表达IL-24基因的溶瘤腺病毒对肝癌细胞的抑制作用

目的 研究携带IL-24基因的溶瘤腺病毒对肝癌细胞抑制生长和转移的作用.方法 构建Ad.HS4.AFP.E1A/IL-24,病毒的复制由人甲胎蛋白启动子控制,携带IL-24基因.检测病毒在不同细胞系中的选择性复制,以及对高转移潜能人肝癌细胞系MHCC97-H的生长抑制、诱导凋亡和抑制转移能力(侵袭、运动、黏附)的作用.结果 Ad.HS4.AFP.E1A/IL-24在肝癌细胞SMMC-7721、Hep3B和MHCC97-H中选择性表达,而不影响正常肝细胞L02(P＜0.05).Ad.HS4.AFP.E1A/IL-24可明显抑制MHCC97-H细胞的增殖,诱导其凋亡,并抑制其体外运动、侵袭和黏附能力(P＜0.01).RT-PCR和明胶酶谱显示其抑制肝癌作用与抑制MMP-2的表达有关.结论 携带IL-24基因的溶瘤腺病毒能够选择性抑制肝癌细胞的增殖并抑制其转移.

Abstract：

Objective To investigate the selective oncolytic role and antitumor action of a novel recombinant adenovirus containing E1A and IL-24 on hepatocellular carcinoma cell(HCC). Methods The recombinant adenovirus expressing IL-24 (Ad. HS4. AFP. E1A/IL-24) was constructed by using modified human alpha-fetoprotein (HS4-AFP) promoter to drive adenovirus E1A gene and II-24 gene.Cell Counting Kit-8 were performed to test the selective cytotoxicity of the virus in hepatocellular carcinoma cell lines SMMC-7721, Hep3B, MHCC97-H and hepatocyte cell line L02 . The mRNA and protein expression of IL-24 gene were detected by RT-PCR and western blot. Cell growth curves and Annexin V/PI assay were used to study cell proliferation and apoptosis of MHCC97-H. The anti-metastatic effects of the recombinant adenovirus were evaluated in cell adhesion, migration, and cell motion. Matrix metalloproteinase-2 (MMP-2) expression was examined by RT-PCR and zymography.Results Selective replications of Ad. HS4. AFP. E1A/IL-24 adenovirus were observed in over expression AFP cell line MHCC97-H, a highly metastatic potential HCC cell line but not in hepatocyte cell line L02. The mRNA and protein of IL-24 were also over expressed in MHCC97-H. This recombinant adenovirus also showed the significant oncolytic action on MHCC97-H but not on L02 (P＜0. 05). Besides, the recombinant adenovirus significantly inhibited MHCC97-H metastatic potential such as cell adhesion, migration and invasion as well(P＜0.01). Conclusion The selective oncolytic adenovirus expressing E1A and II-24 has a selective antitumor effect and play an inhibitory role in metastasis of HCC.     

一氧化氮对组织特异性溶瘤腺病毒转染膀胱肿瘤细胞的影响

目的:研究一氧化氮(NO)对肿瘤组织特异性溶瘤病毒转染过程的影响及对外源基因表达的调节作用。方法:构建组织特异性溶瘤腺病毒,常规培养膀胱肿瘤BIU-87和5637细胞株,以硝普钠作为外源性NO的供体。应用PTIO和L-NMMA分别作为内源性NO的清除剂和诱导型一氧化氮合酶(NOS)的抑制剂。采用Nitrate/Nitrite Assay Kit检测NO和(或)病毒作用前后膀胱肿瘤细胞培养液中的NO水平。应用四甲基偶氮唑盐(MTT)法检测NO对重组病毒抗肿瘤细胞增殖的影响;透射电镜观察腺病毒颗粒进入细胞情况和亚细胞结构变化。结果:膀胱肿瘤细胞BIU-87和5637本身培养液中NO水平很低,给予外源性NO供体后NO水平随时间延长而升高。重组病毒Ad-UPⅡ-E1A能通过E1A基因发挥溶瘤作用。NO能够促进该病毒转染入BIU-87、5637细胞。50μmol/L和100μmol/L的NO联合Ad-UPⅡ-E1A 30MOI作用后能够促进肿瘤细胞的增殖,而200μmol/L的NO联合重组腺病毒作用后则促进肿瘤细胞的死亡。NO对Ad-UPⅡ-E1A的作用具有时间依赖性。透射电镜观察发现,重组病毒Ad-UPⅡ-E1A能够进入并在膀胱肿瘤细胞内复制,而NO能够提高病毒的转染效率并引起肿瘤细胞自吞噬和凋亡。结论:NO能够促进溶瘤腺病毒Ad-UPIIE1A转染膀胱肿瘤细胞的效率,但NO因其浓度不同对溶瘤腺病毒的溶瘤效果具有双向调节作用,低剂量的NO能够下调重组病毒E1A的表达从而促进肿瘤细胞增殖,而高剂量的NO通过上调E1A的表达因而发挥溶瘤效应。     

Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder

OBJECTIVE: The insertion of absent or underexpressed genes into cancer cells to alter their malignant phenotype is an important potential application of available gene therapy technology. One of the more common viral vector systems that has been extensively studied for this purpose are the replication-deficient adenoviruses (Ad). Adenoviral infection of cells is mediated through a complex pathway, initiated following viral-cell attachment. Adenoviral-cell attachment occurs following interactions with a 46-kDa transmembrane protein with high affinity for both the Coxsackie and adenovirus, designated the CAR (Coxsackie and adenoviral receptor). Additional important cell-viral interactions that occur involve the alpha(v)-based integrins, specifically alpha(v)beta3 and alpha(v)beta5. The purpose of the present study was to determine the extent of expression and localization of the known Ad receptor proteins (CAR, alpha(v)beta3, and alpha(v)beta5) in normal and cancerous human bladders. MATERIAL AND METHODS: Frozen tissue samples of normal bladder and invasive transitional cell cancers of the bladder were evaluated. Tissue blocks containing muscle-invasive transitional cell carcinoma (TCC) were obtained following radical cystectomy, which were performed at our institution. Thirty-two invasive transitional cell bladder tumors were evaluated, each with a matched sample of histologically normal-appearing bladder used as a control. Four additional samples of normal bladder were obtained from patients with no evidence of disease of the bladder and served as further controls. Three additional cases of invasive bladder cancer with no matching normal tissue were also evaluated. Identification of the CAR receptor was performed using the anti-CAR mouse monoclonal antibody designated RmBC. The integrins alpha(v)beta3 and alpha(v)beta5 were identified using the mouse monoclonal antibodies designated LM609 and P1F6 respectively. All slides were evaluated by two of the authors (M.B., B.B.) without knowledge of the clinical and pathological data. RESULTS: Normal bladder: Normal bladder mucosa demonstrated a marked positivity for CAR in 29/35 (82.8%) cases. In contrast, normal transitional epithelial cells were uniformly negative when tested for the integrins alpha(v)beta3 and alpha(v)beta5. Subepithelial tissues, specifically the connective tissue components of the lamina propria and deep muscle wall of the bladder, were positive for alpha(v)beta3 and for alpha(v)beta5 in 61 and 75% of samples, respectively. Endothelial cells associated with the various layers throughout the bladder uniformly expressed both integrins and served as a consistent internal control for both antibodies. An almost identical staining pattern of the endothelium was observed using LM609 and P1F6 in all samples tested. Bladder transitional cell carcinoma: CAR immunoreactivity against TCC cells was uniformly decreased compared to normal transitional cells. Nine tumors exhibited a weak positivity for CAR while the remaining samples were negative. In some cases, the absence of CAR positivity was associated with histological evidence of carcinoma in situ. In 6 cases, it led to the identification of small regions of carcinoma in situ that were not noted on primary pathological evaluation. Peritumoral connective tissue expressed both integrins in the majority of cases, similar to the pattern described above for normal bladder. Transitional cell cancers demonstrated a similar pattern of expression of alpha(v)beta5, in which all tumor cells exhibited minimal or no staining. CONCLUSIONS: The success of all viral-mediated gene therapy strategies relies on the ability of the vector to efficiently deliver its genetic material to a target cell population. In the current study, we demonstrate that the bladder epithelial layer consistently expresses high levels of CAR. Deeper layers of the epithelium also express CAR, including the basal layer cells. A decrease in the expression of CAR appears as an early event in bladder carcinogenesis. We observed that both alpha(v)beta3 and alpha(v)beta5 are strongly expressed in muscle cells surrounding the neoplastic cells, as well as within the peritumoral connective tissue. In cases of invasive bladder cancer that have lost CAR expression, an adenoviral vector may still be utilized through the less efficient interactions with the integrins. Bladder tumor tissue may be less susceptible to an adenoviral-mediated gene therapy approach in which a significant percentage of tumor cells require transduction. Adenoviral uptake by tumor or peritumoral cells with subsequent gene transfer could be predicted by the level of CAR and alpha(v)-based integrin expression. This would enhance our ability to identify those patients whose tumors would be more susceptible to Ad-mediated gene delivery as part of an antitumor treatment.     

