Urokinase and the urokinase receptor: association with in vitro invasiveness of human bladder cancer cell lines.

BACKGROUND AND PURPOSE: The plasminogen activators urokinase and tissue plasminogen activator are enzymes that degrade proteins in tissue basement membranes and the extracellular matrix (a biomolecular complex surrounding individual cells in tissues that serves as a barrier between the cells and the vascular and lymph systems). The action of these enzymes allows tumor cells to escape their local environment and metastasize. Plasminogen activator activity can be influenced by the urokinase receptor, which is expressed on the surface of cells, and by the plasminogen activator inhibitors 1 and 2. Because bladder tumors differ in their propensity to invade local areas and distant sites, we studied the expression of both plasminogen activators, the two plasminogen activator inhibitors, and the urokinase receptor in four human bladder cancer cell lines (RT4, 253J, EJ, and T24) to see if there was an association between the expression of these proteins and tumor cell invasiveness in vitro. METHODS: The expression of urokinase, tissue plasminogen activator, and the two inhibitors was measured by enzyme-linked immunosorbent assays of serum-free, cell-conditioned media (i.e., culture fluids). Cell surface expression of the urokinase receptor was assayed by flow cytometry, using an anti-receptor monoclonal antibody (Mab3936). The invasive capacity of untreated cells and of cells exposed to exogenous, high-molecular-weight urokinase was analyzed by use of Matrigel invasion chambers. RESULTS: The four bladder cancer cell lines demonstrated differential expression of both plasminogen activators and both inhibitors; three of the cell lines (T24, EJ, and 253J) expressed the urokinase receptor. The four cell lines differed in their invasive potential in vitro. Neither expression of tissue plasminogen activator nor production of the inhibitors appeared to influence Matrigel invasion. EJ cells and 253J cells produced the highest levels of urokinase and demonstrated the greatest propensity for invasion; T24 cells, which produced only small amounts of urokinase, exhibited a low invasive potential. Pretreatment of T24 cells with exogenous high-molecular-weight urokinase markedly increased their invasiveness. Similar pretreatment of EJ and 253J cells increased their invasiveness as well. RT4 cells, which lacked urokinase receptor expression but produced moderate amounts of urokinase, were not invasive and did not become so after exposure to exogenous high-molecular-weight urokinase. Binding of Mab3936 to urokinase receptors inhibited Matrigel invasion. CONCLUSIONS: To our knowledge, this is the first study demonstrating that bladder tumor cells express the urokinase receptor and that both receptor expression and urokinase expression are required for bladder tumor cell invasion in vitro.

Effect of canarypox virus (ALVAC)-mediated cytokine expression on murine prostate tumor growth.

BACKGROUND: Canarypox virus, ALVAC, does not replicate in infected mammalian cells and has potential as a vector for gene therapy in the treatment of cancer. PURPOSE: Recombinant viruses carrying DNA sequences encoding interleukin 2 (ALVAC-IL-2), interferon gamma (ALVAC-IFN gamma), tumor necrosis factor-alpha (ALVAC-TNF-alpha), or the co-stimulatory molecule B7-1 (ALVAC-B7-1) were investigated as agents for the treatment of a newly defined mouse prostate tumor model. METHODS: RM-1 mouse prostate cancer cells, which are syngeneic (i.e., same genetic background) to C57BL/6 mice, were used. The expression of foreign gene products in vitro in infected RM-1 cells was measured by immunoprecipitation, bioassay, or flow cytometry. The effects of foreign gene product expression on RM-1 tumor cell growth in C57BL/6 mice were measured after subcutaneous injection (in the back) of 5 x 10(5) uninfected or infected cells; measurements included determinations of time to a measurable tumor size, tumor size as a function of time, and survival. The induction of protective immunity by uninfected and infected RM-1 cells was tested by injection of lethally irradiated (70 Gy) cells and subsequent challenge with uninfected cells. The generation of cytotoxic T cells was monitored by use of a 51Cr release assay. Severe combined immunodeficient (SCID) mice were used to determine whether T or B lymphocytes were involved in ALVAC vector-mediated antitumor responses. Data were analyzed by use of Pearson's modification of the chi-squared test and Kaplan-Meier survival methods. Reported P values are two-sided. RESULTS: The level of foreign gene product expression in ALVAC-infected RM-1 cells was dependent on the multiplicity of virus infection used; a multiplicity of five viruses per infected cell was chosen for subsequent experiments. RM-1 tumor growth in C57BL/6 mice was not affected by tumor cell expression of IL-2 alone, IFN gamma alone, or B7-1 alone; however, expression of TNF-alpha alone significantly delayed tumor growth at early time points (compared with parental ALVAC-infected tumors, P = .0001 at day 21 and P = .037 at day 28). Tumor cell expression of both TNF-alpha and IL-2 completely inhibited tumor growth in 60%-100% of treated mice. No protection against subsequent tumor challenge was detected in mice previously exposed to RM-1 cells expressing both TNF-alpha and IL-2. Cytotoxic T-lymphocyte activity toward RM-1 cells was not observed in C57BL/6 mice that rejected tumors. Tumor cell expression of TNF-alpha and IL-2 also resulted in tumor growth inhibition in SCID mice. CONCLUSIONS: RM-1 mouse prostate cancer cells are readily infected by ALVAC vectors, and foreign gene products are efficiently expressed. Inhibition of RM-1 tumor growth by tumor cell expression of TNF-alpha and IL-2 appears to involve nonspecific antitumor activity.

