Human Bladder Transitional Cell Carcinoma Research Articles

Semi-quantitative analysis of telomerase activity in exfoliated human urothelial cells and bladder transitional cell carcinoma.

To determine the utility of detecting telomerase activity in transitional cell bladder cancer, using the telomeric repeat amplification protocol (TRAP) assay, and thus provide a test for the detection and monitoring of bladder cancer, especially low-grade tumours. Telomerase activity was assayed in bladder cancer tissues and the exfoliated urothelial cells from 29 patients with bladder cancer, 10 with benign disease, and in 10 healthy subjects using the TRAP assay. The levels were assessed semiquantitatively by calculating the ratio to an internal telomerase assay standard. Telomerase activity was very low in the exfoliated urothelial cells from all healthy subjects and patients with benign disease, with a mean (SD) ratio of 0.25 (0.03) and 0.33 (0.04), respectively. A threshold ratio of 0.4 was calculated as the mean +/- 2 SD of the telomerase activity level of the exfoliated urothelial cells from patients with benign disease. Using this threshold, telomerase activity was negative in exfoliated urothelial cells from all benign cases (100% specificity) and positive in all 26 bladder cancer tissue samples assayed. In tumour tissue, telomerase activity was not associated with tumour grade, size or stage. Telomerase activity in exfoliated urothelial cells from patients with bladder cancer was positive in 25 of 29 samples (86% sensitivity). The sensitivity of telomerase activity in exfoliated cells was seven of nine in G1, 10 of 12 in G2 and all eight G3 tumours; the corresponding sensitivity for voided urine cytology in G1, G2 and G3 tumours was two of nine, six of 12 and six of eight, respectively. These results indicate that telomerase activation occurs as an early step in carcinogenesis and the semi-quantitative analysis of telomerase activity in exfoliated urothelial cells could be a minimally invasive and useful method for detecting bladder cancer, even in low-grade tumours.

Pharmacologic effects of paclitaxel in human bladder tumors.

The goal of this study was to determine whether paclitaxel, when given by a 2-h treatment, produces significant cytotoxic effects in human bladder transitional cell carcinoma and hence qualifies as a candidate drug for intravesical treatment. Histocultures of surgical specimens from patients (n = 16) were used. Paclitaxel produced partial inhibition of DNA precursor incorporation in about 70% of tumors and induced apoptosis in about 90% of tumors, while these effects were minimal or not detectable in the remaining tumors. In the responsive tumors, the average maximal inhibition of DNA synthesis was 60% and the average maximal apoptotic index was 15%. Resistance to antiproliferative and apoptotic effects was not always found in the same individual tumors, and no relationship was found between the magnitude of antiproliferative and apoptotic effects in individual tumors. The maximal apoptotic index correlated with the LI for the untreated control (r2 = 0.42, P < 0.01). More than 95% of apoptotic cells were labeled by DNA precursor, whereas not all labeled cells were apoptotic. The pharmacologic effects of paclitaxel in bladder tumors were qualitatively equivalent to those previously found in human head and neck tumors and in human prostate tumors after treatment for longer periods of 24 to 96 h. These results indicate that a 2-h paclitaxel treatment was sufficient to produce antiproliferation and apoptosis in 70-90% of human bladder tumors, and the apoptotic effect appeared to be linked to proliferation and occurred after DNA synthesis.

Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1) human bladder washings and transitional cell carcinoma of the bladder; and (2) human sperm and male infertility

Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied. These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma. Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 μM). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 μM and in infertile samples at 100- and 200-μM doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.

Interferons Modify in Vitro Proliferation of Human Bladder Transitional Cell Carcinoma in the Presence of Doxorubicin and Mitomycin C

