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1 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, via S. Pansini 5, 80131 Naples, Italy
2 Laboratorio di Citogenetica Medica e Genetica Molecolare, Istituto Auxologico Italiano, Milano, Italy
3 Dipartimento di Biologia e Genetica per le Scienze Mediche, Università degli Studi di Milano, Italy
4 Neurochirurgia, Ospedale San Raffaele, Milano, Italy
5 NOGEC (Naples Oncogenomic Center)-CEINGE, Centro di Biotecnologie Avanzate, via Comunale Margherita 482, 80145, Naples, Italy
(Requests for offprints should be addressed to A Fusco; Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, via Pansini 5, 80131 Naples, Italy, and NOGEC (Naples Oncogenomic Center)-CEINGE, Centro di Biotecnologie Avanzate, via Comunale Margherita 482, 80145, Naples, Italy; Email: afusco{at}napoli.com)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Recently, our group suggested a critical role for high-mobility group A2 (HMGA2) in pituitary oncogenesis. It has been shown that transgenic mice overexpressing the HMGA2 gene develop growth hormone- and prolactin-secreting adenomas (Fedele et al. 2002). The HMGA protein family consists of a group of small nuclear non-histone chromatinic proteins. They are involved in the regulation of chromatin structure (Fashena et al. 1992) and play an important role in the assembly of a multi-protein transcriptional complex that regulates the transcription of the target genes (Grosschedl et al. 1994). HMGA proteins play a crucial role in the process of cancerogenesis. In fact, chromosomal translocations of 12q13-15, involving the HMGA2 gene, leading to rearrangements and dysregulated expression of the HMGA2 gene, have been frequently detected in benign human tumors of mesenchymal origin (Ashar et al. 1995, Schoenmakers et al. 1995). Conversely, malignant neoplasias show an abundant expression of the HMGA2 gene that is required for malignant cell transformation (Berlingieri et al. 1995, Giancotti et al. 1985, 1987, Abe et al. 2003). Consistently with the development of prolactin adenomas in HMGA2 transgenic mice, induction of HMGA2 expression was observed in human prolactinomas in association with amplification and/or rearrangement in most of the tumor samples analyzed (Finelli et al. 2002), whereas the HMGA2 gene was not expressed at all in normal pituitary gland.
The aim of the present work has been to assess the putative involvement of the HMGA2 gene in another pituitary adenoma subtype, such as the NFPA. NFPAs are benign neoplasias that differ from prolactinomas in showing more frequently a normal karyotype and a lower frequency of trisomy 12 when the karyotype is aberrant (Finelli et al. 2000). Therefore, we analyzed HMGA2 gene expression and possible cytogenetic alterations in a representative panel of 18 NFPAs. Results obtained by fluorescence in situ hybridization (FISH) analysis and reverse transcriptase (RT)-PCR expression show that the majority of NFPAs express HMGA2, which, at odds with prolactinomas, is not associated with over-representation of the HMGA2 region, and only in a few cases is driven by rearrangement of the gene.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The NFPA tissue samples were obtained at transsphenoidal surgery from 18 patients, 11 of whom had undergone surgery for visual defects (pituitary adenomas (PAs) 80, 84, 86, 92, 100, 105, 107, 109, 114, 116 and 120), five for prevention (PAs 82, 93, 99, 103 and 120) and two for an increase in tumor size (PAs 110 and 112). The non-functioning secreting pituitary adenomas were clinically and hormonally characterized on the basis of standard endocrinological criteria; the tumor subtype was confirmed by routine immunohistochemistry analysis (Table 1
). Seven of 18 tumors (PAs 80, 84, 103, 110, 114, 115, 112) presented with invasion of cavernous sinus. Most of the patients had not received any chemotherapy or radiation therapy before surgery. Histological analysis was performed as described previously (Finelli et al. 2000). PAs 82 and 86 were described previously (Finelli et al. 2002).
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The primary pituitary cell cultures were set up as described elsewhere (Bettio et al. 1997). The phytohemagglutinin (PHA)-stimulated peripheral blood cultures were set up according to standard procedure. The Q-banding of fluorescence using Quimacrine (QFQ) banding technique was used for cytogenetic analysis, and the International System for Human Cytogenetic Nomenclature was adopted (Mitelman 1995).
