4-HYDROXYBUTYL(BUTYL)NITROSAMINE (OH-BBN)- INDUCED URINARY BLADDER CANCERS IN MICE: CHARACTERIZATION...

CARCIN-2004-00378.R1

4-HYDROXYBUTYL(BUTYL)NITROSAMINE (OH-BBN)- INDUCED URINARY BLADDER CANCERS IN MICE: CHARACTERIZATION OF FHIT, SURVIVIN AND COX-2 EXPRESSION

AND CHEMOPREVENTIVE EFFECTS OF INDOMETHACIN

Ronald A. Lubet1, Kay Huebner2, Louise Y.Y. Fong2, Dario C. Altieri3, Vernon E. Steele1, Levy Kopelovich1, Claudine Kavanaugh1, M. Margaret Juliana4, Seng-jaw Soong4 and Clinton J. Grubbs4

1Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 3 Cancer Center, University of Massachusetts Medical School, Worcester, MA, and 4 Departments of Surgery, Genetics, and Medicine, University of Alabama at Birmingham, Birmingham, AL. Keywords: Urinary Bladder, Cancer, Indomethacin, NSAIDs, FHIT, Survivin, Chemoprevention Correspondence to: Ronald A. Lubet, Ph.D., National Cancer Institute, Executive Plaza North, Suite 2110, 6130 Executive Blvd., Bethesda, MD 20852. Telephone: (301) 594-0457; FAX: (301) 402-0553; E-mail: [email protected] Running Title: OH-BBN urinary bladder model, efficacy of indomethacin

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ABSTRACT

The administration of OH-BBN to male B6D2F1 mice yielded a high incidence of large palpable urinary bladder cancers that were characterized for expression of certain cancer related genes (FHIT, survivin and COX-2). FHIT was expressed at high levels in normal bladder epithelium, but was minimally expressed in cancers. The antiapoptotic protein survivin was not expressed in normal bladder epithelium, but was variably expressed in bladder cancers. Finally, COX-2 was minimally expressed in both normal bladder and cancer cells. Based on previous positive results, we further explored the chemopreventive effects of the NSAID indomethacin. Continual indomethacin treatment (20 mg/kg diet) was initiated either prior to or following OH-BBN dosing for 12 weeks. Palpable bladder masses developed in 32% of carcinogen-treated only mice by 8 months, while mice administered indomethacin either prior to or after OH-BBN developed palpable masses in 3 and 6% of the animals. In Experiment II, mice were treated with indomethacin beginning 1 week after OH-BBN either for 12 weeks or for 28 weeks. Continual treatment resulted in a 77% decrease in palpable bladder masses and an 82% decrease in all cancers (palpable and microscopic), while limited treatment decreased palpable masses by 48% but failed to decrease the number of bladder cancers (palpable plus microscopic). In Experiment III, OH-BBN treated mice were followed for 14 months. Palpable masses developed in 66% of control mice, while 26% of mice treated with indomethacin continually from one week after OH-BBN developed palpable masses. A separate group treated with indomethacin beginning when 5% of the mice had palpable bladder masses continued to develop new masses for an additional 4 weeks. By 6 weeks after beginning indomethacin treatment, however, these animals showed a profound decrease in the development of additional masses.