Modulation of fibronectin-mediated Bacillus Calmette-Guérin attachment to murine bladder mucosa by drugs influencing the coagulation pathways.

Adjuvant intravesical Bacillus Calmette-Guérin (BCG) has proved to be an effective treatment for superficial bladder cancer. Intraluminal attachment of BCG organisms via binding to the extracellular matrix protein, fibronectin (FN), appears to be required for expression of the antitumor efficacy of BCG against a murine bladder tumor. Initial studies demonstrated that radiolabeled FN localized to the acutely injured urothelium but not to intact urothelium. These studies also demonstrated that exogenous administration of FN enhanced BCG attachment to the injured but not to the intact urothelium. Because FN has been shown to be an integral part of clot formation at sites of urothelial injury, drugs known to affect fibrin clot formation were tested for their effects on BCG attachment and antitumor efficacy in a murine bladder tumor model. A stabilizer of fibrin clot formation was shown to enhance both BCG attachment and antitumor efficacy in the same model. An increased number of BCG organisms were also retained in the lymph nodes and spleens of mice receiving fibrin clot stabilizers, suggesting indirectly that immunological mechanisms are involved in the antitumor efficacy of BCG. The data presented herein provide further support for the hypothesis that BCG attachment to the injured bladder is mediated by FN. Furthermore, modulation of BCG-FN attachment is demonstrated to be possible with drugs influencing the coagulation pathway. This attachment is shown to be required for the antitumor efficacy in a murine bladder tumor model, and thus modulation of BCG-FN attachment appears to have significant influence on the antitumor efficacy of BCG in the murine bladder tumor model.

Comparison of the fibronectin-binding ability and antitumor efficacy of various mycobacteria.

Although the mechanism by which Bacillus Calmette-Guerin (BCG) exerts an antitumor effect on superficial bladder tumors is not fully understood, recent evidence has implicated binding of BCG organisms to fibronectin (FN) as requisite for this antitumor efficacy. Various substrains of BCG and other mycobacteria were tested in vitro for their relative capacities to bind both matrix and soluble FN. A substrain of Mycobacterium kansasii, designated the "high-binding strain," was found to bind FN more readily (P less than 0.05) in in vitro studies, when compared to commercially available substrains of BCG (Tice, Connaught, and Armand Frappier). The binding by the three commercial strains of BCG to FN in vitro appeared to be equivalent. The high-binding strain was further demonstrated to attach more readily in vivo to the acutely injured murine bladder (P less than 0.005) than the Armand Frappier substrain. Finally, using the MB49 murine bladder tumor model, an enhanced antitumor effect (P less than 0.05) was noted in mice treated with intravesical high-binding strain, in comparison to the Armand Frappier substrain, during five weekly treatments. It appears not only that the commercial substrains of BCG bind FN in an equivalent manner but also that the relative binding capacities of the substrains correlate directly with antitumor activity. A substrain of M. kansasii appears to have been identified which may prove more clinically effective than the currently available strains of BCG.

