Nuclear Matrix Protein Research Articles

Rapid detection of bladder cancer using a point-of-care nuclear matrix protein 22 BladderChek control kit: a multicenter clinical trial

Objective To evaluate the clinical significance of nuclear matrix protein 22 (NMP22) test in rapid diagnosis of bladder cancer.Methods From February 2009 to November 2011,803 patients and 200 healthy individuals were included in this multicenter study from four urology centers in China.They provided a urine sample for test of NMP22 protein using a point-of-care NMP22 BladderChek control kit (colloidal gold method).All tests were evaluated in a blinded fashion.Patients were divided into three groups.Group 1 included 392 patients of bladder cancer; group 2 included 411 patients who had benign urinary disease with non-bladder cancer history or non-bladder cancer; group 3 included 200 healthy individuals.The sensitivity and specificity of NMP22 test were analyzed compared with pathologic results using Pearson Chi-square test.Results The overall sensitivity and specificity of the NMP22 test were 69.6％ (273/392,95％ CI 64.9％-74.0％) and 84.9％ (519/611,95％CI 81.1％-87.6％),respectively.In group 1,337 patients had pathologic stage data,and the sensitivities of the NMP22 test for each stage were Ta-T1 69.6％ (165/237) and T2-T4 82.0％ (82/100),respectively; 322 patients had pathologic grade data,and the sensitivities for each grade were G1 60.5％ (72/119),G2 66.2％ (47/71) and G3 90.9％ (120/132),respectively.The specificities for patients of benign urinary disease,of non-bladder cancer history or nonbladder cancer and healthy individuals were 87.8％ (324/369),69.0％ (29/42) and 97.5％ (195/200),respectively.Conclusion NMP22 BladderChek control kit had high sensitivity and specificity in detection of bladder cancer,and it can be used in early detection and rapid diagnosis of bladder cancer. Key words: Nuclear matrix-associated proteins; Urinary bladder neoplasms; Diagnosis; Reagent kits, diagnostic; Multicenter studies

Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout.

Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins. Differential packaging has been related to early or late transcription and also to differential susceptibility to genotoxic damage. Genes located in the more accessible HDNA could be more susceptible to injuries than those located in PDNA, being potential biomarkers of paternal DNA damage. Fish sperm chromatin organization is much diversified, some species lacking protamines and some others totally depleted of histones. Analyzing genotoxic damage in a species homogeneously compacted with some sperm nuclear basic protein type, could help in deciphering the clues of differential susceptibility to damage. In the present study we analyzed in rainbow trout the differential susceptibility of nine genes to UV irradiation and H2O2 treatment. The absence of histones in the sperm nuclei was confirmed by Western blot. The chromatin fractionation in sensitive and resistant regions to PvuII (presumably HDNA-like and PDNA-like, respectively) revealed that the nine genes locate in the same resistant region. The number of lesions promoted was quantified using a qPCR approach. Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy. UV irradiation promoted similar number of lesions in all the analyzed genes and a homogenous distribution of 8-OHdG within the nuclei. 8-OHdG was located in the peripheral area of the nucleus after H2O2 treatment, which promoted a significantly higher number of lesions in developmental-related genes (8.76–10.95 lesions/10 kb) than in rDNA genes (1.05–1.67 lesions/10 kb). We showed for the first time, that differential susceptibility to damage is dependent on the genotoxic mechanism and relies on positional differences between genes. Sensitive genes were also analyzed in cryopreserved sperm showing a lower number of lesions than the previous treatments and a predominant peripheral distribution of oxidative damage (8-OHdG).

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.

The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity.

Background:NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated.Methods:Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity.Results:Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex).Conclusions:The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.

Gold nanoparticles enhanced electrochemiluminescence of graphite-like carbon nitride for the detection of Nuclear Matrix Protein 22

A novel label-free electrochemiluminescence (ECL) immunosensor was developed for the detection of Nuclear Matrix Protein 22 (NMP 22) based on the graphite-like carbon nitride (g-C3N4) and gold nanoparticles (AuNPs). Graphite-like carbon nitride was firstly combined with AuNPs, which promoted the electrons transfer and enhanced the ECL intensity of g-C3N4 largely. Then, anti-NMP 22 was immobilized onto the electrode through Au–NH2 without the usage of crosslinking agents. The connection between AuNPs and antibody influenced the conductive performance of AuNPs, leading to some decrease of ECL intensity. Then, bovine serum albumin (BSA) solution was dropped on the surface to block nonspecific binding sites. Finally, after the specific immunoreaction between NMP 22 and anti-NMP 22, the ECL intensity decreased further, because the protein on the electrode hindered the diffusion of luminescent reagents and electrons toward the electrode surface. Therefore, a linear relation between ECL intensity and the logarithm of NMP 22 concentration was obtained from 50.0pgmL−1 to 2.0ng/mL (R2=0.988) with the detection limit of 10.0pgmL−1. The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay strategy for protein detection, which might be promising for clinical application.