FISH studies
FISH analysis on nuclei was performed by using the following alphoid probes: pZ8.4 (D8Z8) and pDMX1 (DXZ1; Archidiacono et al. 1995), and pBR12 (D12Z3; Baldini et al. 1990). YAC 882a10, which maps on chromosome 5p13, was from the CEPH YAC library, as described by Finelli et al. (2000), while HMGA2 BAC clones (698i6 and 669g18), encompassing the 5' (5' untranslated region and exons 1–3) and the 3' (exons 3–5 and the 3' untranslated region) portions of HMGA2 gene are described in previous works (Finelli et al. 2002, Pierantoni et al. 2003).
The procedure described by Lichter et al. (1990) and Lichter & Cremer (1992), with some modifications, was used for dual-color FISH experiments on interphase nuclei from direct tumor preparations or short-term culture tumor preparations. Briefly, the probes were labeled by nick-end translation with biotin or digoxygenin (Roche Molecular Biochemicals, Germany). For each in situ hybridization experiment, 200 ng labeled alphoid probe and/or 500 ng labeled YAC/ BAC probes were used in a 10 µl volume of hybridization solution. The FISH procedure, detection of biotin- and digoxygenin-labeled probes, nuclei/chromosome counterstaining and digital-image analysis are described elsewhere (Finelli et al. 2000). The images were edited using Adobe Photoshop 7 (Adobe System, Mountain View, CA, USA). As described previously, scoring was based on >200 nuclei per each tumor sample and for reference purposes the background percentage of nuclei with more or less than two signals and the percentage of nuclei with a split hybridization signal were calculated. PHA-stimulated lymphocytes from healthy individuals were hybridized in parallel.
RNA extraction and RT-PCR analysis
Pituitary adenomas were dissected rapidly, frozen on dry ice and stored at –80 °C. Total RNA was extracted using TRI reagent solution (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s protocol. 5 µg total RNA, digested with RNase-free DNase, were reversetranscribed using random hexanucleotides as primers (100mM) and 12 units avian myeloblastosis virus RT (Promega). The cDNA was amplified in a 25 µl reaction mixture containing 0.2mM dNTP, 1.5mM MgCl2, 0.4mM of each primer and 1 unit Taq DNA polymerase (Perkin-Elmer). After a denaturing step (95 °C for 2 min) the cDNA was further amplified in 20 PCR cycles (95 °C for 1 min, 58 °C for 30 s and 72 °C for 30 s). The following primers were used to amplify the HMGA2 transcript: forward primer, 5'-CGAAAGGTGCTGGGCAGCTCCGG- 3' , which maps onto the first exon; reverse primer, 5'-CCATTTCCTAGGTCTGCCTCTTG-3' , which maps onto the third exon; reverse primer II, 5'-CTAGTCCTCTTCGGCAGACTC- 3' , which maps onto the fifth exon.
Expression of the GAPDH gene was used as an internal control for the amount of cDNA tested. The specific primers were: forward, 5'-ACATGTTCCAATATGATTCC- 3' ; reverse, 5'-TGGACTCCACGACGTACTCA- 3' (corresponding to nucleotides 195–215 and 355–335, respectively). The reaction products were analyzed on a 2% agarose gel, and transferred to GeneScreen plus nylon membranes (Dupont, Boston, MA, USA). The membranes were hybridized with a HMGA2 cDNA probe. cDNA probes obtained by PCR were labeled with [32P]dCTP using random oligonucleotide primers (Ready-To-Go; Pharmacia) at a specific activity equal to or higher than 7 x 108 c.p.m./µg.
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Results |
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Table 1
shows a list of the 18 NFPA samples analyzed in our study. Their immunohistochemical patterns are also specified. All the tumors were examined using conventional cytogenetics, either on direct or short-term culture chromosome preparations, with successful karyotyping of 14 tumors. An abnormal karyotype was found in five adenomas (PAs 92, 93, 99, 100 and 105), while an apparently normal karyotype was observed in the remaining nine tumors (Table 1, third column). Peripheral blood cells from healthy individuals were also analysed (C1–C4 in Table 1).