Dynamics and genetics of a disease-driven species decline to near extinction: lessons for conservation.

Amphibian chytridiomycosis has caused precipitous declines in hundreds of species worldwide. By tracking mountain chicken (Leptodactylus fallax) populations before, during and after the emergence of chytridiomycosis, we quantified the real-time species level impacts of this disease. We report a range-wide species decline amongst the fastest ever recorded, with a loss of over 85% of the population in fewer than 18 months on Dominica and near extinction on Montserrat. Genetic diversity declined in the wild, but emergency measures to establish a captive assurance population captured a representative sample of genetic diversity from Montserrat. If the Convention on Biological Diversity's targets are to be met, it is important to evaluate the reasons why they appear consistently unattainable. The emergence of chytridiomycosis in the mountain chicken was predictable, but the decline could not be prevented. There is an urgent need to build mitigation capacity where amphibians are at risk from chytridiomycosis.

Early detection of prostate cancer.

Prostate cancer is unique among the potentially lethal human malignancies in the wide discrepancy between the high prevalence of histologic changes recognizable as cancer and the much lower prevalence of the clinical disease. Despite the availability of effective tests for early detection and of effective treatment for cancers so detected, the diagnosis usually is not established until the tumor is locally advanced or metastatic. Yet, physicians hesitate to use these tests for fear that many cancers found would be latent, of little threat to the life or health of the host, and treatment could introduce inappropriate morbidity. Latent or "clinically unimportant" cancers can be distinguished from those that are clinically important by the larger volume, higher grade, and greater invasiveness of the latter. The available tests can detect only those cancers large enough to be palpable, visible on ultrasound, or capable of elevating the serum level of prostate-specific antigen. Such cancers are clinically important and should be treated for cure if the life expectancy of the patient is sufficiently long and the morbidity rate of therapy is low. Early detection of prostate cancer using the tests that are available today may widen the window of opportunity so that treatment indeed becomes possible in those for whom it is necessary.

Bilateral renal cell carcinoma with metastasis to thyroid.

A case of bilateral synchronous renal cell carcinomas with metastases to the regional lymph nodes and later to the thyroid gland was treated with aggressive surgical extirpation and adjuvant gamma interferon. The patient continues to have an excellent performance status sixteen months after initial diagnosis despite a large tumor burden at presentation.

Bropirimine immunotherapy of upper urinary tract carcinoma in situ.

OBJECTIVES: Bropirimine has been shown to be effective in treating approximately 50% of patients with carcinoma in situ (CIS) of the bladder in recent clinical trials. Patients with upper tract CIS were treated with bropirimine to determine whether this oral drug might be effective in that setting. METHODS: Twenty-four patients with negative radiographic findings and positive cytologic evidence for upper tract CIS in one or both ureters received bropirimine (3.0 g/day orally) for 3 consecutive days each week for up to 1 year. Ureteral collection of urine or barbotage for cytologic analysis was performed quarterly thereafter. RESULTS: Ten (48%) of 21 evaluable patients had a negative ureteral cytologic analysis after 12 weeks (5 patients) or 24 weeks (5 patients). Of these 10 patients, 8 continue to have negative cytology for a period of 3 to 30 months (median, more than 9 months). In 2 patients, negative cytology reverted to positive at 6 and 9 months, respectively, during therapy. Twelve (50%) of the 24 patients reported no toxicity. Three patients stopped treatment at 2, 3, and 3 weeks due to pruritic rash, nausea and vomiting, and severe bone pain, respectively. Therapy was stopped in 1 additional patient between 4 and 5 months because of transient liver enzyme elevations, yet this patient has had a continuous negative cytologic analysis for more than 9 months. CONCLUSIONS: Orally administered bropirimine may be effective therapy for CIS of the ureter or renal pelvis, with acceptable toxicity in most patients. Further efforts to better define this activity as well as the possible need for maintenance or intermittent long-term therapy are warranted.

Oral bropirimine immunotherapy of bladder carcinoma in situ after prior intravesical bacille Calmette-Guérin.

