Epstein-Barr virus infection is associated with the nuclear factor-kappa B p65 signaling pathway in renal cell carcinoma.

BACKGROUND: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors. METHODS: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations. RESULTS: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type. CONCLUSIONS: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion.

Development of Novel PSMA Ligands for Imaging and Therapy with Copper Isotopes.

Prostate-specific membrane antigen (PSMA)-binding tracers have been shown to be promising agents for the specific targeting of prostate tumors. On labeling with the short-lived isotopes 18F and 68Ga, excellent molecular imaging performance is achieved. This potential could be further exploited using long-lived isotopes. Because of the favorable half-life of 64Cu, tracers labeled with this PET nuclide could solve logistic problems. Moreover, this isotope provides a theranostic pair with the therapeutic copper isotope 67Cu. Hence, 9 novel tracers that combine dedicated copper chelators with the PSMA-specific urea-based binding motif were developed. Methods: The precursors were obtained by solid-phase synthesis. The purity and molecular weight of the PSMA ligands were confirmed by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The compounds were labeled with 64Cu, with a radiolabeling yield of more than 99%. Competitive cell binding assays and internalization assays were performed with C4-2 cells, a subline of the PSMA-positive cell line LNCaP (human lymph node carcinoma of the prostate). In vitro serum stability, the stability of 64Cu-CA003 in blood, and the in vivo fate of neat 64Cu-chloride or 64Cu-CA003 were determined to prove whether the stability of the radiolabeled compounds is sufficient to ensure no significant loss of copper during the targeting process. For PET imaging and biodistribution studies, a C4-2 tumor-bearing mouse model was used. Results: The radiolabeled 64Cu-PSMA ligands showed high serum stability. All PSMA ligands showed high inhibition potencies, with equilibrium inhibition constants in the low nanomolar range. 64Cu-CA003 and 64Cu-CA005 showed high internalization ratios (34.6% ± 2.8 and 18.6% ± 4.4, respectively). Both the in vitro serum stability determination and the in vivo characterization of the main radiolabeled compounds confirmed that, except for 64Cu-PSMA-617, all compounds showed high serum stability within the observation period of 24 h. Small-animal PET imaging demonstrated high tumor uptake within 20 min. Organ distribution studies confirmed high specific uptake in the tumor, with 30.8 ± 12.6 percentage injected dose (%ID)/g at 1 h after injection. Rapid clearance from the kidneys was observed-a decrease from 67.0 ± 20.9 %ID/g at 1 h after injection to 7.5 ± 8.51 %ID/g at 24 h after injection (in the case of CA003). The performance of CA003, the compound with the best preclinical properties, was assessed in a first patient. In line with its preclinical data, PET imaging resulted in clear visualization of the cancer lesions, with high contrast. Conclusion: The 64Cu-labeled PSMA ligands are promising agents to target PSMA and visualize PSMA-positive tumor lesions as shown in preclinical evaluation by small-animal PET studies, organ distribution, and a patient application. Most importantly, the images obtained at 20 h enabled delineation of unclear lesions, showing that the compounds fulfill the prerequisite for dosimetry in the course of therapy planning with 67Cu. Thus, we suggest clinical use of copper-labeled CA003 for diagnostics and radiotherapy of prostate cancer.

Preclinical Evaluation of 203/212Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203Pb-L1-203Pb-L5 were performed on mice bearing PSMA(+) PC3 PIP and PSMA(-) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212Pb-L2 was evaluated in PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor models and in the PSMA(+) luciferase-expressing micrometastatic model. 212Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203Pb-L1-203Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203Pb-L1-203Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203Pb-L5 (31.0, 15.2) and lowest for 203Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203Pb-L3 (3.2) and 203Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(+) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(+) flank tumor model. 212Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion:203Pb-L1-203Pb-L5 demonstrated favorable pharmacokinetics for 212Pb-based TRT. The antitumor efficacy of 212Pb-L2 supports the corresponding 203Pb/212Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

Prospective Evaluation of PSMA-Targeted 18F-DCFPyL PET/CT in Men with Biochemical Failure After Radical Prostatectomy for Prostate Cancer.

