Gary Wedemeyer

Improved growth of human urothelial carcinoma cell cultures.

From January, 1981 through June, 1982 specimens from 21 patients with bladder (urothelial) cancer were placed in tissue culture, and one long term cell line was established (5%). From July, 1982 through February, 1984, using an improved culture medium, seven long term cell cultures were established from 21 patients (33%). In addition, one long term culture from a patient with a bladder melanoma was established using the standard culture medium. The nine cell cultures were derived from the following types of tumors: transitional cell carcinoma (6), adenocarcinoma (1), squamous cell carcinoma (1) and melanoma (1). All of the cell lines have produced tumors in athymic nude mice except for one transitional cell carcinoma. All of the cultures demonstrate aneuploidy. Homogeneously staining regions have been seen in some cell cultures. A common marker chromosome has not been identified.

Evidence for Urothelial Cell Activation in Interstitial Cystitis

Bladder biopsy samples from 17 interstitial cystitis patients and 20 controls were evaluated for urothelial cell activation using a panel of monoclonal antibodies to HLA-DR, intercellular adhesion molecule 1, interleukin 1 alpha and tumor necrosis factor alpha. Urothelial cells in the majority (13 of 16, 81%) of the biopsies from patients with interstitial cystitis showed increased expression of HLA-DR, while fewer samples were positive for intercellular adhesion molecule 1 (3 of 16, 19%), interleukin 1 alpha (2 of 17, 12%) or tumor necrosis factor alpha (1 of 15, 7%). No urothelial cell expression of intercellular adhesion molecule 1, interleukin 1 alpha or tumor necrosis factor alpha was detected in the controls, and only 1 of 20 control samples contained HLA-DR positive urothelial cells. These results suggest that an unusual type of cellular activation is present in interstitial cystitis. In vitro studies with cultured normal urothelial cells indicated that cells activated with gamma interferon and tumor necrosis factor alpha expressed intercellular adhesion molecule 1 and HLA-DR, although increases in intercellular adhesion molecule 1 expression occurred earlier. Urothelial cells in interstitial cystitis patients may be defective in ability to express intercellular adhesion molecule 1. Alternatively, the differential expression of HLA-DR and intercellular adhesion molecule 1 in interstitial cystitis specimens may represent a functional subset of interstitial cystitis or reflect different stages of the disease. Urothelial cell activation in interstitial cystitis may result in aberrant immune responses and immune activation within the bladder. Because HLA-DR can be detected in paraffin-embedded tissues, evaluation of urothelial cell HLA-DR expression, although not specific for interstitial cystitis, may become a useful tool in the pathological evaluation of biopsy tissues from patients with this disease.

Uptake of 2-Deoxy, 2-(18F) Fluoro-D-Glucose in Bladder Cancer: Animal Localization and Initial Patient Positron Emission Tomography

An orthotopically transplanted, locally metastasizing rat bladder tumor model was developed to evaluate the extent of uptake of fluoro-deoxy-glucose (FDG) in bladder cancer. Significant uptake of FDG in localized bladder tumors in rats was shown, with an average tumor-to-blood ratio of 39 at 2 hours after intravenous FDG administration. Metastases (3 nodal and 1 peritoneal) also showed significant uptake of FDG, with an average metastasis-to-blood ratio of 21.7, and tumor involved-to-normal lymph node ratio of 5.3. Because FDG is excreted in the urine, urinary FDG potentially could prevent the use of FDG/positron emission tomography (FDG/PET) scanning for localized bladder cancer. Bladder lavage successfully reduced the retention of FDG in the normal rat bladder, with an estimated uptake ratio of tumor-to-normal bladder of 13.1 after 5 ml. saline irrigation. Based on these data, we performed an FDG/PET scan of a patient with biopsy proved recurrent intravesical bladder cancer after radiation therapy. Computerized tomography (CT) of the pelvis showed abnormalities consistent with radiation scarring and extravesical tumor. Due to the scarring, the extent of tumor growth could not be determined. The patient also had pulmonary opacities seen on chest radiography. The FDG/PET scan of this patient showed significant extravesical uptake in the pelvis, confirming the abnormality noted on CT. Good images of the clinically apparent metastases in the chest also were obtained. These preliminary data indicate that FDG/PET imaging of bladder cancer is feasible and it may provide new information for the diagnosis and staging of patients with bladder cancer.

