OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2) in bladder transitional cell carcinoma tissues, and understand its clinical significance.

METHODSReversal transcription polymerase chain reaction (RT-PCR) was used to assess the expression of COX-2 mRNA in 52 cases of bladder transitional cell carcinoma tissues and 17 cases of normal bladder tissues far from neoplasm; Western blot was used to assess the expression of COX-2 protein in 49 cases of bladder cancerous tissues and 17 cases of normal tissues.

RESULTSPositive expression of COX-2 mRNA was detected in 83% (43/52) of bladder cancer tissues and in 29% (5/17) of normal tissues by RT-PCR and there was significant difference in expression of COX-2 mRNA between cancer tissues and normal tissues. Western blot analysis showed that expression of COX-2 protein was correlation with the stage and grade of cancer.

CONCLUSIONCOX-2 is overexpressed in bladder transitional cell carcinoma. COX-2 maybe play a certain role in carcinogenesis and progression of bladder cancer and turn into a useful target of chemoprevention of bladder cancer.

Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Carcinoma, Transitional Cell ; enzymology ; pathology ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Urinary Bladder ; enzymology ; Urinary Bladder Neoplasms ; enzymology ; pathology

The enzyme telomerase maintains a constant telomere length in immortalized cells, allowing unlimited cell proliferation. Almost all cancer cells express telomerase activity. However, little data is available regarding the role of telomerase activity in the detection of bladder cancer with a bladder wash specimen. We detected telomerase activity in a bladder wash specimen of bladder cancer and normal tissues, and compared them with final pathologic diagnosis. Twenty-three patients with transitional cell carcinoma (TCC) of the bladder were enrolled in our study. A bladder wash specimen was obtained before transurethral resection of the bladder (TURB) and normal and cancer tissues from the same patients during TURB. Telomerase activity was analyzed in each specimen a using telomeric repeat amplification protocol (TRAP) assay based on polymerase chain reaction (PCR) technique. Cytologic diagnosis was performed using Papanicolaou's stain with cytocentrifuged cytology preparation. We observed telomerase activity in 95.7% (22/23) of bath cancer tissues and bladder wash specimens; only one case did not express telomerase activity. Telomerase activity was undetected in all normal tissues except one, which was obtained from a patient with carcinoma in situ. A total of 69.6% (16/23) of wash specimens were positive in cytopathologic diagnosis. The accuracy of cytopathologic diagnosis in pathologic grade 2 or 3 was relatively high (83.3%, 15/18). However, in five cases of grade 1 TCC only 20% (1/5) of cytologic diagnosis was positive whereas the telomerase activity of wash specimens was detected in 80% (4/5). Our data demonstrates that not only the majority of human bladder cancer tissues, but also the bladder wash specimens obtained from patients with TCC, expressed telomerase activity. It indicates that telomerase activity may be a reliable marker in detecting bladder cancer especially in cases with a low grade that bladder wash cytology can miss.
Aged ; Aged, 80 and over ; Bladder Neoplasms/enzymology* ; Carcinoma, Transitional Cell/metabolism* ; Female ; Human ; Irrigation ; Male ; Middle Age ; Telomerase/metabolism* ; Tumor Markers, Biological

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide on telomerase activity in bladder cancer cells.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by telomerase PCR ELISA kit. hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and hTERT protein by immunohistochemistry and flow cytometry.

RESULTSTelomerase activity was decreased in T24 cells 48 h after treatment with AS PS-ODN, and was significantly inhibited at 72 h.

CONCLUSIONAS PS-ODN can significantly inhibit telomerase activity by down-regulating hTERT mRNA and protein expression in bladder cancer cells.

Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; enzymology ; pathology

OBJECTIVETo study the relationship between genetic polymorphism of NAT2 and susceptibility to bladder cancer.

METHODSNAT2 genotypes were determined by PCR-RFLP method in 69 patients with bladder transitional cell carcinoma and 88 healthy controls.

RESULTSThe frequency of NAT2 slow genotypes was 26.1% (18/69) in patients compared with 14.8% (13/88) in controls (P < 0.05). Bladder cancer risk in patients with NAT2 slow genotypes was 2 fold as high as that in patients with NAT2 rapid genotypes. When NAT2 rapid genotypes/non-smoker were used as reference, bladder cancer risk increased to 5.8-fold (P < 0.05). Among the smokers with PY higher than 10, the patients showed a higher frequency of NAT2 slow genotype than controls (P < 0.05). It was also shown that the patients with slow NAT2 genotypes were more likely to have high grade tumor (P < 0.05) and advanced stage tumor (P < 0.01).

