<原著>培養軟骨細胞の分化機能発現と細胞増殖動態に関する実験的研究

The present study was undertaken to investigate the relationship among cell morphology, proliferation, and maturation of chondrocytes in primary cultures. Chondrocytes were isolated from the growth cartilages of the rat ribs and cultured for 6 days. In situ DNA cytofluorometry using an inverted epi-illumination cytofluorometer (Nikon P1-I) and 3H-thymidine autoradiography were carried out for the correlated analysis of cell morphology and proliferation. Cytoskeletal staining with fluorescent phalloidin and 35S-sulphate autoradiography were also performed. In addition, in situ hybridization to c-myc mRNA was carried out using DNA probe. According to the results obtained, the cultured chondrocytes were composed of mixed populations of large, polygonal cells and of small, round cells. The round cells showed a significantly higher 35S uptake than the polygonal cells. The cytoskeletal staining clearly revealed stress fibers in the cytoplasm of the polygonal cells, whereas only a fine filamentous structure was shown in the cytoplasm of the round cells. In situ DNA cytofluorometry clearly demonstrated that cell proliferative activity was high in the polygonal cells and low in the round cells. In addition, 3H-thymidine autoradiography with cumulative labeling method revealed that the polygonal cells were changing into the small, round cells. C myc mRNA signals were detected in the cytoplasm of over a half of the round cells, whereas no evidence of c-myc expression were found in the polygonal cells. From these results, it appears that as the shape of the cultured chondrocytes shifts from polygonal to round, the cell proliferative activity decreases in association with cell differentiation. It was also suggested that c-myc mRNA is amplified in the well differentiated round chondrocytes, and not in the proliferative polygonal cells.従来の培養軟骨細胞を用いた研究から, 軟骨細胞の形態と分化機能発現の聞には, 関連性のあることが示されている. 著者らは, 成長軟骨細胞の培養系において細胞形態, 機能が明らかに異なっている2種類の細胞が存在することを見いだした. 本研究では, この培養系を用い, 軟骨細胞の形態・細胞増殖動態・分化機能発現の3者の関連性を総合的に把握することを目的とした. このための方法論として, 細胞形態別増殖動態解析には, in situ DNA 顕微蛍光測光法と3_H-サイミジンオートラジオグラフィーを行い, 分化機能の検索には35_S オートラジオグラフィーを用いた. また, FITC-ファロイジン染色法により, 軟骨細胞の形態と細胞骨格の関係についても調べた. 更に, 本研究では, 悪性腫瘍以外に, 胎生期の細胞や分化途上の細胞にも出現し, 細胞の分化・増殖に深く関係があると考えられている c-myc 遺伝子の発現の有無を, in situ DNA- mRNA hybridization 法を用いて検索した. 実験には, ラット肋軟骨から分離・培養した成長軟骨細胞を用いた. 培養開始4 - 6日目頃の成長軟骨細胞は, 大型多角形の扁平な胞体を持ち, 大きな核を有する細胞(以下, 多角形細胞と略す)と, 比較的小型で類円形ないし球状の胞体と小さな核を有する細胞(以下, 円形細胞と略す)の2種類の細胞から構成されていた. in situ DNA 顕微蛍光測光法による細胞増殖動態解析の結果, 多角形細胞は, 活発な増殖性を示す2倍体細胞と少数の4倍体から構成されているのに対し, 円形細胞は, ほとんど増殖活性を持たない2倍体細胞から構成されていることが判った. 3_H-サイミジンの30分標識の結果から, 多角形細胞の標準率は11%, 円形細胞の標識率は0. 5%であり, その標識率の経時的変化はほとんど認められなかった. 3_H-サイミジンの持続標識実験の結果から, 多角形細胞が円形細胞に形態的に変化することが示唆された. また, 35_S オートラジオグラフィーより, 多角形細胞は, 軟骨基質の産生能が低く, 他方, 円形細胞では, 基質産生が亢進していることがわかった. FITC-ファロイジン染色によるアクチンの細胞内分布パタンを, 両細胞で比較したところ, 多角形細胞ではストレスファイバーがよく発達しているのに対し, 円形細胞には, 分断された線維性構造のみが観察された. 以上の結果をまとめると, 培養軟骨細胞の形態・増殖・分化の3者の間には, たがいに密接な関連が有り, 多角形細胞から円形細胞への形態変化に伴って, 増殖活性が低下し, 分化機能が発現されることが判明した. 次に, c-myc 遺伝子の発現の有無を in situ hybridization 法を用いて検索したところ, 円形細胞の過半数に, c-myc mRNA のシグナルが検出された. このことから, 軟骨細胞では, 分化機能発現と関連して c-myc 遺伝子が発現される可能性が示唆された

The characterization of the human Siah-1 promoter11The nucleotide sequence of the Siah-1 gene promoter can be found in the DDBJ and GenBank databases with the following accession number: AB072970.