OBJECTIVES: Bropirimine is an oral immunomodulator that has demonstrated anticancer activity in transitional cell carcinoma in situ (CIS) in both the bladder and upper urinary tract. Activity also has been documented in patients after prior therapy with bacille Calmette-Guérin (BCG). To more accurately estimate bropirimine's efficacy in BCG-resistant bladder CIS, a Phase II trial was performed. A separate analysis was performed in additional patients intolerant of BCG toxicity. METHODS: Patients received bropirimine 3.0 g/day by mouth for 3 consecutive days, weekly, for up to 1 year. Bladder biopsies and cytologic examination were performed quarterly. Complete response (CR) required negative biopsy and cytology results. RESULTS: Twenty-one of 86 patients entered were not evaluable. CR was seen in 21 (32%; 95th percentile confidence interval [CI], 21% to 44%) of 65 evaluable patients, including 14 (30%, CI 17% to 43%) of 47 BCG-resistant, and 7 (39%, CI 16% to 61%) of 18 BCG-intolerant patients. Overall, by intent-to-treat analysis, CR was thus seen in 21 (24%) of 86 subjects. Most BCG-resistant patients were failures to BCG without relapse, and had received 12 to 36 (median 12) BCG treatments; intolerant patients had received 4 to 11 treatments (median 6). Response duration ranged from 65 to 810 days, with median not yet reached (but greater than 12 months). Thirteen (15%) of 86 stopped bropirimine due to toxicity. Progression to invasive or metastatic disease during or immediately after therapy was documented in only 4 patients (6%), all nonresponders. CONCLUSIONS: Bropirimine may be an alternative to cystectomy for some patients with bladder CIS who have failed or have not tolerated BCG. Further evaluation to improve responses and durability is warranted.

Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies.

OBJECTIVES: Clinical staging of prostate cancer is inaccurate, often with significant upstaging on final pathologic review. We previously demonstrated the ability to predict extraprostatic extension of cancer by use of the Gleason score and serum prostate-specific antigen (PSA) measurements. Herein we present an interim analysis of data from an ongoing multi-institutional study to determine the predictive power of an enhancement of microvessel density analysis in combination with Gleason score and serum PSA to predict extraprostatic extension. METHODS: We evaluated a total of 186 randomly selected biopsy samples and matched totally embedded radical prostatectomy samples with preoperative PSA concentrations and patient demographics. Gleason score and optimized microvessel density (OMVD) were determined from the needle biopsy samples; pathologic stage was verified by independent review of the radical prostatectomy samples. An automated digital image analysis system measured microvessel morphology and calculated the OMVD in the biopsy samples (Biostage; Bard Diagnostic Sciences, Seattle, Wash). RESULTS: Prediction of extraprostatic extension was increased significantly when OMVD analysis was added to Gleason score and serum PSA concentration (P = 0.003). CONCLUSIONS: Optimized microvessel density analysis significantly increases the ability to predict extraprostatic extension of cancer preoperatively when combined with Gleason score and serum PSA concentration. This method appears to be a useful tool that can assist with treatment decisions in selected patients.

Improved detection of recurrent bladder cancer using the Bard BTA stat Test.

OBJECTIVES: To evaluate the BTA stat Test in the detection of recurrent bladder cancer. METHODS: Sensitivity and specificity were determined using frozen voided urine samples from patients with recurrent bladder cancer, volunteers, patients with nonurologic conditions, and patients with a history of bladder cancer but free of disease. Results of cytology and the original BTA Test were compared with the sensitivity of the BTA stat Test in a large subgroup of the patients with cancer. RESULTS: The BTA stat Test detected 147 (67%) of 220 recurrent cancers. For those urine samples with previous cytologic and BTA Test results available, cytology had a sensitivity of 23%, the BTA Test 44%, and the BTA stat Test 58% for detection of recurrent cancer (P < 0.001, stat versus cytology). The specificity of the BTA stat Test was 72% for benign genitourinary disease and 95% in healthy volunteers. CONCLUSIONS: The BTA stat Test has high sensitivity and is significantly superior to voided urine cytologic analysis in the detection of recurrent bladder cancer.

