Human Transitional Cell Carcinoma Lines Research Articles

A Synthetic Polyvalent Ligand for α5β1 Integrin Activates Components of the Urothelial Carcinoma Cell Response to Bacillus Calmette-Guérin

Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin. The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8. The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability. Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.

Bacillus Calmette-Guérin induces p21 expression in human transitional carcinoma cell lines via an immediate early, p53 independent pathway

Purpose Work by our group has shown that bacillus Calmette-Guérin (BCG) induces cell cycle arrest at the G1/S interface in human transitional carcinoma cell lines. This study evaluated the effect of BCG on cell cycle regulatory proteins relevant to this effect. Materials and Methods The effect of BCG on selected cell cycle regulatory proteins, including cyclin D1, p27, and p21, was assessed in 2 human cell lines. After the identification of p21 as a candidate protein, p21 regulation was evaluated using a combination of Western, reverse transcriptase polymerase chain reaction, and promoter-reporter analysis. The immediate early versus delayed nature of p21 induction was determined. Finally, given the known potential for p21 to be regulated by both p53 dependent and independent pathways, the role of p53 in BCG induced expression of p21 was evaluated. Results BCG increased p21 expression 2-fold relative to controls as measured by Western, and promoter-reporter analysis. Inhibition of protein synthesis had no effect on p21 messenger ribonucleic acid induction in response to BCG. T24 cells contained a previously reported mutation in p53. In the p53 wild-type 253J cells, deletion of one or both p53 response elements in the p21 reporter had no effect on BCG induced reporter transactivation. Conclusions BCG up-regulates expression of p21 in human transitional cell carcinoma lines. The transactivation of p21 in response to BCG occurs through an immediate early, p53 independent pathway. The finding of increased p21, together with the observation that BCG induces cell cycle arrest at the G1/S interface, supports a role for this protein in the biologic response to BCG.

Micro-array analysis of the effect of post-transurethral bladder tumor resection urine on transforming growth factor-β1 dependent gene expression in transitional cell carcinoma ☆

Introduction and Objectives Prior studies have shown that bladder trauma occurring during transurethral bladder tumor resection increases urinary levels of the cytokine transforming growth factor (TGF)-β1. This study used complementary deoxyribonucleic acid micro-array technology to identify additional genes in human transitional cell carcinoma (TCC), whose expression is altered as a consequence of increased urinary levels of TGF-β1. Methods The human TCC line 253J was cultured in standard media, or media spiked with either 10% post-transurethral bladder tumor resection urine (PTU), or PTU and anti-TGF-β1 neutralizing antibody. Messenger ribonucleic acid from these conditions, together with messenger ribonucleic acid from stably transfected 253J cells over-expressing TGF-β1, was hybridized with ATLAS® micro-array membranes (Clontech, Palo Alto, CA) containing 588 human genes. Hybridization signal intensity was quantified using phospho-imaging. An analytic strategy based on the variance in the signal intensity ratio of specific housekeeping genes in control and experimental comparisons was used to identify significant changes in gene expression. Reverse transcriptase polymerase chain reaction of target genes was used to confirm gene over-expression and TGF-β1 responsiveness. Results Seven genes were identified on micro-array: v-RAF-1, colony stimulating factor-1 receptor, v-FGR, insulin growth factor-1 receptor, epidermal growth factor receptor, α5 integrin, and interferon receptor-1. Reverse transcriptase polymerase chain reaction confirmed over-expression in the autocrine TGF-β1 producing cell line and increased expression in response to exogenous TGF-β1. Conclusions TGF-β1 in PTU alters the expression of multiple genes in human TCC in vitro. The impact of these changes on the biologic phenotype of the malignant cell and the efficacy of adjuvant therapies requires further evaluation.

