thromboplastin and Carcinoma--Transitional-Cell

Tissue factor (TF), a transmembrane glycoprotein responsible for initiating the extrinsic pathway of blood coagulation plays a key role in cancer growth, metastasis and angiogenesis. Various studies have demonstrated the prognostic potential of TF expression in several cancers. However, its role in bladder cancer is unclear. This study evaluated the prognostic potential of TF expression in muscle-invasive bladder tumors from patients treated with radical cystectomies. Immunohistochemical staining using a monoclonal antibody (mAb) anti-TF was carried out on sections of tissue microarray blocks containing cores of muscle-invasive bladder tumors (4 cores/tumor) from 218 patients. The intensity of the staining was evaluated on a scale from 0 to 3 by two independent observers who were both unaware of the clinicopathological characteristics of the samples. TF was expressed in 77.6% of tumors, independently from baseline characteristics (age, gender, stage and grade) as assessed using the chi(2) and Student t tests. During follow-up (median: 2.6 years), 45.4% of the patients died from the progression of their cancer. Kaplan-Meier survival showed that among the 103 patients with node-negative (N0) transitional cell carcinoma (TCC), those with TF-positive tumors had shorter bladder cancer-specific survival (p = 0.0276). Moreover, multivariate Cox regression analysis showed they had a 3.15-fold greater risk of dying from bladder cancer (95% CI: 1.1-9.0; p = 0.032). In conclusion, TF expression was an independent predictor of disease-specific survival in N0 muscle-invasive TCCs treated by radical cystectomy and therefore, might help identify patients at higher risk of disease progression. These patients could potentially benefit from adjuvant chemotherapy.

Topics: Aged; Analysis of Variance; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cystectomy; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasms, Muscle Tissue; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Protein Array Analysis; Thromboplastin; Urinary Bladder Neoplasms

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends- (5'-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented approximately 1% of the total TF transcripts in normal cells, but constituted 7-10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10-25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Topics: Adenocarcinoma; Alternative Splicing; Base Sequence; Carcinoma, Hepatocellular; Carcinoma, Transitional Cell; Cytoplasm; Exons; Humans; Introns; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Molecular Sequence Data; Neoplasm Staging; Neoplasms; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured; Up-Regulation

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

Topics: Carcinoma, Transitional Cell; Cycloheximide; Factor Xa; Flow Cytometry; Gene Expression Regulation, Neoplastic; Hemoglobins; Humans; Melanoma; Microscopy, Confocal; Neoplasm Proteins; Protein Synthesis Inhibitors; Stimulation, Chemical; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Procoagulant activity of pairs of cell lines, which were derived from the same original cell type but which possess different growth characteristics and metastatic properties, was examined. The following characteristics were considered suggestive of a greater likelihood of metastatic potential: high histological grade; establishment of the line from a metastatic rather than a nonmetastatic cancer; increased tumorigenicity in nude mice; and/or estrogen receptor-negative mammary cancer. Procoagulant activity was evaluated by a two stage clotting assay. Procoagulant activity was highly variable, with up to a 1,300-fold difference, among the cancer cell lines examined. The rate of clot formation was factor VII dependent and was totally inhibited by an anti tissue factor monoclonal antibody, indicating that tissue factor was the only significant procoagulant present in these cancer cells. Tissue factor antigen expression, evaluated by ELISA, correlated with procoagulant activity. In all pairs of cancer cell lines, those with characteristics of increased proliferative potential had increased tissue factor levels compared to cell lines that originated from the same cell type, but which possess less aggressive characteristics. Tissue factor activity in these cancer cells was increased by cell lysis or by exposure of intact cells to a calcium ionophore, similar to results previously obtained in fibroblasts. Tissue factor mRNA was evaluated by northern blot analysis using a specific probe complementary to tissue factor mRNA. The previously described predominant tissue factor mRNA species of 2.2 kb was identified in the majority of cancer cell lines examined, but tissue factor mRNA species of 3.2 to 3.4 kb were also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Blood Coagulation; Blotting, Northern; Breast Neoplasms; Carcinoma, Renal Cell; Carcinoma, Transitional Cell; Cell Line; Gastrointestinal Neoplasms; Gene Expression; Humans; Kidney Neoplasms; Neoplasm Metastasis; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured

Production of procoagulant activity by host and tumour cells may be increased in patients with cancer. Using a simple chromogenic assay, we have determined urinary tissue factor (TF) levels in patients presenting with transitional cell carcinoma of the bladder (TCC, n = 63), normal controls (n = 20) and patients with benign prostatic hypertrophy (BPH, n = 35). In addition, a separate cohort of patients undergoing endoscopic surveillance for superficial bladder cancer were studied to determine whether there was any difference in levels in those with recurrent disease compared to those with normal cystoscopies. Urinary TF activity was higher in TCC compared to controls (p less than 0.001) and patients with BPH (p less than 0.05). In patients undergoing check cystoscopy, those with recurrent disease (n = 32) had higher levels (p less than 0.01) than those with normal examinations (n = 21). It is concluded that urinary TF levels are elevated in bladder cancer and that this reflects disease activity in those at risk of recurrent superficial disease.

Topics: Carcinoma, Transitional Cell; Cystoscopy; Humans; Male; Prostatic Hyperplasia; Thromboplastin; Urinary Bladder Neoplasms