 kDa band, a smaller band (~45 kDa) was also observed, possibly representing non-specific binding or an alternative product (Fig. 2A). Normal pancreas, normal kidney and secondary HPT glands all expressed a ~60 kDa parafibromin-specific product (Fig. 2B).

Mutations and LOH status of MEN1 and HRPT2

Previously published findings of LOH in 1q25 and 11q13 and of HRPT2 mutations (Carpten et al. 2002, Villablanca et al. 2002a) are detailed in Table 3 together with results from MEN1 mutation analysis carried out here. Two MEN1 mutations were identified in two of the tumours (T28 and T39; Table 3, Fig. 3). In case of T28, a 1 bp deletion in exon 2 (c68delC) was detected and in case of T39 the mutation consisted of a 6 bp deletion in exon 10 (c573del6). Both mutations are predicted to inactivate the encoded menin protein through in-frame deletion of two amino acids and protein truncation, respectively. None of the two MEN1-mutated adenomas harboured mutation or LOH of the HRPT2 gene. Similarly, the three tumours with HRPT2 mutations were without involvement of the MEN1 gene.

Loss of parafibromin expression in HRPT2-mutated adenomas

In 40/46 tumours expression of a ~60 kDa parafibromin product was revealed by western blot analysis (Table 3). However, in three tumours expression of parafibromin was not detectable (T20, T41 and T42; Table 3, Fig. 2C). In addition, in two tumours the parafibromin antibody detected protein products of slightly smaller size only (T34 and T38; Table 3, Fig. 2).

The results of immunohistochemical staining of the parathyroid tumours for parafibromin are summarized in Table 3 and exemplified in Fig. 4. In normal parathyroid tissue parafibromin immunoreactivity was highly specific for the parathyroid cells without staining of connective tissue or blood vessels (Fig. 4A and B). All three normal parathyroids studied exhibited clear nuclear localization of parafibromin, and in one of the samples an additional weaker cytoplasmic signal was also observed. The main staining pattern was nuclear immunoreactivity only, which was observed in 34/41 tumours (Fig. 4C–F). Three tumours showed both nuclear staining together with weaker cytoplasmic expression (T6, T21 and T38; Table 3, Fig. 4E and F), while one tumour only exhibited cytoplasmic expression (T34). The intensity of the staining was classified as positive, weakly positive or negative (Table 3) and the proportion of positive cells ranged from 50 to 100%. Finally, in the remaining three tumours (T20, T41 and T42) no parafibromin immunoreactivity was detected. Taken together, parafibromin immunoreactivity was not detected in any of the three tumours with identified HRPT2 mutation (Figs 2 and 4). In addition, one of the three adenomas with aberrant parafibromin expression by western blot analysis showed only weak cytoplasmic expression without a nuclear signal (T34). All the other 37 adenomas studied exhibited nuclear parafibromin expression of varying intensity, in three cases together with a weak cytoplasmic signal.

HRPT2 mRNA expression in parathyroid adenomas

Forty-one of the 46 cystic adenomas and two normal parathyroid biopsies (N1 and N2) were studied by qRT-PCR (Table 3; Fig. 5). Forty of the 41 adenomas studied exhibited positive HRPT2 mRNA expression levels, without gross deviation from the two normal parathyroids (adenomas 0.35–1.57; normals 1). Furthermore, similar results were obtained using assays targeting the 5′ end (exon 2/3) and 3′ end (exon 10/11) of the HRPT2 gene, respectively, on 10 of the tumours and normal parathyroids (Fig. 5, lower panel). Tumour T41 showed a strongly increased HRPT2 expression that was particularly pronounced with the exon 2/3 assay (Fig. 5).

Cyclin D1 expression in relation to parafibromin expression in parathyroid adenomas

Cyclin D1 expression was classified as absent (–), weak (+), moderate (++) or strong (+++; Table 3). In six tumours no cyclin D1 expression was detected, while the remaining 38 parathyroid adenomas showed positive expression of varying intensity (Table 3). No associations were observed between cyclin D1 expression/expression level and parafibromin expression or HRPT2 mutation status (Fig. 2C).

Proliferation index in HRPT2 and MEN1 mutated adenomas

The proliferation index was determined by Ki-67 immunohistochemistry analysis of 19 parathyroid adenomas, including 15 samples from this study and four samples previously identified with MEN1 mutations. In general, all tumours showed a low level of proliferation (range 0.02–0.7%). No statistical significance was found in Ki-67 index between tumours with HRPT2 genetic aberration (mutation/LOH) and those tumours with MEN1 mutations (data not shown).

Discussion

In the present study we demonstrate that parafibromin, encoded by HRPT2, is expressed in normal and neoplastic tissues as a ~60 kDa protein. This finding is well in agreement with the 531-amino acid product predicted from the 1596 bp open reading frame of the 2.7 kb major RNA transcript. In addition, tumours with deleterious HRPT2 mutations specifically showed loss of parafibromin expression by both Western blot and immunohistochemistry.