Antitumor activity of common antibiotics against superficial bladder cancer

Objectives The recurrence rate for superficial bladder tumors treated with complete resection averages 88%. Intravesical chemotherapy decreases the recurrence rate by only 14%; thus, new chemotherapeutic agents are needed. Antibiotics are often used to prevent infections after transurethral resection of bladder tumors. Oral intake of antibiotics results in significantly greater concentrations in the urine than in the serum. Our objective was to evaluate four commonly used urinary antibiotics for their cytotoxic activity against bladder cancer cells at clinically relevant concentrations. Methods Three human transitional cell carcinoma lines—HTB9 (grade 2), T24 (grade 3), and TccSup (grade 4)—were exposed to ciprofloxacin, trimethoprim-sulfamethoxazole, cefazolin, or nitrofurantoin at concentrations from 0 (control) to 1000, 1000, 5000, and 2000 μg/mL, respectively, for 96 hours. Cytotoxicity was evaluated using the MTT colorimetric assay. Six replicates were used for each data point, and the results are reported as the mean ± standard deviation. Results Significant cytotoxicity ( P <0.001) was seen, starting at 12.5 μg/mL (HTB9, TccSup) and 50 μg/mL (T24) for ciprofloxacin, 31.25 μg/mL (HTB9, TccSup) and 62.5 μg/mL (T24) for trimethoprim-sulfamethoxazole, 19.5 μg/mL (HTB9) and 156.3 μg/mL (T24, TccSup) for cefazolin, and 7.8 μg/mL (HTB9, T24, TccSup) for nitrofurantoin. Cytotoxicity was dose dependent for all four antibiotics, and the maximal effect did not differ among antibiotics. Conclusions Commonly used antibiotics exhibit significant dose-dependent cytotoxicity against bladder cancer cells at concentrations achievable in the urine after oral administration. The administration of antibiotics after transurethral resection of bladder tumors might prevent seeding of cancer cells and thereby decrease the recurrence rate. Preclinical data such as these must be considered in the design of clinical trials addressing recurrence after transurethral resection of bladder tumors.

Androgen Dependent Regulation of bacillus Calmette-Guerin Induced Interleukin-6 Expression in Human Transitional Carcinoma Cell Lines

Autocrine expression of interleukin (IL)-6 by transitional cell carcinoma (TCC) in response to bacillus Calmette-Guerin (BCG) may have an important role in promoting BCG adherence to TCC and consequently in BCG treatment efficacy. IL-6 expression in response to BCG requires nuclear factor (NF)-kappaB mediated signal transduction. We evaluated the influence of androgens on BCG induced, NF-kappaB mediated IL-6 expression. Reverse transcriptase-polymerase chain reaction was used to confirm androgen receptor expression in the human TCC lines 253J and T24. A reporter construct containing an androgen response element was used to establish the integrity of androgen mediated signal transduction. Subsequently the dose dependent effect of dihydrotestosterone (DHT) on BCG induced IL-6 expression and NF-kappaB signaling was evaluated. Two pharmacological androgen receptor blockers were used to determine if receptor blockade inhibited the effect of DHT on activation of the androgen response element, NF-kappaB signaling and BCG induced IL-6 expression. The 2 human TCC lines expressed androgen receptor and demonstrated intact androgen stimulated signaling pathways. DHT suppressed BCG induced, NF-kappaB mediated signaling and IL-6 expression in a dose dependent manner. DHT decreased mRNA levels of IL-6, expression of the full-length IL-6 promoter construct and expression of an NF-kappaB specific reporter construct in response to BCG relative to controls. Competitive pharmacological blockade of androgen receptor inhibited the effect of DHT on BCG induced signaling in dose dependent fashion. DHT down-regulates NF-kappaB mediated IL-6 expression by human TCC lines in response to BCG. This effect depends on a functional androgen receptor signaling pathway and it can be blocked by the inhibition of androgen/androgen receptor binding.

Effects of electropermeabilization after the administration of anticancer drugs on transitional cell carcinoma.