CHEMOSENSITIZATION OF BLADDER CARCINOMA CELLS BY BCL-xL ANTISENSE OLIGONUCLEOTIDES

PURPOSE: We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines. MATERIALS AND METHODS: A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test. RESULTS: Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent. CONCLUSIONS: Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.     

Adenoviral Oncolytic Suicide Gene Therapy for a Peritoneal Dissemination Model of Gastric Cancer in Mice

Background

Peritoneal dissemination of gastric cancer is often refractory to systemic therapies. Although adenoviral gene therapy has been reported to be a potentially useful therapeutic modality, the adenovirus itself has a dose-limiting toxicity. A novel system was constructed using adenoviral oncolytic suicide gene therapy targeting carcinoembryonic antigen (CEA), and its therapeutic effect and the possibility to reduce the total viral dose while still preserving the antitumor effect were assessed.

Methods

Three types of adenoviruses were prepared for this novel system: (A) Ad/CEA-Cre, (B) Ad/lox-CD::UPRT for a Cre/loxP system, and (C) Ad/CEA-E1 for conditionally replicating adenovirus. The antitumor effect of the oncolytic suicide gene therapy (A + B + C) was then evaluated in vitro. Mice bearing peritoneal dissemination of human gastric cancer were treated with either this system (A + B + C) or with a tenfold viral dose of suicide gene therapy (A + B). The adverse effects in terms of hepatotoxicity were then evaluated between the two groups.

Results

The current system (A + B + C) demonstrated significantly better cytotoxic effect for CEA-producing cell lines than did suicide gene therapy (A + B) at the same viral dose in vitro. The effect of oncolytic suicide gene therapy was almost equal to that of the tenfold viral dose of suicide gene therapy in vivo. The hepatotoxicity of the two treated groups was also found to be equivalent.

Conclusion

It was possible to reduce the total adenoviral dose of oncolytic suicide gene therapy while still preserving the antitumor effect.     

Gene therapy for meningioma: improved gene delivery with targeted adenoviruses

OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene transfer in meningioma cells. METHODS: The presence of the high-affinity Coxsackievirus and adenovirus receptor (CAR) for adenovirus type 5, as well as endothelial growth factor receptor (EGFR) and alpha, integrins (ITGAVs), were analyzed in primary tumors by using immunohistochemical studies and in primary meningioma cell cultures by using fluorescence-activated cell sorting. Targeting of adenoviruses to EGFR was achieved using bispecific antibodies, whereas targeting of adenoviruses to the ITGAVs was accomplished by insertion of an RGD (arginine-glycine-aspartic acid) motif in the adenovirus fiber H1 loop. Gene transfer efficiency of untargeted and targeted vectors was compared in primary cell cultures and in spheroids derived from patients' resected tumor material. The presence of CARs was observed in all tumors and in all but one of the derived primary meningioma cells. The higher expression of EGFRs and ITGAVs indicated that these receptors could be used as alternative targets to redirect the adenoviruses. Redirection of adenoviruses to the EGFRs or integrins enhanced gene transfer threefold (range two-sevenfold) for EGFRs in primary meningioma cells and ninefold (range three-23-fold) for integrins (p = 0.002, analysis of variance). The effect of adenovirus targeting was confirmed in spheroids composed of primary meningioma cells. CONCLUSIONS: Gene transfer with adenoviruses targeted to tumor-specific receptors is very effective in primary meningioma cells and spheroids. These vectors are promising agents for gene therapy of meningiomas.     