Our purpose is to provide the results of a prospective study evaluating prostate-specific membrane antigen-targeted 18F-DCFPyL (2-(3-{1-carboxy-5-[(6-18F-fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid) PET/CT in patients with biochemical failure after radical prostatectomy for prostate cancer (PCa). Methods: Thirty-one patients with postprostatectomy serum prostate-specific antigen (PSA) levels of at least 0.2 ng/mL and negative conventional imaging results were enrolled in this study and imaged with 18F-DCFPyL PET/CT. A consensus central review identified foci of radiotracer uptake consistent with sites of PCa. Descriptive statistics were used. Results: Twenty-one patients (67.7%) had at least 1 finding on 18F-DCFPyL PET/CT consistent with a site of PCa. Imaging was positive in 59.1% of patients with a PSA level of less than 1.0 ng/mL and in 88.9% of patients with a PSA level of more than 1.0 ng/mL. The median SUVmax across all lesions was 11.6 (range, 1.5-57.6). Conclusion: In this prospective study using the prostate-specific membrane antigen-targeted PET agent 18F-DCFPyL, most patients with biochemical failure after radical prostatectomy had foci of suggestive uptake, even at low serum PSA levels.

Prostate-Specific Membrane Antigen-Guided Surgery.

Since its introduction to the diagnostic pathway for prostate cancer management, prostate-specific membrane antigen (PSMA)-ligand PET has demonstrated great potential. PSMA-ligand imaging is increasingly influencing therapeutic decision making, although its impact on patient outcomes still needs to be defined. One relatively new application, enabled through chemical and engineering efforts, is PSMA-guided surgery. This review highlights the potential of PSMA-guided surgery and discusses its implications in lymph node dissection in primary and recurrent prostate cancer.

Positive correlation between proteasome activity and polyphenols in the telencephalon of adult female mice.

INTRODUCTION: Proteasome regulates proteostasis, and can be compromised in neurodegenerative diseases. Thus, our aim was to correlate the activity of proteasome to the level of polyphenols in telencephalon during murine adulthood. METHODS: Proteasome activity, polyphenols and other variables (glucose and hydroperoxides) were analysed in Balb/c female telencephala (n = 20, age = 4-12 months), using multivariate methods. RESULTS: The following values were found: proteasome activity = 3.1 ± 0.6 FI/µg of tissue proteins, glucose = 0.1 ± 0.0 µg/µg, hydroperoxides = 363.4 ± 96.6 OD/µg, and polyphenols = 0.1 ± 0.0 ng/µg. Polyphenols reduced during aging showed a direct correlation with proteasome (Pearson's coefficient = 0.43, p = 0.0590, and a multivariate linear regressive coefficient = 17.85, p = 0.0216), with glucose and hydroperoxides being not involved (p>0.1). This correlation was confirmed by partial least square regression (beta = 0.67). CONCLUSION: Proteasome activity can be affected during ageing, and promoted by telencephalic polyphenol levels. Thus, these diet compounds might exert benefits in adult brain.

Selective Substrates and Activity-Based Probes for Imaging of the Human Constitutive 20S Proteasome in Cells and Blood Samples.

The proteasome is an enzyme complex critical for maintaining protein homeostasis. Perturbed proteasome function leads to pathologies including cancer and autoimmune and neurodegenerative disease. Therefore, the proteasome constitutes an excellent molecular target for pharmaceutical development. Here, using the HyCoSuL approach, we designed and synthesized novel and selective fluorogenic substrates for each of these three constitutive 20S proteasome activities and applied them to assess inhibition of proteasome subunits by MG-132 and a clinically used inhibitor bortezomib. Our results confirm the utility of designed substrates in biochemical assays. Furthermore, selective peptide sequences obtained in this manner were used to construct fluorophore-labeled activity-based probes and then utilized to detect each constitutive 20S proteasome subunit simultaneously in lysates of HEK-293F cells and red blood cells. Overall, we describe a simple and rapid method useful to measure constitutive 20S proteasome activity in whole human blood samples that could enable early diagnosis of pathological states associated with aberrantly upregulated proteasome activity.

Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

The increased invasiveness of gastric adenocarcinoma is important for progression and metastasis. In recent molecular biological studies, ribophorine II (RPN2) induced epithelial-mesenchymal transition and metastatic activity. However, no studies have evaluated the relationship between RPN2 expression, ability of cancer to invade/metastasis, and patient prognosis in gastric adenocarcinoma. Therefore, we have examined these factors. Immunohistochemical staining was performed to detect RPN2 and p53 in the primary lesion and adjacent normal gastric mucosa of 242 gastric adenocarcinoma patients who underwent resection surgery. We conducted clinicopathologic examinations and analyzed patient prognoses with the Kaplan-Meier method. Further, multivariate analysis was conducted using a Cox hazard model. Also, we analyzed the ability of invasion under inhibited RPN2 expression in vitro. RPN2 expression was observed in 119 of 242 cases of gastric adenocarcinoma patients. RPN2 expression was associated with a higher incidence of depth of wall invasion, lymph node metastasis, lymphatic invasion, venous invasion, peritoneal dissemination, histopathological stage, and p53 expression. In stage II and III curative resection cases, where recurrence is the most serious problem, cases that expressed RPN2 had a significantly lower 5-year survival rate and higher recurrence rate compared to the cases with no RPN2 expression. In the multivariate analysis for prognosis, RPN2 expression was found to be an independent factor. Also, gastric adenocarcinoma cell, had mutant-type p53, reduced the ability of invasion by knockout of RPN2 expression in vitro. RPN2 expression correlates with gastric adenocarcinoma cell invasion and shows promise as a new prognostic factor in human gastric adenocarcinoma.

Proteasome subunit expression analysis and chemosensitivity in relapsed paediatric acute leukaemia patients receiving bortezomib-containing chemotherapy.

BACKGROUND: Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL). METHODS: We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children's Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1) and AML (COG-AAML07P1). Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12) obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test. RESULTS: Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both ß5i/ß5 and ß1i/ß1 subunits (p = 0.004 and p < 0.001). These ratios correlated with therapy response in AML patients; ß1i/ß1 ratios were significantly higher (p = 0.028) between patients who did (n = 4) and did not reach complete remission (CR) (n = 8), although for ß5i/ß5 ratios, this did not reach significance. For ALL patients, the subunit ratios were also higher for patients who showed a good early response to therapy but this relation was not statistically significant. Overall, for this study, the patients were treated with combination therapy, so response was not only attributed to proteasome inhibition. Moreover, the leukaemic blast cells were not purified for these samples. CONCLUSIONS: These first ex vivo results encourage further studies into relative proteasome subunit expression to improve proteasome inhibition-containing therapy and as a potential indicator of bortezomib response in acute leukaemia.

Regulation of Endoribonuclease Activity of Alpha-Type Proteasome Subunits in Proerythroleukemia K562 Upon Hemin-Induced Differentiation.

The proteasome is the main intracellular proteolytic machine involved in the regulation of numerous cellular processes, including gene expression. In addition to their proteolytic activity, proteasomes also exhibit ATPase/helicase (the 19S particle) and RNAse (the 20S particle) activities, which are regulated by post-translational modifications. In this report we uncovered that several 20S particle subunits: α1 (PSMA6), α2 (PSMA2), α4 (PSMA7), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3) possess RNAse activity against the p53 mRNA in vitro. Furthermore, we found that the RNAse activity of PSMA1 and PSMA3 was regulated upon hemin-induced differentiation of K562 proerythroleukemia cells. The decrease in RNAse activity of PSMA1 and PSMA3 was paralleled by changes in their status of phosphorylation and ubiquitylation. Collectively, our data support the notion that proteasomal RNAse activity may be functionally important and provide insights into the potential mechanism of p53 repression in erythroleukemia cells by RNAse activity of the 20S α-type subunits.

