Johan Wejde

The chimeric FUS/CREB3l2 gene is specific for low-grade fibromyxoid sarcoma.

Low‐grade fibromyxoid sarcoma (LGFMS) is a variant of fibrosarcoma that was recognized as a distinct tumor entity only quite recently. We previously described a translocation, t(7;16)(q33;p11), that resulted in a fusion of the FUS and CREB3L2 (also known as BBF2H7) genes in two soft tissue tumors that fulfilled morphologic criteria for LGFMS. To delineate the spectrum of tumors that may harbor the FUS/CREB3L2 gene, we selected 45 low‐grade spindle cell sarcomas for reverse transcriptase polymerase chain reaction (RT‐PCR) and/or fluorescence in situ hybridization (FISH) analyses; none of these tumors had originally been diagnosed as LGFMS. Furthermore, also included were two benign soft tissue tumors and nine high‐grade sarcomas with supernumerary ring chromosomes or 7q3 rearrangement and three tumors diagnosed as LGFMS prior to the genetic analysis. Of the 59 tumors analyzed, 12 were FUS/CREB3L2‐positive, all of which were diagnosed at histopathologic re‐examination as being LGFMS, of both the classical subtype and the subtype with giant collagen rosettes. The breakpoints in the fusion transcripts were always in exons 6 or 7 of FUS and exon 5 of CREB3L2. The results indicated that FUS/CREB3L2 is specifically associated with LGFMS and that RT‐PCR or FISH analysis may be useful for the differential diagnosis.

Fusion, disruption, and expression of HMGA2 in bone and soft tissue chondromas

Soft tissue and skeletal chondromas are rare entities, and only 21 cases with abnormal karyotypes have been reported. A survey of these, and 10 new cases reported herein, showed that the 12q13–15 segment is nonrandomly involved in structural rearrangements in chondromas. The HMGA2 (HMGI-C) locus in 12q15 is frequently rearranged in other benign mesenchymal tumors, and this study aimed at characterizing the expression of HMGA2 in chondromatous tumors. The material consisted of 8 soft tissue and 6 skeletal chondromas, as well as of 14 skeletal chondrosarcomas. All cases had been cytogenetically analyzed. Expression of HMGA2 could be assessed by RT-PCR in 8 chondromas and 13 chondrosarcomas. HMGA2 was expressed in 4of six soft tissue chondromas, all displaying 12q-rearrangements at cytogenetic analysis. A truncated transcript (exons 1–3), but not a full-length (exons 1–5) transcript, was detected in three of them, suggesting activation through an intragenic rearrangement. One soft tissue chondroma had a t(3;12)(q27;q15), and the RT-PCR analysis revealed an HMGA2-LPP fusion transcript, composed of HMGA2 exons 1–3 and LPP exons 9–11. An identical fusion transcript previously has been identified in lipoma and pulmonary chondroid hamartoma. In the fourth soft tissue chondroma, a full-length transcript was detected, indicating expression of at least one intact allele. Both skeletal chondromas expressed HMGA2. In one of them, a full-length transcript was detected, even though 12q was cytogenetically unaffected. A truncated or full-length transcript was found in 8 of 13 chondrosarcomas, 4 of which displayed 12q rearrangements. Possibly, cryptic rearrangements were present among the many complex marker chromosomes in the remaining 4 cases.

Expression of Insulin-Like Growth Factor-1 Receptor (IGF-1R) and p27Kip1 in Melanocyte Tumors: A Potential Regulatory Role of IGF-1 Pathway in Distribution of p27Kip1 between Different Cyclins

Insulin-like growth factor-1 receptor (IGF-1R) has been shown to be important for melanoma cell growth and survival. In this study we first show, using immunohistochemistry, that progression from benign nevi to malignant melanoma is paralleled by an increased expression of IGF-1R and a down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Even though the expression of p27Kip1 was drastically reduced compared to benign tumors, detectable amounts of it could be assayed by Western blotting in cultured melanoma cells. To analyze whether there is a causative relationship between the IGF-1 pathway and p27Kip1 expression, melanoma cells were treated with αIR-3, an antibody blocking the IGF-1 binding to IGF-1R, or Tunicamycin, which inhibits the translocation of IGF-1R to the cell surface. From these studies we could conclude that the overall expression of p27Kip1 is independent of the IGF-1 pathway. In contrast, the association of p27Kip1 with the different cyclins was drastically affected. Both TM and αIR-3 decreased the binding of p27Kip1 to cyclin D1, whose expression was drastically reduced. On the other hand there was an increased binding of p27Kip1 to cyclin E and cyclin A. This redistribution of p27Kip1 may be a mechanism for growth arrest and induction of apoptosis following interruption of the IGF-1 pathway in melanoma cells.