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INTRODUCTION There are two primary chemically induced models of urinary bladder cancers in rodents. Both employ repeated intragastric administration of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN) to induce bladder cancers in either mice or rats (1-3). The bladder cancers typically have a mixed histology showing elements of both transitional and squamous cells. The mice tend to develop large palpable bladder masses that routinely cause bleeding and obstructions; thereby necessitating the sacrifice of the animal. Although this model has been characterized histopathologically and is employed in prevention studies (4-6), a more limited characterization of these lesions on a molecular level has been undertaken. Investigators have found a relatively low frequency of Ras mutation in these cancers (7) which is in accord with results in humans. In addition, roughly 50% of these tumors develop P53 mutations (8) which are similar to those found in humans. There has been further characterization of these tumors for various gene products of the EGFr loop (9). Similar to human bladder tumors, these tumors tend to show over expression of EGFr and amphiregulin. Three gene products that routinely display altered expression in bladder cancers and which are likely to be involved in the carcinogenic process itself are FHIT (10), survivin (11) and COX-2 (12). FHIT is a gene product which has routinely been shown to exhibit decreased expression in a variety of tumors including lung, head and neck, esophagus and urinary bladder. The FHIT locus, in fact, was initially defined as a major site of LOH in these tumors as well as a common site of genetic recombination in renal tumors (13). Recent studies in FHIT knockout mice have shown that these animals are susceptible to a wide variety of spontaneous tumors (14,15). Survivin, in contrast to FHIT, is over expressed in a wide variety of cancers including urinary bladder cancer (11,16). This protein is one member of a class of IAP (inhibitors of apoptosis) and appears to block apoptosis by interfering with the caspase activation pathway. Finally, COX-2 has shown increased expression in a wide variety of human cancers including lung, skin, melanoma, and urinary bladder (17-20). On the basis of this increased expression, inhibition of COX-2 was felt to be a potentially important target for chemopreventive studies in the widest range of organs. Interestingly, our previous studies in this model had shown limited expression of COX-2 in tumor epithelium, but showed high levels of staining in the neovasculature associated with the tumors (1). Nevertheless, both NSAIDS and the COX-2 inhibitor celecoxib were highly effective in decreasing bladder cancers when administered beginning at the time that OH-BBN was given (1, 4, 5). In the present experiments, in addition to characterizing the model for the expression of FHIT, survivin and COX-2, we employed the non-specific NSAID indomethacin to determine a number of questions with regards to the efficacy of NSAIDS in this model. Specifically, we examined: 1) how late in the tumorigenic process can one administer an NSAID and still be effective in preventing bladder cancer formation; 2) is indomethacin an effective therapeutic agent in this model; 3) can limited treatment with an NSAID be effective in preventing bladder cancer; 4) does long term treatment with an NSAID affect expression of genes whose levels are altered in tumor progression (FHIT, survivin, COX-2); and 5) does NSAID treatment lose its efficacy when mice are observed for an extended period of time (14 months).


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MATERIALS AND METHODS

Male B6D2F1 (C57Bl/6 x DBA/2 F1) mice were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) at 28 days of age and were housed in polycarbonate cages (5/cage). The animals were kept in a lighted room 12 hours each day and maintained at 22 + 0.5oC. Teklad 4% mash diet (Harlan Teklad, Madison, WI) and tap water were provided ad libitum. Indomethacin was obtained from Sigma Chemical Co. (St. Louis, Mo). At 56 days of age, mice received the first of 12 weekly gavage treatments with OH-BBN (TCI America, Portland, OR). Each 7.5 mg dose was dissolved in 0.1 ml ethanol:water (25:75). The mice were weighed weekly and checked daily. Indomethacin (20 mg/kg diet) was administered beginning at various times. In Experiment I, mice were administered indomethacin beginning either 1 week prior to the first OH-BBN administration or beginning 1 week following the final treatment with OH-BBN (Table I). Mice (unless sacrificed early because of a large palpable bladder mass) were sacrificed 8 months following the first OH-BBN treatment. In Experiment II (Table II), mice were administered indomethacin beginning 1 week following the final administration of OH-BBN, and remained on this diet either for a limited period of time (12 weeks) or for the duration of the study (28 weeks); Groups 1 and 2, respectively. The study was terminated 10 months after the initial OH-BBN administration. In Experiment III (Table III), mice were administered indomethacin beginning either at 1 week (Group 1) or at 20 weeks (Group 2) following the final treatment with OH-BBN. At the 20 week time-point, roughly 5% of OH-BBN treated only mice (Group 3) had a palpable bladder mass. Mice were sacrificed 14 months following the first OH-BBN administration (unless sacrificed early due to a bladder mass). The Kaplan-Meier test was used to analyze survival data. Urinary bladder cancer multiplicity was analyzed by the Wilcoxon rank-sum test. At necropsy, urinary bladders were inflated with formalin, removed, and observed under a high intensity light for gross lesions. After fixation , each lesion was dissected, processed for routine paraffin embedding, cut into 5 micron sections, and mounted for H and E staining. FHIT Immunohistochemistry. Following deparaffinization and rehydration in graded alcohols, sections were heated in citrate buffer (0.01 M, pH 6.0) in a microwave oven (85-90oC, 3 to 5 min.) before nonspecific binding sites were blocked in goat/rabbit serum. Sections were incubated overnight at 37oC in a humidified chamber with rabbit anti-mouse FHIT antiserum (21) at a 1:2000 dilution, then with biotinylated goat anti-rabbit antibody, and finally with strepavidin horseradish peroxidase. The location of FHIT was visualized by incubation with 3, 4-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, St. Louis, MO). Cells with a brown reaction product in the cytoplasm were defined as positive for FHIT. Survivin immunohistochemistry. Tissue sections were cut from formalin-fixed paraffin embedded bladder specimens, put on high adhesive slides and processed for immunohistochemistry with a rabbit polyclonal antibody (pAb) NOVUS raised against full length recombinant survivin. Tissue sections were initially processed for antigen retrieval by pressure cooking in citrate buffer, and binding of the primary antibody was revealed by addition of goat anti-rabbit IgG using 3,3’-diaminobenzidine as the chromogen. In control experiments, the primary antibody to survivin was substituted with a control non-immune rabbit IgG under the same experimental conditions. Cases were scored as positive when >20% of the tumor cell population exhibited specific cytoplasmic staining for survivin. Specificity was further defined by the absence of reactivity of pAb NOVUS with the infiltrating small lymphocyte population and by the inhibition of pAb NOVUS staining following pre-adsorption of the antibody with saturating concentrations of purifyed recombinant survivin. COX-2 immunohistochemistry. Each lesion was dissected, processed for routine paraffin embedding, cut into 4µm sections, and mounted onto polylysine-coated slides. Sections were dewaxed in xylene, rehydrated in descending alcohols, and blocked for endogenous peroxidase (3% H2O2 in methanol) and avidin/biotin (Vector Blocking Kit). The