Clinicopathologic features and prognostic implications of Gankyrin protein expression in non-small cell lung cancer.

PURPOSE: The expression of Gankyrin, a liver cancer-related oncoprotein, has been observed in several human malignancies including non-small cell lung cancer (NSCLC). However, the clinic relevance of Gankyrin expression in NSCLC remains unclear. METHODS: Gankyrin expression was assessed using immunohistochemical (IHC) methods in 166 paired paraffin-embedded NSCLC specimens and adjacent normal tissues. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were employed to measure the expression of Gankyrin in 24 paired fresh NSCLC specimens and the corresponding normal tissues. The association of Gankyrin expression with clinicopathological parameters was also evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of Gankyrin expression on survival. RESULTS: Data showed that Gankyrin was expressed in 78.3% (130/166) and 28.9% (48/166) of cancer lesions and corresponding adjacent normal tissue, respectively. And the Gankyrin overexpression in tumor tissue occurred in 53.6% (89/166) of patients, while overexpression of Gankyrin in normal tissue occurred only in 4.8% (8/166) of patients (P<0.001). Semi-quantitative RT-PCR and Western blotting showed that NSCLC specimens had increased Gankyrin mRNA and protein expression compared to the corresponding normal tissues. Out of all the clinicopathological factors analyzed, Gankyrin overexpression was significantly correlated with lymphatic metastasis (P<0.001) and p-TNM stage (P<0.001). Gankyrin-overexpressed NSCLC patients had a significantly shorter survival time (P<0.001, Log-rank test), and the prognostic significance of Gankyrin overexpression was apparent in both squamous cell carcinoma patients (P=0.028) and adenocarcinoma patients (P<0.001). Multivariate analysis indicated that Gankyrin overexpression may be an independent prognostic factor in NSCLC (hazard ratio [HR], 1.51; P=0.041). CONCLUSION: Our results indicate that Gankyrin overexpression is of clinical significance and can serve as a prognostic biomarker in NSCLC.

A mass spectrometry platform for a streamlined investigation of proteasome integrity, posttranslational modifications, and inhibitor binding.

The proteasome is responsible for the majority of protein degradation within eukaryotic cells and proteasome inhibitors have gained blockbuster status as anticancer drugs. Here, we introduce an analytical platform comprising reverse phase chromatography, intact protein mass spectrometry, and customized data analysis that allows a streamlined investigation of proteasome integrity and posttranslational modifications. We report the complete mass spectrometric assignment of all subunits of the yeast core particle, as well as of the human constitutive 20S proteasome and the human immunoproteasome, including phosphorylated isoforms of α7. Importantly, we found several batches of commercially available immunoproteasome to also contain constitutive catalytic subunits. Moreover, we applied the method to study the binding mechanisms of proteasome inhibitors, both validating the approach and providing a direct readout of subunit preferences complementary to biochemical methods. Collectively, our platform facilitates an easy, reliable and comprehensive detection of different types of covalent modifications on multisubunit protein complexes with high accuracy.

Homogeneous, bioluminescent proteasome assays.

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect.

BACKGROUND: In mammalian cells, ASPL is involved in insulin-stimulated redistribution of the glucose transporter GLUT4 and assembly of the Golgi apparatus. Its putative yeast orthologue, Ubx4, is important for proteasome localization, endoplasmic reticulum-associated protein degradation (ERAD), and UV-induced degradation of RNA polymerase. RESULTS: Here, we show that ASPL is a cofactor of the hexameric ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast. In addition, ASPL interacts in vitro with NSF, another hexameric ATPase complex. ASPL localizes to the ER membrane. The central area in ASPL, containing both a SHP box and a UBX domain, is required for binding to the p97 N-domain. Knock-down of ASPL does not impair degradation of misfolded secretory proteins via the ERAD pathway. Deletion of UBX4 in yeast causes cycloheximide sensitivity, while ubx4 cdc48-3 double mutations cause proteasome mislocalization. ASPL alleviates these defects, but not the impaired ERAD. CONCLUSIONS: In conclusion, ASPL and Ubx4 are homologous proteins with only partially overlapping functions. Both interact with p97/Cdc48, but while Ubx4 is important for ERAD, ASPL appears not to share this function.