Expression of the HRPT2 gene was demonstrated by RT-PCR in all normal tissues analysed, in agreement with previously published Northern-blotting data (Carpten et al. 2002), thus suggesting a ubiquitous expression of HRPT2 in normal human tissues. Similarly we observed expression of parafibromin protein in different normal tissues (parathyroid, kidney and pancreas). These findings corroborate with the findings by Woodard et al. (2005), where in addition parafibromin expression was demonstrated in adrenal glands, heart and skeletal muscle. Thus HRPT2/parafibromin is normally expressed in tissues where tumours typically arise in the HPT-JT syndrome (parathyroid gland, jaw and kidney), in agreement with the expectations for a tumour-suppressor gene.

By immunohistochemistry, colocalization and coimmunoprecipitation studies, parafibromin has been suggested to be a nuclear protein (Tan et al. 2004, Rozenblatt-Rosen et al. 2005, Yart et al. 2005) and to associate with proteins involved in transcriptional regulation (Rozenblatt-Rosen et al. 2005) as well as in cell proliferation (Woodard et al. 2005). In addition, both nuclear and cytoplasmic localization has also been demonstrated by subcellular fractionation and immunoflourescence analysis in normal parathyroid tissue (Woodard et al. 2005). In agreement with these findings, we observed parafibromin expression in the nucleus, sometimes with an additional weaker cytoplasmatic component. This pattern was observed both in vitro and in vivo as well as in normal and neoplastic tissues.

Given the low frequency of HRPT2 mutations in the selected material, we hypothesized that cystic adenomas could either result from different genetic origin than HRPT2 mutation, or that other mechanisms for parafibromin inactivation are operative, such as epigenetic or regulatory inactivation. For this purpose, we have studied parafibromin and HRPT2 gene expression in these tumours. Positive parafibromin expression was revealed in most of the tumours by western blot analysis and confirmed by immunohistochemistry. Furthermore, no remarkable differences in parafibromin expression level were noted between tumours with LOH at HRPT2 in 1q25 and those without LOH in this region. In contrast, the three tumours with inactivating HRPT2 mutations did not exhibit any parafibromin expression. This finding is well in agreement with the types of HRPT2 mutation involved, which were all predicted to lead to a truncated protein. An additional tumour without demonstrated HRPT2 mutation revealed an aberrantly sized parafibromin protein product and a lack of nuclear parafibromin staining. These findings would further support the suggested tumour-suppressor function of parafibromin, and are consistent with the results of recent studies performed by immunohistochemistry and western blot analysis on sporadic parathyroid carcinomas (Tan et al. 2004, Woodard et al. 2005).

By qRT-PCR we found HRPT2 mRNA expression in all tumours and normal parathyroid glands studied. With one exception all tumours showed comparable levels to the normal tissues. Specifically, the tumours with 1q25 LOH showed similar HRPT2 expression levels as tumours without 1q25 LOH, suggesting the involvement of compensatory mechanisms acting on the transcript level. As these tumours have normal HRPT2 sequence and parafibromin expression, one functional HRPT2 allele is likely to be operative. In contrast, case T20 exhibited a somatic HRPT2 mutation and retained HRPT2 mRNA expression level although no parafibromin expression was detectable. The detected HRPT2 transcript could represent either the mutated or the wild-type allele, and several mechanisms for its inactivation can be considered, including an undetected mutation, epigenetic silencing and dominant-negative effect by the mutated allele. The frequent occurrence of two HRPT2 mutations in parathyroid carcinomas suggests that two inactivating events are required for gene inactivation and tumour development. The demonstration of both wild-type and mutant transcripts in constitutional tissue from hereditary HPT–JT related to an HRPT2 splice mutation would also argue against a dominant effect on the transcript level, at least in non-parathyroid cells (Moon et al. 2005). In case T41 one somatic HRPT2 mutation was detected and the family was linked to the HRPT2 locus (Teh et al. 1998). An additional germ-line HRPT2 mutation would be expected in this patient, but was not identified by sequencing of the coding regions. However, HRPT2 mutations have so far only been demonstrated in about 60% of 1q25-linked HPT–JT kindreds (Carpten et al. 2002). HRPT2 gene expression was strongly increased in T41, while parafibromin was lost in the tumour as compared to the normal rim of the parathyroid tissue.

The oncogene cyclin D1 was expressed in both high and low levels among HRPT2 mutated and non-mutated tumours, suggesting that these events are not mutually exclusive in parathyroid tumour development. This finding is somewhat in contrast to the observed down-regulation of cyclin D1 by parafibromin in in vitro systems (Woodard et al. 2005), indicating that cyclin D1 silencing in the parathyroid cell in vivo can result from additional mechanisms, and that cyclin D1 can still be expressed in the presence of parafibromin.

