Marie-Luise Kremser

Valproate inhibition of histone deacetylase 2 affects differentiation and decreases proliferation of endometrial stromal sarcoma cells

Covalent modifications of histone proteins, in particular deacetylation of lysine residues, are important for the regulation of gene transcription both in normal and malignant cells. These processes are controlled by histone acetyltransferases and histone deacetylases (HDAC) and have up to now not been described in solid mesenchymal tumors. The present study shows differences in the HDAC1 and HDAC2 expression in endometrial stromal sarcomas (ESS) and a cognate cell line (ESS-1) compared with nonneoplastic endometrial stroma. We show for the first time that HDAC2 expression is consistently increased in ESS. In contrast, HDAC1 expression is generally lower than HDAC2 both in nonneoplastic stroma and in ESS, suggesting that these two proteins, although closely related, are regulated in different ways. In vitro experiments with an ESS cell line showed that valproate, an inhibitor of the class I HDACs, led to significant HDAC2 decrease and to cell differentiation. HDAC2 inhibition in ESS-1 cells caused significant changes in the cell cycle by inhibiting G1-S transition and influencing expression of p21WAF1 and cyclin D1. Moreover, in ESS-1 cells, increased expression of the p21WAF1 was associated with reduction of HDAC2 expression after transfection with small interfering RNA directed against HDAC2. Our results suggest that HDAC2 might be considered as potential drug target in the therapy of ESS and that HDAC inhibitors should be further evaluated in clinical trials in ESS. [Mol Cancer Ther 2006;5(9):2203–10]

JAZF1/JJAZ1 Gene Fusion in Endometrial Stromal Sarcomas: Molecular Analysis by Reverse Transcriptase-Polymerase Chain Reaction Optimized for Paraffin-Embedded Tissue

Endometrial stromal tumors are rare uterine neoplasms including benign stromal nodules, low-grade endometrial stromal sarcomas (ESS), and undifferentiated endometrial sarcomas (UES), the latter representing the most aggressive form. Morphological characteristics and cytogenetic abnormalities are heterogeneous, making diagnosis difficult. Recently, a gene fusion on chromosome 7 that includes two zinc-finger genes (JAZF1 and JJAZ1) has been discovered in these tumors. Hitherto only 31 cases, described by three different research groups, have shown JAZF1/JJAZ1 fusion in approximately 50% of all analyzed low-grade ESSs whereas it is less frequent in UESs. In this study we analyzed 20 ESS and 2 UES cases using two-step reverse transcriptase-polymerase chain reaction optimized for formalin-fixed, paraffin-embedded tissue. In our subset of samples, the JAZF1/JJAZ1 fusion transcript occurred in 80% of analyzed ESS cases and in none of two UES cases. In comparison to published data, our results identified the JAZF1/JJAZ1 gene fusion more frequently in endometrial stromal tumors than hitherto presumed. This cytogenetic abnormality was not present in normal endometria, leiomyomas, or leiomyosarcomas or in lung, gastric, or hepatic carcinomas, indicating its specificity for endometrial stromal tumors. In combination with other established methods, accurate reverse transcriptase-polymerase chain reaction analysis of JAZF1/JJAZ1 gene fusion may be useful in diagnosing difficult or unusual ESS/UES cases.

SAHA induces caspase-independent, autophagic cell death of endometrial stromal sarcoma cells by influencing the mTOR pathway

Endometrial stromal sarcomas are rare and molecular mechanisms involved in their pathogenesis are poorly understood. Covalent modifications of histone proteins, in particular de/acetylation of lysine residues, play an important role in the regulation of gene transcription in normal and neoplastic cells, but there are only limited data about these processes in solid mesenchymal tumours. We treated endometrial stromal sarcoma cells (ESS‐1) and non‐malignant human endometrial stromal cells (HESCs) with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. SAHA was able to mediate the cell cycle and expression of genes related to the malignant phenotype of endometrial stromal tumours, eg p21WAF1 and HDAC7. SAHA led to dose‐dependent differentiation and death of ESS‐1 cells but not of HESCs. Exposure of HESCs to SAHA resulted only in slightly decreased cell proliferation. SAHA also increased the p21WAF1 expression and caused significant changes in the cell cycle by inhibiting the G1/S transition in ESS‐1 cells. Recovery experiments indicated that these changes became irreversible when the tumour cells were treated with SAHA for longer than 24 h. In our experimental system, not apoptotic but autophagic processes were responsible for the cell death. Monodansyl cadaverine accumulation in treated ESS‐1 cells and decreased expression of the mTOR and phospho‐S6 ribosomal protein (S6rp) additionally supported this observation. Taken together, our study indicates that HDACs might be considered as potential drug targets in the therapy of stromal sarcomas and that SAHA might be a promising therapeutic agent for endometrial stromal sarcoma. Copyright

Histone deacetylase inhibitor vorinostat suppresses the growth of uterine sarcomas in vitro and in vivo

BackgroundUterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells in vitro and in vivo. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21WAF1 and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma in vivo.ResultsVorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 μM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 μM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21WAF1 expression and apoptosis. Nude mice injected with 5 × 106 MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice.ConclusionsOur data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.

