Proliferation Patterns of N-butyl-N- ( 4-hydroxybutyl ) Nitrosamine ( BBN ) Induced Rat Bladder Tumor Identified by Histone mRNA In Situ Hybridization / 대한비뇨기과학회지

The synthesis of histone mRNA is closely coupled with DNA replication. During the S-phase of the cell cycle, the level of histone mRNA increases over fifty fold then rapidly disappears at the start of G2-phase. The presence, therefore, of abundant quantities of histone mRNA provides a molecular marker of cycling cells. The expression of histone mRNA was investigated by in situ hybridization method in N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced rat bladder tumor to determine the proliferation patterns. This method resulted in intense brown/black cytoplasmic staining of cells containing histone mRNA and extent and intensity of stain were increased in tumorigenesis (normal, simple hyperplasia, nodular or papillary hyperplasia, and transitional cell carcinoma-Ta,T1). The higher S-phase fraction indicated greater biological malignancy based on the fact that the extent and intensity of stain progressively increased with tumorigenesis.

Analysis of Antitumor Mechanism of Intravesical BCG Therapy in Tumorigenesis(II) Study about Memory T Cells and Lymphocyte Homing Receptor Positive Cells / 대한비뇨기과학회지

A study was focused on the analysis of the change of the number and distribution of immunocytes during the tumorigenesis by N-buty1-N-(4-hydroxybutyl)-nitrosamine (BBN) administration to C3H/He inbred mice and intravesical BCG therapy to starch for the subclass of the T cells which could be the major elements in antitumor mechanism of BCG. The number and distribution of memory T(CD44) cells were studied immunohistochemically in the spleen and urinary bladder and those of memory T cells in peripheral lymph nodes and those of lymphocyte homing receptor(Mel-14) positive cells in thymus were studied microflowcytometrically following 4 weekly intravesical BCG instillations. In BBN administered group, changes of immunocytes were not induced by BBN only but as gross tumor developed, increase of memory T cells was observed but was not statistically significant comparing with those of control group. In BCG treated group, memory T cells in bladder and spleen, lymphocyte homing receptor positive cells in thymus and memory T cells in lymph nodes were all increased markedly and de creased with time sequence. In BBN administered and BCG treated group, as the immune cells decreased, gross tumor developed and infiltration of memory T cells to tumor tissues was observed in this group. In conclusion, memory T cells which were activated by BCG can be major element involved in antitumor activity of BCG and the change of the number and the pattern of distribution of immune cells showed us that local reaction as well as systemic cellular immunity seemed to be important in antitumor activity. Also the effect was time limited.

Analysis of Antitumor Mechanism of Intravesical BCG Therapy in Tumorigenesis(I): Study About T Cell Dependency / 대한비뇨기과학회지

A study was carried out to define the role of T cells in tumorigenesis and antitumor activity of BCG treatment for superficial transitional cell carcinoma of urinary bladder using an anima1 mode1. We analysed the change of immunocytes and effect on tumor forming rate of BCG in C3H/He inbred mice with bladder tumors induced by N-butyl-N-(4-hydroxybuty1)-nitrosamine(BBN) and determined the relationship between changes of immunocytes and tumor forming rate. The number and distribution of helper(T4), and suppressor/cytotoxic(T8) T cells were studied immunohistochemically in the spleen and urinary bladder. In BBN administered group, helper T cells were increased. in urinary bladder as gross tumor developed, but it seemed to be not effective in tumor prevention, and there was no significant change observed in suppressor/cytotoxic T cells. Depletion of helper T cells in spleen observed in mice with gross tumors suggested the possibility of systemic response to tumor development In BCG treated group, helper in bladder and spleen were all increased markedly and decreased with time sequence. The change of suppressor T cells were not observed. In BBN administered and BCG treated group, the tumor forming rate was initially reduced by 30-40% comparing to the BBN only group, but the rate was subsequently increased gradually with time sequence. The rate became same between 2 groups by 20 weeks. Local reaction as well as cellular immunity which was mediated by systemic immune system appeared to play an important role in antitumor mechanism of BCG Helper T cell was thought to be the major element involved in antitumor activity. Since our result indicated that effect of BCG was time limited, we have to make an effort to modify current method of BCG treatment accordingly.