Failure of intravesical interferon-alfa-2b for the treatment of patients with superficial bladder cancer previously failing intravesical BCG Therapy.

Twelve patients with superficial bladder cancer who had failed one or two 6-week courses of intravesical bacillus Calmette-Guérin (BCG) therapy were treated with recombinant interferon-alfa-2b (rIFN-alfa-2b). Patients received 12 weekly instillations of rIFN-alfa-2b (100 MU) in 50 cc of normal saline. Those with an initial tumor-free response at the 3-month follow-up received maintenance rIFN-alfa-2b instillations (100 MU) monthly for an additional 9 months. Prior to rIFN-alfa-2b treatment, 6 patients had carcinoma in situ (CIS) with concurrent papillary tumor (pTa or pT I), and 6 had grades I or 2 pTa tumors. Patients were monitored every 3 months with urinary cytologies, cystoscopies, and biopsies when indicated. Median follow-up was 18 months (range 12 to 26 months). At the 3-month follow-up tumor recurrence was noted in 8 (66%) of 12 patients. An additional 3 (25%) patients had tumor recurrence at the 6-month follow-up period, and 3 (25%) patients also developed upper tract tumors during follow-up. Only 1 (8%) patient has maintained a continuous tumor-free response for 24 months. We are unable to demonstrate that rIFN-alfa-2b is likely to induce a tumor-free response in superficial bladder tumor patients who have failed intravesical BCG therapy, including those with CIS.

When intravesical measures fail. Indications for cystectomy in superficial disease.

The indications for intravesical therapy for superficial bladder cancer include multifocal or recurrent tumors, lamina propria invasion, CIS, and superficial involvement of the prostatic urethra. Tumor-free success rates approach 70% in most series. Intravesical therapy is usually administered as a 6-week course with a re-evaluation of the bladder at the 3-month interval. If disease persists, a second 6-week course of therapy is administered. If tumor recurs or persists at the 6-month evaluation, treatment is considered to have failed, and another form of therapy is instituted (Fig. 1). In the 30% of patients failing an adequate course of intravesical therapy, cystectomy may be indicated for uncontrollable superficial disease not amenable to transurethral resection, persistent grade III lesions, lamina propria invasion, persistent CIS, persistent involvement of the prostatic urethra, and persistence of tumor in a nonfunctioning bladder. Rarely, cystectomy may be indicated because of severe adverse effects related to intravesical therapy.

Urokinase (u-PA) and the u-PA receptor. Modulation of in vitro invasiveness of human bladder cancer cell lines.

BACKGROUND: Urokinase (u-PA) and the u-PA receptor (u-PAR) influence tumor invasion and metastasis. PURPOSE: The purpose of this study is to determine whether u-PAR display in 4 human bladder cancer cell lines (RT4, 253J, EJ and T24) can be modulated with substances including phorbol 12-myristate 13-acetate, interferon gamma, epidermal growth factor, and transforming growth factor beta and to correlate changes with u-PAR expression with the ability of these cells to invade artificial basement membrane (Matrigel). METHODS: u-PAR display was determined using flow cytometry and immunohistochemical staining with an anti-u-PAR monoclonal antibody (Mab3936). Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines. RESULTS: The T24, EJ and 253J cells expressed the u-PAR while the RT4 cells did not. The EJ cells (expressing the highest u-PA antigen levels and the u-PAR) invaded Matrigel. The T24 cells, which expressed the u-PAR but do not produce u-PA, invaded Matrigel only when pretreated with high molecular weight urokinase (HMW u-PA). HMW u-PA pretreatment of EJ and 253J cells also enhanced their invasive potential. Blocking u-PA/u-PAR attachment with an anti-u-PAR monoclonal antibody (Mab3936) inhibited invasion. Modulation of u-PAR expression on cell lines displaying the u-PAR directly affected in vitro invasiveness of the cell lines. The RT4 cells which lack the u-PAR were not invasive under conditions tested. Thus, bladder cancer cell lines require both u-PA and the u-PAR to invade Matrigel and modulation of u-PAR display directly affects their in vitro invasive capacity. CONCLUSIONS: This study shows that bladder tumor cells produce u-PA and express the u-PAR and require both for in vitro invasion to occur. When bladder cancer cells express the u-PAR, invasiveness can be enhanced by exogenous u-PA and inhibited by anti-u-PAR antibodies. Modulation of u-PAR expression on the cell surface of bladder cancer cells can also affect their ability to invade Matrigel. IMPLICATIONS: Histologically similar bladder tumors show differences in their propensity to invade locally or metastasize. Intrinsic differences in the tumor cells such as the production of u-PA antigen and u-PA receptor on the cell surface and extrinsic differences in the tumor cell environment such as substances influencing u-PAR display or antibodies blocking the u-PAR may affect the biological potential of human bladder cancers and offer one explanation for the aggressive or indolent tumor behavior observed in individual patients.