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。     

Adenoviral p53 gene transfer in human bladder cancer cell lines: cytotoxicity and synergy with cisplatin

Mutations of the tumor suppressor gene p53 are common in bladder cancer. To determine whether p53 gene transfer would lead to decreased viability of bladder cancer cells, we studied the effect of p53 gene transfer in human bladder cancer cell lines with either mutant or wild-type p53. Bladder cancer cell lines 5637 and J82 (which express only mutant p53) and 253J-BV (which expresses wild-type p53) were transduced with vectors containing the β-galactosidase gene (Ad5-lacZ), wild-type human p53 gene (Ad5CMV-p53), or no foreign gene (DL312 or Ad5-polyA). X-gal staining of cells exposed to Ad5-lacZ showed that the adenoviral vector was capable of transducing each of the cell lines. Increases in p53, p21^waf1/cip1 and bax protein were demonstrated following exposure to Ad5CMV-p53, and there was a dose-dependent increase in the number of apoptotic cells. Cell viability was decreased in all three cell lines, although J82 was less sensitive than either 5637 or 253J-BV. To determine whether cisplatin increases sensitivity of J82 cells to Ad5CMV-p53, we performed median effect analysis for cisplatin combined with Ad5CMV-p53 or DL312. The combination index for cisplatin plus Ad5CMV-p53 revealed synergy, whereas cisplatin and DL312 were only additive. These results suggest that forced p53 gene expression is cytotoxic to human bladder cancer cells with either p53 mutant or wild-type background, and that combination with cisplatin is a potential method for overcoming resistance.     

携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞凋亡的影响

目的 观察携带人mda-7/IL-24的溶瘤腺病毒SG600-IL24对各种不同转移潜能肝癌细胞HepG2、SMMC7721、MHCC97L和正常肝细胞LO2的作用.方法将携带人mda-7/IL-24的溶瘤腺病毒SG600-IL24分别感染人肝癌细胞系HepG2、SMMC7721、MHCC97L和正常肝细胞LO2,逆转录-聚合酶链式反应(RT-PCR)、酶联免疫吸附试验(ELISA)、Western blot检测mda-7/IL-24基因和蛋白的表达,MTT观察肝癌细胞的生长抑制,Hoechst33258和流式细胞仪(FCM)检测细胞的凋亡,流式细胞仪(FCM)检测细胞周期.结果 RT-PCR和Western blot显示mda-7/IL-24在肝癌细胞和正常肝细胞中高表达,ELISA提示细胞培养上清液中mda-7/IL-24蛋白也明显增加.MTT和流式细胞仪提示mda-7/IL-24能明显抑制肝癌细胞的生长(生长抑制率分别为75%±2.5%,86%±3.5%,72%±1.8%,HepG2∶F=5.86,SMMC7721∶F=7.98,MHCC97L∶F=5.13,均P<0.01),促进肝癌细胞的凋亡(凋亡抑制率分别为56.5%±4.0%,34.4%±2.0%,43.3%±2.5%,HepG2∶F=203.4,SMMC7721∶F=130.5,MHCC97L∶F=160.6,均P<0.01),阻滞肝癌细胞在G2/M期(G2/M期阻滞分别为35.4%±4.2%,40.5%±5.0%,42.0%±5.0%,HepG2∶F=112.5,SMMC7721∶F=133.2,MHCC97L∶F=145.5,均P<0.01),而对正常肝细胞没有明显的促进凋亡和增殖阻滞作用.结论 溶瘤腺病毒SG600-IL24能特异性杀伤不同转移潜能人肝癌细胞和诱导凋亡,促进肝癌细胞增殖阻滞,而对正常肝细胞无明显促进凋亡和增殖阻滞作用.