As parafibromin expression has been shown to inhibit proliferation in vitro (Woodard et al. 2005), and parathyroid carcinomas are known to have increased proliferation index, we hypothesized that HRPT2-mutated adenomas would exhibit an increased proliferation index. In our limited samples series we observed similarly low proliferation index in tumours with or without HRPT2 mutation, MEN1 mutation, 1q25 LOH and 11q13 LOH. Thus, clinicopathological differences between HRPT2-and MEN1-mutated adenomas were not strongly reflected on the level of proliferation. As HRPT2 mutants are linked to parathyroid malignancy, an interesting question arises if the tumours presented here with loss of parafibromin expression are potentially malignant. To our knowledge, these patients have not relapsed since their initial parathyroidectomy, performed more than 10 years ago. At histopathological evaluation, one of the three tumours (T42) with HRPT2 mutation exhibited atypical features of the parathyroid adenoma. However, all other 45 tumours including tumour cases T3 and T41 were without atypical changes.

In summary, we demonstrate parafibromin expression in normal human tissues, as well as its absence in parathyroid tumours with structural HRPT2-inactivating mutations. The presented data suggest that in the majority of benign parathyroid tumours the expression of parafibromin remains unaltered, while the loss of parafibromin expression is strongly indicative of gene inactivation through mutation of the HRPT2 gene.

Table 1

Clinical details for the 46 cystic parathyroid adenomas in the study

Preoperative
TumourAge (years)aSexS-CabS-PTHcTumour weight (g)dSporadic/famalial PHPT
FIHP, familial isolated PHPT; 1q, chromosome 1q.
aAge at operation.
bTotal serum calcium (reference range, 2.20–2.60 mM) preoperative.
cIntact serum PTH (reference range, 12–55 ng/l).
dTumour weight was determined after emptying of cysts.
eSerum calcium level originally reported as ionized calcium.
T175Female2.81620.24Sporadic
T272Female2.941181.28Sporadic
T383Female2.861930.50Sporadic
T483Female2.791690.60Sporadic
T555Female3.001050.30Sporadic
T674Female2.731271.22Sporadic
T775Female3.387116.54Sporadic
T876Female2.681391.90Sporadic
T960Female3.12950.78Sporadic
T1078Male3.142282.90Sporadic
T1182Female>3.501591.80Sporadic
T1281Female3.192382.80Sporadic
T1380Female2.711030.62Sporadic
T1447Female2.90550.69Sporadic
T1584Male2.801502.53Sporadic
T1678Male2.651512.19Sporadic
T1753Female2.93610.43Sporadic
T1867Female3.2016811.18Sporadic
T1954Female2.642010.42Sporadic
T2034Male3.181212.11Sporadic
T2166Female2.741248.12Sporadic
T2260Male2.60741.88Sporadic
T2363Male2.85794.44Sporadic
T2468Male3.087310.10Sporadic
T2551FemaleElevatede1890.81Sporadic
T2656Female3.151433.75Sporadic
T2783MaleElevatede1722.09Sporadic
T2881Male2.60670.65Sporadic
T2957MaleElevatede700.79Sporadic
T3070Male2.97970.46Sporadic
T3133FemaleElevatede15001.70Sporadic
T3279Female2.75570.14Sporadic
T3352FemaleElevatede651.11Sporadic
T3485Female3.041141.42Sporadic
T3583Female2.83980.20Sporadic
T3651Male3.091751.98Sporadic
T3758Male2.92711.48Sporadic
T3869Female2.85812.24Sporadic
T3936Female3.08834.86Sporadic
T4051Female2.70621.60Sporadic
T4128Female3.101131.16FIHP, 1q-linked
T4280Male2.871390.33Sporadic
T4354Female2.86930.38Sporadic
T4453Female3.16nd0.35Sporadic
T4550Male2.90550.49Sporadic
T4660Female2.60670.40Sporadic
Table 2

Primers and conditions used for mutation detection of MEN1

ExonPrimerForward (5′ → 3′)PrimerReverse (5′ → 3′)Annealing temperature (°C)
22AF5′-TTAGCGGACCCTGGGAGGAG-3′2AR5′-TCCACGAAGCCCAGCACCAAG-3′58
22BF5′-CCTGTTTGCTGCCGAGCTGG-3′2BR5′-GGCGGCGATGATAGACAGGTC-3′58
22CF5′- AAAGTGCTGGGATTCTAGG-3′2CR5′-GGAGACCTTCTTCACCAGCTCAC-3′58
22DF5′-GCCGTCACCTGTCCCTCTATC-3′2DR5′-CATGGATAAGATTCCCACCTACTGG-3′58
33AF5′-GCACAGAGGACCCTCTTTCATTAC-3′3AR5′-CTTGCCGTGCCAGGTGAC-3′58
33BF5′-CTCGCCCTGTCTGAGGATCATG-3′3BR5′-TGGGTGGCTTGGGCTACTACAG-3′58
44F5′-GGGCCATCATGAGACATAATG-3′4R5′-CTGCCCCATTGGCTCAG-3′58
55F5′-CCTGTTCCGTGGCTCATAACTC-3′5R5′-CTAGGAAAGGATCATAATTCAGGC-3′58
66F5′-GGGTGGCAGCCTGAATTATG-3′6R5′-CTCAGCCACTGTTAGGGTCTCC-3′58
77F5′-GGCTGCCTCCCTGAGGATC-3′7R5′-CTGGACGAGGGTGGTTGG-3′63
88F5′-GTGAGACCCCTTCAGACCCTAC-3′8R5′-TGGGAGGCTGGACACAGG-3′63
99F5′-GGGTGAGTAAGAGACTGATCTGTGC-3′9R5′-TGTAGTGCCCAGACCTCTGTG-3′58
1010AF5′-CGGCAACCTTGCTCTCACC-3′10AR5′-CCAGGCCCTTGTCCAGTG-3′58
1010BF5′-GGGAGTCCAAGCCAGAGGAG-3′10BR5′-GCCCTTCATCTTCTCACTCTGG-3′63
1010CF5′-TGCCAGCACCCGCAGCATC-3′10CR5′-CCCACAAGCGGTCCGAAGTCC-3′63
Table 3

