L-isoleucine and L-leucine: tumor promoters of bladder cancer in rats.

A 4-week assay for screening tumor promoters of bladder cancer has been developed in which increased agglutinability of isolated rat bladder cells with concanavalin A is used as an indicator. On the basis of this assay system, L-isoleucine and L-leucine were suspected of being possible tumor promoters. Results of 40- to 60-week carcinogenesis experiments in which N-butyl-N-(4-hydroxybutyl)nitrosamine was used as an initiator demonstrate that L-isoleucine and L-leucine promote bladder cancer in rats. This finding may be relevant to the high incidence of human bladder cancer in Western countries, where the diet is rich in protein.

Inhibition of rat prostate carcinogenesis by a 5alpha-reductase inhibitor, FK143.

BACKGROUND: Androgen levels in the prostate may influence carcinogenesis in this organ. Inhibitors of the enzyme 5alpha-reductase block conversion of testosterone to the more active androgen dihydrotestosterone. The use of a 5alpha-reductase inhibitor, finasteride, in the chemoprevention of prostate cancer is being evaluated in a clinical trial. PURPOSE: This study was conducted to determine if a 5alpha-reductase inhibitor, FK143, inhibits the development of prostate cancer in rats. METHODS: Male ACI/Seg rats, which spontaneously develop prostate cancer, were randomly assigned at 80 weeks of age to receive one of three diets (n = 35/group) containing 0 (i.e., control group), 20, or 200 ppm FK143. At 140 weeks of age, the animals were killed, and the prostates were removed and examined for histopathologic features in addition to being assayed for androgen concentrations. Two-sided statistical tests were used to calculate all P values. RESULTS: The incidence of prostate carcinoma in the control group was 62.9% (22 of 35 rats); in the group fed the 20 ppm FK143-containing diet, it was 45.7% (16 of 35); and in the group fed the 200 ppm FK143-containing diet, it was 67.6% (23 of 34) (overall, P = .153). The corresponding incidences of macroscopic lesions were 17.1% (six of 35 rats), 0% (none of 35), and 23.5% (eight of 34), respectively (overall, P = .004). The incidence of macroscopic lesions in the prostates of rats in the 20-ppm diet group was significantly lower than that in the control group (P = .029) or that in the 200-ppm diet group (P = .003). Intraprostatic dihydrotestosterone content was significantly lower in rats in the groups fed diets containing 20 or 200 ppm FK143 (mean values: 4.51 and 4.33 pg/mg wet weight of prostate tissue, respectively) than in the control group (6.10 pg/mg) (overall, P<.001); by contrast, testosterone was higher in the 200-ppm diet group (2.09 pg/mg) than in the control group (1.08 pg/mg) or the 20-ppm diet group (1.21 pg/mg) (overall, P<.001). CONCLUSIONS: FK143, when fed to rats at 20 or 200 ppm, significantly reduced the level of dihydrotestosterone in their prostate tissue. However, the incidence of macroscopic cancer in the prostate was suppressed in rats consuming the 20 ppm FK143-containing diet but not in those consuming the 200-ppm diet. The lack of dose dependence for the chemopreventive activity of FK143 may be explained by the reciprocal increase of tissue testosterone in the 200-ppm diet group. IMPLICATIONS: The 5alpha-reductase inhibitor FK143 may, at specific doses, reduce the incidence of spontaneously developing prostate cancer; however, whether these findings in rats will apply to humans remains to be determined.

Further studies on the effect of leupeptin, a protease inhibitor, on induction of bladder tumors in rats by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The effect of a microbial protease inhibitor, leupeptin, on the induction of urinary bladder tumors in W rats by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. Three groups of animals were given 0.01% BBN in their drinking water for 12 weeks. A basal powder diet supplemented with 0.1% leupeptin was given to group A throughout the experiment and to group B when BBN administration was stopped. Group C was given the basal diet without leupeptin throughout the study. The total preservation period was 40 weeks. Results clearly showed that when leupeptin was given during the promotion step of bladder carcinogenesis (as in group B), it increased the size of tumors and the incidences of cancer and invasion. When leupeptin was given throughout the experiments, its effect was counteracted (as in group A).

