Radioimmunoimaging of human bladder tumor xenografts in nude mice by using monoclonal antibodies

Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (111In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The 111In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoimaging and possibly therapy.     

Inhibition of tumor cell growth in vitro by murine monoclonal antibodies that recognize a proliferation-associated cell surface antigen system in rats and humans

The mouse monoclonal antibody (MoAb) B3 raised against a rat bladder cancer cell line and the MoAbs HBJ127 and HBJ98 raised against a human bladder cancer cell line recognize homologous antigens predominantly present on proliferating cells of the corresponding species. Examination of MoAb-defined antigen and epitopes revealed that both HBJ127 and HBJ98 MoAbs defined a human cell surface glycoprotein complex having an apparent molecular weight of 125,000-130,000 which was composed of a heavy subunit of a glycoprotein nature (Mr 90,000-95,000) and a disulfide-linked light subunit of protein nature (Mr 30,000-35,000), but the HBJ127 and HBJ98 MoAbs recognized a protein epitope and a sugar epitope on the heavy subunit, respectively. Likewise, the B3 MoAb recognized a protein epitope on the heavy subunit of a rat cellular glycoprotein complex of similar composition to the HBJ127/HBJ98-defined human antigen. Addition of the B3 MoAb to rat and the HBJ127 or HBJ98 MoAb to human tumor cells inhibited the nucleic acid synthesis or the proliferation of the tumor cells in vitro in a dose-dependent manner. The target tumor cells exposed to MoAb could regrow when they were freed from the antibody, indicating that the effect of these MoAbs on the tumor cells is cytostatic and reversible. These MoAbs did not cause down-regulation of the cell surface antigen and did not arrest the cell cycle in a certain phase. These observations indicate that the Mr 125,000 glycoprotein cell surface component detected in both rat and human systems may play a requisite role for cell proliferation and that our MoAbs could inhibit the function by binding to the functionally proximal region of the component.     

Cytotoxicity of anti-c-erbB-2 immunoliposomes containing doxorubicin on human cancer cells.

We have examined the selective cytotoxicity of immunoliposomes containing doxorubicin (chemoimmunoliposomes, CILs) targeting the c-erbB-2 gene product (gp185) or gp125. Anti-gp185 and anti-gp125 CILs were prepared by conjugation of doxorubicin-containing liposomes with monoclonal antibodies SER4 (IgG) and HBJ127 (IgG) respectively. Both CILs bound to human SKBr-3 breast cancer cells and MKN-7 human gastric cancer cells, which express both antigens in high density. The IC50 of anti-gp185 CILs on protein synthesis by SKBr-3 cells was respectively 2- and 25-fold lower than that of anti-gp125 CILs and unmodified liposomes. Furthermore, anti-gp185 CILs significantly inhibited neither the phytohaemagglutin response of normal lymphocytes nor protein synthesis of gp185-negative T24 bladder cancer. Quantitative analysis of cell-associated doxorubicin revealed that, compared with anti-gp125 CILs, anti-gp185 CILs required, respectively 4.5 and 4.3 times less doxorubicin association in SKBR-3 and MKN-7 cells, for 50% cytotoxicity. In addition, flow cytometric analysis showed that both SKBr-3 and MKN-7 internalised more anti-gp185 CILs and processed them more efficiently than anti-gp125 CILs. These results suggest that anti-gp185 CILs act selectively against gp185-expressing cancer cells and that gp185 is a more sensitive antigen for CIL cytotoxicity associated with endocytosis activity.     

Antibody drug carrier for immunotherapy of superficial bladder cancer: ultrastructural studies.

Superficial bladder cancer represents a promising target for intravesical, antibody-guided therapy. The construction of an optimum antibody-cytotoxic drug conjugate depends mostly on the appropriate selection of a monoclonal antibody (mAb). We have used immunogold labeling and SEM to specifically map the distribution of antigens expressed on three bladder cancer cell lines and on the luminal surface of biopsies from human transitional cell carcinoma of various grades and from normal bladder mucosa. The 48-127 mAb, which recognizes a M(r) 54,000 surface glycoprotein (gp54), was found to be very promising as a potential drug carrier. This antibody reacts with the surface of cells from low- and high-grade tumors; it does not react with the normal urothelium. Labeling of normal bladder mucosa was observed, however, on microvillous intermediate urothelial cells occasionally exposed by small areas of desquamation. The 48-127 mAb could target drugs to all areas of transformed urothelium while avoiding drug delivery to the normal, undesquamated bladder mucosa. Kinetics of gp54/48-127/gold complexes were tested in vitro with T24 and RT4 human bladder carcinoma cell lines incubated in the presence of the 48-127 mAb directly conjugated with 17.7-nm gold particles. Internalization of the gp54/48-127/gold complex was readily demonstrated by transmission electron microscopy. These results suggest that the 48-127 mAb represents a valuable drug carrier for intravesical therapy, allowing specific tumor targeting and internalization of various cytotoxic agents.     