To assess in vitro and in vivo the potential utility of electropermeabilization (EP) as an anticancer drug delivery system for the treatment of transitional cell carcinoma (TCC). To analyse the effects of EP on the internalization of adriamycin and bleomycin by cells, aliquots of a suspension of YTS-1 carcinoma cells (derived from a human TCC line) were mixed with a solution of adriamycin or bleomycin and then exposed immediately to an electric field (1000 V/cm, 1 Hz, 100-micros square wave, eight pulses). After a 2-h incubation the concentration of each drug in the cells was measured by high-performance liquid chromatography (for adriamycin) and by bioassay (for bleomycin). The concentrations of drugs in the cells were compared with untreated cells. To analyse the effects of EP on cytotoxicity, the same treatments were applied to a suspension of cells plus adriamycin or bleomycin and then the cells incubated for 6 h with tritiated thymidine ([3H]-TdR), monitoring the incorporation of [3H]-TdR into the cells. Cells with no electrotreatment acted as controls. To assess the effects on tumours in vivo, YTS-1 cells were transplanted subcutaneously into nude mice; when the tumours had reached 7 mm in diameter, EP was applied (using electrical pulses as before, 10 min after the direct injection of bleomycin into the tumour). Tumours were then measured regularly and compared with sham-treated tumours, tumours treated with electric pulses alone, and tumours treated with bleomycin alone. Survival was also compared. There were no significant differences in the levels of adriamycin between cells with and with no EP, whereas there was a marked difference for bleomycin. Growth inhibition by adriamycin with and with no EP was similar, while the growth-inhibitory effects of bleomycin were almost doubled. There was a reduction in tumour size only in the group treated with bleomycin plus electric pulses and two of five tumours disappeared completely. Survival in this group was also significantly better than in the other groups. EP after administering bleomycin might be an effective treatment for TCC.

Electropermeabilization in bladder cancer chemotherapy.

Electropermeabilization has been used for the introduction of genes into cells. Using this technique, we introduced the cytotoxic drug bleomycin (BLM) into cells and examined whether the technique might be useful for the treatment of bladder cancer. For electropermeabilization in vitro, we used YTS-1 cells, a human transitional cell carcinoma line. Aliquots of cell suspension were mixed with a solution of BLM and immediately exposed to electric pulses. A high-power pulse generator was used to supply square-shaped pulses of 1250 V/cm (100 micros, eight pulses). After a 2-h post-shock incubation, cells were washed and incubated for one further hour. Then the concentration of BLM in the cells was measured using a bioassay. For electropermeabilization of tissue, we used normal male Wistar rats. The bladder was exposed and 10 mg/kg BLM was injected into the caudal vein. A series of eight pulses with a time constant of 100 micros at an electric field intensity of 1000 V/cm was applied. The bladder, liver and lungs were extracted 1 h later and prepared for quantification of the BLM concentration using the bioassay. Electrotreated cells contained significantly higher concentrations of BLM than nonelectrotreated cells. The concentration of BLM 1 h after electrotreatment in bladder tissue was 2.7 times higher than that in nonelectrotreated bladder tissue. The electropermeabilization technique has the potential to serve as a new and effective modality for the treatment of bladder cancer.

Model of Human Transitional Cell Carcinoma: Tumor Xenografts in Upper Urinary Tract of Nude Rat

An in vivo model for the study of human transitional cell carcinoma (TCC) in the urinary tract is desirable. Orthotopic xenografts are useful in order to approximate better the behavior of human tumor cells in situ. Prior models have been described in the urinary bladder of the nude mouse and rat. We have developed the first model of implantation of human TCC in the upper urinary tract of an experimental animal. The kidneys of homozygous nude rats 4 weeks of age were inoculated with 1-5 x 10(6) cells of the RT4 well-differentiated papillary human TCC line through bilateral flank incisions, with transparenchymal injection of tumor cells into either the collecting system (renal pelvis) or the parenchyma. The overall implantation rate was 92% (54/59 kidneys). However, implantation into the collecting system occurred in only 45% (18/45) of the group. Ligation of the ureter prior to inoculation to produce urine stasis improved the mucosal implantation rate. Despite suspect urine cytology findings in the renal pelvis injection group, no distal seeding of the urinary tract was seen. Intraparenchymal tumor growth was less differentiated and had a higher fraction of mitotic cells than mucosal tumors. Vascular and lymphatic invasion were commonly seen; however, distant metastasis was not observed. This model will prove useful in determining the role of seeding in recurrent disease and in developing less invasive modalities for the treatment of upper tract TCC.

