Characteristics of Leiomyosarcoma: Induction of Hematogenous Metastasis by Isolated Uterine Mesenchymal Tumor Stem-like Cells.

BACKGROUND/AIM: Uterine leiomyosarcoma (Ut-LMS) is a refractory tumor that repeatedly recurs with hematogenous metastasis, which may be due to the presence of drug-resistant tumor stem cells. Its treatment is limited to surgical procedures. We previously reported that Ut-LMS spontaneously developed in mice deficient in the proteasome component low-molecular mass polypeptide 2 (LMP2). We showed that LMP2 expression was significantly attenuated specifically in human Ut-LMS. The aim of this study was to investigate the role of LMP2 in hematogenous metastasis using xenograft models with tumor stem-like cells. MATERIALS AND METHODS: We isolated tumor stem-like cells from LMP2-negative primary human Ut-LMS cells established from a human Ut-LMS tissue using the side population (SP) procedure. These cells were used to develop xenograft models with tumor stem-like cells. RESULTS: Human Ut-LMS stem-like cells showed stronger hematogenous metastatic potential than normal Ut-LMS cells. Tumor stem-like cells also had the potential to differentiate into vascular endothelial cells through VEGF-A signaling. CONCLUSION: These results reflect frequent hematogenous metastasis by human Ut-LMS in clinical settings, and may lead to the development of treatments that inhibit hematogenous metastasis in Ut-LMS.

Characterization of Leiomyomatoid Angiomatous Neuroendocrine Tumour (LANT)-like Tumour in the Myometrium with Histopathological Examination.

Leiomyomatoid angiomatous neuroendocrine tumour (LANT) is possibly a new disease entity that was reported as a dimorphic neurosecretory tumour with a leiomyomatous vascular component; it was found in the pituitary. We describe uterine LANT-like malignant tumour in a 45-year-old woman with uterine mesenchymal tumour, diagnosed clinically as uterine leiomyoma. She underwent laparoscopic myomectomy. The tumour consisted of hyalinized vasculature, containing factor VIII-positive endothelium and α-smooth muscle actin-positive vascular smooth muscle cells, and stromal cells, expressing neuroadhesion molecules. Both vascular and stromal components diffusely expressed chromogranin A. Histopathological examinations of uterine LANT-like malignant tumour revealed the common characteristic abnormalities of malignant uterine mesenchymal tumours, i.e. leiomyosarcomas. From our research, defective expression of calponin H1 and proteasome ß9 (PSMB9)/ß1i is observed in uterine LANT-like malignant tumour similarly to immunopathological findings of uterine leiomyosarcoma. These findings meet the definition of uterine LANT-like malignant tumour, and the research findings of our clinical case suggest that LANT is a special type of neuroendocrine neoplasm and is not organ specific.

Molecular Pathology and Novel Clinical Therapy for Uterine Leiomyosarcoma.

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass, with atypical presentations of hypercalcemia and eosinophilia also being reported. Radiographic evaluation with combined positron-emission tomography/computed tomography may assist in diagnosis and surveillance in women with uterine LMS; these are commonly used with stage and tumour grade as prognostic indicators and a recently developed risk-assessment index to predict disease-specific survival. Recent studies have shown that the addition of adjuvant therapy after surgical management does not seem to improve survival, and ovarian preservation does not appear to negatively impact outcome. Experimentally, it is noteworthy that proteasome subunit beta 9 (PSMB9)/ß1i-deficient mice exhibit spontaneous development of uterine LMS, with a disease prevalence of ~37% by 12 months of age. Furthermore, a recent report showed the loss of ability to induce PSMB9/ß1i expression, that is up-regulated by interferon-γ (IFNγ), in human uterine LMS tissues. Here, we reviewed human uterine LMS for genetic mutations in the IFNγ signal cascade, and found serious mutations in three genes, Janus activated kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and PSMB9/ß1i promoter regions. Moreover, molecular experiments demonstrated differential expression of cyclin E and P27/KIP1, that regulate cell-cycle G1 arrest via PSMB9/ß1i expression. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3ζ chain.

Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response. In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3ζ chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3ζ protein. The antibodies were found to be specific and selective for phospho-CD3ζ. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3ζ chain, and for correlating the phosphorylation of specific CD3ζ tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells.