In vitro targeting and cytotoxicity of adriamycin in liposomes bearing monoclonal antibody against rat or human gp125 cell proliferation-associated antigen

Chemoimmunoliposomes (CIL) were prepared by entrapping adriamycin in monoclonal antibody (mAb)-coated liposomes and examined for their binding capacity and cytotoxicity to relevant target tumor cells. Sonicated unilamellar liposomes were coated with B3 and HBJ127 mouse, mAbs, which recognize a rat and a homologous human cell proliferation-associated surface antigen, gp125, respectively, and then adriamycin was entrapped in the liposomes by means of transmembrane Na+/K+ gradients using valinomycin. These CIL selectively bound with relevant target tumor cells bearing the corresponding gp125 antigen, such as BC47 rat bladder cancer, FTL-13 rat thymic lymphoma, T24 human bladder cancer and Molt-4 human leukemia cells, although the binding capacities of the CIL to bladder cancer cells were relatively larger than those to lymphoma cells in both rat and human systems. This difference in the target cell binding was found to be attributable to the amount of gp125 antigen expressed on each target tumor cell, as determined by a Scatchard plot analysis. In accordance with the target cell binding capacities of CIL preparations, the CIL displayed much higher cytotoxic activity to bladder cancers than to lymphomas in both rat and human systems. In conjuction with our previous finding that gp125 antigen is expressed on tumor cells but not on resting normal cells, these findings indicate that CIL composed of anti-gp125 mAb will be useful for tumor therapy and that the antitumor efficacy is dependent upon the extent of the antigen expression on target tumor cells.     

In vitro Targeting and Cytotoxicity of Adriamycin in Liposomes Bearing Monoclonal Antibody against Rat or Human gp125 Cell Proliferation-associated Antigen

Chemoimmunoliposomes (CIL) were prepared by entrapping adriamycin in monoclonal antibody (mAb)-coated liposomes and examined for their binding capacity and cytotoxicity to relevant target tumor cells. Sonicated unilamellar liposomes were coated with B3 and HBJ127 mouse, mAbs, which recognize a rat and a homologous human cell proliferation-associated surface antigen, gp125, respectively, and then adriamycin was entrapped in the liposomes by means of transmembrane Na^±/K^±gradients using valinomycin. These CIL selectively bound with relevant target tumor cells bearing the corresponding gp125 antigen, such as BC47 rat bladder cancer, FTL-13 rat thymic lymphoma, T24 human bladder cancer and Molt-4 human leukemia cells, although the binding capacities of the CIL to bladder cancer cells were relatively larger than those to lymphoma cells in both rat and human systems. This difference in the target cell binding was found to be attributable to the amount of gp125 antigen expressed on each target tumor cell, as determined by a Scatchard plot analysis. In accordance with the target cell binding capacities of CIL preparations, the CIL displayed much higher cytotoxic activity to bladder cancers than to lymphomas in both rat and human systems. In conjuction with our previous finding that gp125 antigen is expressed on tumor cells but not on resting normal cells, these findings indicate that CIL composed of anti-gp125 mAb will be useful for tumor therapy and that the antitumor efficacy is dependent upon the extent of the antigen expression on target tumor cells.     