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sections were permeablized in TNB-BB (0.1 M Tris, pH 7.5) (0.15 M NaCl/0.5% blocking agent/0.3% Triton-X, 0.2% saponin), and incubated with primary antibody at 4oC. The isoform-specific COX-2 polyclonal (PG-27: Oxford Biomedical Research) antisera were diluted to 1:500 in TNB-BB for all tissues. Control sections were incubated with antisera in the presence of 100-fold excess COX-2 protein, or with an isotype-matched IgG normal rabbit serum.


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RESULTS In Experiment I, mice were administered indomethacin (20 mg/kg diet) beginning either one week prior to the carcinogen treatment or one week following the last treatment with OH-BBN (Table I, Figure 1). Mice were sacrificed when they developed a large palpable mass or at 8 months following the initial dose of the carcinogen. In mice treated only with OH-BBN, 22/68 developed palpable urinary bladder masses (which were subsequently diagnosed as cancers) and had to be sacrificed. In contrast, in mice treated early or late with indomethacin, 2/70 and 4/68 mice, respectively, developed palpable masses. The average number of urinary bladder cancers (palpable and microscopic) was 0.53 in the OH-BBN treated-only mice, but only 0.09 and 0.06 in mice treated with indomethacin beginning prior to or after OH-BBN, respectively. This was approximately an 85% decrease of bladder cancers by either treatment regimens of indomethacin. The percentage of mice with non-cancerous lesions (hyperplasias and papillomas) was determined at the end of the experiment in mice without large palpable tumors and was found to be similar in all groups. The number of such lesions could not readily be determined in mice with large palpable cancers since these large lesions made it difficult to score additional lesions. In Experiment II (Table II, Figure 2), mice were administered OH-BBN alone or administered OH-BBN and indomethacin (20 mg/kg diet) beginning one week following the last dose of the carcinogen. Mice received indomethacin either throughout the experiment (28 weeks) or for a limited time (12 weeks). Mice were sacrificed 10 months after the initial dose of the carcinogen. In mice treated with OH-BBN only, 31% developed palpable urinary bladder masses and had to be sacrificed (Figure 2). In contrast, in mice treated continually or for a limited time with indomethacin, 7% and 16% developed palpable bladder masses, respectively. With regard to the total number of cancers (palpable and microscopic), OH-BBN only treated mice had a multiplicity of 0.71, while mice treated continually or for a limited time with indomethacin developed cancer multiplicities of 0.13 and 0.75, respectively. The decrease with continual indomethacin treatment was 82%, which was similar to the decrease observed in Experiment I (Group 2) that used a similar protocol. Thus, demonstrating the reproducibility of this urinary bladder cancer model. Experiment III was performed to determine the long-term effects of treatment with indomethacin on urinary bladder cancer formation (Table III, Figure 3). Mice were treated weekly for 12 weeks with OH-BBN and were observed for a period of 14 months following the first dose of OH-BBN. One group of mice received OH-BBN only while the remaining two groups were administered OH-BBN and indomethacin (20 mg/kg diet) beginning either one week following the last dose of OH-BBN (Week 1) or when approximately 5% of the mice had palpable masses (Week 20 after the last treatment with OH-BBN). In the group that was administered indomethacin beginning at Week 20, urinary bladder masses continued to develop at a similar rate to that of the OH-BBN treated-only mice for approximately four weeks after initiating treatment. After that time, the rate of bladder mass formation was similar in mice administered indomethacin late (Week 20) as it was in mice administered indomethacin early (Week 1). At 14 months, 66% of OH-BBN treated-only mice had developed a palpable bladder mass. In contrast, 26% of mice given indomethacin beginning early (Week 1) and roughly 42% of mice given indomethacin late (Week 20) had developed palpable bladder masses. The histopathology of the cancers that developed in the indomethacin-treated mice was similar to that observed in cancers which developed in the OH-BBN treated-only group. The average number of cancers (palpable and microscopic) in the various groups at the end of the study were: OH-BBN treated-only, 0.83; indomethacin (Week 1), 0.31; and indomethacin (Week 20), 0.51. Urinary bladder cancers were collected from OH-BBN treated-only mice as well as from those mice treated with OH-BBN and indomethacin. The cancers were examined by immunohistochemistry for expression of FHIT, survivin, and COX-2. As can readily be seen in


