Direct action of estradiol on rat mammary tumors.

PIP: This study reports a direct action of 17-beta-estradiol on protein synthesis by 7, 12-dimethylbenz (alpha) anthracene (DMBA) induced rat mammary tumors. Sprague-Dawley female rats were given 20 mg of DMBA in sesame oil by gastric tube. Mammary tumors developed. When tumors reached 1.5 to 2 cm in diameter, animals were ovariectomized. At 4-7 days later animals were killed and tumors removed. Microscopic examination confirmed the tumors to be carcinoma of adenoid cystic variety with regressive changes. 14 tumors from 14 animals were found suitable for study. In vitro treatment with 17-beta-estradiol gave rise to a 7% increase in the rate of 3H-leucine incorporation, expressed as dpm/mg of TCA-insoluble protein. This increase was considered statistically significant (p.001). A large variation among different tumors was noted. Also results varied with differences in time after ovariectomy. Under the same incubation conditions the same percentage of increase in the rate of 3H-leucine incorporation had been observed in the study of the effect of estrogen on the uterus of ovariectomized rats. In other mammalian tissues studied those containing high levels of estrogen receptor were able to respond to direct stimulation of estrogen. It was concluded that estrogen directly stimulates protein synthesis in the mammary tumors. This supports the view that these tumors are estrogen responsive tissues.^ieng

Multiple autotransplantation of rat mammary induced by 7,12-dimethylbenz[a]anthracene: brief communication.

Mammary tumors induced in outbred Sprague-Dawley rats by 7,12-dimethylbenz]a]anthracene were excised, cut into 1- to 2-mm3 pieces, and then autotransplanted sc along the mammary line at six sites. Following an average period of 20--30 days, these autografts grew to approximately 2 cm in diameter in 32 of 48 rats (67%). Autografts in the other 33% of the rats remained dormant. Mammary tumors transplanted into allogeneic hosts failed to grow. Tumors derived from autotransplantation were indistinguishable from their primary tumors with respect to their histologic features, the nature of hormone dependency, the content of estrogen receptors, and their ability to incorporate [3H]leucine. Furthermore, autotransplanted tumors derived from a single primary tumor varied little with regard to the preceding parameters; thus they provided an opportunity for serial sampling of individual tumors for repeated morphologic and biochemical evaluations.

Absorption and protein binding of N-2-fluorenylacetamide and its metabolites in the bladder of rabbit.

To assess the reactivity of a bladder carcinogen, the absorption by the rabbit (male New Zealand White) bladder mucosa of N-2-acetylaminofluorene (AAF), N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF), and the N-O-glucuronide of AAF (N-OGI-AAF), as well as binding to the protein and RNA of bladder mucosa, was measured in vivo and in vitro. Mucosal pieces incubated for 3 hours in medium containing a carcinogen demonstrated that the fluorene nucleus of both AAF and N-CH-AAF bound equally with cellular proteins, while N-OGI-AAF binding was lower. In the presence of an excess of beta-glucuronidase, however, N-OGI-AAF showed binding equivalent to its metabolic precursor. After a 3-hour instillation into the bladder lumen of radioactive carcinogens suspended in urine in vivo, transmural absorption of AAF and N-OH-AAF (90%) was substantial, while N-OGI-AAF was absorbed less (55%). The renal excretion during this period varied from 18 to 52% of the instilled radioactivity. There was little reactivity of these carcinogens with the mucosal RNA, both in vivo and in vitro. The metabolism of N-OH-AAF and N-OGI-AAF was such, both in vitro and in vivo, that the acetyl group was not included in the final protein-carcinogen complex in what appeared to be an enzyme reaction.

Suppression of dibutylnitrosamine-induced bladder carcinomas in hamsters by dietary indole.

