Brian C.-S. Liu

Sensitivity and specificity of NMP-22, telomerase, and BTA in the detection of human bladder cancer☆

OBJECTIVES The recent introduction of novel molecular markers into clinical urology has created a need to evaluate the efficacy and utility of these potential markers. The ideal assay for bladder cancer should be noninvasive, sensitive, specific, and cost-effective. We compared the Matritech nuclear maxtrix protein (NMP)-22 assay, telomerase activity, and the Bard bladder tumor antigen (BTA) assay for the detection of human bladder cancer. METHODS A single voided urine sample was obtained from patients with hematuria without bladder cancer and from patients with known bladder cancer before any treatment. Approximately 50 to 100 mL of voided urine sample was collected and aliquotted for the various assays. The results were compared to single cytologic results and ultimately to pathologic findings. RESULTS In 47 patients with bladder cancer, the overall sensitivity was 81% for NMP-22, 80% for telomerase, 40% for BTA, and 40% for cytology. For Ta tumors (n = 31), sensitivity was 81% for NMP-22, 70% for telomerase, 32% for BTA, and 26% for cytology. For T1 or higher stage tumors (n = 13), sensitivity was 82% for NMP-22, 91% for telomerase, 64% for BTA, and 64% for cytology. The remaining 3 patients had carcinoma in situ (CIS). When tumors were stratified by tumor grade, grade I tumors (n = 16) were detected at 69% with NMP-22, 65% with telomerase, 13% with BTA, and 6% with cytology. Grade II tumors (n = 14) were detected at 86% with NMP-22, 72% with telomerase, 36% with BTA, and 36% with cytology. Grade III tumors (n = 14) were detected at 93% with NMP-22, 93% with telomerase, 79% with BTA, and 79% with cytology. Patients with CIS (n = 3) were detected at 67% with NMP-22, 100% with telomerase, 33% with BTA, and 67% with cytology. In 30 patients with hematuria but without bladder cancer, the overall specificity of the assays was 77% for NMP-22, 80% for telomerase, 73% for BTA, and 94% for cytology. CONCLUSIONS In the population tested, NMP-22 and the telomerase assays gave similar sensitivity and specificity for the detection of bladder cancer, and appear to offer a greater sensitivity than the BTA assay and/or conventional cytology.

Mechanisms of human bladder tumor invasion : role of protease cathepsin B

To study the biochemical mechanisms of bladder tumor invasion, we analyzed specimens of invasive transitional cell carcinoma cell line EJ and non-invasive transitional cell carcinoma cell line RT4 which had been implanted into the bladders of nude mice. Using immunoprobes specific to basement membrane laminin, we observed that superficial but not invasive tumors were surrounded by intact laminin. With immunoprobes specific to cathepsin B, a cysteine proteinase which has the ability to degrade laminin, we demonstrated that cathepsin B is localized in discrete cytoplasmic granules in the non-invasive tumors, and in a more diffuse cytoplasmic pattern in the invasive tumors. Subcellular fractionation followed by immunoblot analysis and enzymatic analysis confirmed that the invasive EJ cells had active cathepsin B localized to its plasma membrane, while non-invasive RT4 cells had cathepsin B confined to lysosomes. Furthermore, immunoblot analysis revealed that invasive EJ cells had the mature form of cathepsin B with a molecular weight of 25 kD, while non-invasive RT4 cells had predominantly precursor forms with molecular weights between 30 and 35 kD. In vitro degradation assays with plasma membrane fractions isolated from invasive EJ cells and non-invasive RT4 cells demonstrated that the plasma membrane of EJ cells but not that of the RT4 cells had the ability to degrade purified laminin, and that the degradative products were similar to those obtained with purified cathepsin B. We conclude that invasive tumor cells have enhanced cathepsin B in their plasma membranes which may be used to degrade basement membrane components such as laminin and thereby facilitate tumor invasion.