Assessment of proteasome concentration and chymotrypsin-like activity in plasma of patients with newly diagnosed multiple myeloma.

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many haematologic malignancies, including multiple myeloma. Under conditions of rapid cell turnover and growth rate, proteasomes are returned into circulation. The measurement of their levels or activity could offer a new approach to diagnosis, prognosis and monitoring of anticancer treatment in carcinoma patients. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in the plasma of 64 patients with a newly diagnosed multiple myeloma and 30 healthy volunteers. The values were found to be significantly higher in the studied patients and advanced disease stages compared to the control group, and decreased significant after chemotherapy. Both proteasome concentration and ChT-L activity correlated with adverse prognostic factors, such as lactate dehydrogenase and ß2-macroglobulin. We also showed that proteasome concentration positively correlates with IL-6 level, as opposed to proteasome ChT-L activity. Of note, higher proteasome ChT-L activity, unlike the concentration, was proved to be an indicator of a shorter progression free survival, constituting thereby an important prognostic marker.

Assessment of cytokine-modulated proteasome activity.

This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cytokine action and inflammation are clear, the assessment and interpretation is not always straightforward. Therefore, we also discuss the pitfalls of the standard fluorogenic assay, particularly in the interpretation of results obtained, and the advantages of the newer, ProCISE assay.

Expression of the oncoprotein gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumor.

OBJECTIVE: The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. METHODS: The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. RESULTS: Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. CONCLUSION: Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors.

Polyubiquitinated proteins, proteasome, and glycogen characterize the particle-rich cytoplasmic structure (PaCS) of neoplastic and fetal cells.

A particle-rich cytoplasmic structure (PaCS) concentrating ubiquitin-proteasome system (UPS) components and barrel-like particles in clear, cytoskeleton- and organelle-free areas has recently been described in some neoplasms and in genetic or infectious diseases at risk of neoplasia. Ultrastructurally similar particulate cytoplasmic structures, interpreted as glycogen deposits, have previously been reported in clear-cell neoplasms and some fetal tissues. It remains to be investigated whether the two structures are the same, colocalize UPS components and polysaccharides, and have a role in highly proliferative cells such as fetal and neoplastic cells. We used immunogold electron microscopy and confocal immunofluorescence microscopy to examine human and mouse fetal tissues and human neoplasms. Fetal and neoplastic cells both showed colocalization of polyubiquitinated proteins, 19S and 20S proteasomes, and polysaccharides, both glycogen and chondroitin sulfate, inside cytoplasmic structures showing all distinctive features of PaCSs. Poorly demarcated and/or hybrid (ribosomes admixed) UPS- and glycogen-enriched areas, likely stages in PaCS development, were also seen in some fetal cells, with special reference to those, like primary alveolar pulmonary cells or pancreatic centroacinar cells, having a crucial role in organogenesis. UPS- and glycogen-rich PaCSs developed extensively in clear-cell neoplasms of the kidney, ovary, pancreas, and other organs, as well as, in infantile, development-related tumors replicating fetal patterns, such as choroid plexus papilloma. UPS-mediated, ATP-dependent proteolysis and its potential energy source, glycogen metabolism, may have a crucial, synergic role in embryo-/organogenesis and carcinogenesis.

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity.

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.

Development and function of cortical thymic epithelial cells.

The thymic cortex provides a microenvironment that supports the generation and T cell antigen receptor (TCR)-mediated selection of CD4(+)CD8(+)TCRαß(+) thymocytes. Cortical thymic epithelial cells (cTECs) are the essential component that forms the architecture of the thymic cortex and induces the generation as well as the selection of newly generated T cells. Here we summarize current knowledge on the development, function, and heterogeneity of cTECs, focusing on the expression and function of ß5t, a cTEC-specific subunit of the thymoproteasome.