Tamoxifen-induced cell death in malignant melanoma cells: possible involvement of the insulin-like growth factor-1 (IGF-1) pathway

Recent data indicate that the estrogen receptor (ER) blocker tamoxifen (TAM) can induce cell death in malignant melanoma cells. However, as shown in the present study and several other studies melanoma cells usually do not express classical ERs. In the present study we investigated whether the cytotoxic effect of TAM on melanoma cells could depend on interference with the expression or function of the insulin-like growth factor-1 receptor (IGF-1R), a plasma membrane receptor important for cell survival in this tumor cell type. Several melanoma cell lines were included in the analysis. Administration of TAM at a concentration of 15 microm or more resulted in cell death of the melanoma cells within 48 h. TAM treatment was correlated to a slight to moderate inhibition of IGF-1 binding to IGF-1R. Since it has been reported that TAM can increase the release of IGF binding proteins (IGFBPs) we then investigated whether this mechanism could underly the decreased IGF-1 binding. However, we could demonstrate that the amount of released IGFBPs were unchanged or decreased in TAM-treated cells. Whereas TAM did not have any strong effect on IGF-1 binding and the expression of IGF-1R at the cell surface, it was was found to efficently block tyrosine phosphorylation of IGF-1R beta-subunit. Taken together, our data suggest that TAM-induced cytotoxicity of malignant melanoma cells can be due to inactivation of IGF-1R.

Evidence for protein dolichylation

Labeling of human colon carcinoma cells with [3H]dol, followed by extensive delipidation and removal of dol‐P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and >140 kDa. HPLC purification of proteolytic products of [3H]dol‐ and [35S]cys‐labeled proteins revealed a hydrophobic peak containing both dol and cysteine. The dol/cys‐labeled products were clearly separated from GG‐cys, and exhibited a hydrophobicity between that of dol‐P and dol. In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds. After HPLC purification of released dol‐like lipids, these were subjected to mass spectrometry. This demonstrated molecular ions with the same mass as that of dol. Taken together our data provide evidence for the existence of proteins covalently modified with dol.

Reverse Transcriptase Polymerase Chain Reaction on Fine Needle Aspirates for Rapid Detection of Translocations in Synovial Sarcoma

OBJECTIVE To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. STUDY DESIGN We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericytoma, one was a poorly defined tumor, and three were suspected synovial sarcomas. FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. RESULTS The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. CONCLUSION Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

Detection of EWS/FLI-1 by Immunostaining. An Adjunctive Tool in Diagnosis of Ewing's Sarcoma and Primitive Neuroectodermal Tumour on Cytological Samples and Paraffin-Embedded Archival Material.

Purpose. Recently we showed that the 68-kDa fusion protein derived from the EWS/FLI1 hybrid gene can be specifically detected by Western blotting using a polyclonal antibody to the C-terminal of FLI1 on biopsy material from Ewings sarcoma. The aim of this study was to investigate whether this antibody also could be used for immunocytochemistry and immunohistochemistry in diagnosis of Ewings sarcoma. Methods. Immunostaining on paraffin-embedded archival material, fine-needle aspirates and tumour touch imprints from Ewings sarcomas and primitive neuroectodermal tumours (PNET) for detection of the fusion protein was performed. Most cases were also analysed by Western blotting.Tumours of differential diagnostic importance were also included. Results. Eighty per cent (12/15 cases) of the Ewing tumours exhibited a positive immunoreactivity for the FLI1 antibody. The signal was mainly localised in the nuclei of the tumour cells, which seems reasonable since EWS/FLI1 is a transcription factor. The signal was found to be specific since it did not appear when the blocking peptide was added to the antibody solution.Moreover, two other types of small-round cell tumours (i.e. neuroblastoma and alveolar rhabdomyosarcoma) were negative as well as most normal tissues. Discussion. Immunostaining of histological and cytological specimens with the FLI1 antibody can be of diagnostic relevance in Ewing tumours carrying t(11;22).The absence of immunoreactivity in non-Ewing cells is most likely due to a low expression of the wild-type FLI1 protein.