Antibody arrays--an emerging tool in cancer proteomics.

Cancer is a result of complex changes that occur in normal cells as they transform to become malignant and further when they become metastatic. These changes are not a consequence of a single protein but rather involve multiple proteins that function in pathways and networks. Thus, profiling cancer-associated changes requires simultaneous measurement of many proteins in a single sample. Identifying these changes may lead to the discovery of cancer-associated biomarkers that may assist in diagnosis, prognosis, patient monitoring and possibly for therapeutic purposes. Antibody arrays are a relatively new technology that enables one to perform multiplex high-throughput protein expression profiling. This review describes current technologies in antibody array and assay design, and presents a survey of the current literature on the use of these arrays in cancer research.

Panorama Ab Microarray Cell Signaling kit: a unique tool for protein expression analysis.

Antibody arrays are a promising new tool for mass analysis of protein level changes in cells responding to different stimuli. Here we describe a novel antibody array system called Panorama Ab Microarray Cell Signaling, that contains 224 antibodies spotted on FAST nitrocellulose-coated slides that can detect protein levels as low as a few nanograms per mL. The antibodies spotted are specific for proteins important in various areas of cell signaling such as phosphorylation, cell cycle, apoptosis, nuclear signaling and cytoskeleton proteins. Furthermore, for some of the protein targes, the Panorama Ab Microarray can detect phosphorylated and nonphosphorylated forms of the traget protein. We found that treatment of the slides post-spotting is important for the array performance (ratio of signal to background) and its stability. Panorama Ab Microarray was used to analyze changes in protein expression in F9 embryonic carcinoma cells stimulated to differentiate by all-trans retinoic acid. We found that the level of several proteins, among them cell cycle regulators and kineases, was either up- or down-regulated. For more than ten protein targets, the results obtained by the Panorama Ab Microarray were confirmed by immunoblotting.

Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis.

Inositol 1,3,4,5-tetrakisphosphate (IP(4)) has been linked to a potential role in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP(4) remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP(4) receptor, GAP1(IP4BP), affects [Ca(2+)](i) following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1(IP4BP) appear to have no effect on Ca(2+) mobilization or Ca(2+) entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1(IP4BP) production on [Ca(2+)](i). In HeLa cells in which the endogenous level of GAP1(IP4BP) has been suppressed by approximately 95%, we failed to observe any effect on Ca(2+) mobilization or Ca(2+) entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1(IP4BP) in intact HeLa cells, we have been unable to observe any detectable effect of GAP1(IP4BP) on [Ca(2+)](i).

Regulation of S33/S37 phosphorylated beta-catenin in normal and transformed cells.

A novel phosphorylation-specific antibody (alphapbeta-catenin) was generated against a peptide corresponding to amino acids 33-45 of human beta-catenin, which contained phosphorylated serines at positions 33 and 37. This antibody is specific to phosphorylated beta-catenin and reacts neither with the non-phosphorylated protein nor with phosphorylated or non-phosphorylated plakoglobin. It weakly interacts with S33Y beta-catenin but not with the S37A mutant. pbeta-catenin is hardly detectable in normal cultured cells and accumulates (up to 55% of total beta-catenin) upon overexpression of the protein or after blocking its degradation by the proteasome. Inhibition of both GSK-3beta and the proteasome resulted in a rapid (t1/2=10 minutes) and reversible reduction in pbeta-catenin levels, suggesting that the protein can undergo dephosphorylation in live cells, at a rate comparable to its phosphorylation by GSK-3beta. pbeta-catenin interacts with LEF-1, but fails to form a ternary complex with DNA, suggesting that it is transcriptionally inactive. Immunofluorescence microscopy indicated that pbeta-catenin accumulates in the nuclei of MDCK and BCAP cells when overexpressed and is transiently associated with adherens junctions shortly after their formation. pbeta-catenin only weakly interacts with co-transfected N-cadherin, although it forms a complex with the ubiquitin ligase component beta-TrCP. SW480 colon cancer cells that express a truncated APC, at position 1338, contain high levels of pbeta-catenin, whereas HT29 cells, expressing APC truncated at position 1555, accumulate non-phosphorylated beta-catenin, suggesting that the 1338-1555 amino acid region of APC is involved in the differential regulation of the dephosphorylation and degradation of pbeta-catenin.