SPHINCTERIC EVENTS DURING PENILE VIBRATORY EJACULATION AND ELECTROEJACULATION IN MEN WITH SPINAL CORD INJURIES

PURPOSE We investigate internal and external sphincter responses during penile vibratory stimulation and electroejaculation in men with spinal cord injury. MATERIALS AND METHODS Ejaculation induction with simultaneous recording of external and internal sphincter pressures was performed in 9 spinal cord injured men. Of the patients with upper motor neuron lesions 3 underwent penile vibratory stimulation and 3 underwent electroejaculation. In 3 men who did not respond to PVS, including 1 with upper motor neuron and 2 with lower motor neuron lesions, penile vibratory stimulation and subsequent electroejaculation were performed. RESULTS In successful penile vibratory stimulation and electroejaculation upper motor neuron cases external sphincter pressure first reached a peak (average 180 cm. H2O) and subsequently decrease followed in 3 to 10 seconds by a peak in internal sphincter pressure (average 178 cm. H2O), which exceeded external sphincter pressure and ejaculation occurred. During electroejaculation, the pattern progressed, despite complete discontinuation of electrical stimulation. In electroejaculation, there was a trend for a more rapid return of external sphincter pressure greater than internal sphincter pressure, which may explain the electroejaculation retrograde fraction. In nonresponders external sphincter pressure never increased to more than 105 cm. H2O in response to penile vibratory stimulation and no ejaculation was induced. In nonresponders to penile vibratory stimulation, electroejaculation induced a typical sustained increase in internal sphincter pressure and external sphincter pressure but at lower peak pressures. CONCLUSIONS Forceful contraction of the external sphincter followed by contraction of the internal sphincter always precedes ejaculation during electroejaculation and penile vibratory stimulation. Similarities between penile vibratory stimulation and electroejaculation suggest that the latter induces ejaculation via a complex neurological pathway rather than by simple direct end organ stimulation. The sustained nature of the response to electroejaculation suggests that electrical stimulation should be stopped completely during ejaculation to allow more relaxation of the external sphincter, as this may lead to a decrease in the retrograde fraction.

Comparison of Antigen Expression on Normal Urothelial Cells in Tissue Section and Tissue Culture

Antigenic characterization of urothelial cells cultured from normal adult ureter was performed. These cells were cultured using a simplified isolation and culture technique and a commercially available serum-free medium. The cells growing in these cultures had epithelioid morphology and normal quantities of DNA. The antigen expression on these cultured normal urothelial cells was evaluated using a panel of monoclonal antibodies: 5G6.4, AN43, URO-5, anti-keratin and anti-blood group antibodies, and 425 (anti-epidermal growth factor receptor). Lower levels of anti-A and AN43 binding on cultured cells were observed than are seen on urothelial cells in sections of normal ureter, while the binding of anti-blood group H, 5G6.4, and URO-5 was unchanged. Binding of anti-epidermal growth factor receptor antibody 425 was improved if the cells were grown in medium lacking epidermal growth factor. These results confirm the urothelial origin of these cultured urothelial cells but indicate that some antigenic differences between cultured normal urothelial cells and urothelial cells in situ in the normal ureter exist.

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.

Evaluation of Multiple Drug Resistance in Human Bladder Cancer Cell Lines

We evaluated multidrug resistance (MDR) in human bladder cancer cell lines UM-UC-2, UM-UC-6, UM-UC-9 and the UM-UC-6dox subline induced to doxorubicin resistance by in vitro doxorubicin exposure. We compared the profile of multidrug resistance in these cell lines with that of the UM-UC-3 human renal cancer cell line. Of these cell lines, UM-UC-2 was most sensitive to both doxorubicin and etoposide, while UM-UC-6, UM-UC-9 and UM-UC-3 showed 1.5-, 2.1-, and 5.4-fold more resistance to doxorubicin than UM-UC-2 cells. These cell lines were also more resistant to etoposide than UM-UC-2. Addition of verapamil at 10 microM. reduced the doxorubicin resistance in UM-UC-6 and UM-UC-6dox cells, but UM-UC-9 cells showed little change in doxorubicin sensitivity in the presence of verapamil. In a model of intravesical (short-term) treatment verapamil increased the doxorubicin sensitivity of UM-UC-6dox but not that of UM-UC-6 cells. This effect in UM-UC-6dox cells was enhanced by continuously treating with verapamil after doxorubicin had been removed. Western blot analysis with rabbit anti-human P-glycoprotein polyclonal antibody demonstrated a distinct increase in P-glycoprotein in the resistant cell lines as compared with UM-UC-2. P-glycoprotein expression was roughly proportional to the degree of resistance to both doxorubicin and etoposide, but did not always correlate with the effect of verapamil on decreasing doxorubicin resistance. These results suggest that multidrug resistance is an important phenomenon in bladder cancer and that more than one pathway of multidrug resistance may be present in human bladder cancer cell lines.

