The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma

PURPOSE: Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. RESULTS: Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. CONCLUSIONS: Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.     

Optimizing chemotherapy for transitional cell carcinoma by application of bcl-2 and bcl-xL antisense oligodeoxynucleotides

OBJECTIVE: Therapy failure after intravesical and systemic chemotherapy for transitional cell carcinoma (TCC) is still high. Antiapoptotic proteins such as Bcl-2 and Bcl-xL have been reported to promote chemoresistance in TCC. Targeting bcl-2 and bcl-xL messenger ribonucleic acid with antisense oligodeoxynucleotides (AS-ODNs) may enhance the cytotoxic effects of chemotherapeutic agents. Therefore, we investigated the effects of bcl-2 and bcl-xL AS-ODNs in combined treatment with conventional and new chemotherapeutic agents to evaluate the cytotoxic effects in comparison to monotreatment. METHODS AND MATERIALS: Western blot analysis or immunohistochemistry verified Bcl-2 and Bcl-xL expression in a panel of human TCC cell lines that had been monotreated with cisplatin, gemcitabine, mitomycin C, and paclitaxel. In addition, bcl-2 or bcl-xL AS-ODNs were applied in combination with each chemotherapeutic agent. Cell viability was determined using a standard MTT assay and Neubauer hemocytometry. RESULTS: All cell lines responded to chemotherapeutic monotreatment in a dose-dependent manner. Maximum cell death rates after monotreatment were 47.4% (cisplatin), 39.0% (gemcitabine), 83.4% (mitomycin C), and 54.8% (paclitaxel). After combined treatment with chemotherapy and bcl-2 or bcl-xL AS-ODNs, cell death rates were significantly higher (e.g., 30.3% vs. 87.2% in HT 1197 cells for monotreatment vs. the combination of paclitaxel and bcl-xL AS-ODNs). Three-way analysis of variance revealed that combined treatment had a significant effect on all cell lines. CONCLUSIONS: Our study confirms that the addition of bcl-2 and bcl-xL AS-ODNs enhances the cytotoxic potential of chemotherapeutic agents in TCC cell lines as a result of combined effects. Further trials in ex vivo and in vivo models have to be performed to promote clinical application in patients.     

LIN28A和LAMP1在膀胱癌细胞系中的表达及意义

目的:探讨LIN28A和LAMP1在膀胱癌细胞系中表达情况,以及两者之间的关系,推测其可能临床意义及对肿瘤进展的影响。方法:采用RT-PCR检测膀胱癌细胞系LIN28A、LIN28B和LAMP1表达,免疫荧光检测LIN28A和LAMP1二者蛋白表达定位;LIN28A敲减后通过qRT-PCR检测LAMP1的mRNA表达变化。结果:5个癌细胞系T24、UM-UC3、J82、5637和SW780和正常移行上皮细胞系SV-HUC-1均表达LIN28A,其中J82也表达LIN28B;5个癌细胞系均表达LAMP1,SV-HUC-1不表达LAMP1;LIN28A和LAMP1蛋白均定位在胞浆;LIN28A敲减后对LAMP1的mRNA表达变化无明显影响,相应蛋白变化需要进一步验证。结论:4个膀胱癌细胞系T24、5637、UM-UC3和SW780可以用于LIN28A与肿瘤相关的机制研究,而J82可用于LIN28B的机制研究。LIN28A对肿瘤细胞和干细胞的调控方面可能具有相似性,敲减后对其靶点mRNA表达量无明显影响,LAMP1蛋白可能对肿瘤细胞侵袭转移具有抑制作用。     

Epidermal growth factor receptor targeting of replication competent adenovirus enhances cytotoxicity in bladder cancer