Abstract：

Objective To investigate the effect of oncolytic adenovirus vector SG600-IL24expressing human melanoma differentiation associated gene-7 (mda-7/IL-24) on hepatocellular carcinoma cell lines with different metastatic potential of HepG2, SMMC7721, MHCC97L and normal liver cell line LO2. Methods The oncolytic adenovirus SG600-IL24 which carrying mda-7/IL-24 gene was transfected into hepatocellular carcinoma cell lines and normal liver cell line. The mRNA and protein expression of mda7/IL-24 in HepG2, SMMC7721, MHCC97L and LO2 cell lines was confirmed by RT-PCR,ELISA assay and Western blot respectively. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst33258 and flow cytometry were studied to indicate the apoptosis effects. Results It was confirmed by RT-PCR, ELISA assay and Western-blot that the exogenous mda-7/IL-24 gene was highly expressed in HepG2, SMMC7721, MHCC97L and LO2 cell lines. MTT and apoptosis detection indicated that MDA-7/IL-24 can induce the growth suppression (the inhibition rate was 75% ±2. 5% ,86% ±3. 5% ,and promotes apoptosis ( the apoptosis rate was 56. 5% ± 4. 0% , 34. 4% ± 2. 0% , 43. 3% ± 2. 5%cell lines at G2/M phase ( the blocking rate was 35. 4% ± 4. 2% , 40. 5% ± 5. 0% , 42. 0% ± 5. 0%metastatic potential hepatocellular carcinoma cell lines but not in normal liver cell line.Conclusions Oncolytic adenovirus vector SG600-IL24 can selectively induce growth suppression, promote apoptosis in hepatocellular carcinoma lines in vitro but not in normal liver cell LO2.     

携带人mda-7/IL-24基因溶瘤腺病毒SG600-IL24构建及其选择性杀伤肝癌细胞的机制研究

目的:探讨携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞选择性杀伤效应的机制。方法:构建携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24后,用其分别感染肝癌细胞株HepG2、HCCLM3和正常肝细胞株L02。用RT-PCR和Westernblot分别检测感染后,各细胞株STAT3以及其信号通路下游相关信号分子基因与蛋白的表达变化,以及磷酸化STAT3(p-STAT3)蛋白的表达变化。结果:成功构建携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24载体。SG600-IL24感染后,3种细胞的mda-7/IL-24基因及蛋白表达均明显升高(均P0.05)。两种肝癌细胞感染后,STAT3的表达水平明显下调,其下游信号分子c-myc、Bcl-xl、Bcl-2、cyclin D2、survivin、MMP-2、MMP-9、XIAP、OPN、VEGF的表达水平明显下调,Bax表达明显上调,均呈一定的时间依赖趋势(均P0.05);p-STAT3蛋白在感染后上升,并在感染2 h达到峰值,随后下降。感染后,L02细胞中未见STAT3及其下游信号分子表达的变化(均P0.05)。结论:携带人mda-7/IL-24基因溶瘤腺病毒SG600-IL24选择性杀伤肝癌细胞的机制可能与其选择性抑制肝癌细胞STAT3信号通路,而对正常肝细胞不产生明显影响。     

Alpha(v)beta3 and alpha(v)beta5 integrin expression in meningiomas

OBJECTIVE: Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two alpha(v) integrins, i.e., alpha(v)beta3 and alpha(v)beta5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS: Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of alpha(v)beta3 and alpha(v)beta5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the alpha(v)beta3 ligand vitronectin. RESULTS: Alpha(v)beta3 and alpha(v)beta5 integrins were expressed by neoplastic vasculature and cells. Alpha(v)beta3 and alpha(v)beta5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. Alpha(v)beta3 was more strongly expressed than alpha(v)beta5 in two cases of histologically benign meningiomas with aggressive clinical behavior. Alpha(v)beta3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION: Our data demonstrate the expression of alpha(v)beta3 and alpha(v)beta5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly alpha(v)beta3, in meningioma angiogenesis.     

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.     

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。     

肺癌中5型腺病毒受体表达与腺病毒感染效率的关系

目的 观察肺癌细胞中5型腺病毒(Ad5)及改良Ad5(Ad5F35)受体CAR和CD46的表达,探讨它们与Ad5、Ad5F35型腺病毒感染效率的关系.方法 采用流式细胞术检测人肺癌细胞株及10例原代培养肺癌细胞中CAR和CD46的表达水平.以带有绿色荧光蛋白(GFP)标记的Ad5和Ad5F35型腺病毒按10和50的感染强度(MOI)感染上述细胞,48 h后通过流式细胞术检测GFP表达阳性细胞百分率.结果 所检测的4株肺癌细胞和10例原代肺癌细胞中CD46的表达水平均明显高于CAR.Ad5-GFP(MOI=50)感染48 h后,A549、Z793、QG56和NCI-H520细胞GFP阳性表达率分别为39%、37%、45%和4%;同样浓度的Ad5F35-GFP感染48 h后,A549、Z793、QG56和NCI-H520细胞GFP阳性率分别为78%、75%、75%和54%.10例原代肺癌细胞中Ad5F35-GFP感染后GFP阳性细胞比例也明显高于Ad5-GFP.结论 Ad5及Ad5F35型腺病毒的基因转移效率与细胞中CAR和CD46的表达水平密切相关.肺癌细胞中CD46的表达水平普遍高于CAR,而Ad5F35对肺癌细胞的感染能力明显优于Ad5.     

Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro

BACKGROUND: Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells. METHODS: Bispecific antibodies, constructed as conjugates between an anti-adenovirus fiber knob Fab' fragment and anti-EpCAM or anti-PSMA monoclonal antibodies, were incubated with an eGFP-expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non-prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined. RESULTS: Receptor-specific transduction was obtained for both EpCAM and PSMA. PSMA-retargeting was shown to be selective for the prostate cancer cell lines. CONCLUSIONS: PSMA serves as a tissue-specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer.     

Potent oncolytic activity of human enteroviruses against human prostate cancer

BACKGROUND: Oncolytic virotherapy offers a unique treatment modality for prostate cancer, especially stages that are resistant to current therapies, with the additional benefit of preferentially targeting tumor cells amongst an environment of healthy tissue. Herein, the low pathogenic enteroviruses; Coxsackievirus A21 (CVA21), as well as a bio-selected variant of Coxsackievirus A21 (CVA21-DAFv) and Echovirus 1 (EV1) are evaluated as novel oncolytic agents against human prostate cancer. METHODS: The surface expression of viral receptors required for enterovirus cell attachment/entry, including intercellular adhesion molecule-1 (ICAM-1), decay-accelerating factor (DAF) and integrin alpha(2)beta(1) on a number of human prostate cancer lines was assessed by flow cytometry. Susceptibility to viral oncolysis was determined via in vitro cell lysis assays performed on cell monolayers cultured in micro titer plates. The in vivo oncolytic efficacy of the enteroviruses was assessed using xenograft models in immune compromised SCID-mice following systemic challenge. RESULTS: The majority of prostate cancer lines tested expressed surface ICAM-1 and/or DAF, or alpha(2)beta(1), facilitating significant degrees of oncolysis following in vitro viral challenge. Systemic delivery of each of the three viruses induced reduction of xenograft tumor burdens in vivo, and a therapeutic dose-response was demonstrated for escalating doses of EV1 in the LNCaP animal model. CONCLUSION: Enteroviruses CVA21, CVA21-DAFv, and EV1 are potentially potent oncolytic agents against human prostate cancer.     

Adhesion of rat glomerular epithelial cells to extracellular matrices: role of beta 1 integrins.

Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.     

Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering

The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MHICI) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Ad(beta)-gal adenovirus, encoding for the reporter gene E. coli beta-galactosidase, in the presence of various polybrene concentrations. We determined the amount of beta-gal positive cells by X-gal staining and the beta-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Ad(beta)-gal resulted in a marked increase of beta-gal positive cells and beta-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.     

Nectin and nectin-like molecules: biology and pathology

Nectins and nectin-like molecules (Necls) are structurally related transmembrane proteins primarily involved in cell adhesion. Nectins and afadin, the adaptor or anchoring protein, stabilize the epithelium and endothelium and establish apical-basal polarity of epithelial cells, independently or in cooperation with other cell adhesion molecules. Necls facilitate cell-cell communication implicated in cell movement and proliferation, immune responses, and cancer cell phenotypes. Necls interact with nectins and specific ligands at cell-cell contacts, whereas Necls associate with integrin alpha v beta 3 and growth factor receptors on the same cell surface. Besides their roles in cell adhesion, nectins regulate the activities of Rho family small G proteins which play critical roles in maintaining the apical junctions of epithelial cells through reorganization of the actin cytoskeleton. Since mice lacking the Rho GDP-dissociation inhibitor (GDI)alpha show massive proteinuria and degeneration of renal epithelial cells, nectins and other cell adhesion molecules may play roles in the structural and functional aspects of renal diseases. Here we summarize our knowledge of nectins and Necls and discuss cell adhesion biology in the kidney.