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Biomedical subjects

G Wedemeyer

Publications and source records attributed to G Wedemeyer.

At least 19 recordsLinked to original sources
PubMed1997

Expression of mal is associated with urothelial differentiation in vitro: identification by differential display reverse-transcriptase polymerase chain reaction.

We have developed an in vitro urothelial differentiation model. In this model, differentiated urothelial cells assemble desmosomes and E-cadherin at cell-cell junctions and stratify and show antigenic and functional evidence for tight junctions. Using this urothelial differentiation model with the differential display reverse-transcriptase polymerase chain reaction (ddRT-PCR), we identified two independently isolated gene fragments that showed near identity with the reported sequence for a human cDNA clone named mal. Differential expression of mal mRNA during urothelial differentiation was confirmed by RT-PCR using two other sets of PCR primers. Furthermore, uncultured urothelial cells from tissues also express mal mRNA, as indicated by RT-PCR. Mal was originally identified in a subtracted cDNA library as a human T-cell differentiation-associated gene and was thought to be T-cell specific. Our results identify mal as a gene also expressed in urothelial cells during differentiation and demonstrate the power of ddRT-PCR for analysis of gene expression under these controlled conditions.

Base Sequence↗
PubMed1994

Loss of co-localization of alpha 6 beta 4 integrin and collagen VII in bladder cancer.

In normal epithelial cells, the alpha 6 beta 4 integrin co-localizes with the hemidesmosomal anchoring complex on the basolateral surface of basal cells. We studied the co-expression of alpha 6 beta 4 integrin with collagen VII, a component of the hemidesmosomal anchoring complex, in normal bladder tissues and in bladder cancers. In normal bladder, the alpha 6 beta 4 integrin co-localized with collagen VII at the junction of the basolateral surface of the basal urothelial cells and the lamina propria. In five of six noninvasive bladder cancers, the localization of collagen VII remained unchanged, found at the junction of the basal cells and the papillary connective tissue. However, in these tumors the alpha 6 beta 4 expression was not polarized and was expressed on suprabasal as well as basal cells. In invasive bladder cancers, the majority (25 of 30) showed either loss of alpha 6 beta 4 and/or collagen VII expression or showed a lack of co-localization of alpha 6 beta 4 and collagen VII. Our results show derangement of the co-localization of these two components of the hemidesmosomal anchoring complex is a consistent event in bladder cancer. Furthermore, the degree of derangement increases in invasive cancers. Loss of co-expression and co-localization of alpha 6 beta 4 and collagen VII may predispose cancer cells to local invasion and may facilitate metastasis as well.

Antigens, Neoplasm↗
PubMed1994

Expression of the VLA beta 1 integrin family in bladder cancer.

Integrins are a family of transmembrane heterodimers, many of which function as receptors for extracellular matrix molecules and play a role in adherence to and motility on matrix components. Because of these functions, integrins are suspected of participating in metastatic processes. We investigated the expression of beta 1 integrins in human bladder cancer cell lines and tissues. Expression of beta 1 integrins on cultured bladder cancer cell lines was evaluated by flow cytometry, of 8 cell lines tested, alpha 1 was found in 4, alpha 2 and alpha 3 in all 8, alpha 4 in 1, and alpha 5 in 3. These results were in sharp contrast to the expression detected by immunostaining tissues containing normal urothelium and low stage (noninvasive) and high stage (invasive) bladder cancers. All normal urothelial tissues tested expressed alpha 2 and alpha 3 and none expressed alpha 1, alpha 4, or alpha 5. Similarly, a majority (77%) of low stage (noninvasive) bladder cancers stained positively for alpha 3, whereas only 6 of 13 expressed alpha 2 and none expressed alpha 1, alpha 4, or alpha 5. Among invasive bladder cancers, alpha 1 was detected in 7%, alpha 2 in 24%, alpha 3 in 68%, alpha 5 in 10%, and alpha 4 was not found in any samples. These results indicate that integrin expression in cultured human bladder cancer cell lines does not represent expression observed in tissue samples and may reflect adaption to or selection during tissue culture conditions. A progressive loss of alpha 2 expression is seen from normal urothelial cells through invasive bladder cancers. This loss may contribute to an invasive phenotype by a loss of the cell-cell adherence function mediated by the alpha 2 beta 1 and alpha 3 beta 1 integrins.