PURPOSE: We evaluated the delivery and oncolytic potential of targeted replication competent adenoviruses in bladder cancer lines. MATERIALS AND METHODS: Seven established human bladder cancer tumor lines (5637, SW800, TCCsup, J82, Scaber, T24 and 253J) were studied for the expression of integrins alpha(v)beta3, alpha(v)beta5, Coxsackievirus and adenovirus receptor, epidermal growth factor receptor (EGF-R) and epithelial cell adhesion molecule antigens using flow cytometry analysis. Bispecific single chain Fv fragments were used to target replication deficient luciferase reporter adenovirus to EGF-R (425-s11) or to epithelial cell adhesion molecule (C28-s11) antigens. Moreover, a fiber modified adenovirus targeting alpha(v)-integrins was studied. Replication competent serotype-5 adenoviruses attenuated to replicate specifically in retinoblastoma pRb (Ad5-d24) or p53 deficient (Ad5-d55K) cells were tested in vitro for oncolytic properties. RESULTS: Low to absent Coxsackievirus and adenovirus receptor expression was found in 5 of the 7 tumor lines (SW800, J82, T24, 5637 and Scaber). EGF-R expression was found in all cell lines, whereas elevated epithelial cell adhesion molecule expression was seen in 3 (5637, Scaber and TCCsup), alpha(v)beta3-integrin was found in 1 (Scaber) and alpha(v)beta5-integrin was found in 3 (TCCsup, 253J and T24). EGF-R targeting using 425-s11 improved transgene expression in all cell lines from 2.1 to 12.5 times over nontargeted viruses. Epithelial cell adhesion molecule and integrin targeting was inferior to EGF-R targeting with a maximal increase in transgene expression of 2 times for epithelial cell adhesion molecule in 5637cells and 1.6 times for integrin targeting in T24 cells. Comparison of the wild-type replication competent virus with conditionally replicating adenoviruses (Ad5-d55K and Ad5-d24) showed superior oncolytic activity for the latter 2 in all lines. Furthermore, improved cytotoxicity (29% to 33%) was obtained in 4 of the 7 lines after pre-incubation of Ad5-d24 with 425-s11. CONCLUSIONS: EGF-R directed bispecific single chain antibodies enhance adenovirus mediated transgene expression and oncolysis in bladder cancer lines.     

LIN28在膀胱癌和膀胱癌细胞系中的表达及意义

目的：探讨LIN28在膀胱癌组织和细胞系中表达情况,以及与mieroRNA初级Let-7g（pri—Lev7g）之间关系,推测其可能临床意义及对肿瘤进展的影响。方法：采用常规RT-PCR、miRNA转录、免疫荧光和免疫组化方法,检测LIN28mRNA和pri-Let-7g表达,以及LIN28蛋白表达定位。结果：2例膀胱癌细胞系均表达LIN28mRNA,T24表达较强,免疫荧光显示这两个细胞系均表达LIN28蛋白,阳性部位位于细胞胞质,T24荧光强度强于5637。所选10例膀胱癌和相应癌旁组织均表达LIN28mRNA,二者并无明显不同,与临床分级也无明确关系。免疫组化显示癌组织LIN28表达阳性并定位于胞质,而癌旁正常组织LIN28表达为阴性。此外,两个细胞系pri—Let-7g表达较强,而癌和癌旁组织的pri—Let-7g表达强度无明显差异,需进一步检测其成熟Let-7g在这些组织中是否存在不同,以明确这些miRNA是否发生生物合成的转录后阻断。结论：明确T24和5637两个膀胱癌细胞系均可作为研究LIN28、Let-7与其相应靶基因关系的体外实验模型。尽管并不确定膀胱癌和癌旁组织LIN28、Let-7g表达强度与临床分级是否相关,但至少明确LIN28／LIN28在膀胱癌中表达,为探讨LIN28和Let-7在泌尿系统来源的其他恶性肿瘤中的作用提供借鉴和实验依据。     

Enhanced sensitivity of bladder cancer cells to tumor necrosis factor related apoptosis inducing ligand mediated apoptosis by cisplatin and carboplatin