CONCLUSIONThe results suggest that NAT2 genetic polymorphism is associated with bladder cancer susceptibility. People with NAT2 slow genotype have higher bladder cancer risk.

Arylamine N-Acetyltransferase ; genetics ; Carcinoma, Transitional Cell ; enzymology ; genetics ; pathology ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Smoking ; Urinary Bladder Neoplasms ; enzymology ; genetics ; pathology

Animals ; Carcinoma, Squamous Cell ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme Activation ; Esophageal Neoplasms ; genetics ; Genome-Wide Association Study ; Head and Neck Neoplasms ; genetics ; Humans ; Neoplasms ; chemically induced ; enzymology ; genetics ; Phosphoinositide Phospholipase C ; chemistry ; genetics ; metabolism ; physiology ; Signal Transduction ; Skin Neoplasms ; chemically induced ; enzymology ; Stomach Neoplasms ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology ; ras Proteins ; metabolism

OBJECTIVETo study the killing effect of human herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system combined with allitride and the possible apoptosis mechanism in BIU87 cells.

METHODSThe cytotoxicity after combination were estimated by theamine blue tetrazolium bromide (MTT). The morphological changes were observed with inverted microscope and in-situ cell apoptosis detection kit. Changes of apoptosis rate and cell cycle were assessed by flow cytometry. B-cell lymphoma-2 (bcl-2), bax, caspase-3 (cysteine aspartate specific proteinase) mRNA changes were detected by reverse transcriptase polymerase chain reaction, and caspase-3 activity was estimated with colorimetry.

RESULTSFor combination group, the cell killing rate was raised to 72.50% to compare with 35.00% of GCV and 37.00% of allitride separately and there was a synergistic effect between these two drugs. The cell apoptosis was induced in all three groups and for the combination group the time of S-phase and G(2)-phase arrest were earlier than other two groups. Both drugs could inhibit the expression of bcl-2 and promote the expression and activity of caspase-3.

CONCLUSIONSThe combination of HSV-TK/GCV system with allitride can inhibit the proliferation of BIU87 cells congenerously through apoptosis, which may be correlated with S- and G(2)-phase arrest, down-regulation of bcl-2 and increased caspase-3 expression and its activity.

Apoptosis ; drug effects ; Drug Synergism ; Ganciclovir ; pharmacology ; Genetic Therapy ; Herpesvirus 1, Human ; enzymology ; genetics ; Humans ; In Vitro Techniques ; Sulfinic Acids ; pharmacology ; Thymidine Kinase ; genetics ; Transfection ; Urinary Bladder Neoplasms ; pathology ; therapy

BACKGROUNDTelomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.

RESULTSAS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-alpha while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.

CONCLUSIONSAS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.

Apoptosis ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Oligonucleotides, Antisense ; therapeutic use ; RNA, Messenger ; analysis ; Telomerase ; analysis ; antagonists & inhibitors ; genetics ; Thionucleotides ; therapeutic use ; Tumor Necrosis Factor-alpha ; physiology ; Urinary Bladder Neoplasms ; enzymology ; pathology ; therapy

OBJECTIVETo investigate the relationship of protein kinase C-alpha (PKCalpha) expression/activation with tumor differentiation and resistance to chemotherapy drugs in superficial bladder carcinoma.

METHODSExpression of PKCalpha was measured by Western-blot analysis in 76 samples including tumor and normal tissues, respectively. A human RT4 bladder cancer cell line stably expressing green fluorescent protein (GFP)-PKCalpha (RT4/PKCalpha) was established. The sensitivity of the RT4/PKCalpha and parental cells to adriamycin (ADM) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The change of sensitivity of the RT4/PKCalpha to ADM were observed under the conditions of PKC activation and inhibition, respectively.

RESULTSTotal level of PKCalpha expression and the ratio of the amount of PKCalpha expression or PKC activity in membrane to that in cytosol (M/C) were all more higher in cancerous tissues than in normal tissues (P < 0.01); With the increase of tumor grade, the relative level of PKCalpha expression significantly increased in membrane (P < 0.01) and decreased in cytosol (P < 0.01), M/C of PKCalpha was significantly elevated (P < 0.01), and total relative level of PKCalpha expression significantly increased (P < 0.01). Thirty-eight cases recurred during the follow-up period in total seventy cases. Multivariate analysis showed that high M/C of PKCalpha was independent prognostic factor for tumor recurrence after standard ADM treatment in the 2-year follow-up (RR = 3.98, 95% CI 1.22-5.68, P = 0.03). Transfection of PKCalpha increased resistance of RT4 cells to ADM [resistance index (RI): 6.97, t = 3.24, P < 0.01]. PKCalpha activation further greatly promoted the resistance (RI: 148.11, t = 5.18, P < 0.001) while inhibition of PKCalpha did conversely (RI: 1.6, t = 1.29, P > 0.05).

