Analysis of human prostate cancers and cell lines for mutations in the TP53 and KLF6 tumour suppressor genes.

A recent report suggests that the KLF6 gene encoding the Krüppel-like factor 6 protein is a frequently mutated, putative tumour suppressor gene in prostate cancer. The aims of the present study were to confirm these initial findings by determining the frequency of exon2 KLF6 mutations in a cohort of European prostate cancer patients, and to investigate whether there was evidence for mutational inactivation of both the KLF6 and TP53 tumour suppressor loci in some tumours. We examined 32 primary prostate tumours and three prostate tumour cell lines for mutations by PCR amplification and direct dideoxy sequencing (KLF6), and by oligonucleotide microarray (p53GeneChip) analysis and dideoxy sequencing (TP53). Whereas TP53 mutations typical of prostate cancer were found at a frequency consistent with the literature, no KLF6 mutations were found in any of the tumour samples nor in the three prostate cancer cell lines.

[Retroperitoneoscopy. Technique and experiences with the first 100 patients]. / Retroperitoneoskopie. Technik und Erfahrungen mit den ersten 100 Patienten.

Retroperitoneoscopic procedures were already being performed in the late 1970s. The clinical breakthrough of retroperitoneoscopy, however, was initiated in 1992 by the balloon-dissecting technique of Gaur, together with the experience gained with transperitoneal laparoscopic procedures in the upper retroperitoneum. We have modified the balloon-dissecting technique into a hydraulic video-optically controlled balloon dissection of the retroperitoneal space, and this procedure was recently further simplified. From December 1992 to December 1995 we performed 100 retroperitoneoscopic procedures in 98 patients (aged 4-82 years). Twenty-two patients had undergone previous abdominal surgery, 16 patients had been operated on for kidney and ureter problems. We performed 28 simple procedures (6 renal biopsies, 17 renal cyst resections, 4 ureterocutaneostomies, 1 foreign body retrieval), 65 difficult operations (47 nephrectomies, 5 nephroureterectomies, 5 nephropexies, 4 ureterolithotomies, 2x ureterolysis). There were also 9 complicated cases (5 heminephrectomies, 2 tumor nephrectomies, 2 dismembered pyeloplasties). Operating time, complications and conversion rate to open surgery mainly depended on the difficulty of the procedure and the personal learning curve, resulting in 50-90 min, 0% and 3.8%, respectively, for an easy retroperitoneoscopy, 90-210 min, 12% and 9.2% respectively for a difficult operation, and 180-390 min, 22% and 11%, respectively, for a very difficult procedure. Excluding the 17 patients with complications or conversion (unclear anatomy, extremely difficult dissection), the mean postoperative need for analgesics was 0.7 vials, and the mean hospital stay was 4.7 days. We have now passed most of our learning curve, and retroperitoneoscopy has become a standardized procedure that is a part of the training program for the experienced urologist.

Spiral-reinforced ureteral stent: an alternative for internal urinary diversion.

Internal urinary diversion of chronic ureteral obstruction is not sufficient in as many as 60% of patients because of tumor compression, catheter kinking, or a small stent lumen. To prevent such problems, we developed a new ureteral stent that is stable in form in spite of a large lumen. This catheter is a thin polyurethane tube supported by a built-in metal spiral wire. We have attempted to use this stent in 16 cases of chronic ureteral obstruction. Stent placement was successful in 14. Other than urinary tract infection in two patients, bladder urgency in one, and stent dislocation in another, there were no complications. Hydronephrosis disappeared soon after stent application in 12 of the 14 patients, and in the remaining two, hydronephrosis was decreased but not totally eliminated. Catheters were left in place for an average of 6.5 weeks (range 2.5-8.5 weeks). A change of catheter because of blockage was necessary in only one patient. This spiral-reinforced stent enables better internal urinary drainage, especially in cases of malignant ureteral obstruction.

Basic experimental studies on corpus cavernosum electromyography and smooth-muscle electromyography of the urinary bladder.