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Figure 4, FHIT was highly expressed in normal bladder epithelium, but was not stained in the cancers. Interestingly, this gene was also under-expressed in bladder papillomas as well (data not shown). Survivin, an antiapoptotic protein, is not routinely expressed in most normal adult tissues, including normal urinary bladder epithelium. Expression of survivin was observed in all of the bladder cancers induced in this study (Figures 5 A-F). One should note, however, that this expression was quite variable; being relatively high in certain cancers and weakly expressed in others. The expression of COX-2 was also determined in normal bladder epithelium and in cancers. There was minimal COX-2 expression in normal bladder epithelial cells and in bladder cancer cells (data not shown). In addition, altered expression of these genes in cancers obtained from mice treated long term with indomethacin was examined. Obviously, there were fewer tumors in the mice treated with indomethacin since it was a highly effective preventive agent. Nevertheless, substantial differences in gene expression relative to cancers from OH-BBN treated-only mice were not observed. Thus, FHIT expression was low, survivin expression remained higher (but somewhat variable), and finally COX-2 expression was not altered in the cancers.


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DISCUSSION OH-BBN is a potent urinary bladder carcinogen in both mice and rats, and has been employed in a wide variety of studies examining prevention of cancer by various agents. Genetic characterization of these cancers showed that mutations in Ras were uncommon (<10%), while mutations in P53 were frequent (>50%). These results corresponded to observations in human urinary bladder cancers. The primary gene expression change that has been examined in this model has dealt with the EGFr pathways. Marjou, et al (9) found that OH-BBN induced bladder cancers displayed higher levels of EGFr, amphiregulin, etc. than surrounding normal bladder epithelium. In order to further examine the relevance of the OH-BBN induced mouse model of carcinogenesis, we examined for altered expression of three specific genes whose expression is altered in many human cancers, including those of the urinary bladder (10-13). The three specific genes were FHIT, survivin, and COX-2. FHIT is under-expressed in a wide variety of tumors including lung, head and neck, esophagus, kidney, and urinary bladder. The gene was initially examined because it fell within a region of chromosome 3 in humans that undergo LOH in a wide variety of cancers. The exact mechanism whereby FHIT contributes to cancer initiation and progression is not known. However, transgenic mice where FHIT has been knocked out in the germline have an increased incidence of both spontaneous and chemically induced cancers (15,21). FHIT expression is strongly decreased in roughly 60% of bladder tumors in humans as assessed by IHC. This decrease in FHIT expression appears to occur in the later stages of urinary bladder cancer in humans (22); in contrast to its relatively early alteration in expression in lung cancer and cancers of the head and neck (23). In the present study, OH-BBN induced cancers had a decreased expression of FHIT; however, this alteration was observed much earlier during tumor progression with clear loss in small cancers and apparent loss in certain pre-invasive lesions. Decreased expression of FHIT is often associated with methylation of the promoter region in many cancers although in bladder cancers decreased expression of FHIT appears to be significantly more common than methylation of the promoter region (15). Preliminary studies employing cancers derived from this model showed altered methylation in the promoter regions of these bladder tumors (24 ). The second gene examined was survivin which is a member of the family of IAPs. These genes inhibit apoptosis and, thereby, contribute to cancer growth and progression. In fact, survivin appears to inhibit the caspase cascade. Survivin is minimally expressed in normal adult tissues, but is over- expressed in the widest range of human cancers; including breast, lung, esophagus, and urinary bladder. In an unselected examination of genes which were substantially over-expressed in a wide variety of human tumors, as contrasted with normal epithelium, survivin proved to be strongly increased in expression (16). As shown in the present study (Fig. 5), survivin was virtually not expressed in normal urinary bladder epithelium, but was expressed in bladder cancers. The expression of survivin in cancers was somewhat variable; being strongly expressed in certain cancers, but substantially less expressed in others. It was also found that those more limited numbers of cancers which did grow out in the presence of extended indomethacin treatment (breakthrough tumors) demonstrated similar expression of FHIT and survivin to bladder cancers from OH-BBN treated-only mice. This result might be expected since certain of these alterations may be necessary for the tumor process itself. These studies show that two of the genes, FHIT and survivin, which are commonly associated with bladder cancer in humans are also altered in expression in this chemically-induced model. These results give further support to the relevance of the model, and may make the model useful for testing preventive or therapeutic strategies specifically aimed at either of these two genes or their downstream effect or genes. In fact, to parallel studies in humans, we are presently examining the use of survivin levels in urine as a marker of urinary bladder carcinogenesis and to monitor the potential therapeutic effects of preventive interventions.


