Retinoblastoma protein in human renal cell carcinoma in relation to alterations in G1/S regulatory proteins

The retinoblastoma gene product (pRb) is the main substrate for cyclin-dependent kinases (CDKs) during the G1/S transition. Aberrations in cell cycle regulatory proteins, which have been observed in many malignancies, can theoretically cause increased phosphorylation of pRb due to unbalanced CDK activities. The expression and phosphorylation of pRb and potential associations to cell cycle aberrations in renal cell carcinomas (RCC) has only partly been clarified. We therefore evaluated the presence of pRb and the level of pRb-phosphorylation in 216 RCCs arranged in tissue microarrays by using different pRb-antibodies, including pRb-phosphospecific antibodies. Most RCCs (95%) expressed pRb, while cases with the low pRb levels, potentially indicative for pRb-inactivation, were few. In order to detect secondary alterations to a potential pRb-inactivation, the p16 expression was also monitored. None of the tumors exhibited increased p16 levels, confirming that pRb-inactivation is rare in RCC. Phosphorylated pRb was detected in approximately 50% of the RCCs, using Western blotting or immunohistochemistry. The immunohistochemical ppRb(ser807/811) levels were associated with high proliferation, cyclin D1, cyclin E and p27 protein content. Surprisingly, there was no association between pRb-phosphorylation and clinicopathological data. In summary, pRb seemed to be functional and aberrations in G1/S-regulatory proteins were associated with increased phosphorylation of pRb and proliferation. The data supports that pRb might be one of the main cell cycle regulators in RCC.     

Growth inhibition and sensitization to cisplatin by zoledronic acid in osteosarcoma cells

Since osteosarcoma is a drug-resistant disease, the aim of the present study was to explore the possible interest of therapeutic approaches including nitrogen-containing biphosphonate zoledronic acid using osteosarcoma cell lines with different genetic backgrounds. Parental p53⁺/pRb⁺ U2-OS, p53-mutant U2-OS (U2-OS/175) and p53⁻/pRb⁻ SAOS were sensitive to zoledronic acid with no significant differences in IC₅₀ values. Analysis of cell cycle distribution revealed a time-dependent shifting of U2-OS cells towards G2 phase with cell cycle arrest in G2 phase at 96 h of exposure to the compound. Conversely, U2-OS/175 and SAOS cells responded to treatment with transient cell accumulation in S phase up to 48–72 h, respectively. Cell lines were exposed to increasing concentrations of cisplatin alone or combined with sub-toxic doses of zoledronic acid. A growth inhibitory effect was seen after combined treatment in U2-OS, otherwise resistant to cisplatin up to 100 ng/ml. Zoledronic acid did not efficiently sensitized U2-OS/175 and SAOS to cisplatin, thereby suggesting that different behavior may depend on p53 mutation. This data was confirmed in U2-OS cells where p53 expression was downregulated by RNA interference. Present findings indicate occurrence of sensitization to cisplatin by zoledronic acid in wild-type p53 osteosarcoma cells but not in p53-null cells nor in cells expressing a dominant-negative form of p53, supporting that wild-type p53 is required for synergistic interaction of cisplatin and zoledronic acid.     

钙结合蛋白S100A2对肝癌细胞生长增殖的抑制作用

目的 探讨钙结合蛋白S100A2对肝癌细胞生长增殖的抑制作用。方法 构建绿色荧光蛋白－S100A2融合基因的重组表达载体,经脂质体介导转入QGY7701肝癌细胞中进行表达,应用荧光显微镜直接观察融合蛋白的表达和定位;通过肿瘤细胞集落形成实验,观察外源性S100A2表达对体外培养细胞增殖能力的影响;以流式细胞仪分析S100A2对肿瘤细胞增殖周期的影响;通过裸鼠接种实验,观察S100A2对肿瘤细胞在体内增殖能力的影响。结果 绿色荧光蛋白－S100A2融合蛋白表达、定位在胞浆和胞核,而单一的绿色荧光蛋白（载体对照）则只定位在胞浆中;细菌在液体和半固体培养基中培养的细胞集落形成率,实验组（QGY7701／A2）明显低于载体对照组（QGY7701／pc）和细胞对照组（QGY7701）;细胞周期分析显示,实验组细胞有G1和G2期阻制,DNA合成量明显减少;实验组裸鼠体内移植瘤重量明显低于两个对照组。结论 外源性S100A2可抑制QGY7701肝癌细胞在体外的集落形成和在裸鼠体内的增殖能力,并对细胞周期有阻滞作用。     

Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G₁ phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.     

p53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1

Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G₁ phase cell cycle arrest. At high doses (>25 μM), SFN dramatically induces the expression of p21^CIP1 while significantly inhibits the expression of the G₁ phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 μM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21^CIP1 gene expression, and G₁phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21^CIP1 and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21^CIP1 play opposing roles in G₁ phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G₁ phase cell cycle arrest in HT-29 cells.     