Fibronectin Distribution in Normal and Malignant Urothelium

Fibronectin is a glycoprotein that mediates the attachment of BCG to the murine bladder. To assess the potential role of fibronectin on bladder cancer cells as a specific substrate for BCG binding in man, a semi-quantitative method was employed to evaluate the presence of fibronectin on normal urothelium and bladder cancer. Monoclonal anti-fibronectin binding to normal and malignant urothelial tissues was evaluated by an immunoperoxidase assay. Human tumor cell lines were evaluated with mixed hemadsorption and immunoperoxidase assays. In both systems, immunoreactive fibronectin had low expression on unfixed normal and malignant urothelium. With fixation, immunoreactive fibronectin decreased on supporting stroma and increased in normal and malignant urothelium. Fibronectin distribution did not show tumor specificity either with fixed or unfixed specimens.

The Expression of Epidermal Growth Factor Receptor on Human Bladder Cancer: Potential Use in Radioimmunoscintigraphy

Monoclonal antibody 425, which binds to an extracellular domain of the epidermal growth factor receptor, was used to evaluate the expression of this antigen on bladder cancer cells. Epidermal growth factor receptor was found on all bladder cancer cell lines tested. Immunoperoxidase staining of fourteen invasive human bladder cancers with monoclonal antibody 425 demonstrated that ten showed strong staining, one showed weak staining and three were negative. Five noninvasive tumors were similarly examined. Four of these were negative and one showed weak staining. Biodistribution experiments with human bladder tumor xenografts in athymic nude mice using radiolabeled monoclonal antibody 425 and an isotype matched control antibody demonstrated specific tumor localization at five and seven days following antibody injection. Successful imaging of a human bladder tumor xenograft was achieved five days post antibody injection. These data confirm that epidermal growth factor receptor expression correlates with bladder cancer stage and suggests that epidermal growth factor receptor may serve as a target antigen for radioimmunoscintigraphy.

CANINE MODEL OF INFERTILITY AFTER SPINAL CORD INJURY: TIME COURSE OF ACUTE CHANGES IN SEMEN QUALITY AND SPERMATOGENESIS

PURPOSE We established a canine model of subfertility after spinal cord injury and examined the time course of acute changes in semen quality and spermatogenesis after spinal cord injury. MATERIALS AND METHODS Seven dogs underwent surgical T7 spinal cord injury. Six dogs were used as controls. Electroejaculation and testicular fine needle aspiration were performed at baseline and twice weekly for 3 weeks after spinal cord injury. Semen quality change was examined by standard semen analysis. Spermatogenesis was assessed by flow cytometry of testicular fine needle aspiration in all dogs as well as by testicular histology at study conclusion in 4 controls and 4 spinal cord injured dogs. RESULTS No significant changes in spinal cord injured dogs were noted before 3 weeks after injury. From baseline to 3 weeks after injury certain changes were evident in spinal cord injured dogs. Mean antegrade sperm motility decreased from 62.9% to 20.1% (p = 0.008), mean total sperm (antegrade plus retrograde total sperm) decreased from 423 to 294 x 106 which was not statistically significant, and the incidence of testicular haploid cells decreased from 75.6% to 48.3% (p = 0.028). No significant change in any parameter was present in control dogs. The mean number of mature spermatids per cross-sectional tubule on final testicular histology was significantly decreased in spinal cord injured dogs compared with controls (13.6 versus 43.9, p = 0.02). CONCLUSIONS In the canine model tested the dogs readily survived spinal cord injury, electroejaculation was effective for obtaining ejaculate and fine needle aspiration allowed serial examination of spermatogenesis. Three weeks after spinal cord injury but not before 3 weeks sperm motility and spermatogenesis were significantly decreased. However, at the same point this decrease in spermatogenesis was not yet reflected in the total ejaculated sperm count.