The Changes of Histopathology, Proliferation Activity, and Nuclear DNA Content N-butyl-N-(4-hydroxybuty1) Nitrosamine(BBN) Induced Bladder Cancer in Rats / 대한비뇨기과학회지

To evaluate the process of tumor progression in chemical carcinogenesis of the bladder cancer. 0.05% BBN was administered to female Wistar rats for 12 weeks. The rats were divided into six groups and sacrificed every or every two weeks from the 12th to the 20th weeks. Cellular touch imprints of urinary bladder for DNA content analysis by image analyzer and mean AgNORs count per nucleus were performed immediately after sacrifice. Thereafter, the urinary bladder was embedded in paraffin for histopathological examination.On histopathological findings, simple hyperplasia was found in all cases after 12 weeks therapy of BBN. Atypical hyperplasia of the bladder, indicative of a precancerous state, was found in 88.9% of the 12 weeks group, 83.3% of the 13 weeks and in all cases after 14 weeks therapy of the BBN. Bladder cancer was found in 33.3% of the 13 weeks, 55.6% of the 14 weeks, and 100% of the above 16 weeks therapy group. The nuclear DNA content of 21 cases of atypical hyperplasia was diploid in 19 cases(91.5%) and aneuploid in 2 cases(9.5%). DNA aneuploidy was found in 18 cases(66.7%) among the 27 cases of the cancer group. Mean AgNORs count per nucleus and proliferation index by flowcytometry were higher in atypical hyperplasia and cancer group than these of simple hyperplasia and control group, but those differences according to histologic type were not statistically significant. And there was statistically significant correlation between proliferation index and mean AgNORs count per nucleus(r=0.57, p<0.05). These data suggest that the change of nuclear DNA content might occur during the early phase of the carcinogenesis in BBN-induced bladder cancer of rats.

Immunohistochemical Study of Ras and Epidermal Growth Factor Receptor Expression in BBN Induced Bladder Tumor of Rat / 대한비뇨기과학회지

There is increasing evidence that genes encoding for signal transduction pathway, including ras and epidermal growth factor receptor( EGFR), are involved not only in normal cellular growth and differentiation but also in development and progression of malignant tumors. N-butyl-N- (4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder tumor is an ideal model to study the time related gene expression in stepwise bladder epithelial carcinogenesis. We herein investigated the roles of ras and EGFR by immunohistochemical study in bladder tissues of Sprague-Dawley rats after administration of BBN. Sequential epithelial changes consisting of normal, simple hyperplasia, nodular or papillary hyperplasia, papilloma and superficial transitional cell carcinoma(TCC) were observed in rat bladders in parallel with the duration of BBN administration. The expressions of both ras and EGFR were weak in bladder epithelia of non-BBN-administered rats, whereas, immunoreactivity and number of reactive cell layer were increased in bladder epithelia of BBN-administered rats in parallel with the duration of BBN administration and progression of bladder epithelial changes. These results further support the hypothesis that ras and EGFR may have important roles in development of bladder TCC.

Detection of C-erbB-2 Internal Domain in Bladder Tumor / 대한비뇨기과학회지

c-erbB-2 overexpression appears to play a role in determining the malignant potential of some human cancers. Using a specific monoclonal antibody to c-erbB-2 internal domain, 25 transitional cell carcinoma and I squamous cell carcinoma specimens of the human bladder, and 30 cases of BBN induced rat bladder tumors were analyzed immunohistochemically to detect the expression of this putative transmembrane receptor. c-erbB-2 internal domain was not expressed in rat urinary bladder carcinomas induced by BBN, but in 32% (8/25) of human transitional cell carcinomas. Although c-erbB-2 was expressed in human bladder tumor, there was no correlation between expression of this oncoprotein and characteristics of bladder tumor. Positivity for c-erbB-2 oncoprotein was easily identifiable as an intense red staining localized predominantly at the cell membrane, supporting the view that c-erbB-2 protein is a transmembrane molecule. In most of the positive tumors, the distribution of staining is heterogenous. This suggests that small populations of tumor cells that have elevated levels of c-erbB-2 oncoprotein can be detected by staining. The availability of antisera that detect many oncoproteins in paraffin embedded tissue makes it feasible to design retrospective and prospective studies on selected groups of patients.