稳定转染HBx基因的HepG2细胞对raf-1表达的影响

 【摘要】　目的　探讨乙型肝炎病毒x（HBx）蛋白致癌变的分子机制。方法　构建HBx 基因真核表达载体pCDNA3.1(+)-x，脂质体转染入HepG2 细胞（HepG2X 细胞），以pCDNA3.1空载体为对照（HepG2X0细胞），G418选择培养，克隆扩增转染细胞，并用Western blot检测X蛋白的表达；MTS实验检测细胞增生情况；流式细胞仪检测细胞周期变化；检测转染组及对照组raf-1的表达变化。结果　转染组X细胞在相对分子质量为21×103位置有X蛋白的表达，而对照组HepG2X0细胞及未处理的HepG2细胞未见X蛋白的表达。MTS结果显示X细胞增生能力显著高于其他两株细胞（P＜0.01）。流式细胞仪结果显示转染后的细胞系凋亡率增高，G1／G0期细胞减少，G2期细胞增多。Western blot结果示转染组X细胞raf-1表达明显较其他两组细胞增高。结论　HBx 有推进细胞周期的作用，使细胞快速进入G2期。其机制可能是HBx增加细胞周期蛋白raf-1的表达。     

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G₁ phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G₁ cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.     

细胞周期蛋白A、B1、D3、E在同步化及非同步化G1期MOLT-4细胞中的表达

背景与目的我们已经利用流式细胞术(flowcytometry,FCM)的多参数分析方法证实,用“双thymidine阻滞”获得的同步化细胞,其细胞周期蛋(Cyclins)A、B1、D3、E的表达严重失衡。但是,由于当时技术条件的限制,不可能对同期同步和非同步化的细胞中CyclinsA、B1、D3、E进行对照研究。本研究目的是用新建立的分选后的免疫印迹法(postsortingWesternblot),比较CyclinsA、B1、D3、E在同期同步及非同步化G1期MOLT鄄4细胞中的表达,证实双thymidine阻滞法诱导细胞同步化用于分析正常细胞周期的不合理性。方法以MOLT鄄4细胞为模型,用双thymidine阻滞法将细胞同步在G1/S转换期起始处的G1期,应用流式细胞术分选出同期非同步化生长的G1期细胞,采用DNA/Cyclins双参数分析法及免疫印迹法(Westernblot),检测同期同步化及非同步化G1期细胞中CyclinsA、B1、D3、E的表达。结果在分选的非同步化的G1期细胞中CyclinsA、B1几乎不表达,而在同期同步化的G1期细胞中具有明显的表达,CyclinsD3、E在同步化的G1期细胞中的表达明显高于同期分选的非同步化的G1期细胞,利用FCM的多参数分析方法和免疫印迹法所获得的结果一致。结论用双thymidine阻滞干预方法获得的同步化细胞其CyclinsA、B1、D3、E并不能代表正常细胞内的表达水平,因而不是分     

The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

Metastatic colorectal cancer (mCRC) remains a major public health problem, and diagnosis of metastatic disease is usually associated with poor prognosis. The multi-kinase inhibitor regorafenib was approved in 2013 in the U.S. for the treatment of mCRC patients who progressed after standard therapies. However, the clinical efficacy of regorafenib is quite limited. One potential strategy to improve mCRC therapy is to combine agents that target key cellular signaling pathways, which may lead to synergistic enhancement of antitumor efficacy and overcome cellular drug resistance. Protein kinase D (PKD), a family of serine/threonine kinases, mediates key signaling pathways implicated in multiple cellular processes. Herein, we evaluated the combination of regorafenib with a PKD inhibitor in several human CRC cells. Using the Chou-Talalay model, the combination index values for this combination treatment demonstrated synergistic effects on inhibition of cell proliferation and clonal formation. This drug combination resulted in induction of apoptosis as determined by flow cytometry, increased PARP cleavage, and decreased activation of the anti-apoptotic protein HSP27. This combination also yielded enhanced inhibition of ERK, AKT, and NF-κB signaling. Taken together, PKD inhibition in combination with regorafenib appears to be a promising strategy for the treatment of mCRC.     