Identification of patients with microscopic hematuria who are at greater risk for the presence of bladder tumors using a dedicated questionnaire and point of care urine test--a study by the members of Association of Urooncology, Turkey.

In patients with microscopic hematuria there is a need for better identification of those who are at greater risk of harbouring bladder tumors. The RisikoCheck® questionnaire has a strong correlation with the presence of urothelial carcinoma (UC) of the bladder and in combination with other available tests may help identify patients who require detailed clinical investigations due to increased risk of presence of bladder tumors. This study aimed to evaluate the efficacy of RisikoCheck® questionnaire together with NMP-22® (BladderChek®) as a point-of-care urine test in predicting the presence of bladder tumors in patients presenting with microscopic hematuria as the sole finding. In this multi-institutional prospective evaluation of 303 consecutive patients without a history of urothelial carcinoma (UC), RisikoCheck® risk group assessment, urinary tract imaging and cystourethroscopy as well as urine cytology and Nuclear Matrix Protein-22 (NMP-22 BladderChek) testing were performed where available. The sensitivity, specificity, negative predictive value (NPV), and positive predictive values (PPV) for the risk adapted approach were calculated. All patients underwent cystoscopy, and tumors were detected in 18 (5.9%). Urine cytology and NMP-22 was positive for malignancy in 9 (3.2%) and 12 (7.5%) of patients, respectively. A total of 43 (14%) patients were in the high risk group according to the RisikoCheck® questionnaire. The sensitivity and specificity of the questionnaire in detecting a bladder tumor was 61.5 % and 84.0 % in the high risk group. In patients with either a positive NMP-22 test or high risk category RisikoCheck®, 23.6% had bladder tumors with a corresponding sensitivity of 54.2% and specificity of 88.6%. If both tests were negative only 3.3% of the patients had bladder tumors. The results of our study suggest that the efficacy of diagnostic evaluation of patients with microscopic hematuria may be further enhanced by combining RisikoCheck® questionnaire with NMP-22.

Genetic analysis of matrin 3 gene in French amyotrophic lateral sclerosis patients and frontotemporal lobar degeneration with amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are adult-onset neurodegenerative diseases with overlapping clinical characteristics. They share common genetic causes and pathologic hallmarks such as TDP-43 neuronal accumulations. Recently, exome analysis identified mutations in matrin 3 (MATR3) gene in patients with familial ALS, suggesting a role for this gene in the pathogenesis of the disease. MATR3 is a nuclear matrix protein with DNA and RNA binding domains that interacts with TDP-43. To confirm the contribution of MATR3 to ALS, we studied a French cohort of 153 familial ALS or ALS/FTLD patients, without finding any variant. We conclude that mutations in MATR3 are rare in French familial ALS and ALS with FTLD patients.

Preparation of Au–Pt nanostructures by combining top-down with bottom-up strategies and application in label-free electrochemical immunosensor for detection of NMP22

A novel label-free amperometric immunosensor for sensitive detection of nuclear matrix protein 22 (NMP22) was developed based on Au–Pt bimetallic nanostructures, which were prepared by combining top-down with bottom-up strategies. Nanoporous gold (NPG) was prepared by “top-down” dealloying of commercial Au/Ag alloy film. After deposition of NPG on an electrode, Pt nanoparticles (PtNPs) were further decorated on NPG by “bottom-up” electrodeposition. The prepared bimetallic nanostructures combine the merits of both NPG and PtNPs, and show a high electrocatalytic activity towards the reduction of H2O2. The label-free immunosensor was constructed by directly immobilizing antibody of NMP22 (anti-NMP22) on the surface of bimetallic nanostructures. The immunoreaction induced amperometric response could be detected and negatively correlated to the concentration of NMP22. Bimetallic nanostructure morphologies and detection conditions were investigated to obtain the best sensing performance. Under the optimal conditions, a linear range from 0.01ng/mL to 10ng/mL and a detection limit of 3.33pg/mL were obtained. The proposed immunosensor showed high sensitivity, good selectivity, stability, reproducibility, and regeneration for the detection of NMP22, and it was evaluated in urine samples, receiving satisfactory results.