Purpose The aim of the present study was to investigate whether interferons with their known antitumor activity modify the response of human bladder carcinoma cells to antitumor drugs. Materials and Methods We investigated the in vitro effect of doxorubicin, mitomycin C and the interferons alpha and gamma on cell proliferation in human bladder carcinoma cell lines as measured by 5-bromo-2'-deoxy-uridine (BrdU) incorporation. Results Exposure of RT 112 (but not EJ 28) cells for 2 hours to doxorubicin (500 ng./ml.) and mitomycin C (200 ng./ml.) reduced the proliferation rate to 85.9 plus/minus 3.3 percent (n = 4) and 89.3 plus/minus 4.0 percent (n = 4) of control. Treatment for 2 days with interferon alpha and gamma up to the highest concentration (200 U/ml.) showed no effect. The combination of 100 U/ml. interferon alpha and doxorubicin decreased proliferation significantly. At 50 ng./ml. the proliferation rate was decreased to 88.0 plus/minus 5.7 percent of control and at 500 ng./ml. to 67.7 plus/minus 3.1 percent. Thus interferon alpha seems to increase the sensitivity of the cells to doxorubicin. Cells treated with 20 ng./ml. mitomycin C after pretreatment with interferon alpha showed a dramatic decrease in cell proliferation (from 98.8 plus/minus 2.1 percent to 80.2 plus/minus 4.0 percent of control). This decrease was similar in the presence of 200 ng./ml. mitomycin C. Thus mitomycin C seems to render cells more sensitive to the antiproliferative action of interferon alpha. Interferon gamma had only minor effects on the response of the cells to doxorubicin or mitomycin C. Conclusions These studies suggest that exposure to interferon alpha increases the efficacy of anticancer drugs in vitro, probably by several mechanisms. Potential consequences of this finding for the therapeutic regime employed for treatment of bladder carcinoma are discussed.

Interferons Modify in Vitro Proliferation of Human Bladder Transitional Cell Carcinoma in the Presence of Doxorubicin and Mitomycin C

Interferons Modify in Vitro Proliferation of Human Bladder Transitional Cell Carcinoma in the Presence of Doxorubicin and Mitomycin C

Development of Secondary Structure, Growth Characteristics and Cytogenetic Analysis of Human Transitional Cell Carcinoma Xenografts in Scid/Scid Mice

Purpose The growth of human bladder transitional cell carcinoma (TCC) in scid/scid mice was examined. Materials and Methods Cystocopically obtained TCC biopsies were implanted in scid/scid mice, and successful xenografts were compared with original tumors for growth and genetic characteristics. Results Low grade papillary tumors formed fluid-filled pseudobladders lined with malignant urothelium and papillary fronds containing fibrovascular cores recruited from the murine host. High grade xenografts grew without these secondary structures. When compared with the patient tumors, xenograft growth fractions, as measured by proliferating cell nuclear antigen, p53 expression and ploidy, were similar in each case. Conclusions The scid/scid xenografts maintain phenotype and architecture. This model may be useful for studying factors determining tumor grade, angiogenesis and tissue organization.

Photodynamic therapy of photofrin II and excimer dye laser on experimental tumors.

Photodynamic therapy (PDT) by combination of photofrin II and excimer dye laser was evaluated for its usefulness in experimental tumors. High antitumor effects of PDT on sarcoma 180 solid type were obtained when PDT was performed with laser irradiation at an energy of 50 or 100 J/cm2 (40 or 80 Hz, 4 mJ/pulse) 48 h after i.v. administration of photofrin II at a dose of 25 mg/kg. Under the same conditions, the antitumor effects of PDT on murine Lewis lung carcinoma, human fibrosarcoma HT-1080 and human bladder transitional cell carcinoma KK-47 were also observed. These results suggest that clinical application of PDT with photofrin II and excimer dye laser might be useful.

Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder

Somatic allelic loss is regarded as a hallmark of tumour-suppressor gene (TSG) inactivation. Thirty-one human bladder transitional cell carcinomas (TCCs) were examined for allelic loss at five chromosome 18q loci, including the DCC gene (deleted in colorectal carcinoma) and at chromosome 11p15 in a restriction fragment length polymorphism analysis. Allelic loss was observed at one or more 18q loci in 9/26 (35%) samples, associated with muscle-invasive disease (P < 0.02). Allelic loss was observed at DCC in 8/24 (33%) samples, associated with muscle-invasive disease (P = 0.05). Three out of the five evaluable recurrent TCCs exhibited allelic loss at DCC, two of which were superficial. No allelic losses were detected at other 18q loci in tumours which retained both DCC alleles. Allelic loss was observed at 11p15 in 5/20 (25%) tumours. These data suggest the presence of a late-acting TSG located on 18q in TCC bladder cancer. DCC is a candidate gene since it lies within the region of most common deletion (18q21.3-qter).