Effect of leupeptin, a protease inhibitor, on induction of bladder tumors in rats by n-butyl-n-(4-hydroxybutyl)nitrosamine.

Leupeptin, isolated from Actinomycetes, is a potent and specific inhibitor of proteases. We found that the administration of leupeptin enhanced that size of urinary bladder tumors induced in rats by the oral administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). BBN was given as a 0.05% solution in the drinking water for 6 weeks, and then animals were fed a diet with or without 0.1% leupeptin for 30 weeks. The average weight of the bladders with tumors in rats fed a leupeptin diet was about eight times that of rats on a diet without leupeptin, though the incidences and average numbers of tumors in the bladders were similar in the two groups.

Loss of heterozygosity on chromosomes 1p and 11p in sporadic pheochromocytoma.

We used 29 polymorphic DNA markers to analyze tumor DNA samples from six patients with sporadic pheochromocytoma for possible loss of chromosomal heterozygosity; four had benign disease and two had malignant disease. Loss of heterozygosity was observed on four chromosomes: 1p (three of four patients), 2p (one of one), 5q (two of six), and 11p (three of five). Chromosomes 1p and 11p frequently had allelic deletions in these tumors, and these deletions may play an important role in the development of pheochromocytoma.

Peritoneal tuberculosis with gastric cancer: a case report.

This is a report of a case with both peritoneal tuberculosis and gastric cancer. Physicians should have a high index of suspicion of peritoneal tuberculosis if the patient is febrile with a past history of tuberculosis.

Agglutination of bladder cells by concanavalin A during the early phase of treatment of rats with N-butyl-N-(4-hydroxybutyl)nitrosamine.

N-Butyl-N-(4-hydroxybutyl)nitrosamine was given to male Wistar rats at a dose of 0.05% in the drinking water for one to five weeks, and agglutination of cell isolated from their bladder by concanavalin A (Con A) was determined at intervals during and after treatment. Mucosal cells were isolated from everted bladder by ethylenediaminetetraacetate treatment and sonication. As early as one week after the start of treatment, Con A caused some agglutination of isolated bladder cells, and this agglutination increased with time, reaching an almost constant value from the third week. Con A agglutination of bladder cells induced by N-butyl-N-(4-hydroxybutyl)nitrosamine treatment for only one week appeared to be irreversible, and it was still observed two weeks after the end of treatment. Scanning electron microscopy showed that microvilli developed on the luminal surface of mucosal cells in situ at the time when the isolated cells became agglutinable with Con A. Measurement of agglutinability of isolated bladder cells with Con A might be a useful way of detecting very early changes in bladder carcinogenesis.

Inhibition of N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat urinary bladder carcinogenesis by alpha-difluoromethylornithine.

