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A Paju

Publications and source records attributed to A Paju.

9 recordsLinked to original sources
PubMed2006

Genome-controlled reverse transcriptase-polymerase chain reaction for targeted gene-expression analysis.

OBJECTIVE: Although gene-expression profiling has an important part to play in the classification of tumours and premalignant conditions, reproducibility of the present polymerase chain reaction (PCR)-based quantitative techniques needs to be improved for diagnostic purposes and to enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. We have developed reverse transcriptase-PCR-based technology for quantitative assessment of the relative content of multiple mRNA transcripts in small tissue or cell samples. MATERIAL AND METHODS: A multiplexed sequence modifying cDNA synthesis reaction is performed with this technique to create a 4-5 degrees increase in the melting temperature of subsequent short (56-64 bp) PCR amplicons. Each cDNA template is competitively co-amplified with genomic DNA, which serves as a universal internal standard. The relative amounts of cDNA and genomic DNA-derived amplicons are quantified in-tube by homogeneous melting curve analysis. RESULTS: The dynamic range of the assay was three orders of magnitude, while the detection limit was 100 cDNA molecules. A prototype assay, consisting of the analysis of eight genes, displayed good reproducibility (inter-assay CV 5-20 %) compared to the TaqMan assay (inter-assay CV 7-43 %). Gene-expression analysis could be performed in 20 of 20 (100 %) archival frozen samples, in 30 of 35 (86 %) archival FFPE samples and in 26 of 27 (96 %) endoscopic biopsies. CONCLUSIONS: We demonstrate that this new technique enables accurate analysis of mRNA expression in cultured cells and endoscopic tissue biopsies. Sensitive analysis FFPE tissue is also possible thanks to the short PCR amplicons.

Biopsy↗
PubMed2006

Differential expression of trypsinogen and tumor-associated trypsin inhibitor (TATI) in bladder cancer.

Tumor-associated trypsin inhibitor (TATI) is a marker of mucinous ovarian carcinoma, but it is also widely expressed in other malignant tumors and normal human tissues. Elevated serum concentrations of TATI are of prognostic value in ovarian, kidney, and bladder cancer. Tumor-associated trypsin is co-expressed with TATI in many malignancies and is thought to be involved in tumor invasion. TATI mRNA has been shown to be overexpressed in bladder cancer. We therefore studied whether trypsinogen expression also can be detected in bladder cancer and how this and TATI expression are associated with the clinicopathological characteristics of the tumors. We used RT-PCR, in situ hybridization and immunohistochemistry to detect trypsinogen- and TATI mRNA and protein in tissue samples from 28 bladder cancer patients and ten benign urothelia. TATI expression was detected in all benign tissues and non-invasive tumors. However, the expression was lower in the muscle-invasive tumors (pT2; n=5), whereas trypsinogen expression was seen in all but one non-invasive tumor. We conclude that trypsinogen is expressed in both malignant and benign bladder epithelium, whereas TATI expression decreases with increasing stage and grade of the tumor. This may suggest that a balanced expression of TATI and trypsinogen is required in normal tissue and that this balance is disrupted during tumor progression.

Adult↗
PubMed2002

The free beta-subunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma.

The free beta-subunit of human chorionic gonadotropin beta is expressed in several nontrophoblastic tumours and this is usually associated with aggressive disease. Little is known about human chorionic gonadotropin beta expression in renal cancer. We determined the pretreatment levels of human chorionic gonadotropin beta in serum of patients with renal cell carcinoma, and studied whether elevated levels predicted the clinical outcome. Serum samples were collected before surgery from 177 patients with renal cell carcinoma and from 84 apparently healthy controls. Human chorionic gonadotropin beta in serum was measured by a highly sensitive time-resolved immunofluorometric assay. The prognostic value of human chorionic gonadotropin beta, and of usual clinical and pathological variables was analyzed by the Kaplan-Meier method, the log rank test and Cox multiple hazard regression. The serum concentrations of human chorionic gonadotropin beta were increased in 23% of the renal cell carcinoma patients and they were significantly higher in patients with renal cell carcinoma than in controls (P<0.0001). The concentrations did not correlate with clinical stage and histopathological grade, but patients with increased human chorionic gonadotropin beta levels had significantly shorter survival time than those with levels below the median (cut-off 1.2 pmol l(-1), P=0.0029). In multivariate analysis human chorionic gonadotropin beta, tumour stage and grade were independent prognostic variables. The serum concentration of human chorionic gonadotropin beta is an independent prognostic variable in renal cell carcinoma. The preoperative value of human chorionic gonadotropin beta in serum may be used to identify patents with increased risk of progressive disease.

Adult↗
PubMed2002

MMP-9 activation by tumor trypsin-2 enhances in vivo invasion of human tongue carcinoma cells.