Effect of dietary indole on the urinary bladder tumorigenesis by chronic dibutylnitrosamine (DBN) treatment was evaluated in hamsters. In the first experiment, in which DBN-water and diet were given ad libitum, dietary indole significantly suppressed bladder tumor incidence. The inhibitory effect was more pronounced in males. In the second experiment, in which consumption of both diet and DBN-water was rigidly controlled by pair-feeding, dietary indole again significantly suppressed bladder tumor incidence; its effect was similar in both males and females. This suppressive effect of indole on bladder tumorigenesis contrasted markedly with its failure to suppress tumors at other sites such as nasal sinuses, trachea, esophagus, and fore-stomach.

Neoplasms of urinary bladders of hamsters treated with 2-acetylaminofluorene and indole.

Tested in hamsters was the effect of neonatal treatment with 2-acetylaminofluorene (AAF) followed by prolonged feeding in combination with indole on the development of bladder tumors. Neonatal males and females were given intraperitoneal injections of AAF, 5 mg/100 g body weight, 3 times weekly until weaning. They were then fed a synthetic diet containing 0.06% AAF and 1.6% indole. The 26 hamsters surviving 10-12 months developed transitional cell carcinomas of the bladder, all but 2 of which were invasive. No hamsters had malignant tumors in the liver. Seven hamsters developed peliosis of the spleen.

Human colon adenocarcinoma cells. II. Tumorigenic and organoid expression in vivo and in vitro.

A human colon epithelial tumor cell line (LS174T) recultured in vitro following passage through hamsters displayed differences in its cell doubling time and synthesis of carcinoembryonic antigen when compared with the cells grown solely in vitro. These animal-passaged cells more closely resembled the parent tumor cell line (LS180) derived from the primary tumor than LS174T, the trypsinized variant of LS180. Analysis of lactate dehydrogenase isoenzymes indicated that the tumor cells recovered from the hamsters were free of xenogeneic host tissue. Furthermore, LS174T grafted to athymic (nude) mice grew as a mucinous adenocarcinoma microscopically resembling the original tumor. The altered growth potential of LS174T was also demonstrated on confluent feeder monolayers of normal cells and by uninhibited multiplication in vitro. These results suggest that, at least in this one case, short-term passage of long-term cultured cells into xenogeneic hosts may effect a phenotypic reversion such that the cells regain properties observed in the primary tumor and the initial in vitro explant.

Conversion from low grade to high grade of rat urinary bladder carcinomas.

The present study was conducted to test if low-grade carcinomas induced by a single dose of N-methyl-Nitrosourea (MNU) can be converted to high-grade carcinomas by a second identical dose of the carcinogen. The heterotopically transplanted rat urinary bladder system was used. Four wk after heterotopic bladder transplantation, the recipient male Fischer 344 rats were divided into 2 groups. The first group received 0.25 mg of MNU into heterotopically transplanted rat urinary bladder; the second group (controls) received 0.9% NaCl solution. At week 29 of the experiment, 1/3 of the animals from each group were killed for histological examination of the heterotopically transplanted rat urinary bladders. The remaining animals from each group were divided into 2 subgroups, the first receiving 0.25 mg MNU and the second, 0.9% NaCl solution. All animals were killed at 50 wk of the experiment. MNU-induced carcinomas at week 29 were all of low histological grade and were noninvasive. Longer follow-up without a second carcinogen administration resulted in both an increase in tumor incidence (P less than 0.005) and more tumors per bladder (P less than 0.001), but high-grade invasive carcinomas were rare. The second dose of MNU administered at the stage when low-grade carcinomas were prevalent (week 29) resulted in a significant increase in invasive high-grade carcinomas (P less than 0.01). Our data are consistent with the view that the second carcinogen administration induces a new mutation(s) within low-grade carcinomas which leads to invasive carcinomas.

Transferrin as a growth factor for rat bladder carcinoma cells in culture.