Sphingosine 1-Phosphate Counteracts the Effects of Interleukin-1β in Human Chondrocytes

Objective The lipid mediator sphingosine 1-phosphate (S1P) is found in the synovial fluid of osteoarthritis (OA) patients. S1P protects bovine cartilage by counteracting the effects of interleukin-1β (IL-1β). This study was undertaken to examine the interaction of S1P and IL-1β in human OA chondrocytes. Methods Human cartilage was obtained from patients undergoing total knee joint replacement. Chondrocytes were cultured in monolayer and treated with IL-1β and S1P. Expression of S1P receptor subtypes and genes involved in cartilage degradation was evaluated using real-time polymerase chain reaction, immunohistochemistry, and Western blotting. S1P signaling was evaluated using inhibitors of S1P receptors and small interfering RNA (siRNA) knockdown of the S1P2 receptor. Phosphorylation of MAP kinases and NF-κB in response to IL-1β and S1P was detected by Western blotting. Results S1P2 was identified as the most prevalent S1P receptor subtype in human OA cartilage and chondrocytes in vitro. S1P reduced expression of inducible nitric oxide synthase (iNOS) in IL-1β–treated chondrocytes. Reduction of ADAMTS-4 and matrix metalloproteinase 13 expression by S1P correlated with S1P2 expression. Pharmacologic inhibition of the S1P2 receptor, but not the S1P1 and S1P3 receptors, abrogated the inhibition of iNOS expression. Similar results were observed using siRNA knockdown. S1P signaling inhibited IL-1β–induced phosphorylation of p38 MAPK. Conclusion In human chondrocytes, S1P reduces the induction of catabolic genes in the presence of IL-1β. Activation of the S1P2 receptor counteracts the detrimental phosphorylation of p38 MAPK by IL-1β.

Regulation of MMP3 by laminin alpha 4 in human osteoarthritic cartilage.

The degradation of cartilage is characterized by a loss of extracellular matrix (ECM) and the appearance of clusters of hypertrophic chondrocytes (1). The transmembrane proteoglycan syndecan is inv...

The Immunosuppressant FTY720 (Fingolimod) enhances Glycosaminoglycan depletion in articular cartilage

BackgroundFTY720 (Fingolimod) is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P) receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction.MethodsBovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P) in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining.ResultsFTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner.ConclusionsOur results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.

A5.9 Sphingosine-1-phosphate counteracts interleukin-1β in human chondrocytes.

Background and Objectives The lipid mediator Sphingosine-1-phosphate (S1P) is found in the synovial fluid of osteoarthritis (OA) patients. S1P protects bovine cartilage by counteracting interleukin-1β (IL-1β). Here we examine the interaction of S1P and IL-1β in human OA chondrocytes. Materials and Methods Human cartilage was obtained from patients undergoing total knee joint replacement. Chondrocytes were cultured in monolayer and treated with IL-1β and S1P. Expression of S1P receptor sub-types and genes involved in cartilage degradation was evaluated using real-time PCR, immunohistochemistry and western blot. S1P signalling was evaluated using inhibitors of S1P receptors and siRNA knock-down of the S1P2 receptor. Phosphorylation of MAP kinases (MAPK) and NF-κB in response to IL-1β and S1P was detected by western blot. Results S1P2 was the most prevalent S1P receptor subtype in human OA cartilage and chondrocytes in vitro. S1P reduced iNOS expression in IL-1β treated chondrocytes. Reduction of ADAMTS4 and MMP-13 expression by S1P correlated with S1P2 expression. Pharmacological inhibition of the S1P2 receptor but not S1P1 and S1P3 receptors abrogated inhibition of iNOS expression. Similar results were observed using siRNA knock-down. S1P signalling inhibited IL-1β induced phosphorylation of p38MAPK. Conclusions In human chondrocytes, S1P reduces the induction of catabolic genes in the presence of IL-1b. Activation of the S1P2 receptor counteracts the detrimental phosphorylation of p38MAPK by IL-1β.