MR urography versus retrograde pyelography/ureteroscopy for the exclusion of upper urinary tract malignancy.

AIM: To evaluate the diagnostic performance of magnetic resonance urography (MRU) versus retrograde pyelography and/or ureteroscopy (RPU) in the detection of upper urinary tract neoplasms. MATERIALS AND METHODS: This retrospective study included 35 patients with suspected upper urinary tract malignancy who underwent MRU and RPU within 6-months in our institution during the study period (February 2002 to January 2007). MRU and RPU reports were reviewed and results recorded. For each patient, the urinary tract was sub-divided into four regions for analysis: left kidney/renal pelvis, left ureter, right kidney/renal pelvis, and right ureter. MRU and RPU results for each patient were compared to a reference standard and the diagnostic performance of both techniques was compared. RESULTS: A total of 113 regions were analysed on MRU and 90 regions on RPU. Nineteen neoplasms were identified. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of urinary tract neoplasms were 63, 91, 60, and 92% for MRU, respectively, and 53, 97, 83, and 88% for RPU, respectively. These differences were not statistically significant (p>0.05). CONCLUSION: The high negative predictive value of MRU in the present series supports its use as a non-invasive screening examination for excluding the presence of upper urinary tract malignancy.

Preoperative serum acid phosphatase and alkaline phosphatase are not predictors of pathological stage and prostate-specific antigen failure after radical prostatectomy.

OBJECTIVE: To examine the utility and prognostic significance of enzymatic serum acid phosphatase (total acid phosphatase, TAP, and prostatic fraction of acid phosphatase, PFAP) and alkaline phosphatase (ALP) for staging, grading and outcome of patients who underwent radical retropubic prostatectomy (RRP) after the introduction of prostate-specific antigen (PSA) testing. PATIENTS AND METHODS: In all, 180 consecutive patients with clinically localized prostate cancer who underwent RRP with standard obturator lymph-node dissection between 1 January 1990 and 31 December 1995 were evaluated. Levels of TAP of > 5.4 IU/L, PFAP of > 1.2 IU/L and ALP of > 120 IU/L were classified as abnormally high. The relationship between abnormally high values and prostate cancer stage, grade and time to recurrence after RRP were calculated. The median follow-up was 86 months (approximately 7 years). RESULTS: Of the 180 patients, information about preoperative TAP, PFAP and ALP were available in 164, 163 and 154, respectively; TAP was abnormal in seven (4%), PFAP in 33 (20%) and ALP in only 13 (8%). None of the markers examined was associated with any variables of disease severity, as measured by pathological stage, Gleason score, perineural invasion, capsular penetration, positive margins, seminal vesicle involvement, and lymph node involvement. Abnormal TAP, PFAP or ALP were not associated with recurrence (P = 0.96, 0.45 and 0.41, respectively). In contrast, a PSA level of > 4 ng/mL was predictive of recurrence after RRP (P < 0.001). In the sample overall, 25 (14%) of the patients had recurrence and only one died from prostate cancer. CONCLUSIONS: Preoperative enzymatic serum TAP, PFAP and ALP levels are not predictors of the severity of disease or PSA disease-free recurrence after RRP.

Clinical management of prostatic intraepithelial neoplasia as diagnosed by extended needle biopsies.