Humans↗
PubMed1993

Fiberoptic microtransducer pressure technology: urodynamic implications.

The FST 200 is a novel commercially available pressure measurement system that combines microtechnology and fiberoptics and is particularly well suited for invasive urodynamic studies. Pressure recording with this system is highly accurate and reproducible. The pressure curves obtained parallel those from standard water cystometry. Being small and portable, the system lends itself to invasive transurethral monitoring in the privacy of an examination room. Additionally, the 5F catheter size allows the performance of leak point pressures and pressure/flow voiding studies with minimal urethral stenting artifact often seen with larger catheters. The technical aspects of this system are presented.

Calibration↗
PubMed1993

Synchronous cystoscopy and cystometry in the management of neurogenic bladder dysfunction.

The physical difficulties associated with cystoscopy and cystometry in the spinal cord injury patient led to a clinical trial of synchronous cystoscopy and cystometry in these individuals. Following a disappointing experience with external transducer methodology, a recently available fiberoptic microtransducer system was used and an effective system was developed. A total of 20 individuals with spinal cord injury underwent a standard water cystometrogram on an examination table followed by synchronous flexible cystoscopy and cystometry using a 5F fiberoptic microtransducer while seated in a chair. The results showed both pressure tracings to be clinically identical. Uninhibited contraction spikes, compliance curves and leak point pressures were essentially the same by both methods and artifact was not a problem. Given the speed, ease and reproducibility of this method, the difficulties with patient transfer for these studies have been virtually eliminated. Two urethral instrumentations have been replaced by 1. Examination room time was decreased from 60 to approximately 10 to 15 minutes with half of the required personnel. Perhaps more importantly, these occasionally problematic individuals clearly prefer this streamlined approach and patient compliance with followup has improved.

Cystoscopes↗
PubMed1993

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

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.

Adult↗
PubMed1993

The monoclonal antibody BQ16 identifies the alpha 6 beta 4 integrin on bladder cancer.

Monoclonal antibody BQ16, raised against UM-UC-9, a human bladder cancer cell line, exhibited strong reactivity with most bladder carcinoma tissue samples and cell lines. In normal urothelium, BQ16 stained only the basal surface of urothelial cells at the junction with the lamina propria. BQ16 immunoprecipitated two protein bands of approximately 140 and 180 kDa (under non-reducing conditions), while on Western blots, BQ16 identified only the 140 kDa protein indicating that BQ16 binds to one chain of a dimeric protein complex. The dimeric structure, molecular size, and basal orientation of the BQ16 antigen prompted a comparison with the alpha 6 beta 4 integrin identified by monoclonal antibody UM-A9. In most tissues BQ16 and UM-A9 produced identical staining patterns. However, normal lymphocytes and certain bladder cancer cell lines were BQ16 positive but failed to react with UM-A9, indicating that the BQ16 and UM-A9 epitopes can be expressed independently. Pulse-chase immunoprecipitation experiments showed that the alpha 6 subunit was more prominent in early BQ16 precipitates and the beta 4 subunit was more prominent in early UM-A9 precipitates. Furthermore, preclearing cell extracts with the anti-alpha 6 antibody GoH3 removed all BQ16 reactivity and in UM-A9-negative, BQ16-positive cells, BQ16 precipitated the alpha 6 beta 1 complex. We conclude that BQ16 identifies the alpha 6 integrin subunit and that alpha 6 beta 4 integrin is strongly expressed in most bladder cancers.

Antibodies, Monoclonal↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

The uptake and efflux of doxorubicin by a sensitive human bladder cancer cell line and its doxorubicin-resistant subline.