Results of the HRPT2, MEN1, parafibromin and cyclin D1 studies in the cystic parathyroid adenomas

LOH dataParafibromin expression
Tumour1q2511q13HRPT2sequenceMEN1sequencemRNAaWesternIHCCyclin D1expression
IHC, immunohistochemistry; ni, not informative; nd, not done; wt, wild type; N, nuclear staining; C, cytoplasmic staining; −, absent; +, weak; ++, moderate; +++, strong.
aRelative HRPT2 expression compared to normal average.
bThe alteration was present also in constitutional DNA of the patient.
cThe protein products were of slightly smaller size only.
Nucleotide and amino acid positions are numbered starting from the initiation codon.
T1NoNowtwt0.35PositiveWeak positive/N+
T2NoNowtwt0.52PositivePositive/N
T3NoNoN272Sbwt0.54PositivePositive/N+
T4NoNowtwt0.55PositiveWeak positive/N+
T5NoNowtwt0.62Positive80% positive/N++
T6NoNowtwt0.64PositivePositive/N +C+
T7NoNowtwt0.72PositivePositive/N+++
T8NoNowtwt0.73PositivePositive/N
T9NoNowtwt0.73Positivend+++
T10NoNowtwt0.74PositivePositive/N+++
T11NoNowtwt0.79PositiveWeak positive/N++
T12NoNowtwt0.80PositivePositive/N+
T13NoNowtwt0.83Positive50% Positive/N+++
T14NoNowtwt0.84Positivend+
T15NoNowtwt0.85Positive70% Positive/N++
T16NoLOHwtwt0.85PositivePositive/N+
T17NoNowtwt0.89PositivePositive/N+
T18NoNowtwt0.90PositivePositive/N++
T19NoNowtwt0.90PositivePositive/N+++
T20NoNo53delTwt0.91NegativeNegative++
T21LOHLOHwtwt0.92PositiveWeak positive/N +C
T22NoNowtwt0.94Positive50% Positive/N+++
T23NoNowtwt0.96Positive80% Positive/N++
T24NoNowtwt0.97Positivend+++
T25NoNowtwt1.02Positivend+
T26LOHNowtwt1.02Positive70% Positive/N++
T27NoNowtwt1.03PositiveWeak positive/N
T28Noniwt68delC1.04PositivePositive/N+
T29NoNowtwt1.04PositivePositive/N+
T30NoNowtwt1.07PositiveWeak stain/N++
T31NoNowtwt1.10Positive50% Positive/N++
T32NoNowtwt1.11Positivend++
T33NoNowtwt1.19PositivePositive/N+
T34NoNowtwt1.22AberrantcNegative N/positive C+
T35NoNowtwt1.25PositivePositive/N
T36NoNowtwt1.27PositiveWeak positive/N++
T37LOHLOHwtwt1.33PositivePositive/Nnd
T38NoNowtwt1.38AberrantcPartly positive/N +C+++
T39niniwt572del61.48PositivePositive/N
T40NoLOHwtwt1.57PositivePositive/N+++
T41NoniW43Xwt5.86NegativeNegative+++
T42LOHni126del24wtndNegativeNegative+++
T43NoNowtwtndPositive70% Positive/N+
T44NoNowtwtndPositivePositive/N++
T45NoNowtwtndPositivePositive/Nnd
T46LOHLOHwtwtndPositivePositive/N++
Figure 1
Figure 1