Modulation of N-myc expression alters the invasiveness of neuroblastoma.

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.

LAK Cell Mediated Apoptosis of Human Bladder Cancer Cells Involves a pH-Dependent Endonuclease System in the Cancer Cell: Possible Mechanism of BCG Therapy

Intravesical bacillus Calmette-Guerin (BCG) is an effective treatment for superficial bladder cancer. However, its mechanism has been only partially elucidated. We studied whether LAK cell killing of human bladder cancer cells occurs via apoptosis (programmed cell death) or necrosis. Fluorescent dye labeled T24 cells were observed to undergo morphologic changes associated with apoptosis in the presence of LAK cells when analyzed under a fluorescence microscope. Furthermore, analysis of the DNA isolated from the cytotoxic assay confirmed that the LAK cell induced death of the T24 cells occurred via apoptosis. By pretreating the LAK cells with antifibronectin antibodies, we were able to significantly inhibit the LAK cell killing of the T24 cells. The percentage of cytotoxicity was reduced from 50% to 13% (p = 0.001), and the apoptotic pattern seen on agarose gel electrophoresis was significantly diminished. There was no significant change in the viability of the LAK cells following treatment with the antibodies. Endonuclease isolation from human bladder cancer T24 cells demonstrated that these cells express a pH-dependent and not a Ca++/Mg++ dependent endonuclease. Significant degradation of a target DNA was observed only in pH 4 to pH 5.6 buffers containing endonuclease from T24 cells and not in pH 6 to pH 8 buffers containing endonuclease from T24 cells. The presence or absence of Ca++/Mg++ in the various pH buffers did not alter the endonuclease activity. Finally, we demonstrated that death of T24 cells can be induced by altering the intracellular pH of the cells to 5.6 or lower with the proton ionophore nigericin. We conclude that LAK cells induce T24 cells to undergo apoptosis and that this process involves the fibronectin molecule present on the LAK cell membrane. Furthermore, the cleavage postulate that, in vivo, LAK cells activated by IL-2 produced by BCG activated CD4+ cells may induce bladder cancer cells to undergo apoptosis. This may partially explain the mechanism whereby BCG achieves its therapeutic effect.

Bacillus Calmette-Guérin Interacts with the Carboxyl-Terminal Heparin Binding Domain of Fibronectin: Implications for BCG-Mediated Antitumor Activity

Intravesical bacillus Calmette-Guérin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder. The mechanisms by which BCG achieves this effect remain unclear. Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility. In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain. Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells. By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells. Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces. However, the numbers of receptors are not significantly different in the two cell populations. We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place.

Bacillus Calmette-Guerin abrogates in vitro invasion and motility of human bladder tumor cells via fibronectin interaction.

Intravesical bacillus Calmette-Guerin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder (TCC). The mechanisms by which BCG limits tumor cell activity have thus far been unclear. We investigated the interaction between BCG and invasive human TCC cell line EJ in an in vitro invasion assay. We observed that BCG inhibited the invasion of EJ cells through an artificial basement membrane. In terms of the steps involved in tumor cell invasion, i.e. attachment, proteolysis, and motility, BCG was found to limit tumor cell motility. Attachment and proliferation of tumor cells were not affected by BCG. The effects of BCG on tumor cell migration were mediated by fibronectin (FN), a basement membrane glycoprotein component. Abrogation of BCG-FN-tumor cell interactions with anti-FN antibodies eliminated the ability of BCG to block tumor cell invasion. Fibronectin appears to link BCG and tumor cells via independent FN binding receptors to separate domains of the FN molecule. The molecular mechanism by which BCG may limit tumor cell motility may be its ability to protect against the formation of specific FN sequences as a result of protease cathepsin B digestion. A 31 kD and 27 kD FN band were absent from purified or tumor cell associated cathepsin B digestion when incubated in the presence of BCG, but present in the absence of BCG. Furthermore when purified from SDS polyacrylamide gel electrophoresis, the fragments were shown to have motility stimulating activity for the invasive EJ cells. These findings suggest that BCG functions as a potent inhibitor of tumor cell invasion. We conclude that BCG-fibronectin-tumor cell interactions may have a direct influence on the invasive mechanisms, such as motility, of tumor cells.