In contrast to the extensive work on in vitro experiments for elucidation of the electrical properties of smooth musculature, the acquisition of knowledge on electrical signal behaviour from smooth-muscle cells in an in vivo situation remains very limited and rare. Smooth-muscle electromyographic recording from the smooth musculature of the genitourinary tract, in particular from the penile cavernous bodies and the urinary bladder, has recently become one of the most interesting issues in both impotence research as well as neurophysiological assessment of the urinary bladder. However, the inadequate available data on corpus cavernosum and EMG recordings remains controversial due to the significant discrepancy between basic physiology of the smooth musculature, technical prerequisites and the expected clinical impact from the smooth-muscle EMG of genitourinary organs. This article is an attempt to describe the fundamentals of smooth-muscle EMG signal behaviour and the technical prerequisites for data acquisition and analysis of electrical activity from smooth-muscle cells of the cavernous bodies and urinary bladder. A description is given of the technical aspects, including methodology and interpretation of the recorded data, and also of the possible interference by artefacts (endogeneous and exogeneous) that might limit the clinical relevance of this encouraging method. The advantages, pitfalls and limitations of online analogous data registration and the possibility of computer-assisted smooth-muscle electrical activity recording and analysis are demonstrated by basic in vivo studies on cavernous bodies and also the detrusor muscle.

[Ureteral stent reinforced with a spiral--an alternative to internal drainage of urine]. / Sonde urétérale renforcée par une spirale--une alternative pour le drainage interne de l'urine.

Internal urinary diversion of chronic ureteral obstruction is not sufficient in up to 60% of the cases. Factors for this high failure rate are tumor compression, catheter kinking or a small stent lumen. To prevent such problems we have developed a new ureteral stent which is stable in form in spite of a large stent lumen. This catheter is made of a thin polyurethane tube which is supported by a built-in metallic spiral wire. We have used this stent in 16 cases of chronic ureteral obstruction. Stent placement was successful in 14 cases. Other than urinary tract infection in two cases, bladder urgency in one case and stent dislocation in another case there were no complications. Hydronephrosis disappeared soon after stent application in 12 out of 14 cases. In the remaining two cases hydronephrosis was decreased, but not totally eliminated. Catheters were left in place for an average of 6.5 weeks (2.5-8.5). A change of catheter due to catheter blockage was necessary only in one case. In conclusion, this spiral reinforced stent enables a better internal urinary drainage especially in cases of malignant ureteral obstruction.

[Internal drainage of urine in cases of complicated urinary stasis caused by pregnancy]. / Le drainage interne de l'urine en cas de stase urinaire compliquée provoquée par une grossesse.

In 14 women with sympthomatic hydronephrosis due to pregnancy (calyx diameter 1-2,6 cm) an internal urinary drainage was carried out during the second half of pregnancy. Retrograde stenting was placed per cystoscopy in all cases. Follow-up examinations were taken weekly for the first two weeks and further on biweekly. Primary stenting was possible in 12 out of 14 cases, and in two patients dilatation of the ureteral orifice was necessary. 11 out of 14 patients suffered from complications such as severe dysuria (9), urinary tract infection (7), persisting lumbar pain plus catheter lumen obstruction (6 each) as well as catheter migration (3). Long-term follow-up showed that urinary tract obstruction was relieved by stenting in only 6 out of 14 patients. Sufficient urinary drainage by so called double-J ureteral stents was achieved in less than half of the cases. Moreover, there was a complication rate of more than 75%. Taking these results into consideration, internal drainage of complicated pregnancy hydronephrosis needs careful evaluation.

[Internal urinary diversion in complicated pregnancy-induced hydronephrosis]. / Untersuchungen zur internen Urinableitung bei komplizierter schwangerschafts-bedingter Harnstauung.

In 14 women with sympthomatic hydronephrosis of pregnancy (calix diameter 1-2.6 cm) an internal urinary drainage was carried out during the second half of pregnancy. Retrograde stenting was performed following cystoscopy in all cases. Follow up examinations were taken weekly for the first two weeks and further on biweekly. Primary stenting was possible in 12 out of 14 cases and in 2 patients dilatation of the ureteral orifice was necessary. 11 out of 14 patients suffered from complications consisting of severe dysuria (9x), urinary tract infection (7x), persisting lumbar pain plus catheter lumen obstruction (6x each) as well as catheter dislocation (3x). Long term follow up showed that urinary tract obstruction was relieved by stenting in only 6 out of 14 patients. Sufficient urinary drainage by so called double-J-ureteral stents was achieved in less than half of the cases. Moreover, there was a complication rate of more than 75%. Taking these results into consideration, internal drainage of complicated pregnancy hydronephrosis needs careful evaluation.

Penile revascularization in nonresponders to intracavernous injections using a modified microsurgical technique.