OBJECTIVE: To examine the results of the clinical management of patients with high-grade prostatic intraepithelial neoplasia (PIN), as diagnosed by extended needle biopsies. PATIENTS AND METHODS: The clinical data were reviewed from a cohort of 387 men who underwent > or = 10 core prostate needle biopsies between 1 January 1996 and 31 December 1997 by one urologist (W.C.D.). Two study groups were identified; the first comprised 47 patients with only high-grade PIN and the second was a control group of 137 patients with only benign findings on their biopsies. Those patients with cancer, atypia or a prostatic biopsy with fewer than 10 cores were excluded. The clinical and histological data were evaluated. The criteria for re-biopsy were two successive increases in prostate specific antigen (PSA) level or any change in the findings on digital rectal examination (DRE). All patients were monitored at 6-12 month intervals. RESULTS: Of the 387 patients, 46% had normal findings, 5.2% had atypia, 12.6% had PIN alone, 15 (3.9%) had PIN plus atypia, 6.7% had PIN plus cancer and 32.3% had cancer. There was no significant difference between the PIN and control groups in age, DRE, PSA level, prostate size (by ultrasonography), free testosterone level, number of the cores and time of follow-up (median 34.8 and 36.6 months for the PIN and control groups, respectively). Of the PIN and control groups, 21 (45%) and 43 (31%) respectively had at least one re-biopsy. Five patients (24%) in the PIN and one (2.3%) in the control group developed cancer (P = 0.0124). All these patients had organ-confined disease and were found to have either Gleason scores 3 + 3 or 3 + 4 on surgical specimens. There was no correlation between the original location of PIN and the location of subsequent malignancy. CONCLUSIONS: Patients with one set of extended needle biopsies with high-grade PIN should be followed clinically every 6-12 months, and it may be safe to reserve repeat biopsy for those with changes in PSA level and/or in the DRE.

Long-term remission in a patient with metastatic collecting duct carcinoma treated with taxol/carboplatin and surgery.

Collecting duct carcinoma of the kidney is a rare and aggressive neoplasm of the distal collecting tubules for which there is no established therapy. We describe a young woman with metastatic collecting duct carcinoma who responded to Taxol/carboplatin chemotherapy with an 80% reduction in her tumor burden, including complete regression of lymph node metastases and significant shrinkage of a renal mass. She was rendered free of disease through nephrectomy and has been without a recurrence for 20 months. This suggests that Taxol/carboplatin chemotherapy and surgery should be considered for the treatment of metastatic collecting duct carcinoma.

Salvage intravesical therapy with interferon-alpha 2b plus low dose bacillus Calmette-Guerin is effective in patients with superficial bladder cancer in whom bacillus Calmette-Guerin alone previously failed.

PURPOSE: We determined whether combining low dose bacillus Calmette-Guerin (BCG) interferon-alpha 2B would be effective for patients in whom previous BCG failed. MATERIALS AND METHODS: A total of 40 patients in whom 1 (19) or more (21) previous induction courses of BCG failed received 6 to 8 weekly treatments of 1/3 dose (27 mg.) BCG plus 50 million units interferon-alpha 2B. Additional 3 week miniseries of further decreased BCG (1/10, 1/30 or 1/100) titrated to symptoms without changing the interferon-alpha 2B dose were given at 5, 11 and 17 months. In 12 patients a second induction course was given with 1/10 BCG plus 100 million units interferon-alpha 2B. There was multifocal disease in 39 patients, previous BCG had failed within 6 months in 34, disease was aggressive (stage T1, grade 3 or carcinoma in situ in 31, there had been 2 or more previous recurrences in 25 and disease history was greater than 4 years in 13. RESULTS: At a median followup of 30 months 63% and 53% of patients were disease-free at 12 and 24 months, respectively. Patients in whom 2 or more previous BCG courses had failed fared as well as those with 1 failure. Of the 18 failures 14 occurred at the initial cystoscopy evaluation. Of 22 patients initially counseled to undergo cystectomy 12 (55%) are disease-free with a functioning bladder. Combination therapy was well tolerated. CONCLUSIONS: While longer followup and larger multicenter studies are required to validate these encouraging findings, intravesical low dose BCG plus interferon-alpha 2B appears to be effective in many cases of high risk disease previously deemed BCG refractory. However, early failure while on this regimen should be aggressively pursued with more radical treatment options.

1,4-Disubstituted imidazoles are potential antibacterial agents functioning as inhibitors of enoyl acyl carrier protein reductase (FabI).

1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.

Inhibitors of bacterial enoyl acyl carrier protein reductase (FabI): 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as potential antibacterial agents.

An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus.

