AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes

OBJECTIVE: AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. DESIGN: Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. RESULTS: AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. CONCLUSION: AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.     

Establishment of a bipotent cell line CL-1 which differentiates into chondrocytes and adipocytes from adult mouse

OBJECTIVE: Establishment of a clonal bipotent chondroprogenitor cell line from adult mouse to provide a new tool for the elucidation of chondrogenesis in adult animal. DESIGN: A clonal cell line CL-1 was established from tibia of adult mouse. Differentiation of CL-1 was characterized in monolayer culture. Effects of growth factors (TGF-beta(1), IGF-I, bFGF) and hormones (all trans retinoic acid, 1 alpha.25(OH)(2)D(3), PTH (1-34)) on the growth and differentiation of CL-1 were examined. Bipotency of CL-1 in vivo was examined by transplantation into SCID mice. RESULTS: CL-1 formed alcian blue (pH1.0) positive nodules spontaneously. The nodules were mineralized in the presence of ascorbic acid and beta-glycerophosphate. CL-1 differentiated also into oil red O positive adipocytes spontaneously. CL-1 cells expressed specific genes of chondrocytes (collagen type II, X, aggrecan) and adipocytes (PPAR-gamma(2), aP(2)). Hyaline cartilage and adipose tissue formation was observed also in subcutaneously transplanted CL-1 cells into SCID mice. These data demonstrate that CL-1 has bipotency either in vitro or in vivo. TGF-beta(1)suppressed growth of CL-1 and induced dominant chondrogenesis accompanied with marked suppression of adipogenesis in 10% FCS. IGF-I stimulated both growth (in 3% FCS) and differentiation of CL-1 into both lineages (in 10% FCS). 1 alpha.25(OH)(2)D(3)and all trans retinoic acid acted as negative regulators on proliferation and differentiation of CL-1 in 10% FCS. CONCLUSIONS: CL-1 will be a useful tool for the understanding of chondrogenesis in adult animal. Furthermore, CL-1 can be also a powerful tool for screening of the chondrogenic agent.     

Arachidonic acid stimulates cell growth in an osteoblastic cell line,MC3T3-E1, by noneicosanoid mechanism

Summary Arachidonic acid, added to α-minimum essential medium containing 10% fetal bovine serum at the final concentration of 10⁻⁴ M, significantly increased DNA content of an osteoblastic cell line, MC3T3-E1, along with an increase of DNA synthesis. No growth-stimulatory effect of arachidonic acid was observed under serum-free condition. α-Linolenic acid, which cannot be converted to arachidonic acid, also increased DNA content at 10⁻⁴ M. Additionally, the stimulatory effects of these fatty acids were not inhibited by simultaneous addition of 10⁻⁵ M of indomethacin. Indomethacin, when added to α-minimum essential medium with 10% fetal bovine serum, also significantly increased DNA content of MC3T3-E1 cells. These results suggest that arachidonic acid may potentiate the growth-stimulatory effect of serumderived growth factors probably via noneicosanoid mechanism. Rat osteogenic sarcoma cell line, UMR106, also showed an increase in DNA content with arachidonic acid treatment. Hence, it is suggested that arachidonic acid may stimulate proliferation of cells of osteoblastic lineage. It is also suggested that indomethacin, probably by blocking endogenous prostaglandin E₂ synthesis, stimulates cell growth in MC3T3-E1 cells.     

新型光敏剂2-丁胺-2-去甲氧基竹红菌乙素光动力作用诱导Capan-1细胞凋亡的实验研究

目的 以新型光敏剂2－丁胺－2－去甲氧基竹红菌乙素（2－BA－2－DMHB）为研究对象，研究其光动力作用诱导人胰腺癌细胞（Capan-1)凋亡。方法 利用荧光分光光度计，MTT，细胞凋亡DNA梯带，流式细胞仪等技术研究2－BA－2－DMHB对人胰腺癌细胞的杀伤作用，诱导细胞凋亡的特征。结果 2－BA－2－DMHB在10－15min内被Capan-1细胞摄取，以后基本保持动态平衡。低浓度时主要定位于线粒体，高浓度时也定位于溶酶体。0.5μM,4J/cm^2红光照射下细胞抑制率达50％以上，8μM,4J/cm^2红光照射下细胞抑制率接近90％。在4J／cm^2红光照射下光增强因子在200以上。DNA琼脂糖电泳分析显示特征性细胞凋亡DNA梯带，流式细胞仪检测有明显凋亡发生，透射电镜分析显示特征性的凋亡小体和核着边。结论 2－BA－2－DMHB在低浓度时光动力作用具有较高细胞抑制作用，其光动力作用机制与细胞凋亡有关，是一种有重要应用前景的光敏剂。     

