Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5mmol/L hexamethylene bisacetamide (HMBA). Their nuclear matrix proteins (NMPs) were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy

Expression and Localization of hnRNP A2/B1 during Differentiation of Human Osteosarcoma MG-63 Cells Induced by HMBA

背景与目的:核基质蛋白的差异表达与细胞癌变和增殖分化调控关系密切。本研究观察了hnRNP A2/B1在诱导分化处理前后人成骨肉瘤MG-63细胞核基质中的存在和分布,及其与Actin、Prohibitin的共定位关系。方法:选择性抽提经环六亚甲基双乙酰胺(hexamethylene bisacetamide,HMBA)诱导处理前后的MG-63细胞核基质,并运用双向电泳、质谱分析、蛋白质印记杂交、免疫荧光、激光共聚焦等技术检测hnRNP A2/B1在核基质中的表达与定位变化,及其与相关蛋白的共定位关系。结果:双向电泳及蛋白质印迹杂交结果证实了hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调;免疫荧光显微镜观察显示hnRNP A2/B1定位在核基质上,经HMBA处理后hnRNP A2/B1表达减弱。激光共聚焦显微镜观察结果显示,hnRNP A2/B1与细胞核基质蛋白组分Actin、细胞增殖相关调控因子Prohibitin具有共定位关系,但在诱导处理后细胞内的共定位关系减弱。结论:hnRNP A2/B1在MG-63细胞诱导分化过程中的表达分布,及其与Actin、Prohibitin的共定位关系的改变对MG-63细胞分化具有重要影响,值得进一步探索和研究。BACKGROUND & OBJECTIVE: The differentially expressed nuclear matrix proteins have great effects on canceration and regulation of cell differentiation. This study was to explore the existence and distribution of ribonucleoprotein hnRNP A2/B1 in nuclear matrix and its co-localization with Actin and Prohibitin in human osteosarcoma MG-63 cells before and after hexamethylene bisacetamide (HMBA) treatment. METHODS: The nuclear matrix of MG-63 cells before and after treatment of HMBA were selectively extracted. The expression and localization of hnRNP A2/B1 in nuclear matrix were detected by 2-D PAGE, MALDI-TOF-MS, Western blot, and immunofluorescent staining. The co-localization of hnRNP A2/B1 with Actin and Prohibitin was observed under laser scanning confocal microscope (LSCM). RESULTS: hnRNP A2/B1 was detected in the component of nuclear matrix proteins of MG-63 cells by Western blot and immunogold staining and its expression was decreased after treatment of HMBA. hnRNP A2/B1 was located in the nuclear matrix, and its expression was weakened after HMBA treatment. hnRNP A2/B1 was co-localized with Actin or Prohibitin in MG-63 cells, while the co-localization relationship was weakened during differentiation of MG-63 cells. CONCLUSIONS: hnRNP A2/B1 is a kind of nuclear matrix protein, and localizes in the nuclear matrix. The distribution and expression of hnRNP A2/B1 and its co-localization with Actin and Prohibitin play important roles during the differentiation of MG-63 cells.国家自然科学基金项目(No.30470877)~

Differentiation Induced by Ginsenoside Rg1 Combined Formula Involved the Alteration of Prohibitin Localization and Expression in Human Osteosarcoma MG-63 Cells

选择性抽提经人参皂苷Rg1组合(RCT)诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.Prohibitin is a nuclear matrix protein and associates with various oncogene products.A combined formula with ginsenoside Rg1,cinnamic acid and tanshinoneⅡA(RCT)was used to treat human osteosarcoma MG-63 cells in this study.We found that the prohibitin expression in the nuclear matrix was of decreased following RCT treatment by Western blot.The colocalization of prohibitin with the products of oncogenes or tumor suppressor genes,including c-Fos,c-myc,p53 and Rb was evaluated by immunofluorescence staining and laser scanning confocal microscopy.We observed significant alternations of prohibiitin distribution and the degree of colocalization with the associated gene products after the cells exposed to RCT.The results suggested that prohibitin might be involved in the RCT-induced differentiation in MG-63 cells.Our findings may provide information to expand the applications of Traditional Chinese Medicine formulas in cancer-related studies.国家自然科学基金资助项目(No.30470877)~

The impact of the chemotherapeutic drug cyclophosphamide on rat spermiogenic chromatin remodeling /

Male reproductive health is of growing concern, as male toxicant exposure can affect progeny outcome; sperm chromatin structure integrity may be a contributing factor. The formation of mature sperm involves the expression of numerous proteins involved in organizing and packaging the chromatin in a specific manner; this ensures transmission and participation of the paternal genome in embryogenesis. Exposure of male rats to cyclophosphamide as spermatid chromatin is remodeled has an adverse effect on embryo development. The hypothesis of this thesis is that cyclophosphamide exposure causes genetic damage and alters the sperm proteome, thus disrupting components of chromatin condensation and organization during spermiogenesis. The first objective was to assess the phase specificity of the susceptibility of spermiogenic germ cells to cyclophosphamide-induced DNA damage. Spermatozoa were analyzed for DNA strand breaks using the comet assay. Cyclophosphamide-induced DNA damage was dose-related and accumulated over time. Germ cell phase-specific damage was maximal during midspermiogenesis; this reflects an increased susceptibility of step 9-14 spermatids at a key point in sperm chromatin remodeling, the histone-protamine exchange. The second objective was to examine the sperm chromatin structure and basic proteome. Multiple assays demonstrated that the effects of cyclophosphamide on sperm chromatin structure were also germ cell-phase specific; midspermiogenic spermatids were most sensitive. Sperm were less condensed with reduced total thiol and protamine contents. The sperm basic proteome was also altered; identified proteins are involved in a variety of spermiogenic and fertilization events. The nuclear matrix organizes chromatin into loop domains, and various components of somatic cell matrices are targets for chemotherapeutic agents. Therefore the last objective of this study was to assess the effect of cyclophosphamide exposure on the protein profile of the sperm nuclear matrix. The expression of several nuclear matrix protein components, a number of which were identified for the first time, was altered following drug exposure. Together these results show that cyclophosphamide alters male germ cell chromatin remodeling at both the DNA and protein level; this could alter sperm function and thus explain the adverse effects on early embryo development