Triclosan, a widely used antibacterial agent, possesses potent activity against Staphylococcus aureus. This study reports on an investigation of the antibacterial target of triclosan in this pathogen. A strain of S. aureus overexpressing the enoyl-[acyl-carrier-protein] reductase (FabI), demonstrated by Western immunoblotting, gave rise to an increase in the MIC of triclosan, while susceptibilities to a range of unrelated antibacterials were unaffected. There are approximately 12 000 molecules of FabI per cell in mid-log phase growth. This number increased by approximately three- to four-fold in the S. aureus FabI overexpressor. Triclosan selectively inhibited the incorporation of [(14)C]acetate into TCA-precipitable product, an indicator of fatty acid biosynthesis. Furthermore, it inhibited de novo fatty acid biosynthesis in this organism. In vitro, triclosan inhibited recombinant, purified S. aureus FabI with an IC(50) of approximately 1 microM. The combination of these biochemical and genetic data provide further evidence that the mode of action of triclosan in S. aureus is via inhibition of FabI.

Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.

The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.

Structure-based combinatorial library design: discovery of non-peptidic inhibitors of caspases 3 and 8.

Structure-based design of a combinatorial array was carried out in order to identify non-peptidic thiomethylketone inhibitors of caspases 3 and 8. Five compounds from the designed array were active against caspase 3, and two were active against caspase 8. A 2.5-A resolution co-crystal structure of caspase 3 and a thiomethylketone array member is reported. The structure-based design strategy has proved useful for identifying caspase inhibitors.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Simplified velocity equations for characterizing the partial inhibition or nonessential activation of bireactant enzymes.

The steady state velocity equation for a bireactant enzyme in the presence of a partial inhibitor or nonessential activator, M, contains squared substrate concentration and higher-ordered M concentration terms. The equation is too complex to be useful in kinetic analyses. Simplification by the method of Cha (J. Biol. Chem. 243, 820 825 (1968)) eliminates squared substrate concentration terms, but retains higher-ordered terms in [M]. It is shown that if strict equilibrium is assumed between free E, M, and EM and for all but one other M-binding reaction, a velocity equation is obtained for an ordered bireactant enzyme that is first degree in all ligands in the absence of products. The equation is an approximation (because it was derived assuming only one M-binding reaction in the steady state), but it contains five inhibition (or activation) constants associated with M, all of which can be obtained by diagnostic replots and/or curve-fitting procedures. The equation also provides a framework for obtaining limiting constants (V'max, K'ia, K'mA, K'mB) that characterize the enzyme at saturating M. The same approach is applicable to an enzyme that catalyzes a steady state ping pong reaction.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Early catheter removal in 100 consecutive patients undergoing radical retropubic prostatectomy.

OBJECTIVES: To investigate the outcome of 100 consecutive patients selected for early catheter removal after radical retropubic prostatectomy (RRP), where urethral catheter drainage is used routinely for 2-3 weeks. PATIENTS AND METHODS: The study included 129 consecutive patients with clinically localized prostate cancer who underwent RRP. Catheters were removed in the clinic (with no radiographic studies) 8-9 days after RRP provided there was no evidence of urine leak, pelvic haematoma, rectal injury or severe obesity. The follow-up (mean 21 months) results were available for 118 patients, 100 of whom were candidates for early catheter withdrawal. Their records were reviewed for evidence of complications, including urinary retention, anastomotic stricture formation and urinary incontinence. RESULTS: Urinary retention developed in two of the 100 patients, requiring simple catheter replacement. Nine patients developed bladder neck contracture requiring dilatation or incision. No patients developed anastomotic disruption, urinary tract infection or pelvic abscess. At the mean follow-up of 21 months, 76% of patients were continent and did not require pads; 19% of patients had mild stress urinary incontinence requiring the use of 4 pads/day. CONCLUSION: With appropriate patient selection as described, catheters can be removed in the clinic (with no radiographic studies) 8-9 days after RRP, with no increased incidence of complications, including anastomotic stricture, retention or incontinence.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Association of the Bloom syndrome protein with topoisomerase IIIalpha in somatic and meiotic cells.

Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301-316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611-620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans.

Apoptosis in polycystic kidney disease: involvement of caspases.