Growth-regulated surface glycoproteins of human bladder cancer

Clinical studies of human bladder cancer cells with mouse monoclonal antibodies (mAbs) have revealed two surface glycoproteins (T43 and T138) expressed by aggressive cancers and not by normal cells and a differentiation antigen (T16) expressed by normal and tumor cells of urothelial origin (Y. Fradet et al., Proc. Natl. Acad. Sci. USA, 81: 224-228, 1984; Y. Fradet et al. Cancer Res., 46: 5183-5188, 1986). To investigate further the possible association of these antigenic phenotypes with growth advantage of tumor cells, their expression, according to phases of the cell cycle and growth states (exponential and plateau phase) was studied in the human bladder carcinoma cell line T24. Expression of the p21 ras oncogene product, the Thomsen-Friedenreich antigen and the HLA class I antigen were also studied with mAbs. Multiparameter flow cytometry was used to determine antigen expression and DNA content of cells stained with mAbs and propidium iodide. Two antigens, T16 and T43, showed marked variations of their expression according to the growth status of the cells, although with an inverse profile. T16 was expressed on resting cells up to 12.5 times more than on exponentially growing cells. Conversely, T43 expression increased by a factor of 4.5 on actively proliferating cells. However, there was no preferential expression of either antigen in any one phase of the cell cycle. None of the other antigens studied, including the p21 protein, showed any density variation with either cell cycle or growth states. The results of these studies suggest that T43 may be associated with a growth advantage of tumor cells and that T16 has the characteristics of a differentiation antigen whose expression is induced on resting cells. These findings may have implications for the potential clinical use of these mAbs.     

Identification by monoclonal antibodies of an antigen shed by human bladder cancer cells

The reactivities of two anti-bladder cancer monoclonal antibodies, AN43 and BB369, were characterized. AN43 and BB369 reacted with a majority (greater than 50%) of bladder cancer tissue sections tested by immunoperoxidase staining. When tested against a panel of 27 normal human tissues, AN43 and BB369 reacted only with urothelium and stomach. AN43 and BB369 showed identical binding patterns and competed for binding on bladder cancer cells, suggesting that the two antibodies react with identical or spatially close epitopes. Bound BB369 antibody was rapidly shed from the surface of viable UM-UC-9 human bladder cancer cells. The antigen was found in spent tissue culture medium from the UM-UC-9 human bladder cancer cell line. AN43 and BB369 define a shed bladder tumor-associated antigen with limited distribution on normal tissues. The antigen is different from bladder tumor-associated antigens defined by other monoclonal antibodies and may be useful for the diagnosis and follow-up of patients with bladder cancer.     

MAGE-A3在人类膀胱癌干细胞中的表达

目的：研究肿瘤相关抗原,黑色素瘤抗原家族A成员3（melanoma antigen family A,3;MAGE-A3）,在人类膀胱癌干细胞中的表达情况,并探讨其意义.方法：采用反转录聚合酶链反应（RT-PCR）技术和Western blot技术检测MAGE-A3在人类膀胱癌细胞系T24细胞中及从其中分离出来的具有干细胞特性的T24侧群细胞中的表达情况;采用免疫荧光双标记技术检测MAGE-A3和膀胱癌干细胞的一个标志物CD133在T24侧群细胞中的共表达情况.结果：在mRNA和蛋白水平,MAGE-A3在具有癌干细胞特性的T24侧群细胞中的表达水平明显高于对应的母系T24细胞;MAGE-A3和膀胱癌干细胞标志物CD133在T24侧群细胞中有阳性共表达.结论：MAGE-A3在人类膀胱癌干细胞中有特异性的高表达,有望成为膀胱癌干细胞一个新的、特异性的标志物,及针对膀胱癌干细胞进行免疫治疗的一个新的靶点.     

细胞黏附分子CD44v6对膀胱癌细胞增殖、凋亡及迁移的影响

目的:探讨细胞黏附分子CD44v6对膀胱癌T24细胞生物学行为的影响.方法:免疫细胞化学法检测正常膀胱移行上皮细胞和膀胱癌T24细胞中CD44v6蛋白的表达.用不同浓度的抗CD44v6单克隆抗体封闭细胞24、48和72 h后,MTT法检测细胞的生长抑制率.FCM检测封闭前后细胞凋亡率,并通过免疫细胞化学法检测细胞凋亡相关蛋白Bcl-2和Bax表达水平的变化;Transwell小室检测细胞迁移特性的变化.结果:膀胱癌T24细胞表达CD44v6蛋白,而正常膀胱移行上皮细胞几乎不表达.MTT法检测结果显示,抗CD44v6单克隆抗体可抑制T24细胞的生长(P＜0.01).抗CD44v6单克隆抗体能增加T24细胞的凋亡率(P＜0.01),并且Bcl-2蛋白表达降低,Bax蛋白表达增加(P＜0.01).抗CD44v6单克隆抗体封闭后T24细胞的迁移能力受到抑制,差异有统计学意义(P＜0.05).结论:抗CD44v6单克隆抗体封闭可以抑制膀胱癌细胞的增殖及迁移能力,同时诱导膀胱癌细胞凋亡,其机制可能与Bcl-2和Bax基因表达水平变化有关.     