At certain centers, microsurgical penile revascularization, using different surgical techniques, has gained importance throughout the past years. In general, only patients classified as intracavernous injection nonresponders are subjected to this kind of surgery. Since 1988, revascularization surgery has been performed at our clinic on 19 intracavernous injection nonresponders. The Hauri technique was carried out on the first 6 patients. The last 10 patients underwent modified anastomosis surgery. The inferior epigastric artery and the dorsal penile artery are anastomosed, one behind the other, end-to-side, to the dorsal penile vein. This results in a more simple procedure with assurance of flow. The Virag technique was performed on 3 patients. 18 patients achieved erections with or without the aid of intracavernous injections (at a mean follow-up of 13.4 months). 11 patients were capable of spontaneous erections, whereby it was particularly noted that 8 of the 10 patients undergoing the modified technique achieved spontaneous erections. The results demonstrate that intracavernous injection nonresponders benefit from revascularization surgery.

[Pregnancy-induced changes in adrenergic receptors]. / Schwangerschaftsbedingte Veränderungen adrenerger Rezeptoren.

In the 24th and 26th week of pregnancy and also 6 weeks post partum, n = 32 pregnant women were evaluated for the concentration of adrenergic receptors on blood cells by radioligand binding assay and an isoprenaline stimulation of the beta-receptors. The concentrations of estradiol and progesterone in serum were also determined and nephrosonography was carried out. The stimulation and concentration of the beta-receptors were significantly higher during pregnancy than post partum. The alpha-receptors displayed contrary alterations: Here, the concentration evaluated during pregnancy was considerably lower. No connection was determined between the progesterone level in serum and the extent of the receptor alterations. However, one was found with the estrogen level and the dilatation of the upper urinary tract: Pregnant women with pronounced alterations above the median showed a distinct decrease of estradiol and a marked dilatation of the kidney collecting system. Alterations in the adrenergic receptors can therefore be determined during pregnancy. The extent of the receptor fluctuation is connected to the estradiol concentration in serum and the dilatation of smooth muscle hollow organs.

[Success and failure with double J ureteral stent. Analysis of 107 cases]. / Succès et échecs avec la sonde double J. Analyse de 107 cas.

An internal urinary diversion of the upper urinary tract was planned in 107 patients with acute, stone-related or chronic tumor-induced ureteral obstruction. Acute ureteral obstruction (n = 34, group I): in 30 of 32 cases (94.1%), successful placement of an indwelling stent resulted in complete drainage in 28 of 30 patients (93.7%). In 2 cases, no satisfactory urine diversion was possible, despite several catheter changes and confirmed clearway through the installed catheters. Chronic ureteral obstruction (n = 73, group II): successful placement of the indwelling stent was possible in 63 of 73 (86.3%) cases. Complete urine diversion was achieved in only 25 patients (39.7%). Hydronephrosis was diminished in 28 patients (44.4%) and persisted or increased in 10 cases (15.9%), despite orthotopic positioning of the catheter and numerous stent changes. Complete urine drainage was achieved with an internal urine diversion in only 4 of 10 cases with chronic hydronephrosis. It is assumed that the reason for the high failure rate lies in the catheter construction and, with respect to chronic hydronephrosis, the reduced or non-existent contractility of the upper urinary tract in combination with the inevitable pressure connection between the upper and lower urinary tract.

Circumvention of multidrug resistance mediated by P-170 glycoprotein using calcium antagonists in primary human renal cell carcinoma.

In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance mediated by P-170 glycoprotein in vitro. So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded sufficient dosage. Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept. In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance. Concomitantly, P-170 glycoprotein expression was traced immunohistochemically using moab C 219. Substances derived from piperazine (flunarizine) showed only minor effects in this respect. The calcium antagonists of the papaverine type such as verapamil etc. revealed the strongest reversal of chemoresistance. Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the verapamil activity. All calcium antagonists lead to a significant enhancement of vinblastine cytotoxicity. An obvious link of P-170 glycoprotein to vinblastine chemoresistance was demonstrated. This particular resistance characteristic was detected in 19 of 27 resistant cases, but in none of the tumors displaying a chemoresponse. In particular, the new stereoisomer R-verapamil, which showed strong reversal of chemoresistance but which exerts 10 times lower cardiovascular side effects than racemic verapamil, seems to be suitable for further evaluation with regard to the clinical application.

Cross-resistance patterns related to glutathione metabolism in primary human renal cell carcinoma.