CONCLUSIONThe abnormal activation and expression level of PKCalpha closely correlate with both tumor grade and intrinsic resistance to ADM in patients with superficial bladder carcinoma.

Aged ; Antibiotics, Antineoplastic ; pharmacology ; Carcinoma, Transitional Cell ; enzymology ; pathology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; physiology ; Enzyme Activation ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Protein Kinase C-alpha ; metabolism ; Transfection ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; enzymology ; pathology

CYP1A2, CYP2D6 and N-acetyltransferase activities were estimated in 100 patients with bladder cancer and 84 control subjects from measurements of theophylline, metoprolol and isoniazid and their metabolites in urine, respectively. The frequency of occurrence of slow acetylators of isoniazid and poor metabolizers of metoprolol were 16.7% and 1.2% in the control group and 16.3% and 2.0% in the cancer patient group. These differences were not significant. The recovery ratio of 1-methyluric acid(1-MU) from theophylline was significantly higher in patients with bladder cancer than in control subjects(0.340 +/- 0.016 versus 0.260 +/- 0.020, p< 0.05). The 1-MU recovery ratio was a significant, independent risk factor among the metabolic capacities tested as shown by logistic regression analysis, controlling for N-acetylation of isoniazid, hydroxylation of metoprolol, age, sex, and smoking. We concluded that the capacity for 3-demethylation of theophylline, as a reflection of CYP1A2 activity, is significantly associated with increased risk of nonoccupational urinary bladder cancer.
Acetylation ; Adult ; Aged ; Amines/metabolism ; Bladder Neoplasms/enzymology/*epidemiology ; Carcinoma, Transitional Cell/enzymology/*epidemiology ; Case-Control Studies ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP2D6 ; Cytochrome P-450 Enzyme System/metabolism/*urine ; Disease Susceptibility ; Enzyme Induction ; Female ; Human ; Isoniazid/*pharmacokinetics ; Korea/epidemiology ; Logistic Models ; Male ; Methylation ; Metoprolol/*pharmacokinetics ; Middle Age ; Mixed Function Oxygenases/metabolism ; Mixed Function Oxygenases/metabolism ; Oxidoreductases/*urine ; Smoking ; Support, Non-U.S. Gov't ; Theophylline/*pharmacokinetics ; Uric Acid/analogs & derivatives/urine

To investigate the antitumor effect of herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system on human bladder cancer cells (T24), a retroviral vector with the gene (pLXSN-TK) was transduced into the packaging cell line PA317. A nude mouse model with human T24 was established to examine the in vivo efficacy. The animals were randomly assigned to two treatment groups and two control groups. Treatment I and Treatment II were given in situ injection of virus suspension and PA317/TK respectively, followed by treatment with GCV for 14 days. Control I and Control II were given in situ injection of same volume of normal saline and PA317/TK respectively, followed by treatment with GCV and with normaly physiologic saline respectively for 14 days. The weight and the volume of tumor were measured. HSV-TK mRNA expression was determined by hybridization in situ. Cell apoptosis was evaluated by flow cytometry (FCM) and termininal deoxynucleofidyl transferase-mediated dUTP nick end labelling (TUNEL) technique. The results showed: (1) In vivo, the retrovirus transferred HSV-TK gene can be transduced into human bladder cancer cell T24. The tumors in T24 mice with TK gene transduced were much smaller than those in other groups. (2) After treatment with HSV-TK/GCV, the phenomenon of bladder ceancer cell apoptosis was more conspicuous as compared with that of other groups. Therefore, HSV-TK/GCV system can suppress the growth of T24 in vivo and may relate to "bystander effect". It could be a valuable therapy for human bladder cancer.
Animals ; Antiviral Agents ; therapeutic use ; Ganciclovir ; therapeutic use ; Gene Transfer Techniques ; Genetic Therapy ; Herpesvirus 1, Human ; enzymology ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Random Allocation ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; genetics ; pathology ; therapy