Using the heterotopically transplanted rat urinary bladder system, we previously showed that normal urine has a tumor-enhancing effect on carcinogen-initiated urothelium. In an attempt to isolate a urinary growth-stimulating (tumor-enhancing) factor(s), urine was first fractionated by Bio-Gel P-100 column chromatography, and each fraction was tested for inducibility of ornithine decarboxylase (ODC) and growth-stimulatory activity in a target rat bladder carcinoma cell line, 804G. ODC inducibility was chosen as a marker for tumor-enhancing effect because it is a key characteristic of tumor promoters. There was a single peak demonstrating a strong growth-stimulatory activity as measured by [3H]thymidine incorporation. There were two ODC-inducible peaks, one located at a high molecular weight region and partly overlapped with the growth-stimulatory peak. The other was located at a lower molecular weight region. CM-Sephadex chromatography and subsequent high performance liquid chromatography successfully separated the high molecular weight-ODC activity from the growth-stimulatory activity. The latter component was found to contain transferrin (TF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion with anti-rat TF antibody and was designated as urinary transferrin fraction. The urinary TF fraction and authentic rat TF stimulated growth of several rat bladder carcinoma cells maintained in a serum-free as well as a serum-deficient medium. The response was proportional to the concentration of TF ranging from 0.2 to 5 microgram/ml. Preincubation of the urinary TF fraction or TF with an anti-rat TF significantly reduced their growth-stimulatory effects in 804G cells. The high molecular weight-ODC also stimulated cell growth but to a lesser extent. These results when combined with our previous observations suggest that TF and possibly also ODC-inducible substances may be important urinary components participating in the tumor promotion by urine.

Irreversibility of low-grade superficial rat bladder carcinomas.

The present investigation was conducted to determine: (a) whether the superficial papillary tumors developing in heterotopically transplanted bladders (HTBs) of rats after N-methyl-N-nitrosourea (MNU) initiation and subsequent weekly urine treatment would regress when placed in a urine-free environment; (b) whether tumors would develop in HTBs if MNU initiation is not followed by further manipulation, such as instillation of urine or 2.1% NaCl solution; and (c) whether tumors would develop in HTBs if urine instillation is delayed for as many as 25 weeks after MNU initiation. The results indicate: (a) that low-grade superficial tumors, once developed, do not appear to regress in a urine-free environment; (b) that tumors develop in MNU-initiated bladders even if they receive no further treatment; and (c) that late institution of urine instillation to HTBs still effectively enhances MNU-initiated tumorigenesis. If the current observation is extrapolated to the human situation, our data suggest that low-grade superficial tumors are indeed neoplastic, and spontaneous regression cannot be expected by urinary diversion. It, however, might be effective in controlling progression of at least some of the early neoplastic lesions to overt cancer.

In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure to N-methyl-N-nitrosourea.

The cause of deeply invasive human bladder carcinoma is unknown. Animal studies suggest that a malignant (invasive) conversion is inducible in low-grade noninvasive tumors by further exposure to a chemical carcinogen. To elucidate what molecular mechanism(s) is involved in the conversion, an in vitro system has been established in which conversion from low- to high-grade carcinoma can be induced. A rat bladder carcinoma cell line D44, derived from an N-methyl-N-nitrosourea (MNU)-induced low-grade noninvasive rat bladder carcinoma was used in the present investigation. Cloned D44 cells (D44c) were exposed to MNU, 50 to 400 micrograms/ml, for 1 h at 37 degrees C once a week for up to 6 weeks. After exposure to MNU, cells with altered morphology were cloned. The yield of altered clones was highest after a total dose of 150 to 200 micrograms of MNU used in 1 to 3 doses. Of 21 clones with altered morphology, 4 clones were further treated with MNU at the initial dose once a week for up to 3 weeks and then subcloned. Thirty-three of these subclones were examined for tumorigenicity in athymic nude mice. Twenty-seven formed highly invasive carcinomas, mostly squamous type, whereas the parental D44c cells failed to develop tumors upon inoculation. Pulmonary metastases were observed in 17 of the 27 clones. Plasminogen activator activity was elevated 4- to 9-fold as compared to parent D44c cells. ras p21 mutations at codon 12 were detected in 5 of 30 clones. These results indicate that the in vitro system described here may provide a useful model to study the molecular mechanisms involved in the conversion of noninvasive bladder carcinomas to metastasizing ones.