The effect of oral administration of alpha-difluoromethylornithine (DFMO), an irreversible ornithine decarboxylase inhibitor, on N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN)-induced rat urinary bladder carcinogenesis was investigated. Four-wk-old male Fischer 344 rats, 30-38 per group, were divided into 3 groups; each group was divided into 3 subgroups. In Group A, 6-wk treatment with 0.05% BHBN in drinking water was followed by either 0.5% (A1), 0.2% (A2), or 0% (A3) DFMO in drinking water for 34 wk. In Group B, coadministration in drinking water of 0.01% BHBN and either 0.5% (B1), 0.2% (B2), or 0% (B3) DFMO was continued for 30 wk. Group C consisted of animals receiving 0.5%, 0.2%, or 0% DFMO in drinking water for 34 wk without prior or cocarcinogen treatment. Bladder tumorigenesis was clearly inhibited by DFMO; tumor incidence was 14 of 37 (38%) in A1, 16 of 38 (42%) in A2, and 31 of 35 (89%) in A3, and 7 of 35 (20%) in B1, 14 of 35 (40%) in B2, and 28 of 35 (80%) in B3 (P less than 0.01, DFMO groups as compared to the respective control A3 or B3). The average tumor volume was strikingly reduced in Group A rats given DFMO (3.0 mm3 in A1, 5.0 in A2, and 38.6 in A3). Significant suppression of tumor multiplicity (number of tumors/tumor-bearing bladder) was observed in DFMO-treated subgroups in Group B (1.1 in B1, 1.3 in B2, and 1.8 in B3). In both Groups A and B, however, DFMO failed to suppress hyperplastic changes (simple hyperplasia) or preneoplastic lesions (nodulopapillary hyperplasia). Systematic examination of all pertinent organs excluding the brain showed no adverse effects attributable to DFMO treatment except for decrease in body weight (less than 7%), which was consistently observed in the groups receiving 0.5% DFMO, and reduction in the combined weight of the prostate and seminal vesicles (less than 20%), which was noted in Group B in which exposure to DFMO was started at a younger age. These results indicate that oral administration of DFMO is quite effective in suppressing (or retarding) BHBN-induced carcinogenesis with minimal untoward effects and confirm the similar inhibitory effects demonstrated earlier with the heterotopically transplanted rat urinary bladder system.

Promotional effect of two-generation exposure to a high-fat diet on prostate carcinogenesis in ACI/Seg rats.

Epidemiological studies have shown an association between a high-fat diet and a high mortality rate from breast, colon, and prostate cancer. However, the promotional effect of a high-fat diet on experimental carcinogenesis has not been fully established for the prostate. In this study, the effect on prostatic carcinogenesis of two-generation exposure to a high-fat diet was investigated using ACI/Seg rats, a strain with high incidence of spontaneous prostate cancer. A high-fat diet (20% corn oil) or a low-fat diet (5% corn oil) was given to mother rats during pregnancy and the newborn male rats were fed the same diets for 60 or 100 weeks after weaning. At 100 weeks, atypical hyperplasia and adenocarcinoma of the prostate were respectively found in 73.3% (11/15) and 20.0% (3/15) of the high-fat diet group and in 20.0% (3/15) and 0% (0/15) of the low-fat diet group. There was a significant increase of atypical hyperplasia in the high-fat diet group (P < 0.05). The serum concentrations of sex hormones and the prostatic proliferative activity as measured by flow cytometry or bromodeoxyuridine labeling were not significantly affected by diet. These results showed that feeding a high-fat diet before conception and from the beginning of organogenesis had a marked promotional effect on the early stage of prostate carcinogenesis in rats.

Relationship between papillary and nodular transitional cell carcinoma in the human urinary bladder.

A total of 186 cystectomized specimens were examined by step-sectioning to determine the relation between papillary and nodular transitional cell carcinomas of the urinary bladder. Tumors were classified as papillary (PC), nodular (NC), and carcinoma in situ (CIS) according to their gross and microscopic configurations. These cases, grouped as simple combinations of PC, NC, and CIS, namely, PC, PC + CIS, PC + NC, PC + NC + CIS, NC, NC + CIS, and CIS, were analyzed with respect to (a) the time from the initial symptom to cystectomy, (b) the treatment before cystectomy, (c) the grade, (d) the stage of tumors, (e) the multiplicity of tumors, (f) the presence of papillary structures inside or on the surface of nodular carcinoma, and (g) data on survival after cystectomy. Of the tumors, 17 were classified as CIS and 80 as PC and PC + CIS. Studies on 57 cases suggested an early change from PC to a mixture of PC and NC through papillonodular carcinoma during development, whereas 6 showed late development of NC during repeated recurrence of PC. These courses indicate that some cases of NC developed from PC. On the other hand, 26 cases exhibited direct progression from CIS to NC. Thus nodular invasive carcinomas may develop in two ways: by emergence of a more anaplastic cell population within a preexisting low grade papillary carcinoma; and by de novo development of an invasive nodular carcinoma directly from CIS.

