Comparison of growth of human bladder cancer in tissue culture or as xenografts with clinical and pathological characteristics

Seventy-four biopsies of human bladder carcinoma were assessed by implantation as xenografts in immune-deprived mice and/or by culture of cell suspensions in agar or methylcellulose. The quality of the cell suspensions was assessed immediately after plating in vitro. The results were compared with the pathological stage and grade of the biopsies and with the clinical course of the disease in patients from whom the biopsies were obtained. We found that (a) progressively growing xenografts were generated from 20 of 53 biopsies (38%). These xenografts grew with mean volume doubling times in the range of 1 to 3 weeks; all of them examined histologically were consistent with transitional cell carcinoma. (b) Colony formation occurred from 21 of 49 cell suspensions (43%), and plating efficiency was in the range of 0.0004 to 1.7%. The majority of cell suspensions were found to have residual small clusters of cells. Colony formation sometimes originated from these clusters, an effect that would be expected to introduce artifacts when the in vivo cloning assay is used for chemosensitivity testing. (c) There was no evident correlation between expression of clonal growth in vitro and success of xenografting, and no correlation between the results of either of these experimental procedures with stage, grade, or clinical course of the disease. Further improvements in tissue culture and xenograft technology will be required before these methods can be used as a guide to patient management.     

Fetal fibronectin: a new screening-marker for bladder cancer?

Early detection of transitional cell carcinoma (TCC) of the urinary bladder is essential for effective treatment. While several serum markers have been evaluated, none have been widely accepted for practical clinical use. Thus, urinary markers have been introduced and investigated to detect the evidence of bladder cancer. But sensitivity and specificity range around 80% respectively. In a prospective study we evaluated fetal fibronectin in the urine of patients with TCC of the urinary bladder. The positivity of oncofetal fibronectin was measured in morning urine samples by membrane immunoassay. This FFN membrane immunoassay is a qualitative test, a solid-phase immunogold assay. A positive sample will result in a single spot after binding of the oncofetal fibronectin-immunogold complex to the membrane containing a monoclonal antibody specific to oncofetal fibronectin (FDC-6, which specifically recognizes III-CS region). The morning urine samples were collected from patients with TCC before they underwent transurethral resection (n=40, 34 non-invasive and 6 invasive carcinomas) and healthy controls (n=20). Oncofetal fibronectin was investigated in the surgical samples by immunohistochemistry (antibody FDC-6, APAAP technique). We found a positive result for oncofetal fibronectin in 38/40 patients with transitional cell carcinoma of the urinary bladder. Two patients with a small pTaG1-TCC showed negative results. In the urine of healthy controls no positive results were detected. Thus, there is a sensitivity of 95% and a specificity of 100%. The TCC was demonstrated as a source of oncfn. To our knowledge this is the first study showing that patients with an evident TCC have a demonstrable amount of oncofetal fibronectin in the urine. We conclude that a positive result is common in TCC-patients. The sensitivity and specificity of this test seems to be extraordinarily high. Because of the small number of cases further studies are required.     

RT-PCR法检测尿脱落细胞中XIAP的表达在膀胱癌诊断中的价值

目的：比较膀胱癌患者尿液脱落细胞中XIAP表达的RT-PCR检测法和常规尿脱落细胞病理学检测在膀胱癌诊断中的临床价值。方法：采用逆转录聚合酶链反应技术（RT-PCR）检测51例膀胱尿路上皮癌患者尿液脱落细胞中XIAP-mRNA的表达,同时行常规尿脱落细胞病理学检测,20例非肿瘤人员作为对照组。结果：实验组51例尿脱落细胞XIAP-mRNA RT-PCR检测阳性27例（53%）,尿脱落细胞学病理学检测阳性12例（24%）,对照组20例尿脱落细胞XIAP-mRNA检测阳性1例（5.0%）,对照组尿脱落细胞病理学检测阳性0例（0%）。实验组RT-PCR检测膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的敏感性高于尿脱落细胞病理学检测,差异有极显著统计学意义（P〈0.01）,实验组RT-PCR检测膀胱尿路上皮癌患者尿中XIAP表达的敏感性显著高于非肿瘤对照组,差异有极显著统计学意义（P〈0.01）。结论：膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的RT-PCR检测法较常规尿脱落细胞病理学检测更敏感,临床上作为膀胱癌的筛选方法,有一定的临床价值。     

