Tomoya Matsunobu

Changes in knee alignment after total knee arthroplasty.

Changes in limb alignment after total knee arthroplasty were evaluated in 20 knees replaced with the Miller Galante knee system. The mean follow-up period was 87.4 months. Seventeen of the 20 knees were in the varus position on the initial postoperative radiographs, but the alignment significantly changed to become even more aligned toward varus during the follow-up period. The thickness of the ultra-high-molecular-weight polyethylene (UHMWPE) also decreased significantly in the medial femorotibial joint. The wear of the UHMWPE possibly changed the alignment, and the postoperative alignment had a positive correlation with the wear rate. The components should be implanted so that the mechanical axis intersects the center of the components to prevent worsening of alignment as well as to minimize any such wear.

Antitumor effects of histone deacetylase inhibitor on Ewing's family tumors

A chimeric protein, EWS‐Fli1, identified in most Ewings family tumors (EFTs) has been shown to be associated with the tumorigenicity of EFTs. We have previously reported that p21Waf1/Cip1 expression was inhibited by EWS‐Fli1 in EFTs. Histone deacetylase inhibitors (HDACIs) are known to up‐regulate p21Waf1/Cip1 expression in various cells and show promise as a cancer therapy. Here, we demonstrate the possible involvement of EWS‐Fli1 in the activities of both histone acetylation and deacetylation, as well as the potential use of HDACIs as an antitumor agent for EFTs. A novel HDACI, FK228, strongly induced p21Waf1/Cip1 expression, leading to the hypophosphorylation of retinoblastoma protein (Rb) in EFT cells. Results indicated that EWS‐Fli1 deregulated histone acetylation through both the repression of histone acetyltransferase (HAT) and the enhancement of histone deacetylase (HDAC) activities in EFT cells. FK228 treatment blocked both of the abnormal functions of EWS‐Fli1. Expressions of EWS‐Fli1 protein and mRNA were also inhibited by HDACIs. We suggest that HDACIs might inhibit the expression of EWS‐Fli1 via the suppression of the EWS promoter activity. FK228 demonstrated potent growth inhibitory effects on EFT cells at nanomolar concentrations, as well as an apparent distinction in the apoptotic effects between EFT and normal cells. Moreover, intraperitoneal administration of FK228 significantly inhibited tumor growth and induced apoptosis in EFTs in vivo. These results suggest that HDACI might be a promising reagent for use in molecular‐based chemotherapy against EFTs.

Knee stability in posterior cruciate ligament retaining total knee arthroplasty

Anteroposterior knee laxity was evaluated in 14 patients (19 knees) who had posterior cruciate ligament retaining total knee arthroplasty using the Miller Galante I prosthesis. The followup ranged from 87 to 118 months (average, 105.9 months), and the measurements were done using the KT-2000 arthrometer. The mean anteroposterior displacement with the knees with Miller Galante I prostheses was 10.1 mm at 30 degrees flexion and 8.1 mm at 75 degrees flexion. In the 15 knees with Miller Galante I prostheses with flexion greater than 90 degrees, seven had less stability at 75 degrees than at 30 degrees flexion. These knees were considered to have a nonfunctional posterior cruciate ligament, and they had a worse Knee Society score (81.1) than did the other eight knees with Miller Galante I prostheses (89.9). There were four knees in which the flexion was less than 90 degrees. In this study, approximately half of the knees with posterior cruciate ligament retaining total knee arthroplasty did not have good anteroposterior stability in flexion an average of 9 years after surgery.

The Possible Role of EWS-Fli1 in Evasion of Senescence in Ewing Family Tumors

The chromosomal translocation t(11;22) yields the EWS-Fli1 fusion gene and is associated with oncogenesis of Ewing family tumors (EFT). In this study, using the RNA interference method, we show that EWS-Fli1-targeting small interfering RNAs (siRNA) depleted EWS-Fli1 protein and caused growth inhibition in EFT cells with the accumulation of p27 protein and the down-regulation of Skp2 protein in dose-dependent, time-dependent, and sequence-specific manners. Depletion of EWS-Fli1 subacutely elicited a senescence-like phenotype, but not apoptosis, in EFT cells. Furthermore, not only the knockdown of p27, but also the forced expression of Skp2, reduced the expression levels of p27 protein and partially rescued senescence-like phenotype caused by EWS-Fli1-targeting siRNAs. The accumulation of p27 protein in EWS-Fli1-depleted cells inhibited cdk2 kinase activity and was related to the stability of p27 protein, which resulted from a decrease in Skp2 protein. Immunohistochemical analysis of p27 and Skp2 proteins in EFT samples revealed that there was an inverse relationship between the expression profiles of p27 and Skp2 proteins. These findings indicate an important role of EWS-Fli1 in the prevention of senescence, leading to the unlimited growth and oncogenesis of EFT cells through a decrease in the stability of p27 protein due to increased action of Skp2-mediated 26S proteasome degradation.

