A transgenic mouse model for TEL-JAK2-induced B-cell lymphoma/leukemia.
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Animals↗
Biomedical subjects
N R dos Santos
Publications and source records attributed to N R dos Santos.
The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.
As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.
Acute Disease↗
Molecular mechanisms underlying human synovial sarcoma development.
Synovial sarcomas are rather common among soft-tissue tumors, occurring at any age but affecting mainly young adults. The vast majority of synovial sarcomas carries a t(X;18)(p11.2;q11.2) chromosomal translocation, in about one-third of the cases as the sole cytogenetic anomaly. Several studies have indicated that the t(X;18) translocation arises exclusively in synovial sarcomas, therefore being an excellent tool to diagnose this malignancy. The breakpoint-associated genes were recently isolated: SYT, from chromosome 18, and SSX1 and SSX2, both from the X chromosome. This discovery enabled the detection of SYT-SSX fusion transcripts by specific reverse transcriptase-polymerase chain reactions. This molecular genetics methodology has now been applied to numerous tumor samples and has led to the finding that, in contrast to tumors carrying SYT-SSX2 fusions, SYT-SSX1-positive tumors more often exhibit a biphasic histology, show a higher proliferation rate, and are associated with a poorer clinical outcome. It has also been shown that the SYT and SSX proteins are localized in the nucleus, where they appear to play a role in transcriptional regulation, SYT as an activator of transcription and the SSX proteins as transcriptional repressors. It was also found that SYT interacts and colocalizes in the nucleus with the BRM protein, a transcriptional coactivator, and that the SSX proteins colocalize in the nucleus with polycomb group proteins, which are transcriptional corepressors. Together, these studies have provided mechanistic clues about how the SYT-SSX fusion proteins may trigger synovial sarcoma development.
Amino Acid Sequence↗
Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization.
In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.
Amino Acid Sequence↗
Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.
The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.
Adult↗
Masked t(X;18)(p11;q11) in a biphasic synovial sarcoma revealed by FISH and RT-PCR.
The initial cytogenetic analysis of a biphasic synovial sarcoma showed an apparently normal karyotype. After FISH using chromosome X- and 18-specific probes and RT-PCR using SYT- and SSX-specific primer sets, a cryptic synovial sarcoma-associated t(X;18)(p11;q11) could be revealed. The "masked" nature of the translocation may best be explained by a two-step scenario in which a genuine t(X;18)(p11;q11) has occurred as a first step and a reverse reciprocal X;18 translocation as a second step, leaving the synovial sarcoma-associated SYT-SSX1 fusion intact. The findings further underline our previous suggestion that SYT-SSX1 fusions may correlate with a biphasic nature of the tumor. In addition, our findings indicate that, in analogy to, e.g., the Philadelphia translocation in chronic myeloid leukemia, "masked" translocations may occur in soft tissue tumors and that, as a standard, RT-PCR and/or FISH analyses should be carried out in order to provide karyotypic information that may be relevant to tumor diagnosis and/or prognosis.
Adult↗
Molecular cytogenetics of bone and soft tissue tumors.
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Bone Neoplasms↗
Benign and malignant thyroid lesions show instability at microsatellite loci.
Forty-six benign and malignant tumours and tumour-like lesions of the thyroid were analysed for microsatellite instability (MI) at eight loci, mapping to four different chromosomes, 7 lesions (15%) displayed MI at one or more loci, including 2/13 nodular goitres, 2/15 follicular adenomas, 2/12 papillary carcinomas and 1/4 follicular carcinomas. Two benign and one malignant lesion among the seven unstable cases exhibited this phenotype at three or more loci. We found no mutations in the mismatch repair gene, hMSH2, in the seven affected cases, after screening all the exons by CDGE mutation analysis. At variance with the data on record, these results indicate that, despite being relatively infrequent, MI does occur not only in thyroid carcinomas but also in benign lesions (goitres and follicular adenomas of the thyroid).
Adenocarcinoma, Follicular↗
Nuclear localization of SYT, SSX and the synovial sarcoma-associated SYT-SSX fusion proteins.
Synovial sarcoma is characterized by a prevalent chromosomal translocation, t(X;18)(p11;q11). As a result of this translocation the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome. In this study, we generated polyclonal antibodies against the SYT and SSX2 proteins. These antibodies specifically detected both these proteins and the SYT-SSX fusion proteins in transfected COS-1 cell extracts. Indirect immunofluorescence analysis of COS-1 cells expressing tagged or untagged SYT, SSX2, SYT-SSX1 or SYT-SSX2 indicated that all these proteins are localized in the nucleus, excluding the nucleoli. The SSX2 protein exhibited a diffuse staining pattern whereas both the SYT and SYT-SSX proteins appeared in several nuclear dots. Similar nuclear dots were also detected in primary synovial sarcoma cells growing in a short-term in vitro culture. Double immunofluorescence in conjunction with confocal laser-scanning microscopy revealed that the SYT and SYT-SSX nuclear dots do not co-localize with known nuclear structures as e.g. coiled bodies, SC35 interchromatin granules or PML bodies. The similar nuclear localization patterns of SYT and SYT-SSX suggest that the SYT-SSX fusion proteins are directed to SYT-associated nuclear domains where an abnormal function may be exerted.
Adult↗
Microsatellite instability at multiple loci in gastric carcinoma: clinicopathologic implications and prognosis.
BACKGROUND & AIMS: Microsatellite instability (replication error [RER]-positive phenotype) is a frequent genetic alteration in gastric carcinomas. The clinical relationship between RER-positive and RER-negative gastric tumors is poorly characterized. The aim of this study was to investigate the relationship between the number of altered microsatellite loci and the clinicopathologic features of gastric carcinoma. METHODS: Five or 6 microsatellite loci were analyzed in 61 gastric carcinomas using polymerase chain reaction. RESULTS: Twenty-one carcinomas (34.4%) had microsatellite instability: 7 at 1 locus, 2 at 2 loci, and 12 at multiple loci. The comparison between the three groups (with none, 1 or 2, and more than 2 RER-positive loci) showed that RER-negative carcinomas and carcinomas with 1 or 2 RER-positive loci share features that differ from those of carcinomas with multiple RER-positive loci. The latter were all of the intestinal or atypical subtype and had lower DNA content, more prominent lymphoid infiltration, and less prevalent nodal metastases than carcinomas in the other two groups. The patients with carcinomas showing multiple RER-positive loci had a better prognosis. CONCLUSIONS: The finding of microsatellite instability in a single or few loci does not qualify a case as a mutator phenotype from a clinical standpoint. Gastric tumors with multiple RER-positive loci have a particular clinicopathologic profile leading to a better outcome.
Carcinoma↗