Interphase FISH
To assess the normal/abnormal chromosomal set of tumors where cytogenetic analysis failed (PAs 84, 103, 107 and 114) or only a few metaphases could be analyzed, we performed FISH of centromeric/ pericentromeric probes specific to chromosomes found at increased dosages in previous studies (Finelli et al. 2000), i.e. chromosomes 5, 8, 12 and X. By this approach, we observed the presence of trisomy X in PA 84, trisomy 12 in PA 114, tetrasomy 12 in PA 103 and combined trisomy 12 and X in PA 107, accounting for the four tumors where conventional cytogenetics failed. In addition, we could confirm trisomy 8 in PA 105, trisomy X in PA 93, trisomy 12 in PA 100 and trisomy 12 and an extra X-specific signal in PA 99 (Table 1, fourth column). Based on previous findings in prolactin-secreting adenomas (Finelli et al. 2002), we conducted FISH experiments on 16 non-functioning secreting pituitary adenomas to establish the dosage and putative rearrangements of HMGA2. Interphase dual-color FISH was performed on nuclei from direct/ short-term tumor preparations using HMGA2-specific probes and different combinations of HMGA2-specific BAC probes with a D12Z3-specific alphoid probe. As reported in Table 1, dual-color FISH of HMGA2- specific BACs showed on 12 out of 18 NFPAs two pairs of red/green overlapping spots. This pattern corresponds to that of peripheral blood cells from healthy individuals (C1–C4 in Table 1) in the great majority of nuclei (90–99%). Conversely, an increased dosage of the target region was detected in five tumors, namely PAs 99, 100, 107 and 114, showing HMGA2 trisomy, and PA 103, which showed HMGA2 tetrasomy in 20–96% of nuclei. A heterogeneous pattern of FISH signals was given by PA 99: in fact, this sample displayed a major trisomic clone (53%), characterized by three signals of the same intensity of control cells, a trisomic subclone (21%), with one/two HMGA2- specific signals of reduced intensity, and minor disomic subclones (summing up to 13%) with one regular (red/ green) HMGA2 signal and one split (either red or green) signal (Fig. 1A and Table 1). Signals of decreased intensity as well as split signals are highly suggestive of intra-HMGA2 rearrangements. PA 80 was disomic for HMGA2 in most cells but contained a small subclone (7%) with an additional signal given by the 3' HMGA2 BAC (Fig. 1B). FISH results with HMGA2 BACs on both NFPAs showing subclones with an atypical pattern were confirmed in independent experiments. Co-hybridization of HMGA2 BACs with chromosome 12 alphoid-specific probe was then performed on the two above NFPAs to detect selective overrepresentation of the HMGA2 region, in addition to the trisomy of chromosome 12, when present. These FISH experiments revealed in PA 99 a number of spots higher than that given by the alphoid probe in about 36% of the nuclei. This percentage derived from the sum of the trisomic subclone with signals of reduced intensity (21%) and that of disomic subclones with split signals (13%).