Various human cancer cells express tumor-associated trypsinogen-2 (TAT-2), which can efficiently activate matrix metalloproteinases (MMPs) in vitro. MMP-2 and MMP-9 are particularly associated with the invasive malignant potential of several tumors. To investigate the role of TAT-2 in tumor invasion, we overexpressed TAT-2 in two malignant human squamous cell carcinoma cell lines of tongue and in non-malignant human papilloma virus transformed gingival keratinocytes. The TAT-2 overexpression significantly increased the levels of active MMP-9 in the most malignant cell line. TAT-2-transfected cells intravasated (invaded blood vessels) up to 60% more efficiently than did the control cells in an in vivo chick embryo chorioallantoic membrane invasion model. This increased intravasation was almost completely abolished by a specific tumor-associated trypsin inhibitor (TATI). These results indicate that TAT-2 has a role in the invasive growth of tumors, either alone or in cascade with gelatinases, especially by generating active MMP-9.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

The levels of trypsinogen isoenzymes in ovarian tumour cyst fluids are associated with promatrix metalloproteinase-9 but not promatrix metalloproteinase-2 activation.

Proteolysis mediated by matrix metalloproteinases (MMPs) and serine proteinases is associated with cancer invasion and metastasis. Activation of latent proMMPs, and especially the proforms of the type IV collagen degrading gelatinases A and B (proMMP-2 and proMMP-9), is thought to be a critical step in this process. We have recently found that human tumour-associated trypsin-2 is a potent activator of proMMP-9 and it also activates proMMP-2 in vitro. Trypsinogen, MMP-2, and MMP-9 are expressed in ovarian cancer. To elucidate the function of trypsin in vivo, we studied whether high concentrations of trypsinogen-1, trypsinogen-2, their alpha(1)-proteinase inhibitor (API) complexes, and tumour-associated trypsin inhibitor (TATI) are associated with proMMP-2 and proMMP-9 activation in ovarian tumour cyst fluids. Zymography and immunofluorometric analysis of 61 cyst fluids showed a significant association between high trypsin concentrations and the activation of MMP-9 (P = 0.003-0.05). In contrast, the trypsin concentrations were inversely associated with the activation of MMP-2 (P = 0.01-0.02). Immunohistochemical analysis of ovarian tumour tissue demonstrated expression of trypsinogen-2 and TATI in the secretory epithelium. MMP-2 was detected both in stromal and epithelial cells whereas MMP-9 was detected in neutrophils and macrophage-like cells in stromal and epithelial areas. These results suggest that trypsin may play a role in the regulation of the MMP-dependent proteolysis associated with invasion and metastasis of ovarian cancer.

Adolescent↗
PubMed2001

Tumor associated trypsin inhibitor as a prognostic factor in renal cell carcinoma.

PURPOSE: We analyzed the prognostic significance of pretreatment serum tumor associated trypsin inhibitor in renal cell carcinoma. MATERIALS AND METHODS: Serum samples were obtained before surgery from 188 patients who underwent radical nephrectomy for renal cell carcinoma. Median followup of living patients was 8.5 years. Serum tumor associated trypsin inhibitor was measured by a time resolved immunofluorometric assay. Statistical analysis was performed using the Kaplan-Meier method, log rank and stratified log rank tests. RESULTS: Preoperatively serum tumor associated trypsin inhibitor was elevated with a cutoff 16 microg/l in 48% of the patients with normal serum creatinine. The concentration in patients with cancer was significantly higher than in controls (p <0.0001). The serum level correlated with clinical stage and nuclear grade. Patients with an elevated level had significantly shorter survival time than those with a normal level (p = 0.005). Stratified log rank test demonstrated that tumor associated trypsin inhibitor was a prognostic factor independent of stage and grade in all patients as well as in those with nonmetastatic disease. CONCLUSIONS: Increased preoperative serum tumor associated trypsin inhibitor was associated with poor survival in renal cell carcinoma. The serum level was an independent prognostic variable. Preoperative serum tumor associated trypsin inhibitor appears to be a useful predictive factor that may be used to identify patients at increased risk of aggressive disease.

Adult↗
PubMed2000

Expression and characterization of trypsinogen produced in the human male genital tract.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

Blotting, Northern↗
PubMedDate not supplied

Collagenases (MMP-1, -8 and -13) and trypsinogen-2 in fluid from benign and malignant ovarian cysts.

Proteolytic enzymes, such as matrix metalloproteinases (MMPs) and tumor-associated trypsinogens (TAT), play a pivotal role in tumor invasion and metastasis. Among MMPs, the interstitial collagenases (MMP-1, -8 and -13) can initiate collagenolysis. In this study, we have studied the levels of MMP-1, -8 and -13 in relation to the level of trypsinogen-2 in fluid from benign and malignant ovarian cysts. Elevated MMP-8 levels occur in many ovarian cyst fluids, and high MMP-8 levels are associated with malignancy. The concentrations of trypsinogen-2 correlate with those of MMP-8, but it remains to be shown whether trypsin-2 plays a role as its activator in vivo. The strong expression of MMP-8 over MMP-1 and MMP-13 in malignant ovarian tumors may indicate that MMP-8 participates in the protease cascades associated with the invasiveness of ovarian tumors.

Adolescent↗