Live-cell imaging and autoradiogram of Western blots showing in vitro expression of parafibromin from different HRPT2 constructs. Live-cell imaging of COS cells transfected with (A) HRPT2 ~mGFP and (B) mGFP~HRPT2 demonstrate strong green fluorescence in the nucleus and weak signals in the cytoplasm. (C) A ~60 kDa parafibromin-specific band is seen in COS and HeLA cells transfected with a construct containing the coding part of HRPT2 (T). For comparison, very low parafibromin expression is detected in untransfected cells (UT). Tubulin is shown as a protein-loading control. The size of the markers is given to the right in kDa. M, molecular-mass markers. In (D) the autoradiograms show parafibromin expression in COS cells using the three different constructs. A band of ~60 kDa is detected for cells transfected with the coding part of HRPT2, while the cells transfected with HRPT2 ~mGFP or HRPT2 ~mGFP revealed a protein band of ~90 kDa (left). The band of ~90 kDa was also detected using antibodies against GFP (right).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, 2; 10.1677/erc.1.01058

Figure 2
Figure 2

Western-blot analyses of parafibromin and cyclin D1. Parafibromin protein is demonstrated in total cellular extracts from (A) established cell lines (B) non-tumour human tissues and (C) total cellular extracts from parathyroid adenomas. Positive expression of a parafibromin-specific band is marked by Pos, and lack of expression is indicated by Neg. In (A) the results from cell lines are shown with and without blocking of the antibody, and the blocking peptide used as control refers to the synthetic peptide. The sizes of the markers are given to the left in kDa. 2°HPT, secondary hyperparathyroidism.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, 2; 10.1677/erc.1.01058

Figure 3
Figure 3

Sequencing chromatograms showing heterozygous MEN1 mutations in tumours T28 and T39. After subcloning, the mutated allele revealed a 1-base deletion at nucleotides 202–207 in tumour T28 (bottom left panel) and a 6 bp deletion starting at nucleotide 1730–1731 in tumour T39 (bottom right panel), while the normal wild-type sequence was found in the other allele (top panels). The positions of the nucleotides are numbered from the initiating codon.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, 2; 10.1677/erc.1.01058

Figure 4
Figure 4

Immunohistochemical analyses of parafibromin expression in (A, B) a biopsy of normal parathyroid gland and (C–H) cystic parathyroid adenomas. Each sample is shown in (A, C, E, G) low magnification (original magnification, ×220) and (B, D, F, H) high magnification (×560). (A, B) Strong nuclear staining and a weaker cytoplasmic staining are seen in normal parathyroid gland. In T45 (C, D) the staining pattern represents approximately 80% positive tumour cells with strong nuclear expression, while in T26 (E, F) nuclear parafibromin expression is seen in 70% of the tumour cells. Images (G) and (H) represent the HRPT2-mutated tumour T41 with loss of parafibromin immunoreactivity.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, 2; 10.1677/erc.1.01058

Figure 5
Figure 5

qRT-PCR analysis of the RNA expression levels of HRPT2 mRNA levels in cystic parathyroid adenomas (T1–T41) and in two normal parathyroid samples (N1 and N2). The results are demonstrated for the mean of quintets PCR assays and after normalization against the housekeeping gene 36B4. Top panel: results for all the tumours included in the study using the HRPT2 probe against exon 2/3. Bottom panel: for comparison, an additional probe (against exon 10/11) was included for 10 of the tumours and the two normals.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, 2; 10.1677/erc.1.01058

This work was supported by Swedish Cancer Foundation (010303, 020568), Gustav V Jubilee Foundation, Stockholm Cancer Society, Knut and Alice Wallenberg Foundation (2003.0043), Vera and Emil Cornell Foundation, Swedish Research Council, Stockholm County Council, Foundation of the National Board of Health and Welfare, Swedish Medical Association and Swedish Society for Medical Research. The authors are very grateful to Professor Christer Höög and Dr Anna Kouznetsova for valuable suggestions. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Aldred MJ, Talacko AA, Savarirayan R, Murdolo V, Mills AE, Radden BG, Alimov A, Villablanca A & Larsson C 2006 Dental findings in a family with hyperparathyroidism-jaw tumor syndrome and a novel HRPT2 gene mutation. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 101 212–218.

    • Search Google Scholar
    • Export Citation
  • Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248–254.

    • Search Google Scholar
    • Export Citation
  • Carpten JD, Robbins CM, Villablanca A, Forsberg L, Presciuttini S, Bailey-Wilson J, Simonds WF, Gillanders EM, Kennedy AM, Chen JD et al. 2002 HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumour syndrome. Nature Genetics 32 676–680.

    • Search Google Scholar
    • Export Citation
  • Cavaco BM, Guerra L, Bradley KJ, Carvalho D, Harding B, Oliveira A, Santos MA, Sobrinho LG, Thakker RV & Leite V 2004 Hyperparathyroidism-jaw tumor syndrome in Roma families from Portugal is due to a founder mutation of the HRPT2 gene. Journal of Medical Genetics 89 1747–1752.

    • Search Google Scholar
    • Export Citation
  • Cetani F, Pardi E, Borsari S, Viacava P, Dipollina G, Cianferotti L, Ambrogini E, Gazzerro E, Colussi G, Berti P et al. 2004 Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Medical Genetics 89 5583–5591.

