Toshiko Takahashi

Sialosyl-Lex expression defines invasive and metastatic properties of bladder carcinoma

Two types of transitional bladder carcinoma have been distinguished based on macroscopic morphology: type A papillary carcinomas, with papillomatous surface outgrowth without infiltration into muscular layer, and type B nodular carcinomas, with a nonpapillomatous surface appearance, most of which display infiltrative growth through muscular layer, and some of which display lymphatic or blood‐borne metastasis. However, there is no specific predictor at early stages for later invasive and metastatic clinical outcome of patients with type B tumors.

N-Acetylglucosaminyltransferase V and β1-6 Branching N-Linked Oligosaccharides Are Associated with Good Prognosis of Patients with Bladder Cancer

Purpose:N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes β1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides (N-glycan) of cell proteins. We examined the relationship between GnT-V expression and clinicopathologic features of the patients with bladder cancer. Experimental Design: We immunohistochemically examined GnT-V expression in paraffin-embedded bladder cancer specimen using anti-GnT-V monoclonal antibody. We compared GnT-V expression with cause-specific survival of the patients with bladder cancer treated by radical cystectomy. Kaplan-Meier survival curves were generated to show the cause-specific survival. Univariate and multivariate analyses were carried out to compare GnT-V expression with other clinical and pathologic variables. We also evaluated mRNA expression of GnT-V and N-linked oligosaccharide structure in bladder cancer specimens. Results: Immunohistochemistry revealed that GnT-V expression inversely correlated with tumor grade and stage. The incidence of positive GnT-V expression in bladder cancer was significantly higher in low-grade/superficial cancer than in high-grade/invasive cancer. The patients whose tumor was positive for GnT-V survived significantly longer than those whose tumor was negative for GnT-V. Univariate and multivariate analyses revealed that GnT-V expression was an independent predictor of prognosis of the patient. The expression of GnT-V mRNA determined by reverse transcription-PCR was consistent with the results with immunohistochemistry for tumor samples. Carbohydrate structural analysis revealed that superficial bladder cancer is rich in branched N-linked oligosaccharides, for which biosynthesis GnT-V is responsible. Conclusions: GnT-V and its resultant β1-6 branching N-linked oligosaccharides are closely related to low malignant potential and good prognosis of the patients with bladder cancer.

Inhibition of motility and invasiveness of renal cell carcinoma induced by short interfering RNA transfection of β1,4GalNAc transferase

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and β1,4GalNAc disialyl‐Lc4 (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of β1,4GalNAc transferase (β1,4GalNAc‐T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, β1,4GalNAc‐T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same β1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of β1,4GalNAc‐T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin‐dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.

Gene transfer of α1,3-fucosyltransferase increases tumor growth of the PC-3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC‐3 with α1,3‐fucosyltransferase III (FTIII) to obtain stable transfectants, PC‐3‐FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC‐3‐FTIII cells produced large prostate tumors, while mock‐transfected PC‐3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC‐3‐FTIII cells was inhibited by preincubation of the tumor cells with anti‐sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC‐3‐FTIII cells and mock‐transfected PC‐3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC‐3‐FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock‐transfected PC‐3. Such adhesion was inhibited by preincubation of PC‐3‐FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti‐E‐selectin, anti‐P‐selectin or anti‐L‐selectin antibodies did not inhibit the adhesion of PC‐3‐FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X.

The study of PSA gene expression on urogenital cell lines

Abstract Purpose : Reverse transcriptase–polymerase chain reaction (RT–PCR) offers a potentially more sensitive assay for detecting cells expressing prostate‐specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts.

High-energy Underwater Shock Wave Treatment for Internal Iliac Muscle Metastasis of Prostatic Cancer: A First Clinical Trial

The first clinical trial of high‐energy shock wave (SW) combined with chemotherapy to treat metastasis of prostate cancer in the internal iliac muscle was conducted. The patient, a 57‐year‐old man, diagnosed as having mucin‐producing, poorly differentiated adenocarcinoma of the prostate invading the bladder wall, had been treated by total cystoprostatectomy. Five months later, metastatic tumors were found in the left axillar subcutaneous tissue and the right internal iliac muscles. For the axillar metastasis we performed radiation and left subclavicular arterial infusion of cisplatin 70 mg, THP‐adriamycin (THP) 50 mg and methotrexate 50 mg. For the right internal muscular metastasis, 10,000 to 20,000 shots of SW and simultaneous intravenous injection of carboplatin 100 mg and THP 10 mg were carried out. Neither of the tumors decreased in size, but on magnetic resonance images, the SW‐treated tumor exhibited a central low‐intensity area. The SW‐treated tumor was resected and central necrosis and a collection of mucin in the central area were observed. Hormone‐resistant prostate cancer is well‐known to be a multidrug‐resistant tumor. It is noteworthy that SW and chemotherapy induced necrosis in such a refractory cancer without any significant side effects.