肺耐药蛋白在膀胱移行细胞癌中的表达及临床意义

目的 :研究膀胱移行细胞癌的多药耐药性。方法 :应用免疫组化方法检测 65例膀胱移行细胞癌和 12例正常膀胱黏膜组织中LRP的表达。结果 :65例膀胱移行细胞癌组织LRP阳性表达率为76 92 % ,明显高于正常膀胱黏膜组织 ;LRP在中、低分化肿瘤 (G2 +G3)阳性表达率明显高于高分化肿瘤 (G1)。结论 :LRP可能是引起膀胱移行细胞癌原发性耐药的原因之一     

In vitro antitumor activity of bropirimine against urinary bladder tumor cells

BACKGROUND: Bropirimine is an orally-active immunostimulant that has an antitumor effect on superficial transitional cell carcinoma of the bladder. The mechanism of its antitumor effect has not yet been clarified. MATERIALS AND METHODS: The cytokine-mediated antiproliferative activity of bropirimine was tested against cultured KK-47 and 724 cells using a modified human clonogenic tumor assay. Perpheral blood mononuclear cells (PBMCs) obtained from five healthy volunteers were co-cultured with bropirimine and the levels of various cytokines, including IFN-alpha, gamma, IL-1beta and TNF-alpha in the supernatants, were quantified. RESULTS: Colony formation by both cell lines was directly inhibited by bropirimine in a dose-dependent manner. In co-cultures of bropirimine and PBMCs, the inhibition of colony formation by both cell lines was not enhanced compared with the effect of bropirimine alone. No significant elevation of cytokines was detected in the presence of PBMCs. CONCLUSION: The results obtained suggest that bropirimine is like to have direct antitumor activity rather than a cytokine-mediated antitumor effect.     

尿核基质蛋白22和细胞角蛋白18在膀胱移行细胞癌中的表达及其临床意义

目的 探讨尿核基质蛋白22(NMP22)和细胞角蛋白18(CK18)在膀胱移行细胞癌中的表达及其临床意义.方法 采用酶联免疫吸附法(ELISA)对293例膀胱移行细胞癌、400例非移行细胞肿瘤、105例泌尿系良性病患者进行尿NMP22和CK18蛋白水平的检测.结果 膀胱移行细胞癌患者术前NMP22和CK18表达中位值分别为17.3 U/ml和484.2 U/L,非移行细胞肿瘤患者分别为6.8 U/ml和156.0 U/L,良性疾病患者分别为2.3 U/ml和66.6 U/L,3组之间进行比较,差异有统计学意义(P<0.001).以NMP22 10 U/ml和CK18 120 U/L为界值,其对膀胱移行细胞癌诊断的敏感度分别为79.2%和78.2%,特异度分别为88.6%和82.9%,两者联合检测的敏感度为91.7%.对术后患者动态观察,治疗有效患者的NMP22和CK18表达水平较治疗前明显下降,而复发、转移的患者则上升,差异有统计学意义(P<0.01).NMP22和CK18对膀胱移行细胞癌的检测有显著的相关性(r=0.689 P<0.0001).NMP22和CK18表达水平在不同病理分级和不同分期的患者中比较,差异有统计学意义(均P<0.01).结论 尿NMP22和CK18检测可作为膀胱移行细胞癌术前诊断、病情监测重要指标,两者联合检测可进一步提高敏感度.     

Inactivation of the FHIT gene favors bladder cancer development.