    • Search Google Scholar
    • Export Citation
  • DeLellis RA, Lloyd RV, Heitz PU & Eng C 2004 Pathology and Genetics of the Tumours of Endocrine Organs, WHO Classification of Tumours. Lyon: IARC Press.

  • Dignam JD, Martin PL, Shastry BS & Roeder RG 1983 Eukaryotic gene transcription with purified components. Methods in Enzymology 101 582–598.

    • Search Google Scholar
    • Export Citation
  • Farnebo F, Teh BT, Kytölä S, Svensson A, Phelan C, Sandelin K, Thompson NW, Höög A, Weber G, Farnebo LO & Larsson C 1998 Alterations of the MEN1 gene in sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism 83 2627–2630.

    • Search Google Scholar
    • Export Citation
  • Forsberg L, Björck E, Hashemi J, Zedenius J, Höög A, Farnebo LO, Reimers M & Larsson C 2005 Distinction in gene expression profiles demonstrated in parathyroid adenomas by high-density oligoarray technology. European Journal of Endocrinology 152 459–470.

    • Search Google Scholar
    • Export Citation
  • Haven CJ, Howell VM, Eilers PM, Dunne R, Takahashi M, van Puijenbroek M, Furge K, Kievit J, Tan MH, Fleuren GJ et al. 2004 Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research 64 7405–7411.

    • Search Google Scholar
    • Export Citation
  • Howell VM, Haven CJ, Kahnoski K, Khoo SK, Petillo D, Chen J, Fleuren GJ, Robinson BG, Delbridge LW, Philips J et al. 2003 HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics 40 657–663.

    • Search Google Scholar
    • Export Citation
  • Jackson CE, Norum RA, Boyd SB, Talpos GB, Wilson SD, Taggart RT & Mallette LE 1990 Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: a clinically and genetically distinct syndrome. Surgery 108 1006–1012.

    • Search Google Scholar
    • Export Citation
  • Krebs LJ, Shattuck TM & Arnold A 2005 HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism 90 5015–5017.

    • Search Google Scholar
    • Export Citation
  • Lundgren E, Rastad J, Thrufjell E, Åkerström G & Ljunghall S 1997 Population-based screening for primary hyperparathyroidism with serum calcium and parathyroid hormone values in menopausal women. Surgery 121 287–294.

    • Search Google Scholar
    • Export Citation
  • Moon SD, Park JH, Kim EM, Kim JH, Han JH, Yoo SJ, Yoon KH, Kang MI, Lee KW, Son HY et al. 2005 A novel IVS2-1G>A mutation causes aberrant splicing of the HRPT2 gene in a family with hyperparathyroidism–jaw tumor (HPT–JT) syndrome. Journal of Clinical Endocrinology and Metabolism 90 878–883.

    • Search Google Scholar
    • Export Citation
  • Polak J & Van Noorden S 1986 An introduction to immunocytochemistry: current techniques and problems. In Immunocytochemistry: Modern Methods and Applications, 2nd edn. Bristol: John Wright & Sons.

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Simonds WF, Robbins CM, Agarwal SK, Hendy GN, Carpten JD & Marx SJ 2004 Familial isolated hyperparathyroidism is rarely caused by germline mutation in HRPT2, the gene for the hyperparathyroidism-jaw tumor syndrome. Journal of Clinical Endocrinology and Metabolism 89 96–102.

    • Search Google Scholar
    • Export Citation
  • Tan MH, Morrison C, Wang P, Yang X, Haven CJ, Zhang C, Zhao P, Tretiakova MS, Korpi-Hyovalti E, Burgess JR et al. 2004 Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clincal Cancer Research 10 6629–6637.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Uhles S, Moede T, Leibiger B, Berggren PO & Leibiger IB 2003 Isoform-specific insulin receptor signaling involves different plasma membrane domains. Journal of Cell Biology 22 1327–1337.

    • Search Google Scholar
    • Export Citation
  • Villablanca A, Calender A, Forsberg L, Höög A, Cheng JD, Petillo D, Bauters C, Kahnoski K, Ebeling T, Salmela P et al. 2004 Germline and de novo mutations in the HRPT2 tumour suppressor gene in familial isolated hyperparathyroidism (FIHP). Journal of Medical Genetics e32 1–7.

    • Search Google Scholar
    • Export Citation
  • Villablanca A, Farnebo F, Teh BT, Farnebo LO, Höög A & Larsson C 2002a Genetic and clinical characterization of sporadic cystic parathyroid tumours. Clincal Endocrinology (Oxford) 56 261–269.