Increased agglutinability of bladder cells by concanavalin A after administration of carcinogens.

The agglutination by concanavalin A of isolated epithelial cells of the rat bladder was examined after in vivo treatment of rats with various bladder carcinogens for one week. The carcinogens tested were N-butyl-N-(4-hydroxybutyl)nitrosamine, dibutylnitrosamine, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, 2-acetylaminofluorene, 2-napthylamine, benzidine, N-methyl-N-nitrosourea, and cyclophosphamide, and they were given to male Wistar rats p.o., s.c., intravesically, or i.p. As negative controls, the effects of administration of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, dimethylnitrosamine, N-methyl-N'-nitro-N-nitrosoguanidine, and surgical implantation of glass beads in the bladder were also tested. One week after the start of treatment, epithelial cells were isolated from the bladder by sonication, and agglutination of the isolated cells with concanavalin A was assayed. The observed agglutinabilities of isolated cells were found to be closely correlated with the reported bladder carcinogenicities of these chemicals in rats. Thus, concanavalin A agglutination of bladder cells should be a useful rapid in vivo mammalian system for screening bladder carcinogens.

Maintenance by saccharin of membrane alterations of rat bladder cells induced by subcarcinogenic treatment with bladder carcinogens.

Saccharin is known to have a tumor-promoting effect on bladder cancer in rats, but its mechanism of action is unknown. We demonstrated that the increased agglutinability of isolated epithelial cells of the bladder in the presence of concanavalin A caused by a subcarcinogenic dose of bladder carcinogens disappeared shortly after the end of their administration. However, saccharin maintained the increased agglutinability when given continuously after administration of carcinogen. Moreover, the agglutinability of bladder cells previously exposed to a subcarcinogenic dose of bladder carcinogens increased again when saccharin was given after the agglutinability had disappeared completely.

Sequential changes of mouse bladder epithelium during induction of invasive carcinomas by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Invasive carcinoma of the bladder in humans shows aggressive growth with poor prognosis. Little is known about its preceding lesions. Sequential changes of the bladder epithelium following administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) were studied in mice. Female C3H/He mice were divided into 4 groups. Three groups were given 0.05, 0.01, and 0.005% concentrations of BHBN, respectively, in their drinking water, and the control group was given tap water. The mice were killed at regular intervals over a period of 26 weeks, and their bladder epithelium was examined histologically. Dysplasia, carcinoma in situ, and invasive carcinoma were observed sequentially in the groups treated with BHBN, and the incidences of dysplasia, carcinoma in situ, and invasive carcinoma were dependent on the dose of BHBN. The data indicate that bladder carcinoma in mice is a good model of invasive bladder carcinoma in humans, although it is not fully compatible with the human model because of the complete absence of metastases.

Frequent association of p53 gene mutation in invasive bladder cancer.

Structural alterations of the p53 gene were investigated to elucidate the molecular biological difference between superficial and invasive bladder cancer by polymerase chain reaction single-strand conformation polymorphism analysis. In 25 bladder cancers obtained from 23 patients, p53 gene mutations were investigated in exon regions 4 to 11. Twenty-four were transitional cell carcinomas, and the remaining one was a squamous cell carcinoma. Only one of 13 superficial bladder cancers, including pTis, pTa, and pT1, was found to have p53 gene mutation. However, of 12 invasive bladder cancers with pT2, pT3, and pT4, six primary carcinomas, including a squamous cell carcinoma and one metastatic carcinoma, were found to have p53 gene mutations. The number of cancers examined in Grades 1, 2, and 3 was three, seven, and 15, respectively. p53 gene mutation was not found in any of the ten cancers with Grades 1 and 2, while eight of 15 bladder cancers with Grade 3 were found to have p53 gene mutation. The results indicated that the incidence of p53 gene mutations appeared to be much higher in invasive-type and high-grade bladder cancers than in superficial and low-grade ones. Our results are compatible with the recently published results by Sidransky et al. [Science (Washington DC), 252: 706-709, 1991] showing that p53 gene mutations were frequently found in invasive bladder cancers by sequence analysis on polymerase chain reaction amplified products corresponding to exons 5 to 9. Our results are also compatible with previously reported results by Olumi et al. (Cancer Res., 50: 7081-7083, 1990) showing that the loss of chromosome 17p, revealed by analysis with restriction fragment length polymorphism, was frequent in high-grade bladder cancers. In this study, p53 gene mutations were often found in exon 4 as well as in other exons. Therefore, this region should also be examined for screening of mutations of this gene in bladder cancer. There appeared to be no consistent mutation sites in exons 4 to 11 of the p53 gene and no specific patterns of the mutation in bladder cancer.