In 59 cases of primary human renal cell carcinoma (RCC), cross-resistance and collateral susceptibility patterns were determined in an MTT microculture assay. Concomitantly, the glutathione (GSH) content and the enzymatic activity of gamma-glutamyl transpeptidase (GGT) were measured as distinct resistance characteristics. Resistance or chemoresponse towards Vinca alkaloids and anthracyclines were found to be highly coincident, suggesting that the classical multidrug resistance mechanism is active in human RCC. Strong resistance to platinum complexes combined with relative sensitivity to bleomycin was significantly associated with elevated glutathione levels, providing evidence for another pathway instigating chemoresistance. In contrast, despite substantial enzymatic activity, GGT effects revealed no correlation to the chemoresistance pattern. This result implies that it is the GSH-linked binding and reduction potential rather than the GGT-associated transportation capacity that has an impact on the expression of chemoresistance in human RCC.

Mechanisms and modulation of multidrug resistance in primary human renal cell carcinoma.

Human renal cell carcinomas show a high degree of intrinsic multidrug resistance. In experimental cell lines, the membrane bound P-170 glycoprotein and the glutathione redox cycle seem to contribute to this phenomenon. P-170 may be inactivated by calcium antagonists; the glutathione redox cycle by buthionine sulfoximine. We studied the resistance patterns of 35 human renal cell carcinomas against vinblastine, doxorubicin and carboplatinum in a tetrazolium-based microculture assay. Concomitantly, P-170 expression was traced immunohistochemically using moab C219 and the glutathione content was determined enzymatically. Reversal of multidrug resistance was examined by applying the R-stereoisomer of verapamil and/or by addition of buthionine sulfoximine. A high degree of chemoresistance was seen in 27 tumors against vinblastine, in 30 tumors against doxorubicin and in 31 tumors against carboplatinum. Chemoresponse was found in eight, five or four cases respectively. P-170 was detected in 70% of highly vinblastine resistant and in 63% of highly doxorubicin resistant tumors, but in none of the less resistant cases. Resistance against carboplatinum and doxorubicin was significantly associated with elevated glutathione levels as compared to less resistant renal cell carcinomas. R-verapamil lead to a strong reversal of vinblastine resistance and to a distinct circumvention of doxorubicin resistance, but revealed no effect in carboplatinum resistance. Buthionine sulfoximine overcame carboplatinum resistance and modified doxorubicin resistance, but had no influence on vinblastine resistance. The combined application of R-verapamil and buthionine sulfoximine reversed doxorubicin resistance but did not act synergistically in vinblastine or carboplatinum resistance. Both mechanisms, P-170 and glutathione, occurred independently of each other and may well explain multidrug resistance of human renal cell carcinomas.

Effects of calcium antagonists in multidrug resistant primary human renal cell carcinomas.

Human renal cell carcinomas display a characteristically high degree of intrinsic chemoresistance to a multitude of chemotherapeutic agents. It was suggested previously, that P-170 glycoprotein contributes to this phenomenon in renal cell carcinoma indicated by elevated MDR-1 gene mRNA levels and by the expression of this specific resistance characteristic. The P-170-related efflux mechanism can be inactivated by certain calcium antagonists. P-170 was traced immunohistochemically using monoclonal antibody C 219. Concomitantly, we studied the enhancement of vinblastine cytotoxicity with 4 major classes of calcium-blocking agents in a microculture tetrazolium assay. Seven different calcium antagonists were selected: verapamil (VPM, racemic form), its R-stereoisomer (R-VPM), diltiazem, flunarizine, nifedipine, and its derivatives nimodipine and nitrendipine. Verapamil or R-verapamil causes a significant decrease of viable tumor cells as compared to vinblastine alone (P less than 0.001). Similar effects were found with diltiazem, nifedipine, and its derivatives reaching approximately 70% of the VPM/R-VPM activity. Flunarizine showed only minor enhancement of cytotoxicity. P-170 expression was demonstrated in 18 of 32 tumors, and a relation to chemoresistance was evident. None of the chemoresponders, but 18 of 25 (72%) of the highly resistant tumors, revealed this resistance factor. It was concluded that certain calcium antagonists in combination with chemotherapy may well offer therapeutic options in renal cell carcinoma as they apparently inactivate the underlying mechanism conferring resistance. The new stereoisomer R-VPM, in particular, may be used in clinical trials since it combines strong enhancement of vinblastine drug responsiveness with a 10-fold lower cardiovascular activity as compared to racemic VPM, thus allowing higher concentrations to be applied.

Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).

MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

[Role of calcium antagonists in the treatment of chemoresistant cancer of the kidney]. / Rôle des antagonistes de calcium dans le traitement des cancers chimiorésistants du rein.

