New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: inhibition of endothelial cell growth

The growth of endothelial cells is necessary for angiogenesis, which in turn is required for later steps of tumor progression. In an attempt to purify new modulators of endothelial cell growth from the conditioned medium of human urinary bladder carcinoma cells, we isolated a small and stable oligonucleotide containing 10 to 16 bases. This oligonucleotide inhibited the growth of endothelial cells in vitro and was identified as a fragment of transfer RNA (tRNA). When unfractionated bovine tRNA was added to the cell culture, it specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines. In contrast, ribosomal RNA, total yeast RNA, and single nucleosides from tRNA hydrolysate had no effect. These results demonstrate a new role for tRNA and its fragment as a selective endothelial cell inhibitor in vitro.     

The resistance of epithelia to vascularization: Proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R). contains approximately 6 μg protein/ g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.     

Tenascin-C and carcinoma cell invasion in oral and urinary bladder cancer

Carcinoma invasion is a complex process regulated by genetic and epigenetic factors as well. A relevant supportive condition for cancer cell migration is the reorganization of the extracellular matrix (ECM), which is realized in an orchestrated multicellular manner including carcinoma cells and stromal fibroblasts. An important key player in the process of ECM reorganization is Tenascin-C (Tn-C). The molecule occurs as different isoforms generated by alternative splicing and de novo glycosylation. Large variants of Tn-C are abundantly re-expressed in the invasive front of many carcinoma types. A special role for initiating migration and accompanied epithelial to mesenchymal transition has been suggested. Here, we review the current knowledge concerning the tumor biological importance of Tn-C, the synthesis and alternative splicing during the invasive process in general, and give an overview on the impact of Tn-C in urothelial carcinoma of the urinary bladder (UBC) and oral squamous cell carcinoma (OSCC).     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human unbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA)_and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70°C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

肿瘤条件培养基对人脐静脉内皮细胞增殖、黏附、迁移能力的调节

本文旨在研究肿瘤条件培养基(tumor conditioned medium,TCM)对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)增殖、黏附和迁移能力的影响.采用MTT法测定TCM作用24 h后内皮细胞的增殖水平,实验设对照组、TCM原液(TCM stoc...     

后腹腔镜治疗上尿路移行细胞癌

目的：比较后腹腔镜肾输尿管全切及膀胱袖状切除术与开放手术治疗上尿路移行细胞癌的临床疗效。方法：回顾分析我院2003年3月至2008年10月行后腹腔镜肾输尿管全切及膀胱袖状切除术治疗上尿路移行细胞癌47例,其中后腹腔镜手术26例（A组）,传统的开放手术21例（B组）。对两组的临床疗效及随访结果等进行对比研究。结果：两组在发病年龄、性别、肿瘤大小、手术时间比较差异无显著性意义;A组术中出血量（98.2±28.5）ml、术后（33.6±12.4）h肠功能恢复、（43.5±12.5）h下床活动、应用止痛药（1.5±0.5）d、静脉应用抗生素（3.5±1.1）d、术后住院天数（6.5±1.5）d、（20.0±8.5）d恢复正常工作,明显优于B组,差异有统计学意义（P＆lt;0.05）;A组并发症少于B组。两组平均随访（36.6±16.0）、（38.0±16.7）月,5年无复发生存率和总生存率比较无显著性差异（P＆gt;0.05）。结论：后腹腔镜肾输尿管切除及膀胱袖状切除术与开放手术相比,疗效相当,创伤小、痛苦少、术后恢复快、并发症少,可作为上尿路移行细胞癌的首选治疗方法。     

Claudins and ki-67: potential markers to differentiate low- and high-grade transitional cell carcinomas of the urinary bladder

Updated classification of urothelial cell cancer differentiates low-grade and high-grade cancers, which determines potential clinical outcome. Substantial interobserver variability necessitates new biomarkers to ensure classification. Claudins' specific expression pattern characterizes normal tissues, different tumor types, and defined grades of tumor differentiation. The aim of this study was to examine the expression pattern of claudins and proliferation marker Ki-67 in low-grade and high-grade urothelial cell cancers compared with independent control samples of non-tumorous urothelium, as well as to reveal the predictive usefulness of claudins. The expression of claudins-1, -2, -3, -4, -5, -7, and -10 and Ki-67 was studied with quantitative immunohistochemistry and real-time RT-PCR with relative quantification in 103 samples: 86 urothelial cell cancers (27 low grade, 59 high grade) and 17 non-tumorous urothelia. Results were analyzed regarding overall survival and recurrence-free period as well. High-grade tumors overall showed significantly higher claudin-4 and Ki-67 and significantly lower claudin-7 expression when compared with low-grade ones. High-grade tumors revealed significantly shorter overall survival in Kaplan-Meier analysis. Claudin-4, claudin-7, and Ki-67 might be used as potential markers to differentiate low-grade and high-grade urothelial cell cancers, thereby possibly enhancing accuracy of pathological diagnosis and adding further information to clinical outcome.     

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma

Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation. This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National Bladder Cancer Project and by the Medical Research Service of the Veterans Administration.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.     

Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.     

Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Long-term human breast carcinoma cell lines of metastatic origin: Preliminary characterization

Summary Nineteen human breast carcinoma cell lines have been established as continous cultures during the past 6 years in our laboratory. This preliminary report is designed to list the lines by their designated code numbers (MDA-MB) and present a brief summary of their morphological, cytogenetic and biochemical characteristics. Sixteen of our lines were obtained from pleural effusions, two from brain metastases, and one from pericardial fluid. All lines have been shown to be distinct entities and are uncontaminated by HeLa cells or each other. A lq marker chromosome is present in all but one of the lines examined. This research was sponsored by the National Cancer Institute Contract NO1-CB-23869; Institutional Grant 5S 07 RR 5511-15 awarded by the Division of Research Resources, and a Kelsey-Leary Grant NO 974.     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

Summary We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain. This work was supported in part by Grants 5319 and 5322 in aid for cancer research from the Ministry of Health and Welfare, Japan.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.