PURPOSE: The development and acquisition of multiple drug resistance in cancer cells are a consequence of cancer chemotherapy and remain a major obstacle in treatment. Therefore, there is an obvious need for alternative approaches, such as immunotherapy and gene therapy. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of the tumor necrosis factor ligand families and it selectively induces apoptosis against cancer cells. Several cytotoxic anticancer drugs also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. We reasoned that combination treatment of cancer cells with TRAIL and drugs may overcome this resistance. We evaluated whether bladder cancer cells are sensitive to TRAIL mediated cytotoxicity and whether TRAIL may synergize with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells. MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. RESULTS: Human T24 bladder cancer line was relatively resistant to TRAIL and TRAIL was not cytotoxic against normal bladder cells. Treatment of T24 cells with TRAIL in combination with 5-fluorouracil or mitomycin C did not overcome resistance to these agents. However, treatment of T24 cells with a combination of TRAIL and cisplatin resulted in a synergistic cytotoxic effect. Synergy was also achieved in the cisplatin resistant T24 line (T24/CDDP), 2 other bladder cancer lines and 3 freshly derived bladder cancer cells. The combination of TRAIL and carboplatin resulted in a synergistic cytotoxic effect on T24 cells. However, the combination of TRAIL and trans-diamminedichloroplatinum (II) resulted in an antagonistic cytotoxic effect. The synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Treating T24 cells with cisplatin enhanced the expression of bax but not bcl-2. Incubation of T24 cells with TRAIL increased the intracellular accumulation of cisplatin. CONCLUSIONS: This study demonstrates that combination treatment of bladder cancer cells with TRAIL and cisplatin overcomes their resistance. The sensitization obtained with established cisplatin resistant and freshly isolated bladder cancer cells required low subtoxic concentrations of cisplatin, supporting the in vivo potential application of a combination of TRAIL and cisplatin for treating TRAIL resistant and cisplatin resistant bladder cancer.     

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study.MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines.ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin ?31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer.ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.     

E-cadherin和CAR在人膀胱癌细胞中的表达及其意义

目的检测上皮型钙黏素（E—cadherin）和柯萨奇-腺病毒受体（coxsackie and adenovirus receptor，CAR）在多种人膀胱癌细胞中的表达情况，初步讨论E-cadherin与CAR在人膀胱癌细胞中表达的意义及两者间的关联。方法用Western印迹法测定人膀胱癌细胞中E—cadherin和CAR的表达情况。结果E—cadherin，CAR在RT4、5637细胞中表达较高，在253J细胞中表达较低；E—cadherin在J82、T24细胞中不表达，CAR在J82细胞中微弱表达，在T24中不表达。结论E-cadherin和CAR在人膀胱癌细胞中的表达趋势一致，两者可能相互协作，共同参与膀胱癌的侵袭转移过程。     

Vascular endothelial growth factor antisense pretreatment of bladder cancer cells significantly enhances the cytotoxicity of mitomycin C, gemcitabine and Cisplatin

PURPOSE: Due to unsatisfactory success in the treatment of local and systemic bladder cancer and the low response rates to commonly used chemotherapy (CT) alternative and additive approaches must be found. The function of vascular endothelial growth factor (VEGF) in neo-angiogenesis and, therefore, in solid tumors makes it a promising target for a specific antitumor therapy. We investigated the possibility of sensitizing transitional bladder cancer cell lines to CT by pretreatment with VEGF antisense (AS) oligodeoxynucleotides (AS-ODNs). MATERIALS AND METHODS: The human bladder cancer cell lines EJ28 and 5637 were transiently transfected with 3 antiVEGF AS-ODNs, followed by incubation with 3 doses of mitomycin C, gemcitabine or cisplatin CT. WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche, Mannheim, Germany) was performed to assess effects on cell viability. Apoptosis was examined by Annexin V staining. In all experiments a nonsense ODN served as a control. RESULTS: Each cell line responded in a dose dependent manner to all CTs. Combined treatment with VEGF AS-ODNs and CT resulted in decreased viability compared with isolated CT. VEGF857 plus CT significantly decreased the viability of the 2 cell lines compared with nonsense ODN plus CT for all 3 CT agents (p <0.007). This detected chemosensitization was based on an AS mediated increase in apoptosis. CONCLUSIONS: One of the 3 AS-ODNs tested (VEGF857) significantly sensitizes human transitional cell carcinoma cells to CT. We suggest VEGF as an additional putative target to enhance the therapeutic benefit of, for example mitomycin C and gemcitabine instillation treatment schedules.     