The Effects of Histone Deacetylase Inhibitors on the Induction of Differentiation in Chondrosarcoma Cells

Purpose: Histologically, chondrosarcomas represent the degree of chondrogenic differentiation, which is associated with the prognosis of the disease. Histone acetylation and deacetylation play key roles in the regulation of chondrocytic differentiation. Here, we describe the antitumor effects of histone deacetylase (HDAC) inhibitors as differentiating reagents on chondrosarcomas. Experimental Design: We examined the effects of a HDAC inhibitor, depsipeptide, on the growth of chondrosarcoma cell lines. We also investigated the modulation of the expression levels of extracellular matrix genes and the induction of phenotypic change in chondrosarcoma cells treated with depsipeptide. Finally, we examined the antitumor effect of depsipeptide on chondrosarcoma in vivo. Results: Depsipeptide inhibited the growth of chondrosarcoma cells by inducing cell cycle arrest and/or apoptosis. HDAC inhibitors increased the expression of the α1 chain of type II collagen (COL2A1) gene due to the enhanced histone acetylation in the promoter and enhancer. Depsipeptide also up-regulated the expressions of aggrecan and the α2 chain of type XI collagen (COL11A2) mRNA in a dose-dependent manner. Moreover, long-term treatment with a low dose of depsipeptide resulted in the induction of differentiation into hypertrophic phenotype, as shown by the increment of the α1 chain of type X collagen (COL10A1) expression in chondrosarcoma cells. In vivo studies and histologic analyses confirmed that depsipeptide significantly inhibited tumor growth and induced differentiation into the hypertrophic and mineralized state in chondrosarcoma cells. Conclusions: These results strongly suggest that HDAC inhibitors may be promising reagents for use as a differentiating chemotherapy against chondrosarcomas.

Involvement of P‐glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamily of histone deacetylase inhibitors in the drug‐resistant osteosarcoma and Ewing's sarcoma cells

Despite recent improvements in multimodal therapies for osteosarcoma (OS) and Ewings family of tumors (EFTs), the prognosis of relapsed cases remains very poor because of the resistance to chemotherapy. Histone deacetylase inhibitors (HDACIs), including members of the cyclic tetrapeptide family such as FK228 and apicidin, are novel antitumor agents that can induce cell cycle arrest and apoptosis in various cancer cells. HDACIs also exhibit potent antitumor effects on OS and EFTs. However, to date there have been no studies to our knowledge reporting the effects of HDACIs on drug‐resistant OS and EFTs. Here, we demonstrated that FK228 and apicidin exhibited strong resistance in doxorubicin‐resistant clones of OS and EFTs expressing P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 1 (MRP1) and that P‐gp and MRP1 might play a crucial role in the resistance mechanism to FK228 and apicidin. A P‐gp inhibitor (verapamil) and an MRP1 inhibitor (MK571) could independently reverse the resistance to FK228 and apicidin in the drug‐resistant clones. Moreover, the combination of verapamil and MK571 could enhance HDACI‐induced cell number reduction in drug‐resistant clones to a similar extent as that in their parental clones. Although these findings suggest the difficulty in treating drug‐resistant tumors expressing P‐gp and/or MRP1 with these HDACIs, the combination of P‐gp and MRP1 inhibitors might reverse the resistance to the HDACIs in the treatment of those tumors. Because HDACIs are potent and promising antitumor drugs and seem to be close to clinical use, it is necessary to pay attention to the resistance mechanisms against HDACIs.

The Prognostic and Therapeutic Relevance of p27kip1 in Ewing’s Family Tumors

Purpose: Ewing’s family tumors (EFTs) display the characteristic fusion gene EWS-Fli1. We have reported EWS-Fli1 may promote the cell cycle progression accompanied by the suppression of the expression of cyclin-dependent kinase inhibitor p27kip1 in EFT cells. Here, we describe the prognostic and therapeutic relevance of p27 in EFTs. Experimental Design: We examined tumor samples taken from 21 patients with primary EFTs for the expression of p27 protein immunohistochemically and evaluated its correlation with clinical outcome. We also investigated the usefulness of p27 as a therapeutic strategy in vitro and in vivo using p27 expression adenovirus. Finally, we examined the process of EWS-Fli1-mediated reduction of p27 expression. Results: Immunohistochemical analysis showed that a low expression level of p27 protein was related to poor event-free survival in an univariate analysis and that the expression level of p27 correlated more significantly with patient survival than several clinical factors in a multivariate survival analysis. Overexpression of p27 with the adenoviral vector remarkably inhibited the cell growth in all EFT cells tested and further induced apoptosis in the wild-type p53 EFT cells. In vivo studies demonstrated a reduction in tumor growth of EFT xenograft in nude mice treated with the intratumoral injection of p27-expressing adenovirus. EWS-Fli1 did not significantly affect the p27 promoter activity and p27 mRNA levels. However, the challenge of the proteasome inhibitor caused accumulation of p27 protein in EFT cells. These data strongly suggest EWS-Fli1 might attenuate p27 protein level via activation of the proteasome-mediated degradation pathway. Conclusions: Our findings provide the first evidence of the prognostic relevance of p27 expression in EFTs. We propose p27 as a novel and powerful therapeutic factor for the molecular target therapy of EFTs.