Expression of brdU in BBN induced rat bladder tumor / 대한비뇨기과학회지

We labeled-S-phase cell in BBN induced rat bladder tumors using anti-bromodeoxyuridine anti- body. The thymidine analogue, bromodeoyuridine (BrdU), was incorporated into DNA during the S -phase or the cell cycle. Anti-BrdU monoclonal antibody can be detected by the immunohistochemical method for an in vivo labeling study. Sequential cellular change (normal, simple hyperplasia, nodular or papillary hyperplasia, and transitional cell carcinoma-Ta, T1) was observed in rat urinary bladder according to the duration of BBN administration. The BrdU labeling index, S phase fraction, was determined by counting the number of BrdU labeled cells in the tissue section. The average labeling index obtained using this method in normal, simple hyperplasia(SH), nodular or papillary hyperplasia(NPH), and bladder tumor (Ta. T1) was 1.7, 5.6, 11.2, and 18.9 (14.9, 21.9), respectively. Also, the higher S phase fraction was found in grade II than in grade I .The higher S phase fraction indicated greater biological malignancy based on the fact that mean labeling index progressively increased with tumorigenesis. However, mean labeling index at NPH did not significantly differ from that of Ta, which implies that NPH might represent the similar cellular characteristics.

Scanning electron microscopic findings in changes of bladder epithelium induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rat / 대한비뇨기과학회지

N-buty1-N-(4-hydroxybutyl)nitrosamine(BBN) is known to have a strong. specific carcinogenic action on the urinary bladder of rats. Rat bladder lesions induced by BBN are very similar to those of Pusan histopathologically. To investigate the surface change in the urinary bladder epithelium induced by 0.05% BBN scanning electron microscopic examination was done. Normal bladder epithelium was formed by relatively uniform sized. flat polygonal cells and clearly defined with intercellular ridges. in high magnification, the luminal surface of superficial cell consisted of hexagona1 areas and concave plaques between microridge giving a honeycomb appearance but no microvilli was visible. On BBN administration for 12 weeks, the superficial cells varied in size and shape and covered by numerous uniform round microvilli forming cobble stone appearance. On BBN administration for 12 weeks and water for 6 weeks, the superficial cells were covered with numerous microvilli completely. In some areas numerous pleomorphic microvilli were seen. Although pleomorphic microvilli are not a specific marker for neoplastic transformation, their presence is an indicator. of a markedly abnormal proliferative response.

Effect of intraperitoneal injection of single chemotherapeutic agent on rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine / 대한비뇨기과학회지

Chemotherapeutic agents were evaluated for their effect on the development of urinary bladder tumors, induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female Sprague-Dawley rats. For 16 weeks, 0.05% BBN was administered orally, and then chemotherapeutic agents (adriamycin and cisplatin) were injected intraperitoneally for 12 weeks. In our preliminary experimental series, an experimental bladder tumor model induced by BBN was useful in evaluating the effectiveness of chemotherapeutic agents. Cisplatin appears to be effective in inhibiting precancerous histologic changes and carcinoma in rat urinary bladder mucosa by BBN. However, the effectiveness of adriamycin was inconclusive.

Effects of Vitamin A and Bacillus Calmette -Guerin (BCG) Combination on Experimental Bladder Cancer / 대한비뇨기과학회지

The effects of BCG and vitamin A acetate, either alone or in combination were studied on rats with bladder tumors induced by N-butyl -N-(4 -hydroxybutyl) nitrosamine. Therapy was started at week 12 and all rats were sacrificed at week 20. Vitamin A acetate therapy significantly reduced the mean number of tumors and the incidence of bladder cancer. The combination of vitamin A acetate and intraperitoneal BCG therapy, more effectively reduced the mean number of bladder tumors and the incidence of bladder cancer than vitamin A acetate therapy alone did. BCG therapy also significantly reduced the mean number of tumors and the incidence of bladder cancer. The difference between the effect of intraperitoneal BCG injection and that of intravesical BCG was not statistically significant. The combination therapy of BCG and vitamin A acetate more effectively reduced the mean number or tumors than BCG therapy along did. The incidence of bladder cancer was also reduced though the statistical significance was not definite (p =0.07). The above results suggest that vitamin A acetate and BCG therapy after initiation of precancerous lesions, alter favorably the course of the experimental bladder cancers in rats and the combination of the two does more effectively.