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RT-PCR analysis, using primers specific for exons 1 and 3 of HMGA2 gene, was performed in parallel to check the HMGA2 expression in the NFPAs evaluated by cytogenetics and interphase FISH. 15 tumors could be investigated together with PAs 82 and 86, used as negative controls in our previous study (Finelli et al. 2002). Insufficient material has been obtained to evaluate the HMGA2 expression in PA 93. As shown in Fig. 1C, most tumors (12/15) showed HMGA2- specific mRNA, whereas only PAs 100, 103 and 109 were negative. Notable differences in the levels of HMGA2 mRNA could be appreciated among tumors, with a group that expresses high levels of HMGA2 mRNA (PAs 112, 114, 115 and 120) and one that expresses HMGA2 at low levels (PAs 84, 92, 105, 110 and 116). As expected, HMGA2 was not expressed in normal pituitary gland (Fig. 1C, lane NP; Zhou et al. 1995). To verify the presence of truncated transcripts of the HMGA2 gene, we evaluated the expression of the entire HMGA2 transcript in 15 of the NFPA tumours. To this end, we have utilized primers specific for exons 1 and 5 of the HMGA2 gene, which amplify the entire coding sequence. Eight adenoma samples, PAs 84, 107, 110, 112, 114, 115, 116 and 120, showed HMGA2 gene expression, indicating the presence of a standard-sized transcript, whereas in the other samples, PAs 80, 82, 86, 99, 100, 103 and 109, no amplification was observed (Fig. 1D). The results from PAs 82, 86, 100, 103 and 109 confirm those obtained by using the other HMGA2 primer pair. The results obtained for PAs 80 and 99 were consistent with the FISH results, where we observed a hybridization pattern suggestive of intra-HMGA2 rearrangements. For all PCR assays the amplification was also performed with non-reverse-transcribed RNAs to exclude DNA contamination (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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). Despite the fact that HMGA2 rearrangement was monitored only in a fraction of cells in tumors PAs 80 and 99, RT-PCR analysis showed only aberrant transcripts. This suggests that microrearrangements of the gene or sequence alterations, not detectable by FISH analysis, also affected the majority of tumor cells with apparent integrity of the HMGA2 region by FISH analysis. Interestingly, one of the two rearranged NFPAs, PA 99, showed a consistent fraction of cells trisomic for chromosome 12, which represents likely a primary genetic event that might facilitate the occurrence of further rearrangements. Indeed, evidence has been provided that polysomy promotes structural instability in tumor-cell chromosomes through asynchronous replication and breaks within late-replicating regions (Kost-Alimova et al. 2004). The HMGA2 region falls within a G-dark band, which likely corresponds to a late-replicating region that may become a preferential site of structural rearrangements in the unstable polysomic chromosome 12. As already demonstrated in a high number of benign tumors of mesenchymal origin (Schoenmakers et al. 1995) and for two cases of prolactinomas (Finelli et al. 2002), the rearrangement of the HMGA2 gene in PAs 80 and 99 results in a break in the large intervening sequence (IVS3) that separates the third from the fourth exon. This would induce the oncogenic potential of the HMGA2 protein because of the loss of its C-terminal tail, as demonstrated previously (Fedele et al. 1998, Battista et al. 1999).
As demonstrated previously for prolactinomas, the sole trisomy 12 is not sufficient for HMGA2 expression, which is associated with overrepresentation of the HMGA2 region and/or rearrangement. In fact, PAs 100 and 103 were trisomic and tetrasomic for chromosome 12, but they did not have overrepresentation of the 12q14 region (data not shown) and did not express HMGA2. Since overrepresentation of HMGA2 region, via trisomy or tetrasomy, is quite common in prolactinomas (Finelli et al. 2002), and rare in NFPAs, we retain that the polysomy rearrangement is a major contributor to HMGA2 activation in prolactinomas, while it is implicated less in NFPAs, most of which have a diploid karyotype. Since FISH experiments have shown the common lack of an extra chromosome 12 and rearrangements only in a small percentage of cells that would trigger new rearrangement in the polysomic chromosome, and most NFPAs express high levels of HMGA2 transcript, other mechanisms able to activate HMGA2 expression should occur in human NFPAs. Alternative HMGA2-activating mechanisms, among which sequence alterations or dysregulation by cryptic rearrangements, need further study. In fact, it cannot be excluded that theHMGA2overexpression may be due to the same, still mainly unknown, mechanisms responsible for HMGA2 overexpression in malignant neoplasias.
In conclusion, the findings reported here extend those obtained in prolactinomas by confirming the involvement of HMGA2 in pituitary oncogenesis. Since HMGA2 transgenic mice never develop NFPAs, and since rearrangements of HMGA2 are rare in this subtype, we can also hypothesize that whereas in most human prolactinomas HMGA2 overexpression would represent one of the initial and causal events, in most NFPA subtypes HMGA2 overexpression might represent a secondary event that occurs independently of the specific initializing event and might be responsible for tumor progression. However, this hypothesis needs to be validated by future work.
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Acknowledgements |
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Footnotes |
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References |
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