Can erythema nodosum and reactive arthritis be a sequel to Shigella flexneri gastroenteritis?

Erythema nodosum and seronegative arthritis associated with diarrhoea was previously regarded as implying the presence of inflammatory bowel disease. Recently, however, erythema nodosum has been described as a sequel to diarrhoea caused by infection with Salmonella typhimurium, Yersinia enterocolitica and Campylobacter jejuni. We report two patients in whom erythema nodosum and reactive arthritis followed acute Shigella flexneri gastroenteritis, a rare association.

Effect of adjuvant radiation therapy on prostate specific antigen following radical prostatectomy.

A total of 21 patients received adjuvant radiation therapy after radical prostatectomy for a persistently detectable prostate specific antigen value (more than 0.6 ng. per ml.) postoperatively. Adjuvant radiation therapy decreased serum prostate specific antigen values to the undetectable range in 6 of 21 patients (29%) all of whom have remained free of tumor recurrence with a mean followup of 12.6 months (range 6 to 30). Three patients initially showed a decrease in serum prostate specific antigen to undetectable levels but they subsequently demonstrated an increasing level within 12 months after adjuvant radiation therapy. Additionally, 7 of 13 patients whose prostate specific antigen values remained in the detectable range despite adjuvant radiation therapy have had clinical evidence of tumor recurrence. Further followup will be required to determine what ultimate impact adjuvant radiation therapy will have on survival free of tumor.

Clinical use of prostate specific antigen in patients with prostate cancer.

The clinical use of prostate specific antigen as a screening test for prostate cancer, as a preoperative determinant for staging of prostate cancer and to monitor response to therapy in prostatic cancer patients was evaluated in 168 men with benign prostatic hyperplasia and 231 men with prostate cancer. Only 3% of the men with benign prostatic hyperplasia had prostate specific antigen levels greater than 10 ng. per ml. compared to 44% of the men with proved prostate cancer. Preoperative prostate specific antigen levels increased with higher clinical stages of prostate cancer but there was substantial overlap among stages. Among patients with stage A1 prostate cancer who were followed expectantly none had an elevated prostate specific antigen value or metastatic disease during a followup of 15 to 120 months. After radical prostatectomy serum prostate specific antigen values decreased to undetectable levels (less than 0.6 ng. per ml.) in 89% of the patients with organ-confined disease, in 87% of those with microscopically positive margins only but in only 34% with seminal vesicles or lymph node involvement. Failure of the prostate specific antigen levels to decrease to the undetectable range after radical prostatectomy was associated with a greater likelihood of subsequent tumor recurrence. Only 3 of 18 patients (17%) treated with definitive radiation therapy had post-irradiation prostate specific antigen values of less than 0.6 ng. per ml., while in 39% the prostate specific antigens values remained greater than 4 ng. per ml. and in 4 of 18 (22%) the values were greater than 10 ng. per ml. Of patients with previously untreated stage D2 prostate cancer the mean pre-treatment prostate specific antigen value was 63.7 ng. per ml. compared to a post-hormonal therapy mean value of 31.1 ng. per ml. Of 32 patients treated with hormonal therapy 14 had stable disease, including 13 with prostate specific antigen levels of less than 10 ng. per ml. In contrast, 18 patients had progressive disease, of whom 16 had prostate specific antigen levels of more than 10 ng. per ml. We conclude that the serum prostate specific antigen assay is most useful clinically to monitor the response to therapy of prostate cancer patients.