The uptake and efflux of doxorubicin (Dox) were investigated in a human bladder cancer cell line (UM-UC-6) and in a multi-drug resistant (mdr) subline (UM-UC-6Dox). Unlike previous reports, the initial uptake kinetics of Dox, and its accumulation and retention to steady-state were modelled mathematically. Cells were incubated with Dox and the amount of Dox in the cellular and medium phases was measured by a specific HPLC method. When monitored for 1 min from 0.02 microM to 25 microM Dox, the uptake was very rapid but was significantly faster in the resistant cell line. The initial rate of uptake at t = 0 followed Michaelis-Menten kinetics yielding Vmax values (the maximal rate of uptake) of 15.0 +/- 1.7 and 12.9 +/- 1.2 nmol/10(6)/min and Km (rate at Vmax/2) of 25.2 +/- 4.7 and 16.4 +/- 2.9 microM for UM-UC-6 and UM-UC-6Dox, respectively. There was no metabolism of Dox by keto-reduction or reductive hydrolysis. At 1.0 microM the uptake of Dox to steady-state was biexponential but there was no difference in total cellular Dox concentration between the two cell lines at equilibrium. A 3 compartment sequential closed model was fitted yielding significantly different values for the intercompartmental and hybrid rate constants, indicating altered intracellular distribution in resistant cells. Verapamil (10 microM), trifluoperazine (10 microM) or Tween 80 (0.005%) had no effect on the uptake or efflux of Dox. The UM-UC-6Dox line appeared to show atypical mdr characteristics since net drug accumulation was not lowered and classic P-glycoprotein inhibitors were not effective. The primary mechanism of Dox resistance is not enhanced metabolism or lowered intracellular concentrations.

Chromatography, High Pressure Liquid↗
PubMed1991

Effect of verapamil on the uptake and efflux of etoposide (VP16) in both sensitive and resistant cancer cells.

The effect of calcium antagonist verapamil on the uptake and efflux of Etoposide (VP16), a semi-synthetic derivative of podophylotoxin and a broad spectrum antineoplastic agent, has been investigated and compared in sensitive (UM-UC-2) and resistant (UM-UC-9) human bladder cancer cells, and L1210 leukemia cells, by using both radioisotope (3[H]-VP16) liquid scintillation and high performance liquid chromatography assay with electrochemical detection. The uptake of VP16 was rapid in all three cell lines, showing an initial rapid linear phase followed by a second slower phase, but at steady state the ratios of intracellular to extracellular VP16 concentrations were only 0.004-0.006. No significant difference in drug uptake was observed in sensitive UM-UC-2 and resistant UM-UC-9 cells at all concentrations studied. Verapamil at a concentration of 10 microM enhanced the intracellular VP-16 levels in all sensitive and resistant cell lines. The increments were 21.5% for UM-UC-2, 11.8% for UM-UC-9, and 31.0% for L1210 cells after 30 minutes incubation with 1 microM VP16. A slower efflux of VP16 was observed in verapamil treated cells in all three cell lines. There was a small increase in the nonexchangeable components in verapamil treated cells, although only 5-10% of VP16 was retained. No peak other than that of VP16 was detected in the HPLC chromatogram of extracts from both cell pellet and influx or efflux medium.

Animals↗
PubMed1990

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.

ABO Blood-Group System↗
PubMed1990

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.

BCG Vaccine↗
PubMed1988

Autologous antibodies to human bladder cancer.

The autologous serologic reactivity of 13 patients with bladder cancer was evaluated using cell lines derived from each individual's own tumor as targets. Protein A and immune adherence assays were employed to determine antibody binding to the tumor targets at varying passage numbers. Autologous reactivity was found in 6 of the 13 cell lines tested. However, the titer was usually low regardless of the passage number. Seven autologous serum/cell line combinations were tested using both low and high passage cells as targets. In six of these combinations, the degree of antibody binding was similar with both low and high passage target cells. The incidence of autologous reactivity in the 12 patients with urothelial tumors was 50%.

Animals↗
PubMed1986

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.

Aneuploidy↗
PubMed1985

Human renal carcinoma: characterization of five new cell lines.

Five human renal carcinoma cell lines have been established in long-term tissue culture. Two of the cell lines, UM-RC-2 and UM-RC-3, produced clear cell tumors in athymic nude mice. The cell lines have been characterized by staining with oil red O, doubling time in vitro, and number of chromosomes. Although protein A assay reactivity of autologous combinations of patient's sera and tumor cells were seen with all five cell lines, similar binding was also found with autologous normal kidney cultures. However, the immune adherence assay demonstrated low titer autologous reactivity with two renal carcinoma cell lines but not with the corresponding normal kidney cultures. This strongly suggest host recognition of tumor-associated antigens. Characterization of cell surface antigens with murine monoclonal antibodies demonstrated shared reactivity between normal kidney tubular cells and renal carcinoma cells. Antibody A68.11 reacted strongly with all five cell lines. Antibody A80 bound to only UM-RC-3 and UM-RC-6.

Adenocarcinoma↗