Hematoporphyrin derivative-induced photodynamic inhibition of Na+/K(+)-ATPase in L929 fibroblasts, Chinese hamster ovary cells and T24 human bladder transitional carcinoma cells.

The photodynamically induced inhibition of Na+/K(+)-ATPase with hematoporphyrin derivative as photosensitizer was studied in murine L929 fibroblasts, Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells. In T24 cells the inhibition of the enzyme activity appeared to be caused by ATP depletion rather than by direct damage from the enzyme itself. In L929 and CHO cells, on the other hand, the inhibition was caused by photodynamic damage from the enzyme molecule. For all three cell lines it was shown that a causal relationship between photodynamically induced reduction in Na+/K(+)-ATPase activity and the loss of clonogenicity is highly unlikely.

HPD-Induced Photodynamic Changes in Intracellular Cyclic AMP Levels in Human Bladder Transitional Carcinoma Cells, Clone T24

Human bladder transitional carcinoma cells, clone T24, were incubated with hematoporphyrin derivative and subsequently exposed to red light. Immediately after light exposure a light-dose dependent increase in intracellular cyclic AMP levels was observed. The concentrations decreased to non-treated levels within 2 h after illumination. Inhibition of cyclooxygenase with indomethacin reduced the increase drastically, whereas the presence of PGE 2 enhanced the production of cyclic AMP. PGE 2, 8′-bromo-cyclic AMP and a direct stimulation of adenylyl cyclase by forskolin reduced the photodynamically induced cell killing. On the other hand, suppression of cyclic AMP by the cyclooxygenase inhibitor indomethacin, enhanced the photodynamically induced cell killing. Therefore, it is suggested that cyclic AMP production in T24 cells is a step in a cellular rescue pathway, resulting in protection against photodynamic cell killing.

Correlation between the expression of oncogenes ras and c-erbB-2 and the biological behavior of bladder tumors

Expression of the ras and the c-erbB-2 oncogene products was investigated in 56 cases of human bladder transitional cell carcinoma and 6 samples of human normal bladder tissue using an immunohistochemical method. Thirty of the 56 cases of bladder tumor were found to be immunohistologically positive with the monoclonal anti-ras p21 antibody, while 19 of 56 cases were positive with the polyclonal anti-c-erbB-2 oncoprotein antibody. All 6 controls were negative with both antibodies. The ras p21 positive staining was found more frequently in the well or moderately differentiated, superficial and non-recurrent tumors than in the poorly differentiated (p < 0.01), muscle invasive (p < 0.05) and recurrent tumors (p < 0.01), while the c-erbB-2 gene product was more commonly detected in high-grade (p < 0.01), invasive (p < 0.01) and recurrent tumors (p < 0.05). Thus, the expression of either ras or c-erbB-2 was closely associated with the histological grade, clinical stage and recurrence of bladder transitional cell carcinomas.

Characterization of the internalization of bacillus Calmette-Guerin by human bladder tumor cells.

Adjuvant intravesical Mycobacterium bovis BCG is the treatment of choice for recurrent superficial bladder cancer. Fibronectin (FN) was previously demonstrated to be necessary for the retention of BCG within the bladder and for the expression of antitumor activity. Recent studies have demonstrated that BCG attach and are ingested by bladder epithelial cells, suggesting the existence of a second bacterial attachment mechanism. We report the characterization of the molecules involved in BCG attachment and internalization by the human bladder transitional cell carcinoma cell line T-24. Pretreatment of T-24 cells with monoclonal antibodies to either alpha 5 or beta 1 integrin subunits significantly inhibited both BCG attachment and ingestion. Exogenous FN was observed to enhance both attachment and ingestion of BCG, and anti-FN was observed to inhibit both phenomena. Latex beads precoated with either FN or laminin (LN) but not BSA were ingested by T-24 cells, but only FN-coated beads inhibited BCG attachment and ingestion. Pretreatment of BCG with FN augmented both attachment and ingestion. The role of bacterial FN binding proteins was evaluated. A monoclonal antibody to a 55-kD FN-binding protein was observed to abrogate attachment and ingestion. These results demonstrate that attachment and ingestion of BCG are mediated in part by the alpha 5 beta 1 integrin receptor and are dependent on FN. These studies demonstrate a mechanism of entrance of mycobacteria into epithelial cells and suggest a second role for FN in the adjuvant antitumor effect of BCG.