Interleukin-6 as a paracrine and autocrine growth factor in human prostatic carcinoma cells in vitro.

Interleukin (IL)-6 plays a significant role in genitourinary carcinomas. The present study was conducted to define the role of IL-6 in the growth of prostatic carcinoma and benign prostatic hyperplasia (BPH). An in vitro experiment was carried out using human prostatic carcinoma cell lines (LNCaP, which is androgen sensitive and slow growing, and DU145 and PC3, which are androgen insensitive and fast growing), and primary human epithelial and stromal cells derived from BPH. Cells were treated with recombinant human IL-6 or conditioned medium (CM) derived from the above cultured cells to identify possible paracrine and autocrine pathways. LNCaP was clearly responsive to exogenous IL-6 and to the CM derived from stromal cells, but not to the CM from LNCaP cells (P < 0.001). DU145 and PC3 were slightly stimulated to grow by exogenous IL-6 and the CM derived from both stromal and respective homologous cells (P < 0.01). In contrast, BPH-derived epithelial cells showed little or no response to IL-6. The stimulatory effect of CM on prostatic carcinoma cells was significantly reduced by the addition of anti-IL-6 antibody to the culture medium. Furthermore, the growth of DU145 and PC3 in serum-free medium was also inhibited by anti-IL-6 antibody (P < 0.001). All cell lines tested, except for LNCaP, secreted IL-6 into the culture medium. Results of reverse transcriptase-PCR analysis indicated that IL-6 receptor mRNA was present in all carcinoma cell lines but not in epithelial cells or stromal cells derived from BPH. These results suggest that IL-6 functions as a paracrine growth factor for LNCaP and as an autocrine growth factor for DU145 and PC3, but it has no stimulatory effect on epithelial cells derived from BPH.

Enhancement by urine of urinary bladder carcinogenesis.

The role of urine as a tumor-enhancing agent in urinary bladder carcinogenesis was investigated by using the heterotopically transplanted rat urinary bladder. Bladders removed from rats initiated with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine in drinking water for 4 or 10 weeks were heterotransplanted to syngeneic rats. These heterotopically transplanted bladders receiving repeated instillations of normal rat urine subsequent to transplantation had a higher incidence of carcinoma than did those receiving 0.9% NaCl solution. These results suggest that normal urine may contain tumor promoter(s).

Inhibitory action of alpha-difluoromethylornithine on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat urinary bladder carcinogenesis.

We previously have shown that urine components capable of stimulating ornithine decarboxylase activity of urothelium can enhance rat urinary bladder carcinogenesis, and that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, suppresses carcinogen-initiated rat urinary bladder carcinogenesis. The present investigation was conducted to determine whether DFMO's suppressive effect is stage specific during carcinogenesis and whether the suppressive effect lasts with its continued use. Following initiation with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine in drinking water for 6 wk, male Fischer 344 rats initially weighing 125 to 150 g were randomly divided into two groups, the first receiving 0.2% DFMO in drinking water ad libitum and the second receiving tap water only. Groups of animals were killed at regular intervals until the completion of the experiment at 75 wk. The effect of DFMO was evaluated by monitoring the incidence of tumors, the mean number of tumors per rat, the mean volume of individual tumors, and the mean total tumor volume per rat. The results showed that continuous treatment with DFMO significantly reduced tumor formation until 60 wk (P less than 0.017). The effect was only of borderline significance (0.017 less than P less than 0.035) at 75 wk. Discontinuation of DFMO treatment at 40 wk resulted in the loss of protective effect in all comparisons except for the borderline effect on the tumor number and total tumor volume per rat. DFMO had no significant effect on the incidence or development of preneoplastic early lesions. Mucosal polyamine (spermidine and spermine) levels were reduced and correlated well with the reduction in tumor growth, suggesting that the reduction in tumor growth rate by DFMO may be due to its ability to reduce polyamine levels in urothelium. There were no side effects attributable to DFMO treatment. DFMO may be a useful chemopreventive agent to retard the recurrence of human superficial bladder cancer.

