Low molecular weight cyclin E is associated with p27-resistant,high-grade,high-stage and invasive bladder cancer

Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.Key words: cyclin E, p27, Cdk2 kinase, bladder cancer, cell cycle     

Tryptic Hydrolysis of Dodecapeptides Possessing Paired Basic Residues

Summary Four dodecapeptides of general formula Tyr-Gly-Gly-Phe-Met-X-X-Tyr-Gly-Gly-Phe-Met-NH₂ (Enk-X-X-Enk-NH₂) possessing X-Arg or Lys have been synthesized and subjected to cleavage by trypsin. The peptide with the sequence containing-Lys-Arg-, depicted as BI-NH₂, represents the 100–111 segment of proenkephalin. The time course of the degradation was followed by high performance liquid chromatography. This method allows one to observe the formation of not only the final but also intermediate peptides. Among the peptides studied, the most susceptible to the cleavage was BI-NH₂. The primary hydrolysis proceeded rapidly at the arginine residue, followed by slow release of arginine. The other peptides (with-Arg-Arg-,-Lys-Lys-and-Arg-Lys-) were cleaved at both possible positions, but the resulting mixture contained Enk-X as a major product, which was the result of both primary and secondary cleavage.     

Detection of bladder cancer using proteomic profiling of urine sediments

We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.     

The capacities of autoleukocyte-labelled scintigraphy in the detection of foci of inflammation and suppuration

The diagnostics of occult inflammation foci up to day remains the major and complicated problem in almost each clinical discipline. Whole-body scintigraphy and SPECT studies with 99mTc-autoleucocytes show silent foci of inflammation and infection. 68 patients (24 females and 44 males) aged 14-59 years with non-localized inflammatory diseases were evaluated. Each patient underwent routine clinical and laboratory investigation. But because of the inadequacy of other diagnostic procedures, the Tc-99m-WBC scans may be of great clinical importance. For the determining of the SPECT of thorax were performed using Tc-99m-HMPAO (hexametylpropilenaminoxime) labeled autoleucocytes. In 1, 3 and 24 hours post injection inflammatory foci were seen as the areas of pathogenic activity. In 24 patients the circumscribed activity seen in relation to heart convincingly demonstrated endo- and/or myocarditis. 16 patients showed acute inflammation in postoperative wound, empyema of pleura and mediastinitis as the consequences of cardiosurgery. In 16 patients whole-body scanning and SPECT of the head revealed chronic infection in nasopharynx and accessory nasal sinuses dental infection and the rest 7 patients had abdominal and pelvic inflammatory diseases, such as ulcerative colitis, intraabdominal and retroperitoneal septic foci, tubo-ovarial inflammatory disease. The data demonstrated that Tc-99m-leucocytes imaging is a highly sensitive and specific for the detection of inflammatory diseases of different localization.     

Whole-Organ Genomic Characterization of Mucosal Field Effects Initiating Bladder Carcinogenesis

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Tryptic Hydrolysis of Dodecapeptides Possessing Paired Basic Residues

Four dodecapeptides of general formula Tyr-Gly-Gly-Phe-Met-X-X-Tyr-Gly-Gly-Phe-Met-NH₂ (Enk-X-X-Enk-NH₂) possessing X = Arg or Lys have been synthesized and subjected to cleavage by trypsin. The peptide with the sequence containing -Lys-Arg-, depicted as BI-NH₂, represents the 100–111 segment of proenkephalin. The time course of the degradation was followed by high performance liquid chromatography. This method allows one to observe the formation of not only the final but also intermediate peptides. Among the peptides studied, the most susceptible to the cleavage was BI-NH₂. The primary hydrolysis proceeded rapidly at the arginine residue, followed by slow release of arginine. The other peptides (with -Arg-Arg-, -Lys-Lys- and -Arg-Lys-) were cleaved at both possible positions, but the resulting mixture contained Enk-X as a major product, which was the result of both primary and secondary cleavage.     

Synthesis and biological evaluation of human preproenkephalin (100-111) and its analogs

The dodecapeptide sequence, Tyr-Gly-Gly-Phe-Met-Lys-Arg-Tyr-Gly-Gly-Phe-Met (BI), which is totally conserved in the primary structures of human, bovine, rat and toad preproenkephalins, has been synthesized by the solid-phase method. Coupling reactions were achieved by using symmetrical anhydrides of tert.-butyloxycarbonylamino acids performed with N-tert.-butyl,N'-methylcarbodiimide. 6-Arg and 7-Lys analogs have also been obtained. The peptides show opiate activity in both GPI and MVD assay, and possess antinociceptive properties as estimated by the hot-plate test in mice when applied intracisternally.