The value of the NMP22 test for superficial bladder cancer diagnosis and follow-up.

In this study, we investigated the efficacy of nuclear matrix protein 22 (NMP22) in the diagnosis and surveillance of bladder cancer. Patients with hematuria or who applied for cystoscopic control of proven bladder cancer were prospectively enrolled in this study. Routine cytologic examination and NMP22 test were performed on the voided urine sample obtained before the cystoscopy. The patients who had been diagnosed with bladder cancer were categorized according to stage, grade, number, size of the tumor and risk of the disease. Then the diagnostic performance of the NMP22 and the cytology test, alone or in combination, were evaluated separately using ROC curves in the diagnosis and surveillance groups. A total of 87 patients (87/136) were investigated because of hematuria. The sensitivity, specificity, positive and, negative predictive values, and positive likelihood ratio (LR+) of the NMP22 test were 70, 80, 68, 81, and 3.42%, respectively. While, the sensitivity, specificity, positive and, negative predictive values, and positive likelihood ratio (LR+) of the cytology examination were 27, 96, 82, 68, and 7.36%, respectively. There were 49 patients in the bladder cancer group. The sensitivity, specificity, positive, and negative predictive values and positive likelihood ratio (LR+) of the NMP22 test in these patients were 33, 76, 31, 78 and 1.37%, respectively. The sensitivity, specificity, positive and, negative predictive values, and positive likelihood ratio (LR+) of the cytology examination were 25%, 97%, 75%, 80% and 9.25, respectively. NMP22 test can be used as an adjunctive tool for the detection of bladder cancer, but its diagnostic performance is limited in surveillance when used alone or in combination with a cytology examination.

Ciz1 is a novel predictor of survival in human colon cancer

Cip1-interacting zinc-finger protein1 (Ciz1) is a nuclear matrix protein associated DNA replication factor which has been implicated in breast and lung cancer progression. However, the clinical significance of Ciz1 expression in colon cancer has not been determined. This study aimed to examine Ciz1 expression pattern and its potential as a biomarker of prognosis in colon cancer. Using quantitative PCR, tissue microarray (TMA), and ELISA, we evaluated Ciz1 mRNA and protein levels in tumor tissues from patients with colon cancer and in paired adjacent normal tissues. Ciz1 mRNA expression was significantly upregulated in 22 of 39 paired samples (P < 0.001). Immunohistochemistry on TMA-containing samples from 203 colon cancer patients indicated that Ciz1 protein expression was significantly higher in tumor tissues than in adjacent normal tissues (Stuart-Maxwell test, P < 0.001). Elevated expression of Ciz1 protein was significantly correlated with T stage (P < 0.001), N stage (P = 0.005), M stage (P = 0.021), and AJCC stage (P = 0.002). Multivariate Cox proportion hazard model analysis revealed that Ciz1 expression is an independent prognostic factor for overall time (OS; hazard ratio (HR): 1.76; 95% confidence interval (CI): 1.04-2.98; P = 0.034) and disease-free survival (DFS; HR: 2.02; 95% CI: 1.14-3.58; P = 0.017) of patients with colon cancer after colectomy. Our data suggested that Ciz1 may be involved in colon cancer progression and could serve as a novel predictor of survival for colon cancer patients.

Using analogy role‐play activity in an undergraduate biology classroom to show central dogma revision

For the study of biology in an undergraduate classroom, a classroom exercise was developed: an analogy role-play to learn mechanisms of gene transcription and protein translation (central dogma). To develop the central dogma role-play exercise, we made DNA and mRNA using paper sheets, tRNA using a wire dress hanger, and amino acids using Lego® blocks (Lego System A/S, Denmark). Students were studying in the course of mathematics, physics, or chemistry, so biology was not among their usual studies. In this exercise, students perform the central dogma role-play and respectively act out nuclear matrix proteins, a transcription factor, an RNA polymerase II, an mRNA transport protein, nuclear pore proteins, a large ribosomal subunit, a small ribosomal subunit, and several amino-acyl tRNA synthetases. Questionnaire results obtained after the activity show that this central dogma role-play analogy holds student interest in the practical molecular biological processes of transcription and translation.

Protein sub-nuclear localization prediction using SVM and Pfam domain information.