Type IV Procollagen mRNA Regulation: Evidence for Extracellular Matrix/Cytoskeleton/Nuclear Matrix Interactions in Human Urothelium

The absence of basement membrane components correlates with tumor stage and progression in human bladder cancers. We have previously shown that invasive tumors possess the ability to degrade basement membrane. However, the presence of basement membrane may be affected not only by its degradation, but by its synthesis and deposition as well. Our results in the present study suggest that while the invasive human transitional carcinoma cell line EJ has an increased amount of type IV procollagen mRNA when compared to the non-invasive RT4 cell line, type IV collagen staining is absent in the invasive EJ cells and intensely present in the non-invasive RT4 cells. Moreover, when EJ cells were grown on an artificial basement membrane (Matrigel), type IV procollagen mRNA expression was down-regulated to the levels seen with the non-invasive RT4 cells. We also discovered that the invasive cells, when grown on Matrigel, appeared morphologically different from the same cells grown on plastic tissue cultures. We conclude that a deficient basement membrane in invasive cancer cells may be due not only to active proteolytic activity but also to an abnormal production and deposition of extracellular matrix components. In addition, we also demonstrated that basement membrane components may have a significant effect on epithelial cell morphology and gene regulation, and that any alterations of the extracellular matrix-cytoskeleton-nuclear matrix interactions can lead to altered gene regulations and cell function.

Tumor Necrosis Factor Mediates Apoptosis Via Ca sup ++ /Mg sup ++ Dependent Endonuclease with Protein Kinase C as a Possible Mechanism for Cytokine Resistance in Human Renal Carcinoma Cells

AbstractPurpose: Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necrosis factor are now being considered for clinical use. In this study, we used 2 renal cancer cell lines, SK-RC-42 and SK-RC-49, and determined the cytotoxic effects of tumor necrosis factor (TNF) and the possible mechanism of TNF resistance.Materials and Methods: Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used.Results: Cytotoxic assays with SK-RC-42 demonstrated that TNF at 50 ng./ml. for 24 hours resulted in 19 percent cytotoxicity of the cells. Similar assay with SK-RC-49 only demonstrated less than 4 percent cytotoxicity. Based on these results, we defined our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resistant cells.To determine whether protein kinase C (PKC), which is involved in the signal transduction pathway of a cell, could regulate endogenous basal TNF mRNA l...

Applications of telomerase in urologic oncology.

SummaryVertebrates have special structures at the ends of their chromosomes, known as telomeres, which are composed of 5- to 15-kb pairs of a guanine-rich hexameric repeat (TTAGGG)n. In normal somatic cells there is a progressive degradation of telomeres with aging., The cell can afford to lose only a finite number of these telomeres before significant sequences of the parent DNA are lost, resulting in chromosomal instability and cell death. However, germ-cell telomeres are maintained despite multiple rounds of replication. This suggests that they produce an enzyme that maintains their telomere length. This enzyme, a ribonucleoprotein, is called telomerase. In this review, we discuss the presence of telomerase activity in various human cancers and, in particular, in urologic tumors. We describe the potential clinical utility of detection of the presence of telomerase activity in cells from voided urine samples of patients with bladder cancer.

Bone extracellular matrix induces homeobox proteins independent of androgens : Possible mechanism for androgen-independent growth in human prostate cancer cells

Differences in gene expression in prostate cells are believed to be secondary to epithelial‐stromal interactions. We theorized that bone matrix may provide a fertile “soil” for prostate cancer by inducing androgen‐dependent genes and allowing for androgen‐independent growth.