The fragile histidine triad (FHIT) gene located on chromosome 3p14.2 is frequently deleted in human tumors. We have previously reported deletions at the FHIT locus in 50% of bladder carcinoma derived cell lines and reduced expression in 61% of primary transitional carcinomas of the urinary bladder. To additionally investigate the role of FHIT alterations in the development of bladder cancer, we used heterozygous and nullizygous Fhit-deficient mice in a chemically induced carcinogenesis model. Results showed that 8 of 28 (28%) and 6 of 13 (46%) of the Fhit -/- and +/-, respectively, versus 2 of 25 (8%) Fhit +/+ mice developed invasive carcinoma after treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine. To explore the possibility of a FHIT-based gene therapy for bladder cancer, we studied the effects of restored Fhit protein expression on cell proliferation, cell kinetics, and tumorigenicity in BALB/c nude mice, with human SW780 Fhit-null transitional carcinoma derived cells. In vitro transduction of SW780 Fhit-negative cells with adenoviral-FHIT inhibited cell growth, increased apoptotic cell population, and suppressed s.c. tumor growth in nude mice. These findings suggest the important role of Fhit in bladder cancer development and support the effort to additionally investigate a FHIT-based gene therapy.     

A new method of implanting orthotopic rat bladder tumor for experimental therapies

We developed a model of orthotopic transplantation of bladder tumor cells in female Fischer rats using a new reproducible technique. After first performing the mechanical abrasion of a portion of the bladder urothelium with an Abrader inserted transurethrally via a catheter, we administered a suspension of 5-40 x 10(6) viable AY-27 tumor cells in sterile phosphate-buffered saline to the bladder cavity. This rapidly led to a tumor growth incidence of approximately 100%. The induced bladder tumors grew expansively into the bladder cavity from the surface (mucosa) and gradually invaded the submucosa, muscles, serosa and surrounding tissue (high-stage invasive transitional cell carcinoma). Size and staging were related to the quantity of tumor cells instilled into the bladder cavity. This model matches the characteristics of human bladder tumor more closely than other bladder cancer models induced with tumor cells. Moreover, it presents many advantages: the method is reproducible, tumors grow rapidly, they are directly attached to the bladder surface and they are always located on the bladder wall, in line with the urethra. This proves especially helpful for evaluating chemotherapeutic agents by different means such as in vivo fluorescence spectroscopy, a noninvasive method used in photodynamic therapy, or other methods designed to detect and treat transitional cell carcinoma.     

反义转化生长因子β1抑制人膀胱癌细胞体内外增殖的实验研究

目的 探讨反义转化生长因子β1(TGFβ1)对人膀胱癌细胞体内外增殖的抑制作用。方法 将携TGFβ1反义基因的逆转录病毒载体pRevTβ-AS转染膀胱癌EJ细胞,观察转染细胞体外及SCID小鼠皮下生长情况,通过免疫组化方法评价细胞增殖活性的改变;通过病理图像分析和电子显微镜技术观察体内生长的EJ细胞形态学和超微结构改变;采用流式细胞分析技术观察转染后细胞周期时相分布的变化。结果 pRevTβ-AS能有效抑制EJ细胞TGFβ1mRNA转录和蛋白表达。体外实验表明,转染反义TGFβ1可使EJ细胞体外增殖活性和S期细胞比例明显降低;体内实验证实,反义组种植肿瘤的形成和生长被有效抑制,DNA异倍体细胞比例和基因复制活跃程度明显低于对照组。结论 转染反义TGFβ1能明显减弱EJ细胞致瘤性,抑制肿瘤细胞体内外增殖活性。     