In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance in vitro. So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded a sufficient dosage. Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept. In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance. Substances derived from piperazine (flunarizine) showed only minor effects in this respect. The calcium antagonists of the papaverine type such as Verapamil etc. revealed the strongest reversal of chemo-resistance. Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the Verapamil activity. All calcium antagonists tested lead to significant enhancement of vinblastine cytotoxicity. In particular, the new stereoisomer R-Verapamil, which showed strong reversal of resistance but which exerts 10 times lower cardiovascular side effects than racemic Verapamil, seems to be suitable for further evaluation with regard to the clinical application.

P-170 glycoprotein, glutathione and associated enzymes in relation to chemoresistance of primary human renal cell carcinomas.

High intrinsic chemoresistance contributes to the dismal outcome of patients with disseminated renal cell carcinoma (RCC). In experimental cell lines, two defined defence mechanisms, P-170 glycoprotein and glutathione metabolism, have been established in multidrug resistance, a cross-resistance to cytotoxic compounds without functional or structural similarities. In 21 primary human RCCs, P-170 expression was examined, glutathione content and activities of related enzymes determined and the results were correlated to the degree of in vitro chemoresistance. P-170 was found in 10 of 17 resistant tumors but in none of the sensitive cases. The glutathione content was significantly higher and the related enzyme distinctively enhanced in resistant RCCs. Both mechanisms occurred independently and may well explain the multidrug resistance of RCC. Therefore, reversal of one or both systems may have a clinical impact on the chemotherapy of RCCs.

[3H]idazoxan and some other alpha 2-adrenergic drugs also bind with high affinity to a nonadrenergic site.

We compared the pharmacological properties of the alpha 2-adrenergic radioligand [3H]idazoxan with those of [3H]rauwolscine in rat and [3H]yohimbine in human renal cortical membranes. The density of "specific" [3H]idazoxan binding sites (defined by 100 microM tolazoline) was twice as high as that of [3H]rauwolscine in rat kidney and four times as high as that of [3H]yohimbine in human kidney. A variety of structurally different drugs fully competed for specific [3H]rauwolscine and [3H]yohimbine binding, with affinities appropriate for the interaction with alpha 2-adrenergic receptors. Specific [3H]idazoxan binding, however, was only partially competed for by the catecholamines epinephrine and norepinephrine in both tissues. Thus, [3H]idazoxan labels both alpha 2-adrenergic receptors and a nonadrenergic site. Clonidine, B-HT 920, moxonidine, phentolamine, prazosin, yohimbine, dopamine, and serotonin also could not compete for this site. However, UK 14,304, guanabenz, indanidine, tolazoline, oxymetazoline, and SK&F 104,078 competed for the additional [3H]idazoxan sites with affinities similar to those at alpha 2-adrenergic receptors. [3H]idazoxan binding substantially in excess of [3H]rauwolscine or [3H]yohimbine binding was also found in human platelets, myometrium, and erythroleukemia (HEL) cells but not in three cell lines lacking alpha 2-receptors (MDCK, BC3H1, and Jurkat cells). Although we have been unsuccessful thus far in defining the precise nature of the additional [3H]idazoxan binding sites, we hypothesize that these sites may be closely affiliated with alpha 2-adrenergic receptors but clearly distinct from the catecholamine binding site of the receptor. The results indicate that care must be taken in the use of [3H]idazoxan or drugs that are recognized at its nonadrenergic site when studying alpha 2-adrenergic effects and receptor subtypes.

[The effect of beta-stimulation and beta-1-blockade on the motility of the upper urinary tract]. / Untersuchungen zum Einfluss von beta-Stimulation und beta-1-Blockade auf die Motilität des oberen Harntraktes.

18 patients with severe congestion of the urinary tract in pregnancy were treated by implantation of ureteral catheters for intermittend exoneration. Before and during the infusion of 2 micrograms/min Fenoterol the basal tone, the frequency and amplitude of the contractions of the renal pelvis were measured after retrograde filling with increasing volumes. 45 min after the i.v. application of 10 mg Metoprolol the measurements were repeated under the same conditions. During the infusion of Fenoterol there was a significant decrease of the frequency and amplitude of the contractions of the renal pelvis. Whereas at low retrograde filling volumes the basal tone in the renal pelvis decreased, at higher filling volumes - because of the concomittant deteriation of the urine transport capacity - an increase of the basal tone could be found. The relaxant effects of the beta-stimulation on the upper urinary tract could be diminished by the application of the beta-1-blocker Metoprolol; a complete compensation however was not possible.