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Finally, the expression of the COX-2 gene was examined. The present results are a repeat and a confirmation of our previous findings that COX-2 did not appear to be highly expressed in the bladder cancer cells in this model, although it was expressed in the neovasculature (1). This is in contrast to two other animal models of cancer which routinely respond to NSAIDS; the UV induced model of squamous cell cancers of the skin (25) and the azoxymethane induced model of colon carcinogenesis (26). In both of these models, we have previously shown substantial expression of COX-2 in the tumor cells. Interestingly, despite the fact that COX-2 appears to be minimally expressed in bladder cancer cells, prior studies in our labs and others have shown that NSAIDs and the COX-2 inhibitor celecoxib were highly effective chemopreventive agents when administered beginning at the time of OH-BBN treatment of male B6D2F1 mice. At that time, we also found that celecoxib was a highly active chemopreventive agent in the OH-BBN induced female Fischer-344 rat bladder model when administered after OH-BBN administration. The present studies imply that the target is likely to be something else other than COX-2 in the cancer cells. Our initial interpretation was that the target may be the expressed COX-2 in the tumor vasculature. Such an interpretation is compatible with the finding that the NSAIDS and COX-2 inhibitors block angiogenesis (27). Although other potential targets for the NSAIDS and COX-2 inhibitors have been discussed, it must be remembered that a wide variety of structurally diverse NSAIDS (e.g., indomethacin, ibuprofen, piroxicam, ketoprofen as well as the COX-2 inhibitor celecoxib) have all been relatively effective in this specific cancer model. Thus, a target which is preferentially affected by only one or two of these agents is unlikely to explain the results. One potential surprise with regards to the repeated and striking results with indomethacin is that this agent is more effective in blocking COX-1 than COX-2. We have employed indomethacin to examine a number of questions. First, whether treatment starting with the first OH-BBN administration or following the last OH-BBN exhibited similar efficacy. At 8 months after the initial OH-BBN treatment (Experiment I), 32% of mice in the OH-BBN treated-only group developed palpable masses, while only 3 and 6% of mice given indomethacin beginning one week prior to or one week after OH-BBN, respectively, developed palpable masses. Examination of all cancers, both palpable and microscopic, showed that the average number of bladder cancers in the OH-BBN treated-only group was 0.83. Indomethacin treatment started either before or after OH-BBN administration reduced the number of bladder cancers to 0.09 and 0.06, respectively. These results imply that virtually all the effects of indomethacin occur during the progression stage and not during the initiation stage since similar results were obtained with either treatment regimen. In the second experiment (Experiment II), we examined whether limited treatment with indomethacin would be effective as a chemopreventive agent. Mice were treated with OH-BBN for 12 weeks and then placed on indomethacin beginning 1 week after OH-BBN for either the duration of the experiment (28 weeks) or for a limited time (12 weeks) . Continual treatment resulted in a 77% decrease in palpable urinary bladder masses and an 82% decrease in total bladder cancers (palpable and microscopic). In contrast, while limited indomethacin treatment decreased palpable masses approximately 50%, it had no effect on the total numbers of cancers; implying that the effects of indomethacin treatment are rapidly lost when administration is stopped. This result is in agreement with our previous data with piroxicam in rat colon (28) and celecoxib in mouse skin (29), which similarly showed that the effects of NSAIDS are rapidly lost. These would also appear to agree with studies in humans showing regrowth of adenomatous polyps following removal of NSAIDS (30). Finally, we examined the effects of indomethacin in an extended 14 months study (Experiment III) to determine: (1) whether urinary bladder cancers continued to develop when mice were observed for a longer time; i.e., 14 months rather than 8 months, (2) whether indomethacin would still be profoundly effective in preventing cancers when examined at this later time point, and (3) whether very late administration of indomethacin would be effective in