膀胱移行细胞癌的分子细胞遗传学研究

目的　分析膀胱移行细胞癌的染色体畸变。方法　采用 7,9,11,17号染色体着丝粒探针对 34例膀胱移行细胞癌患者尿液、30例膀胱冲洗液的脱落细胞核进行荧光原位杂交 (fluorescenceinsituhybridization ,FISH )研究 ,并同时做了细胞学检查。结果　 (1)膀胱癌患者尿液脱落细胞核中 7,9,11,17号染色体数目畸变阳性率分别为 2 3.5 %、38.2 %、14.7%和 11.8% ;冲洗液中各号染色体畸变阳性率分别为 30 .0 %、5 0 .0 %、2 6 .7%和 16 .7%。其中 9号染色体畸变率较高 ,但与膀胱癌分级、分期无明显关系 ;7号染色体数目畸变与膀胱癌的分期密切相关 ;11,17号染色体数目畸变与膀胱癌分级、分期无显著相关性。 (2 )膀胱癌患者尿液组中尿细胞学、FISH阳性率分别为 2 9.4%和 5 5 .9% ,两种方法联合后阳性率达 6 7.6 % ;膀胱冲洗液组中阳性率则分别为 2 7.6 %和 73.7% ,两种方法联合后阳性率达 80 .0 %。结论　膀胱癌的发生发展与染色体的畸变有关。FISH检测膀胱癌患者尿液、冲洗液脱落细胞间期核染色体数目畸变 ,有可能作为膀胱癌诊断、预后判断的一项辅助方法。     

RT-PCR法检测尿脱落细胞中XIAP的表达在膀胱癌诊断中的价值

目的:比较膀胱癌患者尿液脱落细胞中XIAP表达的RT-PCR检测法和常规尿脱落细胞病理学检测在膀胱癌诊断中的临床价值。方法:采用逆转录聚合酶链反应技术(RT-PCR)检测51例膀胱尿路上皮癌患者尿液脱落细胞中XIAP-mRNA的表达,同时行常规尿脱落细胞病理学检测,20例非肿瘤人员作为对照组。结果:实验组51例尿脱落细胞XIAP-mRNA RT-PCR检测阳性27例(53%),尿脱落细胞学病理学检测阳性12例(24%),对照组20例尿脱落细胞XIAP-mRNA检测阳性1例(5.0%),对照组尿脱落细胞病理学检测阳性0例(0%)。实验组RT-PCR检测膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的敏感性高于尿脱落细胞病理学检测,差异有极显著统计学意义(P<0.01),实验组RT-PCR检测膀胱尿路上皮癌患者尿中XIAP表达的敏感性显著高于非肿瘤对照组,差异有极显著统计学意义(P<0.01)。结论:膀胱尿路上皮癌患者尿脱落细胞中XIAP表达的RT-PCR检测法较常规尿脱落细胞病理学检测更敏感,临床上作为膀胱癌的筛选方法,有一定的临床价值。     

膀胱移行细胞癌中MAGE-A3基因的表达及意义

目的：研究膀胱移行细胞癌组织中黑色素瘤抗原家族A成员3（melanoma antigen family A,3,MAGE-A3）的表达及其临床意义.方法：采用反转录聚合酶链反应（RT-PCR）技术检测51例膀胱移行细胞癌患者癌组织（新鲜标本,Ta-T1期30例,T2-T4期21例;G1 22例,G2 16例,G3 13例）及其中10例患者癌旁正常组织的MAGE-A3mRNA表达.采用Western blot技术检测上述组织中MAGE-A3蛋白的表达.在上述部分组织中进一步应用免疫组化技术证实MAGE-A3蛋白的表达情况.结果：51例膀胱移行细胞癌组织中,26例（51％） MAGE-A3 mRNA表达阳性,10例癌旁组织表达均阴性.23例（45％） MAGE-A3蛋白表达阳性,10例癌旁组织表达均阴性.MAGE-A3蛋白为胞浆内染色.肿瘤不同分期、不同分级之间MAGE-A3mRNA及MAGE-A3蛋白表达的差异均无统计学意义（P＞0.05）.结论：MAGE-A3基因在膀胱移行细胞癌中有较高表达,而在癌旁组织无表达,有望成为膀胱移行细胞癌特异性免疫治疗的靶分子.     