    • Search Google Scholar
    • Export Citation
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  • View in gallery

    Live-cell imaging and autoradiogram of Western blots showing in vitro expression of parafibromin from different HRPT2 constructs. Live-cell imaging of COS cells transfected with (A) HRPT2 ~mGFP and (B) mGFP~HRPT2 demonstrate strong green fluorescence in the nucleus and weak signals in the cytoplasm. (C) A ~60 kDa parafibromin-specific band is seen in COS and HeLA cells transfected with a construct containing the coding part of HRPT2 (T). For comparison, very low parafibromin expression is detected in untransfected cells (UT). Tubulin is shown as a protein-loading control. The size of the markers is given to the right in kDa. M, molecular-mass markers. In (D) the autoradiograms show parafibromin expression in COS cells using the three different constructs. A band of ~60 kDa is detected for cells transfected with the coding part of HRPT2, while the cells transfected with HRPT2 ~mGFP or HRPT2 ~mGFP revealed a protein band of ~90 kDa (left). The band of ~90 kDa was also detected using antibodies against GFP (right).

  • View in gallery

    Western-blot analyses of parafibromin and cyclin D1. Parafibromin protein is demonstrated in total cellular extracts from (A) established cell lines (B) non-tumour human tissues and (C) total cellular extracts from parathyroid adenomas. Positive expression of a parafibromin-specific band is marked by Pos, and lack of expression is indicated by Neg. In (A) the results from cell lines are shown with and without blocking of the antibody, and the blocking peptide used as control refers to the synthetic peptide. The sizes of the markers are given to the left in kDa. 2°HPT, secondary hyperparathyroidism.

  • View in gallery

    Sequencing chromatograms showing heterozygous MEN1 mutations in tumours T28 and T39. After subcloning, the mutated allele revealed a 1-base deletion at nucleotides 202–207 in tumour T28 (bottom left panel) and a 6 bp deletion starting at nucleotide 1730–1731 in tumour T39 (bottom right panel), while the normal wild-type sequence was found in the other allele (top panels). The positions of the nucleotides are numbered from the initiating codon.

  • View in gallery

    Immunohistochemical analyses of parafibromin expression in (A, B) a biopsy of normal parathyroid gland and (C–H) cystic parathyroid adenomas. Each sample is shown in (A, C, E, G) low magnification (original magnification, ×220) and (B, D, F, H) high magnification (×560). (A, B) Strong nuclear staining and a weaker cytoplasmic staining are seen in normal parathyroid gland. In T45 (C, D) the staining pattern represents approximately 80% positive tumour cells with strong nuclear expression, while in T26 (E, F) nuclear parafibromin expression is seen in 70% of the tumour cells. Images (G) and (H) represent the HRPT2-mutated tumour T41 with loss of parafibromin immunoreactivity.

  • View in gallery

    qRT-PCR analysis of the RNA expression levels of HRPT2 mRNA levels in cystic parathyroid adenomas (T1–T41) and in two normal parathyroid samples (N1 and N2). The results are demonstrated for the mean of quintets PCR assays and after normalization against the housekeeping gene 36B4. Top panel: results for all the tumours included in the study using the HRPT2 probe against exon 2/3. Bottom panel: for comparison, an additional probe (against exon 10/11) was included for 10 of the tumours and the two normals.

  • Aldred MJ, Talacko AA, Savarirayan R, Murdolo V, Mills AE, Radden BG, Alimov A, Villablanca A & Larsson C 2006 Dental findings in a family with hyperparathyroidism-jaw tumor syndrome and a novel HRPT2 gene mutation. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 101 212–218.

    • Search Google Scholar
    • Export Citation
  • Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248–254.

    • Search Google Scholar
    • Export Citation
  • Carpten JD, Robbins CM, Villablanca A, Forsberg L, Presciuttini S, Bailey-Wilson J, Simonds WF, Gillanders EM, Kennedy AM, Chen JD et al. 2002 HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumour syndrome. Nature Genetics 32 676–680.

    • Search Google Scholar
    • Export Citation
  • Cavaco BM, Guerra L, Bradley KJ, Carvalho D, Harding B, Oliveira A, Santos MA, Sobrinho LG, Thakker RV & Leite V 2004 Hyperparathyroidism-jaw tumor syndrome in Roma families from Portugal is due to a founder mutation of the HRPT2 gene. Journal of Medical Genetics 89 1747–1752.

    • Search Google Scholar
    • Export Citation
  • Cetani F, Pardi E, Borsari S, Viacava P, Dipollina G, Cianferotti L, Ambrogini E, Gazzerro E, Colussi G, Berti P et al. 2004 Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Medical Genetics 89 5583–5591.

    • Search Google Scholar
    • Export Citation
  • DeLellis RA, Lloyd RV, Heitz PU & Eng C 2004 Pathology and Genetics of the Tumours of Endocrine Organs, WHO Classification of Tumours. Lyon: IARC Press.

  • Dignam JD, Martin PL, Shastry BS & Roeder RG 1983 Eukaryotic gene transcription with purified components. Methods in Enzymology 101 582–598.

    • Search Google Scholar
    • Export Citation
  • Farnebo F, Teh BT, Kytölä S, Svensson A, Phelan C, Sandelin K, Thompson NW, Höög A, Weber G, Farnebo LO & Larsson C 1998 Alterations of the MEN1 gene in sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism 83 2627–2630.