AbstractSiah-1, the human homologue of Drosophila seven in absentia, is related to apoptosis and tumor suppression. Although it was reported that the expression of Siah-1 is induced by p53 and p21/WAF1, little is known about the transcriptional regulation of the Siah-1 gene. To investigate the transcriptional regulation, we isolated and sequenced the genomic fragment of the Siah-1 promoter region. The Siah-1 promoter has no typical TATA box or CCAAT box. Transient transfection assays using reporter plasmids in which the promoter region of the Siah-1 gene was deleted or mutated showed that one Sp1 site was responsible for the basal promoter activity. In Northern blotting analysis, the expression of the Siah-1 gene was upregulated by p53, but activation of the reporter plasmid by the p53 co-transfection assay was not shown, suggesting that a p53 responsive element does not exist in the promoter region we examined in this study but might be present in another region

Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma

Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, shortterm acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance

Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane

The prognostic factors of recurrent GCT: A cooperative study by the Eastern Asian Musculoskeletal Oncology Group

Background Giant-cell tumor (GCT) of bone is a common primary benign tumor with high local recurrence and potential distant metastasis or malignant transformation. We haveinvestigated the clinical behavior of recurrent GCT of bone in the extremities. Methods We retrospectively reviewed 110 patients with recurrent GCTs of bone in the extremities treated by the Eastern Asian Musculoskeletal Oncology Group. The factors that affected the number of recurrences and distant metastasis were analyzed. Results The median interval between initial surgery and the first recurrence of GCTwas 16 months (2-180 months). All patients received additional surgery for first recurrence. Twenty-five patients had a second recurrence and 6 patients had a third recurrence. The mean interval between theinitial surgery and the first recurrence correlated withthe eventual number of recurrences-14.1 months for the repeated recurrence groups (two and three recurrences) and 28.3 months for the single recurrence group (p = 0.016). Campanacci grade did not correlate with repeated recurrence (p = 0. 446). The venue of the initial surgery did not correlate with recurrence but did affect preservation of the adjacent joint (chi-squared test; p =0.046). Campanacci grade II and III also correlated withsacrifice of the adjacent joint (p = 0.020). The incidence of lung metastasis and malignant transformation were 7.5% (8 out of 107 patients) and 2.7% (3 out of 110 patients), respectively. Repeat recurrence was associated with lung metastasis (p = 0.018). © The Japanese Orthopaedic Association 2011

Acridine Orange is an Effective Anti-Cancer Drug that Affects Mitochondrial Function in Osteosarcoma Cells

Acridine orange (AO) is an antimalarial drug that accumulates into acidic cellular compartments. Lysosomes are quite acidic in cancer cells, and on this basis we have demonstrated that photoactivated AO is selectively toxic in sarcomas. However, photodynamic therapy is only locally effective, and cannot be used to eradicate systemic residual disease. In this study, we have evaluated the activity of non-photoactivated AO on sensitive and chemoresistant osteosarcoma (OS) cells to be considered for the systemic delivery. Since lysosomes are even more acidic in chemoresistant cells (MDR), we found that AO accumulation was significantly higher in the lysosomes of MDR in respect to parental cells, and in both cell types, therapeutic doses of AO significantly inhibited cell growth. However, the level of growth inhibition was inversely related to the level of lysosomal uptake of AO, suggesting that the main target of this agent is indeed extralysosomal. A significant reduction of intracellular ATP content and of the expression of mitochondrial complex III suggests a mitochondrial targeting. Notably, MDR cells showed a lower mitochondrial activity. Finally, the combined treatment of AO with the anticancer agent doxorubicin (DXR) significantly increased chemotoxicity by promoting DXR mitochondrial targeting, as revealed by the further reduction in ATP intracellular content. In conclusion, AO is able to effectively target both sensitive and resistant OS cells through mitotoxicity