The nucleus is the largest and the highly organized organelle of eukaryotic cells. Within nucleus exist a number of pseudo-compartments, which are not separated by any membrane, yet each of them contains only a specific set of proteins. Understanding protein sub-nuclear localization can hence be an important step towards understanding biological functions of the nucleus. Here we have described a method, SubNucPred developed by us for predicting the sub-nuclear localization of proteins. This method predicts protein localization for 10 different sub-nuclear locations sequentially by combining presence or absence of unique Pfam domain and amino acid composition based SVM model. The prediction accuracy during leave-one-out cross-validation for centromeric proteins was 85.05%, for chromosomal proteins 76.85%, for nuclear speckle proteins 81.27%, for nucleolar proteins 81.79%, for nuclear envelope proteins 79.37%, for nuclear matrix proteins 77.78%, for nucleoplasm proteins 76.98%, for nuclear pore complex proteins 88.89%, for PML body proteins 75.40% and for telomeric proteins it was 83.33%. Comparison with other reported methods showed that SubNucPred performs better than existing methods. A web-server for predicting protein sub-nuclear localization named SubNucPred has been established at http://14.139.227.92/mkumar/subnucpred/. Standalone version of SubNucPred can also be downloaded from the web-server.

A label-free electrochemical immunosensor based on Au@Pd/Ag yolk-bimetallic shell nanoparticles and amination graphene for detection of nuclear matrix protein 22

A sensitive label-free electrochemical immunosensor was developed based on Au@Pd/Ag yolk-bimetallic shell nanoparticles (Au@Pd/Ag NPs) and amination graphene (NH2-GS) for the detection of nuclear matrix protein 22 (NMP22). Au@Pd/Ag NPs were loaded on the surface of NH2-GS (NH2-GS/Au@Pd/Ag) by covalent binding between noble metal NPs and amino groups. A glassy carbon electrode (GCE, 4mm in diameter) was used as the working electrode for the assembly of NPs and NH2-GS. Due to the large specific surface area and excellent electrical conductivity of NH2-GS/Au@Pd/Ag, the electroactivity of K3[Fe(CN)6] as electron transfer mediator was promoted and the amount of NMP22 antibody immobilized was enhanced significantly. Under optimum conditions, the immunosensor exhibited high selectivity and a wide linear range from 0.01 to 18ng/mL with a detection limit of 3.3pg/mL. For real sample analysis, the recovery of standard addition was 97.4–99.3% and the relative standard deviation (RSD) was 4.83–7.21%, which indicated that the proposed method exhibited great potential as a reliable technique for the determination of NMP22 for clinical research and diagnostic applications.

P0144 Diagnostic value of nuclear matrix protein-22 (NMP-22) compared with urine cytology in diagnosis of bladder carcinoma and muscular invasion