Tumour-associated macrophages correlate with poor prognosis in myxoid liposarcoma and promote cell motility and invasion via the HB-EGF-EGFR-PI3K/Akt pathways

Background:Myxoid liposarcoma (MLS) is the second most common subtype of liposarcoma, and metastasis occurs in up to one-third of cases. However, the mechanisms of invasion and metastasis remain unclear. Tumour-associated macrophages (TAMs) have important roles in tumour invasion, metastasis, and/or poor prognosis. The aim of this study was to investigate the relationship between TAMs and MLS.Methods:Using 78 primary MLS samples, the association between clinical prognosis and macrophage infiltration was evaluated by immunochemistry. The effects of macrophages on cell growth, cell motility, and invasion of MLS cell lines were investigated in vitro. In addition, clinicopathological factors were analysed to assess their prognostic implications in MLS.Results:Higher levels of CD68-positive macrophages were associated with poorer overall survival in MLS samples. Macrophage-conditioned medium enhanced MLS cell motility and invasion by activating epidermal growth factor receptor (EGFR), with the key ligand suggested to be heparin-binding EGF-like growth factor (HB-EGF). The phosphoinositide 3-kinase/Akt pathway was mostly involved in HB-EGF-induced cell motility and invasion of MLS. The expression of phosphorylated EGFR in MLS clinical samples was associated with macrophage infiltration. In addition, more significant macrophage infiltration was associated with poor prognosis even in multivariate analysis.Conclusions:Macrophage infiltration in MLS predicts poor prognosis, and the relationship between TAMs and MLS may be a new candidate for therapeutic targets of MLS.

Transactivation of cyclin E gene by EWS-Fli1 and antitumor effects of cyclin dependent kinase inhibitor on Ewing's family tumor cells

Chromosomal translocation t(11; 22)(q24; q12) is detected in approximately 90% of Ewings family tumors (EFTs) including Ewings sarcoma and primitive neuroectodermal tumor. This results in the formation of the EWS‐Fli1 fusion gene, which produces EWS‐Fli1 fusion protein. This chimerical gene product acts as an aberrant transcriptional activator, which may be responsible for the tumorigenesis of EFTs. We have previously reported that cyclin E expression was upregulated in EFT cells and in EWS‐Fli1 transformed fibroblastic cells. However, the mechanism of the overexpression of cyclin E by EWS‐Fli1 is still unknown. In our study, we investigated the mechanism of transactivation of the cyclin E gene in EFT cells. We found that EWS‐Fli1 enhanced the activity of the cyclin E gene promoter partially through E2F binding sites in the promoter. In addition, the basic transcriptional factor, Sp1, might also be involved in the transactivation of the cyclin E gene by EWS‐Fli1. To study the biological significance of cyclin E overexpression in EFT cells, we used flavopiridol, a pan‐cyclin‐dependent kinase (CDK) inhibitor and found that flavopiridol efficiently suppressed the growth of EFT cells in vitro and in vivo by the inhibition of cyclinE/CDK2 kinase activity and the induction of apoptosis. These results suggest that targeting of the cyclin/CDK complex may provide new insight into treatment of EFTs.

Ossifying fibromyxoid tumor presenting EP400-PHF1 fusion gene.

Ossifying fibromyxoid tumor is a rare soft tissue tumor of borderline malignancy and uncertain differentiation. Recently, a novel fusion gene, EP400-PHF1, was discovered in ossifying fibromyxoid tumor; however, its relation to this type of tumor has been uncertain because the EP400-PHF1 fusion gene has been successfully detected in only 1 case. We present an ossifying fibromyxoid tumor case with the EP400-PHF1 fusion gene detected by reverse transcriptase polymerase chain reaction, along with compatible cytogenetic data showing a t(6;12)(p21;q24.3) translocation. Our results suggest that the EP400-PHF1 fusion gene is a reproducible finding in ossifying fibromyxoid tumor.