Identification of epidermal growth factor as a component of the rat urinary bladder tumor-enhancing urinary fractions.

Using the heterotopically transplanted rat urinary bladder, we have shown that normal rat urine has a potent tumor-enhancing effect on bladder carcinogenesis. In an attempt to isolate tumor-enhancing factor(s), urine was fractionated by Bio-Gel P-100 column chromatography and each eluate fraction was examined for inducibility of ornithine decarboxylase (ODC) in a target rat bladder carcinoma cell line, 804G. We have identified two ODC-inducible peaks, one located in a high molecular weight region designated as Fraction I (Fr. I) and the second in a low molecular weight region designated as Fraction II (Fr. II). Fr. I consisted of two principal elements, transferrin and a component which induced ODC. The present investigation was conducted to characterize the ODC-inducible activity in Fr. I and II. Chromatographic analysis of Fr. I by Sephacryl S-200 and Fr. II by Bio-Gel P-10 chromatography separated several ODC-inducible peaks. However, the major ODC inducibility was due to a high concentration (460 ng/mg Fr. I residue, approximate Mr 54,000, and 580 ng/mg Fr. II residue, approximate Mr 6,100) of epidermal growth factor (EGF) as determined by radioimmunoassay. Aliquots obtained from these peaks competed with mouse EGF for EGF receptors in A431 cells. Preincubation of Fr. I and II with rabbit anti-rat EGF IgG significantly reduced ODC inducibility. Transforming growth factor alpha activity as determined by radioimmunoassay was also demonstrated in both Fr. I (34 ng/mg) and Fr. II (9 ng/mg). The results of the present study together with our previous data indicate that the majority of the ODC-inducing activity in the tumor-enhancing urinary components Fr. I and Fr. II is due to EGF itself and EGF-related growth factors of high molecular weight and that Fr. I also contains transferrin.

Epidermal growth factor-responsive and -refractory carcinomas initiated with N-methyl-N-nitrosourea in rat urinary bladder.

We tested the role of epidermal growth factor (EGF) in the development of low-grade superficial bladder tumors by using a heterotopically transplanted rat urinary bladder system. Weekly EGF administration (250 ng/0.5 ml of phosphate-buffered 2.1% NaCl solution) for 28 weeks into heterotopically transplanted rat urinary bladders initiated with a low dose of N-methyl-N-nitrosourea resulted in a significant increase in the incidence (17 of 25 versus 6 of 30 rats; P < 0.001) and the mean number of tumors per bladder (1.08 versus 0.20; P < 0.001) as compared with those for a vehicle-only group. Changing to vehicle without EGF for the last 8 weeks resulted in tumors in 8 of 24 rats (P = 0.02 versus the EGF group), comparable to the rate for controls. Switching from vehicle to EGF for the last 8 weeks resulted in tumors in 15 of 24 rats, comparable to the rate in the 28-week EGF group. When tumors were divided into two groups according to size (>4.2 mm3 and

In vitro induction of ornithine decarboxylase in urinary bladder carcinoma cells.

The induction of ornithine decarboxylase by normal rat urine in bladder cancer cell cultures was tested in view of recent observations that urine acts as a tumor promoter. Addition of urine up to 15% in final concentration to culture medium resulted in a 10-fold increase in ornithine decarboxylase activity over the control. The stimulatory factor(s) contained in urine appears heat stable and may be multiple. 12-O-Tetradecanoylphorbol-13-acetate, a potent promoter in mouse skin carcinogenesis, induced a 39-fold increase in ornithine decarboxylase activity, the best response among the various substances tested. This suggests that it may act as a promoter of bladder cancer.

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.

Enhancement of transformation in vitro of a nontumorigenic rat urothelial cell line by interleukin 6.