Background We aimed to compare the diagnostic value of nuclear matrix protein-22 (NMP22) and urine cytology for the diagnosis of bladder carcinoma and its muscular invasion. Methods Cross-sectional studies were done with 27 urine specimens from patients with suspicion of bladder carcinoma who were admitted to Dr. Kariadi General Hospital, Semarang, between August 2010 and April 2011. Urine cytology and NMP22 tests were done on the samples, and sensitivity, specificity, positive and negative predictive values, and accuracy were compared with histology results from cystoscopy biopsy-TURBT. Table 2X2 and SPSS 17.0 for Windows were used for statistical analyses. Findings Twenty-seven urine samples were investigated; three were excluded because no further cystoscopy biopsy was performed. NMP22 tests were positive in 21 (87.5%) samples, and negative in three (12.5%). Urine cytology were positive in eight (33.3%), and negative in 16 (66.7%) samples. From cystoscopy biopsy-TURBT investigations, two samples were non-bladder carcinoma, and 22 were bladder carcinoma, of which 19 (86.36%) were muscle-invasive bladder carcinomas and three (13.64%) were non muscle invasive. Sensitivity, specificity, positive predictive value, and negative predictive values of NMP22 in detecting bladder carcinoma were: 90.9%, 50%, 95.28%, and 33.3% respectively, compared with 38.1%, 100%, 18.75%, and 45.83%, respectively for urine cytology. Sensitivity, specificity, positive predictive value, and negative predictive value for NMP22 in predicting muscular invasion were 94.73%, 66.67%, 94.73%, and 66.67%, compared with 36.84%, 66.67%, 87.5%, and 14.28% for urine cytology. Interpretation NMP22 had better ability than urine cytology in detecting bladder carcinoma. This investigation could be useful in screening for bladder carcinoma, particularly in patients with high-risk factors, and to monitor recurrences in low grade bladder carcinoma which are undetectable with urine cytology. This investigation is simple, fast, inexpensive, and non-operator dependent. We aimed to compare the diagnostic value of nuclear matrix protein-22 (NMP22) and urine cytology for the diagnosis of bladder carcinoma and its muscular invasion. Cross-sectional studies were done with 27 urine specimens from patients with suspicion of bladder carcinoma who were admitted to Dr. Kariadi General Hospital, Semarang, between August 2010 and April 2011. Urine cytology and NMP22 tests were done on the samples, and sensitivity, specificity, positive and negative predictive values, and accuracy were compared with histology results from cystoscopy biopsy-TURBT. Table 2X2 and SPSS 17.0 for Windows were used for statistical analyses. Twenty-seven urine samples were investigated; three were excluded because no further cystoscopy biopsy was performed. NMP22 tests were positive in 21 (87.5%) samples, and negative in three (12.5%). Urine cytology were positive in eight (33.3%), and negative in 16 (66.7%) samples. From cystoscopy biopsy-TURBT investigations, two samples were non-bladder carcinoma, and 22 were bladder carcinoma, of which 19 (86.36%) were muscle-invasive bladder carcinomas and three (13.64%) were non muscle invasive. Sensitivity, specificity, positive predictive value, and negative predictive values of NMP22 in detecting bladder carcinoma were: 90.9%, 50%, 95.28%, and 33.3% respectively, compared with 38.1%, 100%, 18.75%, and 45.83%, respectively for urine cytology. Sensitivity, specificity, positive predictive value, and negative predictive value for NMP22 in predicting muscular invasion were 94.73%, 66.67%, 94.73%, and 66.67%, compared with 36.84%, 66.67%, 87.5%, and 14.28% for urine cytology. NMP22 had better ability than urine cytology in detecting bladder carcinoma. This investigation could be useful in screening for bladder carcinoma, particularly in patients with high-risk factors, and to monitor recurrences in low grade bladder carcinoma which are undetectable with urine cytology. This investigation is simple, fast, inexpensive, and non-operator dependent.

Regulatory role of nucleophosmin during the differentiation of human liver cancer cells.

Nucleophosmin (NPM, also known as B23), mainly localized in the nucleolus, has been reported to be overexpressed in many types of human cancer, including colon, ovarian, prostate and gastric cancer. NPM was identified while screening the differential nuclear matrix proteins during HMBA-induced differentiation of human liver cancer cells. We investigated the aberrant expression and subcellular localization of NPM in clinical liver cancer tissues and a cell line with the aim of providing more evidence for revealing the roles of NPM on regulating liver cancer cell proliferation and differentiation. In addition, we studied the potential interaction between NPM and several important proteins. Our results revealed that NPM protein was overexpressed in cancer cells, which was in accordance with the overexpressed mRNA in cancer tissues compared to the corresponding non-cancer tissues. We also found a decrease of NPM in protein and mRNA levels upon treatment with the differentiation reagent HMBA. We focused on the aberrant localization of NPM. Immunochemistry and immunofluorescence revealed aberrant cytoplasmic and nucleoplasm localization of NPM in liver cancer tissues and its colocalization with c-Myc, c-Fos, P53 and Rb in the SMMC-7721 cell line. The interactions between NPM and the above proteins were confirmed by GST pull-down assay and co-immunoprecipitation assay. These findings indicate that NPM plays a regulatory role in liver cancer, which deserves in-depth investigation.

Detection of Bladder Cancer by Measuring CD44v6 Expression in Urine With Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

To examine urinary CD44v6 total ribonucleic acid (RNA) expression in patients with bladder cancer using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and evaluate its potential as a novel marker of bladder cancer. We used the bladder cancer cell line T24 and determined CD44v6 expression in cancer cells using in situ hybridization and immunohistochemistry. Subsequently, we obtained urine samples from 21 patients with bladder cancer and 25 patients without bladder cancer (controls). We extracted total RNA from the urine samples, measured CD44v6 total RNA expression in both groups using qRT-PCR, and compared the expression between groups. We also compared the sensitivity, specificity, and concordance rate between CD44v6 total RNA expression analysis by qRT-PCR and cytologic analysis, UroVysion fluorescent in situ hybridization, bladder tumor antigen identification, and nuclear matrix protein 22 measurements. We observed increased CD44v6 expression in bladder cancer cells using in situ hybridization and immunohistochemistry. CD44v6 total RNA expression was significantly higher in the urine samples of patients with bladder cancer than in those of controls. We calculated the cutoff value from the receiver operating characteristic curve and obtained sensitivity and specificity values of 85.7% and 72.0%, respectively, for qRT-PCR analysis. Our results suggest that CD44v6 total RNA levels in urine can serve as a potential noninvasive biomarker of bladder cancer.