Characterization of Fibronectin Attachment by A Human Transitional Cell Carcinoma Line, T24

The ability of transitional cell carcinoma cells to adhere to and invade the extracellular matrix is important in invasion and metastasis. The glycoprotein fibronectin is associated with laminin and Type IV collagen in the bladder basement membrane. We characterized the interaction between T-24 cells, a cell line derived from an invasive transitional cell tumor, and fibronectin. The cells use the α5β1 heterodimeric integrin receptor complex to mediate adherence via the classical (RGDS) fibronectin binding site. The importance of the α5β1 receptor to T-24 function is unknown and under investigation.

The Effects of Recombinant Human Intereron-Gamma on a Panel of Human Bladder Cancer Cell Lines

We have examined the Major Histocompatibility Complex class II antigen inducing capabilities of recombinant human interferon-gamma, on a panel of human transitional cell carcinoma lines which have been raised from original tumours of varying histological grades: RT4 (grade 1), RT112 (grade 2) and MGH-U1 (grade 3). Cells were examined for class II antigens using an indirect immunofluorescent staining method and analysed on a fluorescence activated cell sorter.Twenty percent of RT4 cells constitutively expressed class II antigen. Both RT112 and MGH-U1 were repeatedly found to be negative for this antigen prior to treatment with interferon-gamma. Following treatment with interferon-gamma all three lines showed an increase in class II antigen expression, which was consistently dependent on both the length of incubation and concentration of interferon-gamma. A differential susceptibility was found amongst the three cell lines which may relate to the histological grade of the parent tumor.

The Development of Liposomes Containing Interferon Alpha for the Intravesical Therapy of Human Superficial Bladder Cancer

Current therapy of human superficial bladder cancer includes the intravesical administration of antitumor drugs and immunomodulators. The purpose of these studies was to determine whether phospholipid liposomes that bind to human bladder cancer cells can improve the delivery of interferon alpha (IFN-α) to neoplastic urothelium. The antiproliferative activity of free IFN-α and IFN-α encapsulated in liposomes was assessed in vitro against the human transitional cell carcinoma line 253J. The cells were exposed to free and liposome-encapsulated IFN-α for short periods ranging from 30minutes to four hours, and inhibition of cell growth was determined three days later. The production of >25 percent cytostasis of 253J cells by free IFN-α required four hours of continuous exposure. In contrast, IFN-α encapsulated in liposomes produced 35 percent and 60 percent cytostasis after a 30-minute and four-hour exposure, respectively. Liposome-encapsulated IFN-α was also effective (50 percent cytostasis) against a subline of 253J cells selected for resistance against free IFN-α. Liposomes containing IFN-α were stable in the presence of human urine. In vivo studies in mice showed that intravesical administration of radiolabeled IFN-α or radiolabeled liposomes did not yield significant systemic absorption and deposition in distant organs. Collectively, these results suggest that the encapsulation of IFN-α within multilamellar liposomes may augment its antiproliferative activity, overcome some forms of tumor cell resistance to IFN-α, and prove useful for intravesical therapy of superficial bladder cancer. (J. Urol, 143:1252-1260, 1990)

Growth of Urinary Transitional Cell Carcinoma Cell Lines in Agar, Agarose and Methyl Cellulose

AbstractBecause in vitro cell growth of transitional cell carcinoma expiants and cell lines often fail to adequately proliferate in semisolid media, we have examined the effect of agents used to make media semisolid (methyl cellulose, Bacto-agar, Sea Plaque agarose and Sea Prep 15/45 agarose) on the in vitro growth of 11 transitional cell carcinoma cell lines. The growth of human transitional cell carcinoma lines was supported such that agents permissive for growth ranked as follows:Sea Plaque agarose ∼ Sea Prep agarose > methyl cellulose > Bacto-agar. These observations have important implications for the in vitro study of transitional cell carcinoma cell lines and are relevant to the development of improved chemosensitivity determinations for human transitional cell carcinoma.

UM-UC-1 and UM-UC-2: Characterization of Two New Human Transitional Cell Carcinoma Lines

Two new human transitional cell carcinoma lines have been established in long term tissue culture. UM-UC-1 was derived from a bladder cancer metastasis, and UM-UC-2 originated from a ureteral carcinoma. Both cell lines produce tumors in athymic nude mice. The cell lines have been characterized by isoenzyme phenotype and karyotype. Both of these methods differentiate UM-UC-1 from UM-UC-2.