Toxicity of ribosome-inactivating proteins-containing immunotoxins to a human bladder carcinoma cell line

Immunotoxins were prepared by linking the type I ribosome-inactivating proteins (RIP) momordin I, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6 to the 48-127 monoclonal antibody (MAb) recognising a glycoprotein (gp54) expressed on all human bladder tumours tested and on human bladder carcinoma cell lines, in particular on the T24 cell line. T24 cells required a 2 hr contact with immunotoxins to ensure binding and endocytosis. A time course of exposure, followed by further incubation without the immunotoxins, showed that maximum inhibition of protein synthesis by T24 cells was reached after 2 hr of contact followed by 3 days without the immunotoxins. Under optimal conditions, 48-127/RIP immunotoxins at nanomolar concentrations inhibited by 50% protein synthesis of target T24 cells. No toxicity was observed if (i) target cells were treated with non-conjugated RIP, (ii) target cells were treated with momordin I- or PAP-S-containing immunotoxins made with an irrelevant antibody and (iii) a non-target cell line was treated with the same 2 RIP conjugated to 48-127 antibody. The in vitro selective toxicity of these immunotoxins encourages further studies in view of a possible use in clinical trials for the local therapy of human bladder carcinomas. © 1996 Wiley-Liss, Inc.     

MicroRNA-449a acts as a tumor suppressor in human bladder cancer through the regulation of pocket proteins

Frequent downregulation of microRNA-449a (miR-449a) was detected in 14 human bladder cancer tissues. The restoration of miR-449a inhibited cell growth and induced G1-phase arrest in T24 and 5637 human bladder cancer cells. CDK6 and CDC25a were downregulated after miR-449a treatment, resulting in the functional accumulation of the pocket proteins Rb and p130. The growth of T24 tumor xenografts was suppressed by exogenous miR-449a, and the nuclear proliferation antigen Ki-67 was downregulated in miR-449a-treated tumors. These results suggest a tumor-suppressive role for miR-449a in human bladder cancer.     

Glycosylation in bladder cancer

Complex carbohydrates are major components of the cell membrane and they play crucial roles in cell-cell and cell-extracellular matrix interactions, as well as in signal transduction. They consist of three kinds of molecular species; glycoproteins, proteoglycans, and glycosphingolipids. There is a distinct difference in carbohydrate profiles between normal and tumor tissues. The characteristic carbohydrate expression associated with malignant transformation is caused by "aberrant glycosylation" catalyzed by specific glycosyltransferases and glycosidases. A close relationship between blood type antigens and bladder cancer was first established in the 1960s, using the classic red-cell adherence test. Lectin immunohistochemical staining eventually replaced the red-cell adherence test. In the 1980s, several monoclonal antibodies were raised against complex carbohydrates, and the clinico-pathologic significance of blood type antigens in bladder cancer was investigated using these antibodies. Recent studies have demonstrated the high sensitivity and specificity of immunostaining for Lewis X antigen, a carbohydrate blood type antigen, in exfoliated cells from voided urine samples. Other than blood type antigens, the significance of aberrant glycosylation in bladder cancer has been demonstrated in a number of articles. For instance, overexpression of the ganglioside (an acidic glycosphingolipid which has sialic acid) GM3 induces apoptosis and reduces invasive potential in a bladder cancer cell line. Hyaluronic acid promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. The expression of N-acetylglucosaminyltransferase V and beta,1-6 branching N-linked oligosaccharides is closely related to low malignant potential in bladder cancer. Selectins and galectins, specific ligands for carbohydrate antigens, are also key molecules involved in the apoptosis and metastasis of cancer cells. Thus, proteoglycans, glycoproteins, and glycosphingolipids, and their ligands, play crucial roles in the malignant transformation, invasion, and metastasis of bladder cancer. A novel diagnostic and therapeutic approach may be possible by taking advantage of innovative techniques in glycobiology.     