Antiproliferative effects of interferons against human bladder carcinoma cell lines in vitro

In order to evaluate the antiproliferative effects of recombinant human interferon-gamma 2c (rHu IFN-gamma 2c), recombinant human interferon-gamma (rHu IFN-gamma), natural interferon-beta (IFN-beta), and their combination with cytotoxic agents, 17 different human bladder carcinoma cell lines were tested in vitro. The antiproliferative effects were compared in evaluating the tumor cell inhibiting potency of the different interferon (IFN) classes. It could be demonstrated that interferons have inhibiting effects on the bladder cancer cell multiplication rate, yet there are significant differences in the susceptibility of different IFN preparations on different cell lines. The cell lines BT1, RT4, EJ, 468P, 253J, SD, TCCSUP, and SW1738 can be defined as sensitive, T24, 647V, VM-CUB2, and J82 as semisensitive, HT1376, 5637, VM-CUB1, 639V and SW1710 as resistant upon treatment with IFN. The combination of rHu IFN-alpha 2c, IFN-beta, and rHu IFN-gamma seems to be more effective than treatment with rHu IFN-alpha 2c alone. The cytotoxic effect of doxorubicin on bladder cancer cells can be intensified by combining it with IFN.     

Interferon-α inhibits cyclooxygenase-1 and stimulates cyclooxygenase-2 expression in bladder cancer cells in vitro

The enzymes cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2) catalyze the initial step in the formation of prostaglandins (PGs). PGs are known to be involved in numerous processes, for example inflammation, immune responses, carcinogenesis, and tumor angiogenesis. The formation of PGs is stimulated in various cancers since the expression of Cox-2 is upregulated. Interferon (IFN)-α is used in the treatment of bladder cancer, although not all of the effects of such treatment are thoroughly known. Therefore, we investigated the expression of cyclooxygenases in two bladder cancer cell lines, 5637 and T24, under basal conditions and in the presence of human recombinant IFN-α (100, 1,000, and 10,000 U/ml). The mRNA of Cox-1 and Cox-2 was expressed in both cultured bladder carcinoma cell lines. The level of Cox-1 expression was low in 5637 cells and higher in T24 cells. In contrast, Cox-2 expression was prominent in 5637 cells and low in T24 cancer cells. The highest IFN-α concentration (10,000 U/ml) decreased the expression of Cox-1 to 47 and 28% of the control levels in 5637 and T24 cells, respectively. In contrast, Cox-2 expression increased in both cell lines. In 5,637 cells, Cox-2 expression increased 1.3-fold with 10,000 U/ml of IFN-α. In T24 cells, the maximum effect was achieved by 1,000 U/ml of IFN-α, which increased the expression of Cox-2 up to 2.4-fold. These findings may have relevance in the outcome of patients treated with IFN-α because upregulated Cox-2 expression may suppress the cell-mediated defense system. On the other hand, the inhibition of Cox-1 could be beneficial because Cox-1 is known to stimulate angiogenesis. Received: 5 August 1999 / Accepted: 8 September 2000     

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines

Summary The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [¹²⁵I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P<0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [¹²⁵I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (K _d) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [¹²⁵I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [¹²⁵I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.     

Augmentation of cisplatin sensitivity in cisplatin-resistant human bladder cancer cells by modulating glutathione concentrations and glutathione-related enzyme activities

OBJECTIVES: To investigate the roles of glutathione and glutathione-S-transferase (GST) in cisplatin-resistance mechanisms in human bladder cancer, by using glutathione-depleting or GST-blocking agents. MATERIALS AND METHODS: Cisplatin-resistant human bladder cancer cell lines were established by continuous exposure of T24 cells to increasing concentrations of cisplatin. Buthionine sulphoximine (BSO), ethacrynic acid and indomethacin were used to deplete glutathione or block GST. Intracellular glutathione content, GST activity and cisplatin cytotoxicity were determined after exposing parental and drug-resistant cell lines to these agents. RESULTS: Intracellular glutathione content and GST activity were significantly decreased, and cisplatin cytotoxicity significantly enhanced, in both parental and resistant cell lines by glutathione-depleting or GST-blocking agents. However, the resistance of cisplatin-resistant cell lines did not fully recover to that of the parental cells with combined BSO and indomethacin. CONCLUSIONS: Both increased glutathione content and GST activity are significant in the cisplatin resistance of human bladder tumour cells. Because BSO, ethacrynic acid and indomethacin caused a partial recovery of resistance in the cisplatin-resistant cell line, further studies are needed to investigate their efficacy for treating patients with metastatic bladder carcinoma resistant to cisplatin.     