联合survivin反义寡核苷酸和基因重组融合蛋白对人膀胱癌细胞的抑制作用

 目的 用体外实验探讨联合应用survivin反义寡核苷酸、基因重组融合蛋白TP40是否对膀胱移行细胞癌T24细胞具有协同抑制作用。方法 实验分为对照组、错义寡核苷酸组（MS）、反义寡核苷酸组（AS）、TP40组和联合组（AS+TP40）。RT PCR和Western blot法检测各组细胞survivin的表达；MTT法检测各组细胞生长情况；流式细胞仪检测各组细胞凋亡率；体外成瘤实验检测各组细胞的体外锚定非依赖性生长能力。处理时间为3天。 结果 所合成AS(300nM)对survivin mRNA的表达具有明显抑制作用。分别从转录水平和表达水平证明了联合组下调survivin的能力最强。MTT法显示联合组用药后生长抑制更加明显（P＜0.0001）,第3天细胞存活率仅12.2%，凋亡率达到96.37％。在软琼脂糖体外成瘤实验中，TP40组和AS组的体外锚定非依赖性生长的能力大大降低，联合组（TP40+AS）细胞几乎没有形成克隆。结论 采用survivin反义寡核苷酸封闭survivin的表达，可以增强TP40对膀胱移行细胞癌T24细胞的生长抑制作用，呈现协同效应。     

Simultaneous occurrence of adenocarcinoma and transitional cell carcinoma as two separate primary tumors in the bladder

We report on a 56-year-old man who had two distinct and separate primary tumors of the bladder simultaneously: one mucin-producing adenocarcinoma and the other a transitional cell carcinoma. Despite local extension of the adenocarcinoma beyond the bladder wall, surgical resection has resulted in a 6-year recurrence-free survival of the patient.     

VEGF和 bFGF在浅表膀胱移行细胞癌中的表达及意义

背景与目的：血管内皮生长因子（vascular endothelial growth factor,VEGF)和碱性成纤维细胞生长因子（basic fibroblast growth factior,bFGF)都能促进血管内皮细胞分裂和诱导血管形成,是肿瘤的生长,浸润,和转移过程中非常重要的物质,但它们在单发性和多发性浅表膀胱移行细胞癌中表达的差异则未见报道,本研究主要是探讨VEGF和bFGF在浅表膀胱移行细胞癌中的表达及意义。方法：采用免疫组化法对60例浅表膀胱移行细胞癌组织及10例正常膀胱组织进行VEGF和bFGF的检测,观察单发性和多发性浅表膀胱移行细胞癌组织中VEGF和bFGF表达的关系。结果：多发性浅表膀胱移行细胞癌的VEGF阳性率为55.6%,bFGF阳性率为50.0%,以及VEGF和bFGF共同表达阳性率为50.0%。均明显高于单发者的水平,而高水平表达的多发性肿瘤患者的术后复发率61.1%也明显高于单发组的复发率（单发组的复发率16.7%)。结论：VEGF和bFGF表达水平的高低与浅表膀胱移行细胞癌的生物学行为有关。     

Diabetes mellitus and bladder cancer — An epidemiological relationship?

An epidemiological association between diabetes mellitus and transitional cell carcinoma of the bladder has been proposed. This study looked retrospectively at 125 patients with transitional cell carcinoma of the bladder as a study group and 80 other hospital patients with conditions not specifically associated with diabetes mellitus as a control group. Diabetic patients had an increased, significant odds ratio for bladder cancer compared with non diabetics even after adjustment for smoking and age [OR: 2.69 p=0.049 (95% CI 1.006-7.194)] A history of smoking OR 2.16 p=0.013 (95% C.I. 1.175-3.964) is a significant independent association with transitional cell carcinoma of the bladder as is age: p=0.001 OR 1.07. We propose potential pathogenic pathways for transitional cell carcinoma of the bladder in diabetic patients based on altered integrin and cadherin distribution in urothelial cells in diabetic patients. A larger study is planned to confirm an association between diabetes mellitus and transitional cell carcinoma of the bladder.     