水泡口炎病毒M蛋白对小鼠Lewis肺癌LL/2c细胞增殖与凋亡的影响

背景与目的:水泡口炎病毒(vesicular stomatitis virus,VSV)具有明显的抗肿瘤作用,在人的A549肿瘤模型和小鼠的LL/2c模型中已取得了明显的治疗效果.但复制完全型病毒在临床应用中存在着一定的安全隐患,且研究报道VSV抗肿瘤作用与其基质蛋白(matrix protein,M蛋白)有关.本研究旨在探讨VSV-M蛋白对小鼠的LL/2c肿瘤细胞增殖和凋亡的影响.方法:采用分子克隆技术构建VSV-M蛋白重组真核表达质粒pcDNA3.1-M,经酶切、PCR、测序鉴定得到阳性重组子,体外转染LL/2c细胞,RT-PCR、Western blot检测M蛋白的表达.采用噻唑蓝(MTT)法检测了M蛋白质粒对LL/2c肿瘤细胞增殖的影响.DNALadder、Hoechst33528染色检测LL/2c肿瘤细胞凋亡.结果:构建了VSV-M蛋白真核表达质粒pcDNA3.1-M.重组质粒体外转染LL/2c细胞后,检测到M蛋白的表达;倒置显微镜下观察到细胞形态学的改变,且48 h后细胞生长抑制明显,抑制率达41.3%(P＜0.05),而空载体转染组只有6.5%的抑制率(P＞0.05).琼脂糖凝胶电泳可见DNA ladder形成,Hoechst 33528染色检测到LL/2c细胞凋亡,细胞核改变.结论:VSV-M蛋白可抑制小鼠LL/2c细胞增殖,并诱导细胞凋亡.     

Expression of SKP2 Protein in Non-small Cell Lung Carcinoma

Objective: To study the expressive characteristics of SKP2 protein in non-small cell lung carcinoma and it is affection to NSCLC patients' prognosis. Methods: The expression of SKP2 protein was detected in 89 NSCLC, 5 benign lung neoplasmas, 5 normal bronchus and lung tissues by Tissue Chip and immunohistochemistry technology. Results: The positive rate of SKP2 protein staining was (23.52±13.57)% in NSCLC tissues, significantly higher than that in benign lung neoplasmas, normal brochus and lung tissues (2.91±1.27)% (P=0.0000<0.001). The expressive level of SKP2 protein in NSCLC tissues was closely related to cell differentiation (P1=0.000<0.001), but not to age, sex, smoking history,pathological type, site, size, lymph node metastasis and TNM stage (each P1>0.05). The survival analysis displayed that the NSCLC patients' 5 years survival rate was lower in positive expression group than that in negative expression group (P1=0.042/0.031<0.05; r=-0.186, P2=0.000<0.001). Conclusion: The positive expression of SKP2 protein may play an enhancement role in the occurrence and development of NSCLC. Moreover, it may be a bad indicator to NSCLC patients' prognosis.     

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4-19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCND1 was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.     

蛋白激酶D抑制剂SD-208对非小细胞肺癌细胞增殖及凋亡的影响

目的 探讨蛋白激酶D抑制剂SD-208对非小细胞肺癌细胞增殖、凋亡及细胞周期的影响。方法 采用5、10、20、30 μmol／L SD-208处理非小细胞肺癌细胞A549（设不加SD-208仅添加完全培养基组为对照组）,分别在24、48、72 h 3个不同刺激时间点应用四甲基偶氮唑盐比色（MTT）法检测A549细胞的吸光值并计算增殖抑制率;膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）和碘化丙啶（PI）双染色法结合流式细胞术检测不同浓度SD-208处理A549细胞24、48 h后的细胞凋亡情况,PI染色法流式细胞仪检测分析不同浓度SD-208处理A549细胞48 h后细胞周期时相分布情况;Western blotting检测不同浓度SD-208处理48 h后的细胞周期蛋白A（Cyclin A）、Cyclin D1、Cyclin E、细胞周期蛋白依赖性激酶4（CDK4）和p16蛋白的表达水平。结果 SD-208 可抑制A549细胞的增殖,且呈剂量和时间依赖性（P＜0.05）;SD-208处理后A549细胞的凋亡率及G0／G1期细胞比例均高于对照组,而S、G2／M期细胞比例均低于对照组,各浓度间的差异均有统计学意义（P＜0.05）;与对照组比较,SD-208处理后的Cyclin D1和CDK4蛋白水平降低,p16蛋白水平升高,差异均有统计学意义（P＜0.05）。SD-208处理对A549细胞Cyclin A和Cyclin E蛋白水平无影响,与对照组比较差异无统计学意义（P＞0.05）。结论 SD-208可抑制A549细胞增殖并诱导细胞凋亡及G0／G1期阻滞,其机制可能与其影响细胞周期及相关调控蛋白水平有关。     