膀胱癌转移与Raf激酶抑制蛋白的关系

目的 观察转移性不同的3种膀胱癌细胞株中Raf激酶抑制蛋白(RKIP)的表达,探讨其与膀胱癌侵袭、转移的关系.方法 选取膀胱移行细胞癌细胞株EJ、T24和BIU-87,用Western blot检测各细胞株中RKIP的蛋白表达,用逆转录-聚合酶链反应(RT-PCR)检测各系RKIP的mRNA水平,比较它们的差异.结果 EJ、124和BIU-87三者RKIP蛋白相对表达量分别为1.99±0.24、0.82±0.16、0.15±0.03(P<0.05),mRNA相对表达量分别为1.87±0.33、0.86±0.25、0.29±0.12(P<0.05),均依次降低.结论 RKIP可抑制膀胱癌细胞株的侵袭、转移,其表达下调可能在膀胱癌的进展中具有重要作用,RKIP可能与膀胱癌的侵袭和转移呈负相关.     

赫赛汀联合丝裂霉素C对人膀胱癌T24细胞生长的抑制作用

目的 探讨赫赛汀(Herceptin,HER)联合丝裂霉素C(MMC)对HER-2/neu基因高表达人膀胱癌细胞株生长的抑制作用.方法 应用免疫细胞化学法、逆转录-聚合酶链反应(RT-PCR)法检测膀胱癌T24细胞株HER-2/neu的表达;采用噻唑蓝(MTT)比色法测定不同浓度HER(10、20、40、80、160μg/L)、MMC(4、8、16、32、64μg/L)及联合用药对人膀胱癌T24细胞株体外生长的抑制率并进行比较.结果 应用免疫细胞化学方法及RT-PCR法检测证实HER-2/neu在膀胱尿路上皮癌T24细胞株高表达;单用HER于72 h出现细胞抑制(72 h时各浓度组分别与48 h时比较,均P<0.05);HER和MMC联用在24、48、72 h基本上均表现为协同作用(q值1.264～3.473),在96 h呈相加作用(q值0.913～1.138).结论 HER-2/neu在膀胱尿路上皮癌T24细胞株高表达;HER单药对T24细胞有轻度抑制作用,且起效慢(72 h);与MMC联合应用能协同抑制T24细胞生长.     

端粒酶反义RNA转染促进膀胱癌T24细胞凋亡的研究

目的　探讨端粒酶反义RNA对膀胱癌T2 4细胞恶性表型的抑制及促进其凋亡的作用。　方法　采用脂质体转染法将转录出端粒酶反义RNA质粒导入膀胱癌T2 4细胞。应用PCR ELISA法测定转染后T2 4细胞的端粒酶活性 ;光镜、电镜、MTT及流式细胞术 (FCM )等方法观察端粒酶反义RNA对T2 4细胞生长及凋亡的影响。　结果　端粒酶反义RNA能显著抑制T2 4细胞的端粒酶活性 ,转染T2 4细胞后使其生长受到抑制。形态学观察 ,转染后T2 4细胞出现典型的凋亡现象。FCM检测发现G1期前出现凋亡峰。　结论　转染端粒酶反义RNA能抑制膀胱癌T2 4细胞的恶性表型 ,促进其凋亡     