Polycystic kidney disease (PKD) is characterized by the development of large renal cysts and progressive loss of renal function. Although the cause of the development of renal cysts is unknown, recent evidence suggests that excessive apoptosis occurs in PKD. With the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we have confirmed the presence of apoptotic bodies in cystic kidneys of congenital polycystic kidney (cpk) disease mice carrying a homozygous mutation at 3 wk of age. Apoptosis was localized primarily to the interstitium with little evidence of cell death in cyst epithelium or noncystic tubules. In addition, we observed that the expression of various caspases, bax and bcl-2, was upregulated in cystic kidneys. With the use of various substrates in enzyme activity assays, we have demonstrated a greater than sevenfold increase in caspase 4 activity and a sixfold increase in caspase 3 activity. These data suggest that there is a caspase-dependent apoptosis pathway associated with PKD and support the hypothesis that apoptotic cell death contributes to cyst formation in PKD.

Clinical and pathological characteristics of micropapillary transitional cell carcinoma: a highly aggressive variant.

PURPOSE: We present preliminary clinical, histochemical and molecular findings for 5 patients with micropapillary transitional cell carcinoma of the bladder, a rare histological variant not widely recognized in the urological literature. MATERIALS AND METHODS: The 5 patients were prospectively identified. In 3 cases immunohistochemical staining for expression of CD31, p53, E-cadherin, and alpha, beta and gamma-catenin was performed on paraffin embedded tissue. Sequencing was used to identify point mutations in exons 5 to 9 of p53, and exons 1 and 2 of H-ras. RESULTS: Of the patients 2 died within 1 year of presentation to our institution with rapid local extension along the bladder serosal surface and ureteral sheaths. Another patient had progression to invasive disease within 22 months. In the 3 cases with immunohistochemical staining p53 was negative, despite positive staining of nonmicropapillary transitional cell carcinoma within the same specimen. Stains for the angiotrophic marker CD31 were negative. In all 3 cases normal membrane associated alpha, beta and gamma-catenin expression was present. Examination of p53 sequences revealed a single point mutation in exon 8 of 1 case. In 2 cases different mutations in exon 1 of H-ras were noted. CONCLUSIONS: Micropapillary transitional cell carcinoma is a rare and highly aggressive variant. Paradoxically, our study demonstrated no significant p53 abnormalities. The lacunar histological pattern did not appear to represent invasion of vascular spaces. Rather, these tumors seemed to have the ability to disrupt and replace the normal stromal matrix to achieve rapid nonendothelial extension. Thus, micropapillary histology may predict a lesser likelihood of surgical cure.

Is low serum free testosterone a marker for high grade prostate cancer?

PURPOSE: The association of free and total testosterone with prostate cancer is incompletely understood. We investigated the relationship of serum free and total testosterone to the clinical and pathological characteristics of prostate cancer. MATERIALS AND METHODS: We retrospectively reviewed the clinical records of 117 consecutive patients treated by 1 physician and diagnosed with prostate cancer at our medical center between 1994 and 1997. Low free and total testosterone levels were defined as 1.5 or less and 300 ng./dl., respectively. RESULTS: After evaluating all 117 patients we noted no correlation of free and total testosterone with prostate specific antigen, patient age, prostatic volume, percent of positive biopsies, biopsy Gleason score or clinical stage. However, in patients with low versus normal free testosterone there were an increased mean percent of biopsies that showed cancer (43% versus 22%, p = 0.013) and an increased incidence of a biopsy Gleason score of 8 or greater (7 of 64 versus 0 of 48, p = 0.025). Of the 117 patients 57 underwent radical retropubic prostatectomy. In those with low versus normal free testosterone an increased mean percent of biopsies demonstrated cancer (47% versus 28%, p = 0.018). Pathological evaluation revealed stage pT2ab, pT2c, pT3 and pT4 disease, respectively, in 31%, 64%, 8% and 0% of patients with low and in 40%, 40.6%, 12.5% and 6.2% in those with normal free testosterone (p>0.05). CONCLUSIONS: In our study patients with prostate cancer and low free testosterone had more extensive disease. In addition, all men with a biopsy Gleason score of 8 or greater had low serum free testosterone. This finding suggests that low serum free testosterone may be a marker for more aggressive disease.