Cell surface antigens of human bladder tumors: definition of tumor subsets by monoclonal antibodies and correlation with growth characteristics

Normal human urothelium and tumors of urothelial origin were analyzed with a panel of seven mouse monoclonal antibodies that identify surface antigens of cultured bladder cancer cell lines. Three categories of antigens were defined on the basis of differential expression on normal urothelium versus bladder tumors. Om5 (a category 1 antigen) is a highly restricted, differentiation antigen detected in the normal urothelium of 50-60% individuals. No other normal cell type in Om5- or Om5+ individuals expresses Om5. The incidence of Om5 expression in superficial bladder tumors is significantly higher (88%) than in normal urothelium, whereas its expression in invasive or metastatic tumors is far lower (20%), suggesting Om5 gain/loss in bladder tumors. Paired biopsies of normal urothelium and bladder tumors from the same individuals have shown Om5 induction in the superficial bladder tumors of Om5- individuals and Om5 loss in invasive bladder cancers of Om5+ individuals. Category 2 antigens (T43, T138, T23) are not expressed by normal urothelium or most superficial bladder tumors but are detected on a high proportion of invasive or metastatic bladder tumors, indicating that category 2 antigens are associated with late stages of tumor progression. Category 3 antigens (T16, T87, J143) provide lineage markers for normal or neoplastic cells of urothelial origin, being found on normal urothelium and virtually all bladder tumors. Thus, differential expression of category 1 and 2 antigens divide bladder tumors into distinct subsets, and these subsets correlate with pathological and clinical features of the disease.     

Identification of cell proliferation-associated epitope on CD98 oncoprotein using phage display random peptide library

CD98 is known as a cell surface antigen expressed in proliferating normal tissues and in almost all tumor cells. Although the function of CD98 is not yet fully elucidated, it is suggested that CD98 is concerned functionally in lymphocyte activation, cell proliferation, and malignant transformation. Monoclonal antibody against human CD98 heavy chain (h.c.), termed HBJ127, shows inhibition of lymphocyte activation and tumor cell growth in vitro . These observations suggest that the epitope recognized by HBJ127 may be crucial for CD98 function. In the present study, the authors investigated the epitope recognized by HBJ127 using a phage display random heptapeptide library. Approximately 2.4 × 10⁴-fold amplification of eluted phage titer was obtained after three rounds of panning of the phage library against HBJ127. Seven different heptapeptide sequences were isolated from 30 randomly selected clones of the post-panning phage population. A homology search using ClustalW identified the peptide sequence corresponding to ⁴⁴²AFS⁴⁴⁴ of human CD98 h.c. It was also found that ⁴⁴³F is a human-specific amino acid by comparing sequences of human, rat, and mouse origin. Reduced reactivity of HBJ127 was detected against the phenylalanine-substituted peptide but not detected against the alanine or serine-substituted one. It has been identified that HBJ127 reacts only with human species and the HBJ127 epitope position is predicted in 418–529 of human CD98 h.c. From these results and observations, it was estimated that ⁴⁴²AFS⁴⁴⁴ of human CD98 h.c. may be the HBJ127 epitope. Moreover, ⁴⁴³F may be critical for the binding of HBJ127 against human CD98 h.c. ( Cancer Sci 2007; 98: 1696–1700)     

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high-grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty-eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue-specific expression, KU-BL-1 and KU-BL-2 were identified as immunogenic antigens with restricted tissue expression. KU-BL-1 was found to be a putative human lipoic acid synthetase with a metal-binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU-BL-1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU-BL-2 was found to be a putative human kelch-like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU-BL-2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU-BL-2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA-A24 binding KU-BL-2 peptides. Therefore, KU-BL-1 and KU-BL-2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.     

Antibody epitope peptides as potential inducers of IgG antibodies against CD98 oncoprotein

An epitope is an antibody-recognition site on a target antigen. As such, active immunization of epitope peptides may induce therapeutic efficacy equivalent to the administration of parent antibody medicines. In the present study, we designed peptides based on the epitope recognized by the tumor-suppresive anti-CD98 monoclonal antibody HBJ127, and investigated their efficacy for induction of antitumor immunity. The immune sera showed reactivity against the corresponding peptide–keyhole limpet hemocyanin (KLH) and peptide–bovine serum abumin (BSA) conjugates, although they did not react with CD98-positive HeLa cells or recombinant CD98 heavy chain. To elucidate whether the epitope peptide failed to induce antitumor immunity or not, we constructed the IgG1, κ Fab phage display libraries from spleen cells of immunized mice and tried to retrieve CD98-reactive recombinant Fab (rFab) fragments by panning against either epitope peptide–BSA conjugates or live HeLa cells. RFab fragments retrieved from peptide–BSA panning showed no reactivity to HeLa cells. Their variable-region sequences were different from HBJ127. However, rFab fragments retrieved from HeLa cell panning showed reactivity to CD98 by indirect immunofluorescence and immunoprecipitation. Moreover, they were structurally almost identical to HBJ127. Although the immunogenicity of epitope peptides may be insufficient for induction of expected antitumor activity in vivo , we used antibody phage display to show that IgG antibodies almost identical to HBJ127 were an undetectable population in epitope peptide-induced immune sera. ( Cancer Sci 2009; 100: 126–131)     