Immunohistochemical Study with Carcinoembryonic Antigen in Experimental Bladder Tumor Induced by Administration of N-butyl-N-(4-hydroxybutyl)-nitrosamine / 대한비뇨기과학회지

Immunohistochemical study with carcinoembryonic antigen (CEA) by peroxidase-antiperoxidase (PAP) method in rat bladder tumor induced by administration of N-butyl-N-( 4-hydroxybutyl)- nitrosamine was performed to know the immunological cross reactivity between human bladder tumor and experimentally induced rat bladder tumor and to evaluate, if all, its role as an indicator of malignant potential in rat bladder tumor. The results revealed 6 false positive reactions regardless of their histologic types and gave us no meaningful informations about ongoing disease process.

Effect of Intraperitoneal and Intravesical Bacillus Calmette-Guerin on Bladder Carcinogenesis in Rats Induced by N-butyl-N-(4-hydroxybutyl) nitrosamine / 대한비뇨기과학회지

The effects of intraperitoneal injection and intravesical instillation of viable Bacillus Calmette-Guerin(BCG) as an inhibitor in the development of experimental bladder tumors, were examined in rats administered with N-butyl-N-(4-hydroxybutyl) nitrosamine(BBN). A single intraperitoneal injection of BCG at the initiation of BBN administration inhibited significantly the occurrence of transitional cell carcinoma of the urinary bladder, while the same intraperitoneal BCG 2 weeks prior to the administration of BBN did not significantly suppress the carcinogenesis. A series of additional intravesical BCG instillation of different doses and durations to the initial intraperitoneal BCG did not potentiate significantly the antitumor effect by BCG, however, there was a considerable reduction of tumor incidence. The above results suggest that viable BCG immunotherapy alter favorably the course of the experimental bladder cancer in rats, and the routes, intervals and doses of BCG would critically effect the anti-tumor activity, accordingly.

Effects of Intravesical Mitomycin-C on Bladder Cancer of Rats Induced by N-butyl-N-(4-Hydroxybutyl) Nitrosamine / 대한비뇨기과학회지

The effects of intravesical instillation of mitomycin-C(MMC) as inhibitor of development of experimental bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine(BBN) were morphologically studied. BBN was administered for 12 weeks orally and then MMC was instilled intravesically once a week with different concentration of 2 mg/ml and 5 mg/ml and doses of 4 and 8 times. There was a significant reduction in incidence if papilloma in group V which received MMC 2 mg/ml for 8 times and also there were significant reductions in incidence of carcinoma in all MMC treated groups except group III which received MMC 2 mg/ml for 4 times. These results indicate that intravesical instillation of MMC is an effective method to prevent bladder carcinogenesis. The ultrastructural effects of MMC were studied by transmission electron microscope. Partial nucleolar fragmentation and decrease in number and height of the surface microvilli of tumor cells resulting in overall increase in intercellular space and cellular detachment from the tumor surface might have played a role in reduction of cancerincidence.

Effects of Intravesical Mitomycin-C on Bladder Cancer of Rats Induced by N-butyl-N-(4-Hydroxybutyl) Nitrosamine / 대한비뇨기과학회지

The effects of intravesical instillation of mitomycin-C(MMC) as inhibitor of development of experimental bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine(BBN) were morphologically studied. BBN was administered for 12 weeks orally and then MMC was instilled intravesically once a week with different concentration of 2 mg/ml and 5 mg/ml and doses of 4 and 8 times. There was a significant reduction in incidence if papilloma in group V which received MMC 2 mg/ml for 8 times and also there were significant reductions in incidence of carcinoma in all MMC treated groups except group III which received MMC 2 mg/ml for 4 times. These results indicate that intravesical instillation of MMC is an effective method to prevent bladder carcinogenesis. The ultrastructural effects of MMC were studied by transmission electron microscope. Partial nucleolar fragmentation and decrease in number and height of the surface microvilli of tumor cells resulting in overall increase in intercellular space and cellular detachment from the tumor surface might have played a role in reduction of cancerincidence.