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fairly established bladder cancers. Indomethacin treatment was initiated either one week following the last dose of OH-BBN (Week 1) or when roughly 5% of the mice had palpable masses (Week 20 after the final dose of OH-BBN). We observed that bladder cancers continued to occur in the OH-BBN treated-only group throughout the experiment despite the fact that the carcinogenic stimulus was stopped at 12 weeks (roughly 20% of the way through the experiment). Sixty-six percent of the mice treated with OH-BBN alone developed palpable masses, while 26% of mice administered OH-BBN and then placed on indomethacin beginning at Week 1 following the last OH-BBN developed palpable masses. Thus, although this NSAID was highly effective, it could not block the outgrowth of all urinary bladder cancers over an extended time period; which represented roughly 40-50% of the life time of the animals. We also observed that once palpable cancers were detected, they tended to grow quite rapidly. Delaying indomethacin treatment until Week 20 still reduced the incidence of palpable bladder masses to 42%. In the animals that were treated with indomethacin beginning Week 20 following the final OH-BBN dose, palpable tumors continued to rapidly develop for roughly 4 weeks (Figure 3). However, following this time cancers developed at a similar rate in mice given indomethacin early (Week 1). This implies that although indomethacin is not a strong therapeutic agent (since tumors still grew for at least 4 weeks), it has a strong effect on developing lesions prior to the time that they become palpable and imply that they may be useful in an adjuvant setting. In summary, the present studies reinforce the use of the OH-BBN model of urinary bladder carcinogenesis showing that it exhibits alterations in expression of FHIT and survivin that parallel known changes in human bladder cancer. Furthermore, the data on the NSAID indomethacin presents additional supporting data that: 1) NSAIDS are effective when administered beyond the time of tumor initiation; 2) NSAIDS by themselves do not appear to be therapeutic; and 3) NSAIDS must be administered continually to have a striking chemopreventive effect.


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ACKNOWLEDGEMENTS The authors wish to thank Ms. Mary Jo Cagle and Ms. Jeanne Hale for secretarial and editorial services. The studies were supported in part by NCI contract number N01-CN-05119-MAO and NCI grant number R01 CA96131.