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AIPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca 2+] i, reaching a peak within 5–15 min after exposure and then returning to basal level (∼ 200 nM). The level of the peak [Ca 2+] i depended on the light fluence, reaching a maximum of 800–1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca 2+] i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca 2+ influx is mainly responsible for elevated [Ca 2+] i. PDT is unique in triggering a cell rescue process via elevated [Ca 2+] i. Other cytotoxic agents, e.g., H 2O 2, produce sustained increase of [Ca 2+] i that is involved in the pathological processes leading to cell death.

An Investigation of Factors Influencing the in Vitro Induction of Lak Activity Against A Variety of Human Bladder Cancer Cell Lines

We investigated the sensitivity of transitional cell carcinoma cells, derived from the human bladder, to lymphokine activated killer cells. Recombinant interleukin-2 activated peripheral blood mononuclear cells were studied for their ability to mediate the cytolysis of a panel of four established human bladder transitional cell carcinoma cell lines. Lymphokine activated killer activity was assessed using a standard four hour chromium release assay. All four bladder cancer cell lines proved to be susceptible to lymphokine activated killer mediated cytolysis. This was found to be dependent upon the dose of cytokine and upon the duration of the activation period. The four cell lines were differentially susceptible to lysis (specific cytotoxicity at effector to target ratio of 40:1; RT112 = 22.9%, RT4 = 49.2%, MGH-U1 = 49.1%, EJ18 = 62.3%). The varying susceptibility of lymphokine activated killer mediated cytotoxicity was found to be independent of the histological grade of the parent tumour or the donor of effector cells. Both interferon-alpha and tumour necrosis factor-alpha also elicited lymphokine activated killer cell activity, although the maximum specific cytotoxicity achieved was considerably lower than that obtained with interleukin-2 alone. Interleukin-2, at optimal concentration, and tumour necrosis factor-alpha were found to behave synergistically in the generation of lymphokine activated killer effectors. However, concentrations of tumour necrosis factor-alpha higher than 100 Uml.-1 resulted in a decrease in specific cytotoxicity. These findings suggest a possible use of adoptive immunotherapy in human bladder cancer and indicate the optimum conditions for the generation of such effector cells.

エキシマ・ダイレーザーを用いた光力学的治療の基礎的検討

The usefulness of photodynamic therapy (PDT) for experimental tumors by using Photofrin II and excimer dye laser was studied. High antitumor effects on Sarcoma 180 graft were obtained when PDT was performed 48 hours after iv administration of Photofrin II at a dose of 25mg/kg following the laser irradiation at energy of 50J/cm2 (80Hz, 4mJ/pulse). Under the same condition, the nonspecific anticancer effects of PDT on murine Lewis lung carcinoma, human fibrosarcoma HT-1080, and human bladder transitional cell carcinoma kk-47 grafts were observed. These results suggest that clinical applications of PDT with Photofrin II and excimer dye laser might be useful.

The Effect of Verapamil on a Multi-Drug ResistantBladder Carcinoma Cell Line and Its Potential as anIntravesical Chemotherapeutic Agent

A human bladder transitional cell carcinoma cell line, MGH-U1R, exhibits reproducible resistance to doxorubicin. We examined the effects on survival of this cell line caused by verapamil, which has been shown to reverse multi-drug resistance in vitro in other neoplastic cell lines. Both MGH-U1R and MGH-U1, the non-resistant parent cell line, were treated with varying concentrations of doxorubicin alone, verapamil alone, or both drugs simultaneously, all for one hour. Cells were then grown in drug-free medium for 10 days, stained, and counted. Standard survival curves were calculated. Verapamil alone had no significant cytotoxicity. Verapamil at concentrations of 16μg./ ml. and 32μg./ml. decreased the IC50 of doxorubicin for MGH-U1R by a factor of 2.5.Using H3-verapamil, we also examined the systemic and local absorption of this drug resulting from intravesical verapamil administration in rabbits. All animals were treated for one hour, and multiple serum samples were drawn during treatment. Verapamil was found in high concentrations in the mucosa, less in the adventitia, and was absent in venous blood.Verapamil effectively reverses resistance to doxorubicin of MGH-U1R in vitro. The intravesical use of verapamil appears to be safe, and may prove to be a useful adjunct in the intravesical therapyof some bladder tumors. (J. Urol., 143: 1053–1056, 1990)

Recombinant human tumor necrosis factor alone and with chemotherapeutic agents. Effect on nude mouse-supported human bladder cancer heterografts.