Volatile N-nitrosamines in the urine of normal donors and of bladder cancer patients.

Volatile N-nitrosamines were detected in the urine of male volunteers with gas-liquid and high-pressure liquid chromatography interfaced to the thermal energy analyzer. Of 50 samples from normal males, 10 contained nitrosodimethylamine (0.02 to 0.10 micrograms/liter), 6 contained nitrosodiethylamine (0.02 to 3.10), 9 contained nitrosomorpholine (0.006 to 0.67), and none contained nitrosodibutylamine. Of 4 samples from bladder cancer patients, 2 contained nitrosodibutylamine (0.35 and 0.66). Cigarette smoking did not appear to be related to the pattern or amount of urinary volatile N-nitrosamines. The possibility that the N-nitrosamines arise from the diet or from endogenous production is considered.

Markedly increased amounts of messenger RNAs for vascular endothelial growth factor and placenta growth factor in renal cell carcinoma associated with angiogenesis.

The presence of mRNAs for vascular endothelial growth factor (VEGF) and a VEGF-related protein, placenta growth factor (PIGF) was examined in 29 cases of renal cell carcinoma tissues and adjacent normal kidney tissues and in 4 human renal cell carcinoma cell lines. Northern blot analysis showed that 26 of 27 hypervascular renal cell carcinoma tissues (96%) exhibited a markedly elevated level (3-13 fold) of VEGF mRNA compared to the adjacent normal kidney tissues. Even tumors of small size, whenever they were hypervascular, overexpressed VEGF mRNA. We also demonstrated that mRNA for PIGF was expressed in 21 of 23 hypervascular renal cell carcinoma tissues (91%) but was not detected in the adjacent normal kidney tissues. Two hypovascular carcinoma tissues neither overexpressed VEGF mRNA nor had PIGF mRNA. VEGF mRNA was detected in four human renal cell carcinoma cell lines, while PIGF mRNA was not. There was no difference in the level of basic fibroblast growth factor mRNA between tumor tissues and normal kidney tissue, although our previous study demonstrated elevated basic fibroblast growth factor protein in the serum of renal cell carcinoma patients (K. Fujimoto et al., Biochem. Biophys. Res. Commun., 180: 386-392, 1991). Taken together, these results suggest that VEGF, PIGF, and basic fibroblast growth factor are cooperatively working to increase the angiogenesis in renal cell carcinoma in vivo.

Asian studies of cancer chemoprevention: latest clinical results.

Chemoprevention trials in Asia, including those already completed and those now ongoing, are reviewed. Information was mainly collected from Japan, Korea and China. Each country features its own characteristics. Cancer chemoprevention trials targeting, from various aspects, hepatocellular carcinoma, gastric cancer and colon cancer have been, and are now being, conducted in Japan. Japan also has a long history of basic carcinogenesis research and carcinogenic research using animal experiments. In Korea, ginseng is the main focus of studies of chemopreventive agents. A large body of information has been collected and prospective studies are also ongoing. In China, the Linxian study, a cooperative study participated in by China and the NCI of the USA, is well known and the results impressive. However, we must exercise caution because, for example, the population of Linxian are chronically deficient in multiple vitamins and trace minerals. This situation may, therefore, differ from that observed in other countries. In any event, chemoprevention studies will be popular from an economical point of view even in Asia because cancer is becoming the number one cause of death in these countries.