Analysis of Dolichols and Polyprenols and Their Derivatives by Electron Impact, Fast Atom Bombardment and Electrospray Ionization Tandem Mass Spectrometry

Dolichols and polyprenols are polyisoprenoid lipids found in all cells. Polyisoprenoids have recently been found covalently bound to cellular proteins constituting a new type of post-translational modification. To study these compounds effectively in biological systems a sensitive mass spectrometric procedure giving molecular weight and structural information is required. In the present study an assessment has been made of possible mass spectrometric procedures. Dolichols and polyprenols have been analysed at the pmol level in their underivatized form and as tert-butyldimethylsilyl ethers by electron ionization mass spectrometry. Sulphated dolichols and polyprenols have been analysed at a pmol level by fast-atom bombardment and at the sub-pmol level by electrospray mass spectrometry. Dolichol phosphates have also been analysed by electrospray mass spectrometry. Collision-induced dissociation spectra of the underivatized and derivatized polyisoprenoids have been recorded. These spectra provide structural information from only pmols of sample.

Dolichol-like lipids with stimulatory effect on DNA synthesis: Substrates for protein dolichylation?

Substantial evidence has suggested that a nonsterol product of mevalonic acid (MVA) is essential for the initiation of DNA synthesis in mammalian cells. Several possible isoprenoid candidates have been suggested, but the identity of this compound still remains unknown. In this study we have isolated and purified MVA products from SV40‐transformed human fibroblasts and identified fractions with a growth‐stimulatory effect. The cells were labelled with [14C]MVA in the presence of inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. After lipid extraction, the [14C]MVA‐labelled lipids were subjected to high performance liquid chromatography and size‐exclusion chromatography, and the effect of the fractionated eluate on the DNA synthesis of arrested MVA‐depleted target cells was tested. Thereby we found a fraction of [14C]MVA‐labelled lipids with a substantial stimulatory effect on DNA synthesis. The chromatographic behavior suggested that the growth‐stimulating fractions contained dolichol‐20. This was confirmed by mass spectrometric analysis. Similar results were obtained when lipids from hepatocellular carcinoma cells and a sample from breast tumor were isolated and analyzed by the same procedure. The mechanisms by which these compounds induce DNA synthesis are unknown. Recent data obtained in our laboratory have provided evidence that dolichyl groups are covalently linked to tumor cell proteins, which implicates a new biological function for long‐chain polyisoprenoid alcohols (Hjertman et al. [1997] FEBS Lett 416:235–238). In this study we demonstrate that tumor cells containing dolichol‐like growth‐stimulatory lipids also contained dolichylated proteins. This raises the question whether the growth‐stimulatory dolichol‐like lipids serve as substrates for the dolichylation reaction. J. Cell. Biochem. 71:502–514, 1998.

Sorafenib inhibits tumor growth and vascularization of rhabdomyosarcoma cells by blocking IGF-1R-mediated signaling

The growth of many soft tissue sarcomas is dependent on aberrant growth factor signaling, which promotes their proliferation and motility. With this in mind, we evaluated the effect of sorafenib, a receptor tyrosine kinase inhibitor, on cell growth and apoptosis in sarcoma cell lines of various histological subtypes. We found that sorafenib effectively inhibited cell proliferation in rhabdomyosarcoma, synovial sarcoma and Ewing’s sarcoma with IC50 values <5 μM. Sorafenib effectively induced growth arrest in rhabdomyosarcoma cells, which was concurrent with inhibition of Akt and Erk signaling. Studies of ligand-induced phosphorylation of Erk and Akt in rhabdomyosarcoma cells showed that insulin-like growth factor-1 is a potent activator, which can be blocked by treatment with sorafenib. In vivo sorafenib treatment of rhabdomyosarcoma xenografts had a significant inhibitory effect on tumor growth, which was associated with inhibited vascularization and enhanced necrosis in the adjacent tumor stroma. Our results demonstrate that in vitro and in vivo growth of rhabdomyosarcoma can be suppressed by treatment with sorafenib, and suggests the possibilities of using sorafenib as a potential adjuvant therapy for the treatment of rhabdomyosarcoma.