反义寡聚核苷酸抑制人膀胱癌T24细胞系端粒酶活性的研究

目的研究反义寡聚核苷酸(asONs)能否抑制人膀胱肿瘤T24细胞系端粒酶活性和增殖活性。方法设计并合成2条针对端粒酶RNA模板区的asONs片段,在其作用于T24细胞第5天时,用7复孔法用聚合酶链反应-酶标法(PCR-ELISA)检测其对端粒酶活性的影响;分别在实验的第1、3、5、7天,用7复孔四唑盐比色试验(MTT)法检测其对细胞增殖活性的影响。结果经不同浓度(0、5、10μmol/L)处理5d后T24细胞端粒酶活性受到抑制,吸光度(A)值分别为0.79±0.10、0.24±0.14;经10μmol/L处理1、3、5、7d,T24细胞的增殖活性逐渐下降,A值分别为0.59±0.11、0.39±0.09、0.21±0.02、0.19±0.07,各组分别比较差异有非常显著性(P＜0.001)。结论asONs能够同时抑制人膀胱肿瘤T24细胞系端粒酶及细胞增殖活性,提示asONs可能通过抑制端粒酶活性达到抑制人膀胱肿瘤T24细胞系的增殖。     

The growth inhibitory effect of p21 adenovirus on human bladder cancer cells

PURPOSE: To evaluate whether p21 (WAF-1/CIP1) should be considered a potential candidate for human bladder cancer gene therapy, we determined: (1) the basal level of p21 expression in bladder cancer cell lines, (2) the response of bladder cancer cells to increased p21 expression following p21 adenovirus infection, and (3) the mechanism of growth inhibition produced by p21 overexpression. MATERIALS AND METHODS: Five established human bladder cancer cell lines and one primary culture derived from an invasive transitional cell carcinoma were used in this study. To examine the effect of p21 protein on the growth of human bladder cancer cells, a recombinant adenovirus vector system containing p21 cDNA, under the control of cytomegalovirus promoter, was constructed. A control virus containing p21 in an antisense orientation was used to eliminate potential artifacts caused by viral toxicity. RESULTS: Human bladder cancer cell lines exhibit variable endogenous p21 levels which correlate with the in vitro growth status. Significant, but highly variable increases in the steady-state level of p21 were detected in p21 adenovirus infected cells. Human bladder cancer cell lines responded heterogeneously to p21 adenovirus infection. Growth of the WH cell line was substantially inhibited in a dose and time-course dependent fashion. The mechanism of p21 growth inhibition was found to be due to G0/G1 arrest and not the induction of apoptosis. In contrast, p21 adenovirus failed to inhibit the growth of T24 bladder cancer cells because T24 cells were resistant to viral infection. The 253J bladder cancer cells exhibited marked sensitivity to adenovirus; substantial growth inhibition was seen with both sense and antisense p21 very early in the time course of infection. CONCLUSIONS: We found significant variation in the basal level of p21 protein expression in several human bladder cancer cell lines. Increased p21 expression as a result of adenoviral infection may be a potent growth suppressor in some human bladder cancer because it elicits cell cycle arrest in G0/G1 stage, but not the induction of apoptosis. Bladder cancer cells exhibit a wide spectrum of sensitivity to adenoviral infection that may be caused by the presence of viral receptor heterogeneity. This wide spectrum of sensitivity has significant basic scientific and clinical implications and warrants further study.     

MiR-519d靶向调控OCT4抑制膀胱癌细胞系5637增殖

目的:探讨miR-519d对膀胱癌细胞系5637增殖的影响及分子机制。方法:通过转染miRNA mimics在膀胱癌细胞系5637中过表达miR-519d,分别利用MTS及细胞集落形成试验,检测膀胱癌细胞系5637增殖能力的改变。通过双荧光素酶报道实验和Western Blot方法验证miR-519d对OCT4的靶向调控。利用OCT4特异性干扰RNA证实miR-519d通过靶向调控OCT4抑制膀胱癌细胞系5673增殖。结果:在膀胱癌细胞系过表达miR-519d后,细胞活性、增殖能力明显下降;双荧光素酶报道实验结果表明,相对于各对照组,共转染OCT4 3'UTR载体与miR-519d组荧光素酶相对活性明显降低(P0.05)。过表达miR-519d后5637细胞中OCT4蛋白表达下调。利用siRNA特异性下调OCT4表达后,5637细胞活性及增殖能力同样受到抑制。结论:miR-519d能靶向结合OCT4 3'UTR序列,通过下调OCT4的表达抑制膀胱癌细胞系5637的增殖。