Characterization of cell lines derived from a multiply aneuploid human bladder transitional-cell carcinoma, UCRU-BL-13

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.     

miR-623在膀胱癌中的表达及靶向Fascin1抑制膀胱癌细胞迁移和侵袭

目的:探讨miR-623在膀胱癌中的表达及通过靶向Fascin1对膀胱癌细胞迁移和侵袭能力的影响。方法:采用实时荧光定量PCR检测正常膀胱上皮组织、膀胱癌组织、正常永生化膀胱上皮细胞系(SV-HUC-1)和膀胱癌细胞系（T24、UMUC3）中miR-623的表达量;采用脂质体瞬时转染miR-623 mimics,划痕实验和Transwell侵袭实验检测miR-623过表达后膀胱癌细胞迁移、侵袭能力的改变;生物信息学预测miR-623的作用靶蛋白。miR-623过表达后Western blot及双荧光素酶报告基因检测其靶点的表达及结合情况;使用Fascin1特异性siRNA观察膀胱癌细胞迁移和侵袭能力的变化,并同时转染miR-623 inhibitor进行恢复实验。结果:miR-623在膀胱癌中的表达水平显著低于在正常膀胱组织中的表达（P＜0.05）,在膀胱癌细胞系（T24、UMUC3）中的表达水平显著低于正常膀胱细胞系(SV-HUC-1)（P＜0.05）;miR-623过表达显著抑制T24和UMUC3细胞的迁移和侵袭能力。生物信息学预测Fascin1为miR-623的靶基因,在T24和UMUC3细胞中过表达miR-623,能够显著降低Fascin1的蛋白水平;荧光素酶报告基因分析结果证实miR-623作用于Fascin1的3'-UTR。下调Fascin1表达能够抑制膀胱癌T24和UMUC3细胞的迁移和侵袭能力,同时抑制miR-623的表达能够提高细胞的迁移和侵袭能力。结论:miR-623在膀胱癌中表达水平降低,是一个抑癌因子;并可能通过靶向Fascin1调节膀胱癌的侵袭和转移能力。     

Relationship of monoclonal antibody binding to estrogen and progesterone receptor content in breast cancer

Three monoclonal antibodies--H59, H71, and H72--which react with human breast cancers have been developed using the estrogen-dependent human breast cancer cell line, ZR-75-1, as the immunogen. H59 bound only to estrogen receptor-positive, estrogen-regulated breast cancer cells in culture, whereas H71 and H72 bound breast cancer cells irrespective of the estrogen receptor content. All three antibodies have minimal cross-reactivity with non-breast tissue culture cell lines. The three antigens appear to be glycoproteins located on the cell surface. H59 and H72 antigens bound preferentially to the apical surface of duct cells and may be secreted; H71 antigen demonstrated no evidence of an apical orientation or secretion. The binding of the antibodies to fixed cryosections from 152 breast cancer and 111 benign breast disease specimens has been evaluated using a radioimmunoassay. Eighty-five % of breast cancer and almost 100% of benign disease specimens were bound by at least one antibody. H59 bound 39%, H71 bound 51%, and H72 bound 65% of cancer specimens. Estrogen receptor and progesterone receptor analyses were obtained on 141 specimens. H59 bound almost exclusively to tumor specimens which contained estrogen and/or progesterone receptor, but not to all receptor-positive tumors. Therefore, the H59 antigen appeared to be present on a subset of estrogen receptor-positive tumors. Considering that it bound only to estrogen-regulated cells in culture, the antigen may be estrogen regulated, and its presence may predict a response to hormone therapy. H71 and H72 recognized cell surface differentiation antigens but bound tumor specimens regardless of the receptor content. These antibodies may be useful as independent variables for predicting response to therapy and prognosis of patients with breast cancer.