Chronic inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. We have shown that acute and chronic inflammation induced by intravesical instillations of killed Escherichia coli strikingly enhances N-methyl-N-nitrosourea (MNU)-initiated rat bladder carcinogenesis. To test the hypothesis that cytokines released during inflammation may be involved in the enhancement of bladder carcinogenesis, we conducted an in vitro experiment. Using soft agar growth as an index of transformation, we examined the effect of inflammation-associated cytokines on the enhancement of MNU-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. In the first experiment, after 1-h exposure to MNU (50 micrograms/ml), cells (5 x 10(4)) were grown in soft agar in the presence of interleukin (IL)-1 alpha, IL-6, IL-8, or tumor necrosis factor-alpha (10 to 100 ng/ml). Colonies consisting of more than 20 cells were counted 4 weeks later. Among the cytokines tested, IL-6 (100 ng/ml) significantly increased colony counts over those for the untreated controls (P < 0.001). In the second experiment, the cells treated with MNU similarly as in the first experiment were cultured with or without IL-6 (100 ng/ml) for 1 week before the cells (5 x 10(4)) were grown in soft agar in the presence or absence of IL-6. IL-6 pretreatment increased colony counts irrespective of subsequent IL-6 treatment (P < 0.05). Moreover, IL-6-stimulated anchorage-dependent growth of MNU transformants far exceeded that of the parental MYP3. However, among the transformants, there was no parallel relationship in response to IL-6 between anchorage-dependent and -independent growth. Our results suggest that IL-6 may provide a selective growth advantage to MNU-initiated bladder epithelial cells in vitro and that it may be a factor accounting for the marked enhancement of inflammation-associated rat bladder carcinogenesis.

Transformation in vitro of a nontumorigenic rat urothelial cell line by hydrogen peroxide.

Chronic infection/inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. The present study examined the hypothesis that hydrogen peroxide (H202) and cytokines released during inflammation are involved in the enhancement of bladder carcinogenesis. Using growth in soft agar and tumorigenicity in athymic nude mice as indices of transformation, we examined the effect of H202 and cytokines on the enhancement of N-methyl-N-nitrosourea (MNU)-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. MYP3 cells pretreated with or without MNU were exposed to H202 (0.001 to 0.1 mM) daily for 1 week in monolayer culture and were then tested for growth in soft agar. A marked increase in colony numbers was observed in the cells that were MNU-initiated and exposed to H202 (P < 0.01). Furthermore, H202 exposure alone at 0.01 mM or 0.1 mM caused colony formation in soft agar. The transformants induced by MNU plus H202 or H202 alone formed high-grade transitional cell carcinomas when injected into nude mice. The growth of these transformants was stimulated by several cytokines (interleukin 1alpha, interleukin 6, and tumor necrosis factor-alpha) better than the parental cells both on a plastic surface and in soft agar. Our results indicate that H202 causes genetic change(s) to induce tumorigenic conversion in urothelial cells and that the transformants are stimulated to grow because of their selective response to several cytokines. We suggest that these mechanisms may be involved in the in vivo carcinogenesis associated with chronic urinary tract infection.

Effects of urine and continued exposure to carcinogen on progression of early neoplastic urinary bladder lesions.

Based on reports of regression of superficial bladder tumors after urinary diversion, a study was designed to measure the effects of urine and continued exposure to carcinogen on the incidence of progression of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced early urinary bladder lesions to invasive tumor. After being fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide diet for 14 weeks, one-half of the male Fischer rats had urinary diversion by ureterosigmoidostomy, and the remainder were sham operated. One-half of each of these two groups was continued on the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide diet while the remaining animals were fed regular chow postoperatively. One-half of each of the four groups was sacrificed at 3 months, and the remainder were sacrificed at 6 months after ureterosigmoidostomy or sham-operation. The incidence and mean number of tumors as well as the incidence of invasive tumor were tabulated. The combined 3- and 6-month data indicate that excreted carcinogen in the urine influences progression of the preinvasive lesions more than urine alone or systemic carcinogen alone. However, urine alone had a significant effect (p < 0.025) on tumor incidence (8 of 19 sham-operated animals with tumor versus 1 of 18 diverted animals with tumor). Urine acts as a promoter in this experimental system. These findings may have clinical applications in the treatment of early transitional cell carcinoma.