Effects of cellular non-protein sulfhydryl depletion in radiation induced oncogenic transformation and genotoxicity in mouse C3H 10T1/2 cells

A study was made of the effects of cellular non-protein sulfhydryl (NPSH) depletion on cytotoxicity, cell cycle kinetics, oncogenic transformation and sister chromatid exchange (SCE) in C3H 10T1/2 cells. Using DL-Buthionine S-R-Sulfoximine (BSO) at a concentration of 0.05 mM to deplete thiols, it was found spectrophotometrically that less than 5% of control NPSH level remained in the cells after 24-hour treatment under aerated conditions. Such NPSH depleted cells, when subject to a 3 Gy gamma-ray treatment, were found to have no radiosensitizing response either in terms of cell survival or oncogenic transformation. In addition, decreased levels of NPSH had no effect on spontaneous or radiation-induced SCE nor were cell cycle kinetics additionally altered. Therefore, the inability of NPSH depletion to alter gamma-ray induced cellular transformation was unrelated to any possible effect of BSO on the cell cycle. These results suggest that, while endogenous NPSH depletion has been considered to play an important role for most radiosensitizers in clinical or preclinical use, such depletion may result in little or no additional oncogenic or genotoxic effects on aerated normal tissues.     

细胞分裂周期7蛋白在肝癌组织的表达及其对肝癌细胞增殖和侵袭的影响

目的 检测细胞分裂周期7(CDC7)蛋白在人肝癌组织的表达,探讨CDC7过表达对肝癌细胞增殖和侵袭的影响。方法 实时定量聚合酶链式反应(Real-time PCR)检测CDC7 mRNA在肝癌组织和癌旁组织的表达,检测CDC7 mRNA在肝癌细胞系和正常肝脏细胞中的表达。Western blot检测CDC7蛋白在肝癌组织和癌旁组织中的表达,检测CDC7蛋白在肝癌细胞系和正常肝脏细胞中的表达水平。利用慢病毒载体构建稳定过表达CDC7的肝癌HCCLM3和SMMC 7721细胞株。细胞克隆实验和CCK-8法检测CDC7过表达对肝癌细胞增殖的影响。Transwell实验和划痕实验检测CDC7过表达对肝癌细胞侵袭迁移的影响。结果 与癌旁组织相比,CDC7 mRNA和蛋白在肝癌组织中表达水平显著升高(P＜0.001);与正常肝细胞相比,肝癌细胞系的CDC7 mRNA和蛋白表达水平显著增高(P＜0.001);CCK-8法、细胞克隆实验说明CDC7过表达会明显增强肝癌细胞的增殖能力;划痕实验、Transwell实验说明CDC7过表达会明显提高肝癌细胞的侵袭能力。结论 CDC7蛋白在人肝癌组织高表达,其过表达促进肝癌细胞的增殖和侵袭能力。     

苦参碱对白血病细胞癌基因和细胞周期调控蛋白表达的影响

目的：探讨在苦参碱诱导下,白血病细胞株K562增殖和分化时相关癌基因及细胞周期调控蛋白表达表达的改变及意义。方法：应用分子生物学Norhern Blot和DotBlot杂交技术检测人红白细胞病细胞株K562在苦参碱作用后的c-myc,N-ras及p53mRN表达;同时用Western BLotting-ECL分析苦参碱作用前、后24、48、72小时K562细胞Cyclin D1,CyclinE,Ddk2,Cdk5的不同表达水平。结果：在增殖的K562细胞（培养48小时）中,伴随着DNA合成增加,CyclinE和Cdk2保持高表达;而在苦参碱作用24小时分化启动的细胞中,随着DNA合成减少,N-ras,p53mRNA表达增强;c-myc mRNA表达明显受抑;同时CyclinD,Cdk5表达增强,结论：若参碱抑制K562细胞增殖并诱导分化可能与相关癌基因表达和CyclinD1/Cdk5,CyclinE/Cdk2不同表达有关。