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TABLE I

EFFECT OF INDOMETHACIN ON OH-BBN INDUCED URINARY BLADDER LESIONS IN MALE B6D2F1 MICE – TIME OF ADMINISTRATION

Experiment I

No.of Percent Palpable Av. No. of Group Mice Carcinogena Treatmentb Masses Cancers

1 70 OH-BBN Indomethacin, 20 mg/kg diet, 3% 0.09 one week prior to OH-BBN 2 68 OH-BBN Indomethacin, 20 mg/kg diet, 6% 0.06 one week after OH-BBN 3 68 OH-BBN Diet only 32% 0.53 ___________________________________________________ a OH-BBN was administered to mice 1x/week for 12 weeks beginning when the animals were 56 days of age. b Study was terminated 8 months after the initial administration of the carcinogen

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TABLE II

EFFECT OF LIMITED TREATMENT WITH INDOMETHACIN ON OH-BBN INDUCED URINARY BLADDER LESIONS IN MALE B6D2F1 MICE

Experiment II No.of Percent Palpable Av. No. of Group Mice Carcinogena Treatmentbc Masses Cancers

1 55 OH-BBN Indomethacin, 20 mg/kg diet, 16% 0.75 (for 12 weeks) 2 54 OH-BBN Indomethacin, 20 mg/kg diet, 7% 0.13 ( for 28 weeks) 3 55 OH-BBN Diet only 31% 0.71 ___________________________________________________ a Mice received 12 weekly intragastric doses of OH-BBN beginning at 56 days of age. b Diet supplementation with indomethacin initiated one week after the last OH-BBN treatment. c Study terminated 10 months after the initial OH-BBN treatment.



TABLE III

EFFECT OF INDOMETHACIN ON OH-BBN INDUCED URINARY BLADDER LESIONS WHEN INITIATED ONE WEEK AFTER THE CARCINOGEN OR WHEN 5% OF THE MALE B6D2F1 MICE HAD A PALPABLE BLADDER MASS

Experiment III

No.of Percent Palpable Av. No. of Group Mice Carcinogena Treatmentb Masses Cancers

1 65 OH-BBN Indomethacin, 20 mg/kg diet, 26% 0.31 one week after OH-BBN 2 61 OH-BBN Indomethacin, 20 mg/kg diet, 42% 0.51 when 5% of mice had a palpable mass 3 60 OH-BBN Diet only 66% 0.83 ___________________________________________________ a OH-BBN was administered to mice 1x/week for 12 weeks beginning when the animals were 56 days of age. b Study was terminated 14 months after the initial administration of the carcinogen.



FIGURE LEGENDS

Fig. 1. Effect of time of administration of indomethacin on survival of B6D2F1 mice that received OH-BBN (Experiment I). Study was terminated 8 months after the initial OH-BBN treatment. The groups were: ●, indomethacin (20 mg/kg diet, one week prior to OH-BBN);■, indomethacin (20 mg/kg diet, one week after OH-BBN); ∆ diet only. Fig. 2. Effect of indomethacin treatment for either 12 or 28 weeks beginning one week after OH-BBN on survival of B6D2F1 mice (Experiment II). Study was terminated 10 months after the initial OH-BBN treatment. The groups were: ●, indomethacin (20 mg/kg diet for 12 weeks); ■ indomethacin (20 mg/kg diet for 28 weeks); ∆, diet only. Fig. 3. Effect of indomethacin treatment beginning either one week after OH-BBN or when approximately 5% of mice had palpable urinary bladder masses on survival of B6D2F1 mice (Experiment III). Study was terminated 14 months after the initial OH-BBN treatment. The groups were: ●, indomethacin (20 mg/kg diet one week after OH-BBN); ■, indomethacin (20 mg/kg diet when 5% of mice had palpable masses); ∆, diet only. Fig. 4. Normal mouse urinary bladder (Top). The epithelium consists of three layers of cells (H&E; x 200). Normal bladder tissue showing strong cytoplasmic FHIT staining (DAB, dark brown) in the epithelium (x200). Mouse bladder carcinoma (H&E; x100) (Bottom). Adjacent tissue section shows reduced staining of FHIT in the tumor areas but still strong FHIT expression in the outer epithelial cells (x100). Fig. 5. Immunohistochemical detection of survivin in mouse urinary bladder tumors. Five micron sections of formalin-fixed, paraffin-embedded bladder specimens were stained with non-immune rabbit IgG (A, C, E, G) or an antibody to survivin (B,D,F,H) using DAB as a chromogen. A and B, normal bladder urothelium negative for surviving expression. Differential expression of survivin in the individual bladder tumors involved both cytoplasmic and nuclear staining. Magnification, x100.


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4-HYDROXYBUTYL(BUTYL)NITROSAMINE (OH-BBN)- INDUCED URINARY BLADDER CANCERS IN MICE: CHARACTERIZATION...

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