Internal Iliac Arterial Infusion Chemotherapy for Rabbit Invasive Bladder Cancer

Background:

Predictive value of N-acetylglucosaminyltransferase-V for superficial bladder cancer recurrence.

PURPOSE GnT-V is an enzyme that catalyzes beta1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides of cell proteins. GnT-V expression has been closely related to malignant potentials in colon cancer, brain cancer and hepatocellular carcinoma. We determined whether GnT-V expression is predictive of superficial bladder cancer recurrence. MATERIALS AND METHODS The cohort comprised 60 consecutive patients with first time superficial bladder cancer treated with transurethral resection. None of the patients received prophylactic intravesical therapy until recurrence. Paraffin embedded tumor specimens were immunohistochemically examined by the avidin-biotin peroxidase method using monoclonal antibody against GnT-V. Kaplan-Meier survival curves were generated to determine disease-free survival. Univariate and multivariate analyses were done to compare GnT-V expression to other clinical and pathological variables. RESULTS GnT-V expression correlated inversely with tumor grade and stage. The positive incidence of GnT-V in G1 to G3 tumors was 7 of 9 (78%), 21 of 43 (49%) and 3 of 8 (38%), respectively. GnT-V was positive in 26 of 44 cases of pTa (60%) and in 5 of 16 of pT1 (31%) disease. The 31 patients with positive GnT-V expression had significantly higher disease-free survival than the 29 with negative GnT-V expression (log rank test p = 0.0034). Multivariate analysis revealed that patient age, pT, grade and negative GnT-V expression were independent predictors of recurrence (p = 0.015, 0.001, 0.019 and 0.011, respectively). CONCLUSIONS Immunohistochemical detection of GnT-V is an independent predictor of superficial bladder cancer recurrence.

Enzyme-linked immunosorbent assay detection of prostate-specific antigen messenger ribonucleic acid in prostate cancer.

OBJECTIVES To develop a rapid, sensitive, reverse transcriptase-polymerase chain reaction (RT-PCR) prostate-specific antigen (PSA) messenger ribonucleic acid (mRNA) detection method by applying colorimetric enzyme-linked immunosorbent assay (ELISA). METHODS Total RNA was extracted from 16 urogenital cancer cells (including PSA-producing LNCaP cells) from pelvic and inguinal lymph node aspiration biopsy samples from patients with prostate, bladder, and penile cancer, as well as from blood samples of 500 patients with urogenital cancer. We used rTth polymerase for RT and PCR. The RNA target was amplified by RT-PCR with dinitrophenyl-labeled primer. The PCR product was denatured and hybridized on a PSA-specific probe-coated microwell plate. RESULTS In 1 6 cancer cell lines, only LNCaP cells expressed especially high PSA mRNA values, with an optical density (OD) greater than 3. In other cell lines, two testicular cells had relatively high ODs, 1.909 and 0.987, respectively. A high PSA mRNA value was obtained by fine needle aspiration from pelvic lymph node specimens of cytologically positive lymph nodes from patients with prostate cancer but not from patients with cytologically proved bladder or penile cancer. Sensitivity and specificity of fine needle aspiration samples were 70% and 100%, respectively. Blood tests obtained from patients with prostate cancer demonstrated high PSA mRNA values. CONCLUSIONS The PSA mRNA RT-PCR ELISA method provides a sensitive photometric enzyme immunoassay for the detection of PSA mRNA, using nonradioactive techniques.

Telomerase activity: Simplification of assay and detection in bladder tumor and urinary exfoliated cells

Detection of telomerase activity can differentiate malignant from benign cells. However, the original telomeric repeat amplification protocol (TRAP) methods had a number of limitations including a radioisotope labeling [α(32)P] dCTP [α(32)P] dGTP system. We developed digoxigenin labeled CX primer to detect telomerase activity without using radioisotope and attempted to detect telomerase activity of bladder tumor and exfoliated cells in bladder cancer patients. Telomerase activity was detected in 5 (71%) of 7 patients diagnosed with grade 1, 31 (97%) of 32 grade 2, and 11 (100%) of 11 grade 3 bladder tumors. In urinary exfoliated cells, 32 (82%) of 39 grades 1 or 2 bladder tumors were positive for telomerase activity but 20 (51%) of 39 were positive for urinary cytology (P < 0.01). Ten (91%) of 11 of grade 3 tumors were positive for telomerase activity and 11 (100%) of 11 were positive urinary cytology. Three of 100 noncancerous patients were positive for telomerase activity. Sensitivity, specificity, and positive predictive value of telomerase activity assay in urinary exfoliated cells were 84%, 97%, and 93%, respectively. Telomerase activity may be a useful diagnostic marker to detect the existence of immortal cancer cells in the urine.