榄香烯对小鼠肝癌腹水瘤细胞系Hca-F25/CL-16A3的视网膜母细胞瘤抑癌蛋白及腺病毒E2启动子结合因子表达的影响

目的：探讨榄香烯对小鼠肝癌腹水瘤细胞系Hca-F25/CL-16A3治疗的作用机制。方法：将22只小鼠分成两组，对照组12只，治疗组10只。从荷瘤次日起，治疗组用榄香烯腹腔注射治疗，对照组用生理盐水治疗。20d后取瘤组织用免疫组织化学染色方法观察荷瘤小鼠两组肿瘤组织的视网膜母细胞瘤抑癌蛋白（pRB）和腺病毒E2启动子结合因子（E2F－1）蛋白的表达情况。结果：对照组肿瘤组织pRB阳性率为50％（6／12），用药组阳性率为100％（10／10），两组间差异有显著性（P＜0．05）。对照组和用药组E2F－1均呈阳性表达，表达率均为100％（12／12）和（10／10），两组间差异无显著性（P＞0．05）。结论：榄香烯对小鼠肝癌腹水瘤细胞系Hca-F25/CL-16A3有明显的抑制作用。其作用机制与pRB蛋白表达有关，与E2F－1无关。     

Effect of a newly synthesized Zn sulfophthalocyanine derivative on cell morphology,viability, proliferation,and cytotoxicity in a human lung cancer cell line (A549)

Photodynamic therapy (PDT) is a photochemotherapeutic process that is used for the treatment of cancer. Photofrin is the most widely used photosensitizer, however, the chemical composition of Photofrin is unclear and it has a low absorption in the therapeutic wavelength (600–900 nm). This factor has stimulated research in synthesis and testing of new photosensitizers. This in vitro study evaluated the effectiveness of a Zn sulfophthalocyanine (ZnPcS_mix) as a potential photosensitizer in the treatment of human lung cancer. Lung cancer cells (A549) were divided into four groups: group 1 was control cells receiving neither light nor drug; group 2 was light control for cells exposed to laser irradiation at a fluence of 4.98 J/cm²; group 3 was drug control for cells incubated with 15.8 μM photosensitizer and not exposed to laser irradiation, while group 4 was cells receiving the experimental treatment with 15.8 μM photosensitizer and irradiation with 4.98 J/cm². Laser irradiations were performed using a 636-nm diode laser with an output power of 110 mW at 4.98 J/cm². Changes in cellular responses were evaluated by cell morphology, viability, proliferation, and cytotoxicity. While control groups 1, 2, and 3 showed no changes in cell morphology, viability, proliferation, or cytotoxicity, group 4 receiving both photosensitizer and irradiation showed changes in cell morphology, a decrease in cell viability and proliferation, and an increase in cytotoxicity, cell death, and cell membrane damage. Irradiation or photosensitizer alone had no effect on the lung cancer cells since the cells remained viable and showed no evidence of damage. However, irradiation in the presence of a photosensitizer induced cell death.     

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim:To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests.Methods:The human embryonic kidney cell line 293T was employed to produce rhZP1,rhZP2 and rhZP3 proteins individually and together by co-expression.Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis.The effect of the recombinant proteins on the human AR was assessed.Results:RhZP2 and rhZP3 were secreted into the culture medium,whereas rhZP1 was found only in the cell lysate.Interestingly,when all zona pellucida proteins were co-expressed in the same cells,rhZP1 was also secreted into the culture medium.However,despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis,biological activity to induce the AR was not observed.Conclusion:RhZP1,rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T.It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell.Although this approach is not satisfactory for producing active human ZP proteins,it makes a significant contribution to the under-standing of the structural and functional characteristics of the ZP proteins.(Asian J Androl 2004 Mar; 6:3-13)     

Expression of embryonic stem cell markers in cultured JKT-1, a cell line derived from a human seminoma

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (αFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2γ and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.     

Establishment and characterization of a novel malignant astrocytoma cell line derived from a tumor removed in a patient with neurofibromatosis type 1

OBJECT: Patients with neurofibromatosis Type 1 (NF1) have a predisposition to development of a variety of benign and malignant tumors including neurofibromas, astrocytomas, pheochromocytomas, and malignant peripheral nerve sheath tumors. The availability of an astrocytoma cell line derived from NF1 would be useful in studies in which sporadic astrocytomas could be compared with NF1-derived astrocytomas. In this article the authors describe a novel astrocytoma cell line, TM-31, that they established from a tumor removed in a 42-year-old woman with NF1. METHODS: The TM-31 cell line was prepared from a surgical specimen of malignant astrocytoma and was serially subcultured over 250 times throughout a 6-year period without showing any sign of cell senescence. Immunocytochemical analyses demonstrated that TM-31 cells are negative for glial fibrillary acidic protein but positive for vimentin and S-100 protein. The TM-31 cells display little neurofibromin expression when subjected to immunoblotting, indicating that there is an NF1 gene mutation. Polymerase chain reaction-single-strand conformational polymorphism analysis revealed that TM-31 cells harbor a p53 point mutation in exon 7, codon 238. Chemosensitivity testing of TM-31 cells revealed a resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, although they are sensitive to cisplatin and etoposide. In addition, TM-31 cells displayed no morphological differentiation after all-transretinoic acid and dibutyryl cyclic adenosine monophosphate treatments. Pharmacological inhibition of farnesyltransferase of the Ras oncoprotein led to decreased proliferative activity and inhibition of anchorage-independent growth of TM-31 cells in soft agar. CONCLUSIONS: The TM-31 cell line is an immortalized astrocytoma cell line derived from a tumor obtained in a patient with NF1. Ras activation may be the major event of proliferative activity and of the transformed phenotype of TM-31 cells, and the farnesyltransferase inhibitor may be potentially important as a novel antiproliferative therapy for NF1-derived astrocytomas.     