Colon Cancer-Specific Antigen-2 May Be Used as a Detecting and Prognostic Marker in Colorectal Cancer: A Preliminary Observation

BackgroundA specific and sensitive serum marker for colorectal cancer (CRC) detection and surveillance is central to effective treatment. It was preliminarily reported that some nuclear matrix proteins may be served as a specific blood based marker for colon cancer. The objective of this study is to evaluate the value of serum CCSA-2 detection in diagnosis, prognostic estimation and surveillance for CRC.MethodSerum CCSA-2 protein was measured in 181 various patient populations and 20 healthy donors before surgery. For 106 CRC patients, it was also measured on day 7 after surgery. Among them, 49 CRC patients' CCSA-2 protein were measured during the follow-up period according to NCCN Guideline.ResultsThe serum CCSA-2 concentration in CRC patients was significantly higher than which in other patients and healthy individuals. Serum CCSA-2, at the cut-off point of 64.10 ng/mL, had a sensitivity of 98.10% and a specificity of 97.90% in separating CRC populations from all other individuals. The CCSA-2 assay was significantly more sensitive than CEA and CA19-9 assay in CRC detection. After surgery, the serum CCSA-2 level of CRC patients declined significantly, but it rebounded to a high level when recurrences occurred. The pre-operative serum CCSA-2 level in patients who had a relapse within the follow-up period was significantly higher than which in patients without relapse.ConclusionsSerum CCSA-2 not only may be a potential biomarker using in screening and surveillance of CRC, but also may be an independent prognostic marker for CRC patients. Further clinical trials need to be performed in a larger population of patients to ulteriorly confirm these results.

Thoc1 inhibits cell growth via induction of cell cycle arrest and apoptosis in lung cancer cells

THO complex 1 (Thoc1) is a human nuclear matrix protein that binds to the retinoblastoma tumor suppressor retinoblastoma protein (pRb). While some studies suggest that Thoc1 has characteristics of a tumor suppressor protein, whether Thoc1 can inhibit lung cancer cell growth is not clear. In the present study, we observed that Thoc1 is lowly expressed in the lung cancer cell lines SPC-A1 and NCI-H1975. Then, we investigated the potential effects of Thoc1 on lung cancer cell proliferation, cell cycle and apoptosis after stable transfection of these lines with a Thoc1 expression vector. We found that overexpression of Thoc1 can inhibit cell proliferation, induce G2/M cell cycle arrest and promote apoptosis. Further investigation indicated that overexpression of Thoc1 is involved in the inhibition of cell cycle-related proteins cyclin A1 and B1 and of pro-apoptotic factors Bax and caspase-3. In vivo experiments showed that tumors overexpressing Thoc1 display a slower growth rate than the control xenografts and show reduced expression of the protein Ki-67, which localized on the nuclear membrane. Taken together, our data show that in lung cancer cells, Thoc1 inhibits cell growth through induction of cell cycle arrest and apoptosis. These results indicate that Thoc1 may be used as a novel therapeutic target for human lung cancer treatment.

The karyosphere capsule in Tribolium castaneum oocytes

Structure and composition of the karyosphere (karyosome) capsule were studied in the oocytes of a laboratory insect, Tribolium castaneum, with the use of electron microscopy and immunoelectron cytochemistry. Basing on the study of nuclear structure dynamics, we distinguished 8 stages that characterize the period of oocyte growth. At the diplotene stage, T. castaneum oocyte chromosomes conjoin early into a compact karyosphere, but a significant chromatin condensation does not occur. The process of karyosphere formation is accompanied by the development of an extensive extrachromosome capsule surrounding chromatin. The capsule consists of a material of different morphological types. Significant molecular components of the T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix including F-actin and lamin B. Besides the structural proteins, the Sm proteins of small nuclear (sn) RNPs and mature 2,2,7-trimethyl guanosine (TMG) 5'-capped snRNAs are revealed immunocytochemically in the karyosphere capsule. The obtained data can form a basis for further expansion of ideas on the functions of the karyosphere capsule as a specialized extrachromosomal nuclear domain of the oocytes. We believe that the T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.

PY RNA1-s2: A Highly Retina-Enriched Small RNA That Selectively Binds to Matrin 3 (Matr3)

The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem −1 and −2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.