The external radiotherapy with three-dimensional conformal boost after the neoadjuvant androgen suppression for patients with locally advanced prostatic carcinoma.

PURPOSE: To analyze the results in patients with locally advanced prostatic carcinoma treated by hormonal therapy followed by external radiotherapy using three-dimensional conformal radiation therapy (3D-CRT) boost. METHODS AND MATERIALS: From 1987 to 1995, 46 patients with histologically proven locally advanced adenocarcinoma of the prostate were treated with 3D-CRT at the National Cancer Center Hospital, Tokyo. The neoadjuvant androgen suppression started immediately after the diagnosis followed by radical radiation therapy, according to the prospective protocol. They were treated with photons of 6-14 MV for wide fields and the boost, of which a multiple-leaf collimator of 2-cm width was available. The boosted dose was delivered with the rotational 3D-CRT, after the delivery of whole pelvis 4-field box from a dose of 40-46 Gy up to 66 Gy. The planning target volume encompassed 1 cm outside throughout the clinical target volume, and the prostate and the seminal vesicles were included in the boost field. RESULTS: The 3D-CRT boost treatment completed as planned in all 46 patients. The median follow-up for all the patients was 60 months (range, 5-120 months). Nineteen of 46 patients died. Of these, 11 patients died of the intercurrent diseases. For all 46 patients, the 5- and 8-year overall survival rates were 61.3% and 42.4%, and the 5- and 8-year cause-specific survival rates were 82.4% and 64.4%, respectively. The prostate-specific antigen (PSA) relapse-free rates for 5- and 8-year were 64.6% and 52.5%, and the clinical local control rates for 5 and 8 years were 75.3% and 69.9%, respectively. The preradiation therapy PSA and the Gleason score were the factors that significantly associated with PSA relapse-free survival. Sixteen of 46 patients (35%) showed at least one form of late toxicities. Of these, 3 patients experienced late complications of Grade 3 (urinary, 2, proctitis, 1). CONCLUSION: The treatment results were fairly good and were consistent with those in Western countries, indicating that this study shows the preliminary status of 3D-CRT for the locally advanced prostate cancer in Japan. Preradiation therapy PSA seems to be a significant predictor of PSA relapse-free survival (p = 0.004) after neoadjuvant androgen suppression.

Prepubertal XY gonadal dysgenesis.

Two children had prepubertal XY gonadal dysgenesis. A 7-year-old girl with clitoral enlargement had a left ovarian tumor that contained a dysgerminoma; the right gonad proved to be a gonadoblastoma. The second child (a 2-year-old girl) showed poor physical development and slight virilization of the genitalia. Her bilateral dysgenetic gonads were removed at exploratory laparotomy. The occurrence of gonadal tumors in XY gonadal dysgenesis is increased. It is probably related to the hypergonadotropinism existing from childhood as well as to genetic predisposition of the cryptorchid testis in the presence of a Y chromosome. Our first patient is one of the youngest who had XY gonadal dysgenesis iwth gonadoblastoma reported. The indication of prophylactic gonadectomy in XY gonadal dysgenesis is emphasized.

Concanavalin A agglutination of bladder cells of rats treated with bladder carcinogens; a rapid new test to detect bladder carcinogens.

N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), which is a potent, specific bladder carcinogen in rats, and its related compounds were orally administered to rats for 1 week. The bladder cells were isolated by the treatment with EDTA and sonication and they were subjected to agglutination assay by concanavalin A (Con A). Bladder cells obtained from rats treated with BBN, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and N-butyl-N-(2-hydroxyethyl)nitrosamine (BHEN) were agglutination-positive, while those cells treated with N-tert-butyl-N-(4-hydroxybutyl)-nitrosamine (t-BBH) and N-butyl-N-(3-hydroxypropyl)nitrosamine (BHPN) were negative. Results obtained by this method were highly correlated with the known carcinogenicity of BBN and its analogues. Therefore, this method could be used for rapid screening of bladder carcinogens.