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.     

LRIG1, a candidate tumour-suppressor gene in human bladder cancer cell line BIU87

OBJECTIVES: To determine the effects of LRIG1 on the growth, migration and invasion of bladder cancer cells and the mechanisms underlying such effects. MATERIALS AND METHODS: The plasmid pLRIG1-green fluorescence protein (GFP) was transfected into BIU87 bladder cancer cells by Lipofectamine2000 (Invitrogen, Groningen, the Netherlands), and the cells that expressed LRIG1 stably were screened out by G418. The changes in LRIG1 and epidermal growth factor receptor (EGFR) protein levels were measured by Western blot; growth curves were estimated by the tetrazolium (MTT) assay; then cell-cell adhesion, cell-matrix adhesion and cell invasion assays were used to measure proliferation, adhesion and invasion in LGIR1-transfected and control cells. RESULTS: The LRIG1 protein level in pLRIG1-GFP transfected cells was significantly higher than that in control cells, while the EGFR protein level was significantly lower. pLRIG1-GFP transfected cells had less proliferation than control cells. Contrasting with non-LRIG1-transfected cells, the invasion and cell-matrix adhesion ability of pLRIG1-GFP transfected cells decreased markedly, and conversely the homotypic cell-cell adhesion ability was significantly higher. CONCLUSIONS: LRIG1 might act as a tumour-suppressor gene, participating in negative feedback control of EGFR expression, which inhibits bladder cancer cells from growth, migration and invasion.     

Gpr177, a novel locus for bone mineral density and osteoporosis,regulates osteogenesis and chondrogenesis in skeletal development

Human genetic analysis has recently identified Gpr177 as a susceptibility locus for bone mineral density and osteoporosis. Determining the unknown function of this gene is therefore extremely important to furthering our knowledge base of skeletal development and disease. The protein encoded by Gpr177 exhibits an ability to modulate the trafficking of Wnt, similar to the Drosophila Wls/Evi/Srt. Because it plays a critical role in Wnt regulation, Gpr177 might be required for several key steps of skeletogenesis. To overcome the early lethality associated with the inactivation of Gpr177 in mice, conditional gene deletion is used to assess its functionality. Here we report the generation of four different mouse models with Gpr177 deficiency in various skeletogenic cell types. The loss of Gpr177 severely impairs development of the craniofacial and body skeletons, demonstrating its requirement for intramembranous and endochondral ossifications, respectively. Defects in the expansion of skeletal precursors and their differentiation into osteoblasts and chondrocytes suggest that Wnt production and signaling mediated by Gpr177 cannot be substituted. Because the Gpr177 ablation impairs Wnt secretion, we therefore identify the sources of Wnt proteins essential for osteogenesis and chondrogenesis. The intercross of Wnt signaling between distinct cell types is carefully orchestrated and necessary for skeletogenesis. Our findings lead to a proposed mechanism by which Gpr177 controls skeletal development through modulation of autocrine and paracrine Wnt signals in a lineage‐specific fashion. © 2013 American Society for Bone and Mineral Research.     

低盐诱导的致密斑细胞环氧化酶2表达与p38丝裂素激活蛋白激酶信号通路

目的 探讨低盐(LS)培养对小鼠致密斑细胞 (MMDD1)环氧化酶2 (COX-2)表达、前列腺素E2(PGE2)释放的诱导作用及p38丝裂素激活蛋白激酶(MAPK)信号通路的调控作用。方法 采用RT-PCR和免疫印迹方法检测正常盐(NS)与LS培养对MMDD1细胞COX-2表达的影响。用ELISA法检测上清液PGE2的含量。用免疫印迹检测细胞内p-p38 MAPK表达的变化。 结果 与NS培养相比,LS培养均能诱导MMDD1细胞COX-2 mRNA和蛋白表达增加(16 h时高峰 mRNA 0.94±0.12比0.26±0.09,28 h时高峰蛋白0.59±0.02比0.25±0.07,P均< 0.01);PGE2分泌各时间点均显著升高,于24 h达高峰 [(644.33±26.54)ng/L比(224.0±18.33)ng/L, P < 0.01。LS培养后,MMDD1细胞内p38MAPK的磷酸化程度显著上调,180 min时较高(从0.17±0.01升至0.28±0.01,P < 0.01)。20 μmol/L p38抑制剂SB-203580下调 LS诱导的COX-2蛋白表达(从0.58±0.01降至0.19±0.02, P < 0.01)。 结论 低盐培养促进MMDD1细胞COX-2的表达和PGE2的分泌。p38 MAPK激活介导了低盐诱导的MMDD1细胞COX-2表达。