Although studies done in tissue culture (in vitro) have shown synergism between recombinant human tumor necrosis factor (rhTNF) and chemotherapeutic agents, whether such synergism exists in a complex in vivo environment with acceptable toxic side effects has not been determined, to our knowledge. The effect of rhTNF alone and in combination with cisplatin, etoposide, doxorubicin, or dactinomycin on the growth of heterotransplants of human bladder transitional cell carcinoma was studied using a modified subrenal capsule assay in athymic nude mice. Only etoposide potentiated rhTNF cytotoxicity; no increase in host toxicity was noted. Variably enhanced toxic side effects were seen with other combinations. It is concluded that rhTNF combined with etoposide may have potential clinically exploitable therapeutic "synergism" in the treatment of advanced bladder cancer.

Antigenic variation and macrophage infiltration of human bladder tumors xenografted into nude mice.

The presence of macrophages both within and around human bladder transitional cell carcinomas xenografted into nude mice has been examined using immunocytochemical staining. Nine different xenograft lines derived from bladder tumors of eight patients were stained for F4/80, MHC II and PGP-1 labelled cells. The results revealed considerable heterogeneity both within and between tumors. All of the tumors generated some macrophage response at the tumor periphery, and this was marked in two instances, UCRU-BL-13, passage 9 and UCRU-BL-17, passage 2. In only two tumors was there substantial penetration of the tumor epithelium by macrophages, though infiltrating Ia+, F4/80+, PGP-1+ cells were sometimes seen spreading along the base of the epithelial sheets. Three of the tumors were characterized by the presence of L3T4+ T lymphocytes at the invasion front. The presence of macrophages or lymphocytes either at the invasion front or within the tumors did not correlate with the pathological grade of the tumors. PGP-1 monoclonal antibodies also stained granulocytes, which were present within the tumor mass in UCRU-BL-15, passage 2, possibly reflecting the production by the tumor of G-CSF. In addition, the PGP-1 antibodies stained some of the bladder tumors cells themselves, and, in some cases, the interstitial structures within the tumors. The significance of this staining is not yet understood. The xenografted tumors will provide a useful model to examine the constraints on tumor therapy using macrophage activating stimuli.

Features of squamous and adenocarcinoma in the same cell in a xenografted human transitional cell carcinoma: evidence of a common histogenesis?

Ultrastructural features of squamous differentiation have been found in adenocarcinomatous cells in a xenografted line (UCRU-BL-17) established in nude mice from a primary human bladder transitional cell carcinoma (grade III, stage T4) with a tetraploid DNA component. The line has been characterized by light and electron microscopy, flow cytometry and immunocytochemistry. The initial xenograft showed predominantly adenocarcinomatous differentiation with mucin secretion, whilst the subsequent passages also contained cells showing squamous differentiation. A xenograft subline established from a cell culture of the initial xenograft shows the emergence of a population of cells with near triploid DNA, which are less differentiated, grow more quickly, show decreased expression of carcinoembryonic antigen, and a change in the distribution of staining with peanut lectin from cell surface to cytoplasm. These lines offer an unusual opportunity to study the histogenetic relationships between the histological subtypes of bladder cancer.

Experimental Studies on the Response of Nude Mouse-Grown Human Urothelial Cancer to High Dose of 1-β-D-Arabinofuranosylcytosine

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on human bladder transitional cell carcinomas (TCC) was evaluated using nude mouse-grown established tumor lines SW-780, SW-1738, TCC-K1, and PR49. In the nude mouse tumors grew subcutaneously and evaluation of response to treatment was based on tumor volume analysis. Ara-C was administered intraperitoneally at a dosage of 250 mg/kg on days 1 to 4. All four tumor lines showed an initial phase of considerable regression during the treatment week. This was followed by a variable period of tumor growth arrest after which a gradual tumor regrowth was observed. The results of the present study suggested that ara-C may alter the growth rate of TCC, its effect becoming apparent during the time of its administration and for a short period thereafter. These observations indicate that more studies are warranted at both the experimental and clinical levels to further evaluate dosage and treatment schedule. The results of such additional studies may determine the importance of ara-C in the management of patients with bladder cancer.