    • Search Google Scholar
    • Export Citation
  • Forsberg L, Björck E, Hashemi J, Zedenius J, Höög A, Farnebo LO, Reimers M & Larsson C 2005 Distinction in gene expression profiles demonstrated in parathyroid adenomas by high-density oligoarray technology. European Journal of Endocrinology 152 459–470.

    • Search Google Scholar
    • Export Citation
  • Haven CJ, Howell VM, Eilers PM, Dunne R, Takahashi M, van Puijenbroek M, Furge K, Kievit J, Tan MH, Fleuren GJ et al. 2004 Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype. Cancer Research 64 7405–7411.

    • Search Google Scholar
    • Export Citation
  • Howell VM, Haven CJ, Kahnoski K, Khoo SK, Petillo D, Chen J, Fleuren GJ, Robinson BG, Delbridge LW, Philips J et al. 2003 HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics 40 657–663.

    • Search Google Scholar
    • Export Citation
  • Jackson CE, Norum RA, Boyd SB, Talpos GB, Wilson SD, Taggart RT & Mallette LE 1990 Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: a clinically and genetically distinct syndrome. Surgery 108 1006–1012.

    • Search Google Scholar
    • Export Citation
  • Krebs LJ, Shattuck TM & Arnold A 2005 HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism 90 5015–5017.

    • Search Google Scholar
    • Export Citation
  • Lundgren E, Rastad J, Thrufjell E, Åkerström G & Ljunghall S 1997 Population-based screening for primary hyperparathyroidism with serum calcium and parathyroid hormone values in menopausal women. Surgery 121 287–294.

    • Search Google Scholar
    • Export Citation
  • Moon SD, Park JH, Kim EM, Kim JH, Han JH, Yoo SJ, Yoon KH, Kang MI, Lee KW, Son HY et al. 2005 A novel IVS2-1G>A mutation causes aberrant splicing of the HRPT2 gene in a family with hyperparathyroidism–jaw tumor (HPT–JT) syndrome. Journal of Clinical Endocrinology and Metabolism 90 878–883.

    • Search Google Scholar
    • Export Citation
  • Polak J & Van Noorden S 1986 An introduction to immunocytochemistry: current techniques and problems. In Immunocytochemistry: Modern Methods and Applications, 2nd edn. Bristol: John Wright & Sons.

  • Rozenblatt-Rosen O, Hughes CM, Nannepaga SJ, Shanmugam KS, Copeland TD, Guszczynski T, Resau JH & Meyerson M 2005 The parafibromin tumor suppressor protein is part of a human paf1 complex. Molecular and Cellular Biology 25 612–620.

    • Search Google Scholar
    • Export Citation
  • Shattuck TM, Välimäki S, Obara T, Gaz RD, Clark OH, Shoback D, Wierman ME, Tojo K, Robbins CM, Carpten JD et al. 2003 Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. New England Journal of Medicine 349 1722–1729.

    • Search Google Scholar
    • Export Citation
  • Simonds WF, Robbins CM, Agarwal SK, Hendy GN, Carpten JD & Marx SJ 2004 Familial isolated hyperparathyroidism is rarely caused by germline mutation in HRPT2, the gene for the hyperparathyroidism-jaw tumor syndrome. Journal of Clinical Endocrinology and Metabolism 89 96–102.

    • Search Google Scholar
    • Export Citation
  • Tan MH, Morrison C, Wang P, Yang X, Haven CJ, Zhang C, Zhao P, Tretiakova MS, Korpi-Hyovalti E, Burgess JR et al. 2004 Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clincal Cancer Research 10 6629–6637.

    • Search Google Scholar
    • Export Citation
  • Teh BT, Farnebo F, Twigg S, Höög A, Kytolä S, Korpi-Hyovalti E, Wong FK, Nordenström J, Grimelius L, Sandelin K et al. 1998 Familial isolated hyperparathyroidism maps to the hyperparathyroidism-jaw tumour locus in 1q21-q32 in a subset of families. Journal of Clinical Endocrinology and Metabolism 83 2114–2120.

    • Search Google Scholar
    • Export Citation
  • Uhles S, Moede T, Leibiger B, Berggren PO & Leibiger IB 2003 Isoform-specific insulin receptor signaling involves different plasma membrane domains. Journal of Cell Biology 22 1327–1337.

    • Search Google Scholar
    • Export Citation
  • Villablanca A, Calender A, Forsberg L, Höög A, Cheng JD, Petillo D, Bauters C, Kahnoski K, Ebeling T, Salmela P et al. 2004 Germline and de novo mutations in the HRPT2 tumour suppressor gene in familial isolated hyperparathyroidism (FIHP). Journal of Medical Genetics e32 1–7.

    • Search Google Scholar
    • Export Citation
  • Villablanca A, Farnebo F, Teh BT, Farnebo LO, Höög A & Larsson C 2002a Genetic and clinical characterization of sporadic cystic parathyroid tumours. Clincal Endocrinology (Oxford) 56 261–269.

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