联合检测尿液Survivin浓度及脱落细胞端粒酶活性在膀胱移行细胞癌中的诊断价值

目的:探讨尿液中Survivin浓度检测联合尿脱落细胞端粒酶活性检测在膀胱移行细胞癌诊断中的应用价值。方法:收集膀胱移行细胞癌患者尿液64例及非膀胱癌对照组患者尿液42例,应用ELISA方法检测各尿液标本中Survivin浓度及脱落细胞端粒酶的活性。结果:尿液中Survivin在膀胱癌诊断中的敏感度为82.8%,特异度为85.7%;尿脱落细胞端粒酶活性检测对膀胱癌诊断的敏感度为84.4%,特异度为69.0%;两种方法联合应用,两者均为阴性时诊断为阴性,否则为阳性,诊断膀胱癌的敏感度为95.3%,特异度为64.3%。结论:应用ELISA方法对尿液中Survivin浓度及脱落细胞端粒酶活性联合检测可显著提高膀胱移行细胞癌诊断的敏感度,而特异度变化不大,因此,两种指标联合检测较单一检测对膀胱移行细胞癌诊断更有价值。     

野生型PTEN基因高表达对膀胱移行细胞癌EJ细胞的抑癌作用

目的 探讨外源性野生型人酪氨酸磷酸酶（PTEN）基因的高表达对膀胱移行细胞癌EJ细胞的抑癌作用。方法 利用携带人PTEN基因的野生型、磷酸酶域突变型质粒体外分别转染人膀胱移行细胞癌EJ细胞。Western blot检测目的基因PTEN的表达,观察细胞形态变化及超微结构变化;MTIO法检测细胞增殖率及转染细胞对吡柔比星（THP）和丝裂霉素（MMC）的敏感性;Western blot法检测bcl-2蛋白的表达。以空载质粒作为对照。结果 质粒转染后,EJ细胞的PTEN蛋白表达上升75．0％。转染野生型质粒后,EJ细胞异型性低,出现典型凋亡小体,细胞增殖率下降40．1％,bcl-2蛋白表达被下调,并提高了对THP和MMC的敏感性。而转染突变型质粒的EJ细胞则无此作用。结论 野生型PTEN基因在体外对膀胱移行细胞癌EJ细胞增殖有明显抑制作用,诱导细胞凋亡,磷酸酶域突变型PTEN基因无此作用。野生型PTEN的抑癌作用可能与其对bcl-2蛋白表达的下调有关。     

Cyclin E overexpression in transitional cell carcinoma of the bladder

We attempted to clarify the relationship between cyclin E to p27(Kip1), Ki-67 and clinicopathologic features in transitional cell bladder carcinoma. Immunohistochemical staining of archival tissue specimens of transitional cell bladder carcinoma obtained from 94 patients was performed by the labeled streptavidin-biotin-peroxidase method. Overexpression of cyclin E protein was observed in 38 of the 94 (40.4%) specimens, and was positively correlated with histological grade, Ki-67 LI and p27(Kip1) labeling index (LI). These data suggest that cyclin E may be associated with aggressive tumor growth, and may have a relationship with p27(Kip1) for the regulation of cell cycle progression in transitional cell bladder carcinoma.     

Human transitional cell carcinoma in the NMRI-nu/nu mouse bladder: a new animal model for the in vivo use of monoclonal antibodies and cytotoxic agents

Human transitional cell carcinoma of the bladder was successfully transplanted into the bladder of the NMRI-nu/nu mouse. Transplantation of a single-cell suspension from a human Grade II transitional cell carcinoma had an acceptance rate of 33%. The tumors showed invasive growth and could be identified by an anti-human monoclonal antibody. This tumor model can be valuable as an in vivo test system for the local use of cytotoxic agents and monoclonal antibodies in diagnosis of and therapy for human bladder carcinoma.