4-Hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancers in mice: characterization of FHIT and survivin expression and chemopreventive effects of indomethacin

Ronald A. Lubet, Kay Huebner1, Louise Y.Y. Fong1, Dario C. Altieri2, Vernon E. Steele, Levy Kopelovich, Claudine Kavanaugh, M.Margaret Juliana3, Seng-jaw Soong3 and Clinton J. Grubbs3

Division of Cancer Prevention, National Cancer Institute, Executive Plaza North, Suite 2110, 6130 Executive Boulevard, Bethesda, MD 20852, USA, 1 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA, 2 Cancer Center, University of Massachusetts Medical School, Worcester, MA, USA and 3 Departments of Surgery, Genetics and Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

4 To whom correspondence should be addressed. Tel: +1 301 594 0457; Fax: +1 301 402 0553; Email: rl57{at}nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The administration of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN) to male B6D2F1 mice yielded a high incidence of large palpable urinary bladder cancers. Since prior studies demonstrated chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs), we further explored the efficacy of the NSAID indomethacin using different treatment regimens. OH-BBN was administered twice per week for 12 weeks (the first week of treatment was designated week 1). In Experiment I continual indomethacin treatment (20 mg/kg diet) was initiated either prior to (week –1) or following (week 13) OH-BBN dosing. Palpable bladder masses (subsequently diagnosed as cancers) developed in 32% of carcinogen-treated only mice by 32 weeks, while mice administered indomethacin either prior to or after OH-BBN developed palpable masses in 3 and 6% of the animals, respectively. In Experiment II mice were treated with indomethacin beginning 1 week after OH-BBN for either 12 weeks (limited treatment, weeks 13–24) or for 30 weeks (weeks 13–42). 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 61 weeks. Palpable masses developed in 66% of control mice, while 26% of mice treated with indomethacin continually from 1 week after OH-BBN (weeks 13–61) developed palpable masses. A separate group in this study 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 cancers. The expressions of FHIT and survivin in normal urinary bladder epithelium and in bladder cancers were determined by immunohistochemical analysis. FHIT was expressed at high levels in normal epithelium, but was minimally expressed in cancers, and even showed decreased expression in papillomas. The anti-apoptotic protein survivin was not expressed in normal bladder epithelium, but was variably expressed in cancers. FHIT and survivin expressions were similar in cancers from indomethacin-treated and non-treated mice.

Abbreviations: COX-2, cyclooxygenase 2; DAB, 3,4-diaminobenzidine tetrahydrochloride; EGFr, epidermal growth factor receptor; FHIT, fragile histidine triad; H&E, hematoxylin and eosin; IAPs, inhibitors of apoptosis; LOH, loss of heterozygosity; NSAIDs, non-steroidal anti-inflammatory drugs; OH-BBN, 4-hydroxybutyl(butyl)nitrosamine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (13). The bladder cancers typically have a mixed histology showing elements of both transitional and squamous cells. Mice tend to develop large palpable bladder masses that routinely cause bleeding and obstructions; thereby necessitating killing of the animal. Although this model has been characterized histopathologically and is employed in prevention studies (46), a more limited characterization of these lesions on a molecular level has been undertaken. Investigators have found a relatively low frequency of Ras mutations in these cancers (7), which is in accord with results in humans. In addition, ~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 epidermal growth factor receptor (EGFr) loop (9). Similar to human bladder tumors, these tumors tend to show overexpression of EGFr and amphiregulin. More recently we have employed gene array analysis to search for genes whose expressions were substantially altered when comparing mouse bladder tumors with normal mouse bladder epithelium (10). The study confirmed the altered expression of EGFr and, furthermore, identified a substantial number of genes related to oncogenesis with altered expression in tumors, including c-Jun, cyclin B, aurora protein kinase and cyclin-dependent kinase inhibitor 2.

Two gene products that routinely display altered expression in bladder cancers and which are likely to be involved in the carcinogenic process itself are fragile histidine triad (FHIT) (11) and survivin (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 was, in fact, initially defined as a major site of loss of heterozygosity (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 overexpressed in a wide variety of cancers, including urinary bladder cancer (12,16). This protein is one member of a class of inhibitors of apoptosis (IAPs) and appears to block apoptosis by interfering with the caspase activation pathway.

Our previous studies in the OH-BBN model have shown limited expression of cyclooxygenase 2 (COX-2) in cancers, but high levels of staining in the neovasculature associated with the cancers (1). However, both non-steroidal anti-inflammatory drugs (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 and survivin, we employed the non-specific cyclooxygenase inhibitor indomethacin to answer a number of questions with regards to the use of NSAIDs in the prevention of urinary bladder cancer. Specifically, we examined: (i) how late in the tumorigenic process can one administer a NSAID and still be effective in preventing bladder cancer formation; (ii) is indomethacin an effective therapeutic agent; (iii) can limited treatment with a NSAID be effective in preventing bladder cancer; (iv) does NSAID treatment lose its efficacy when mice are observed for an extended period of time; (v) do large tumors that develop in control or indomethacin-treated mice exhibit similar expression of FHIT and survivin?


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Male B6D2F1 (C57Bl/6 x DBA/2 F1) mice were obtained from Harlan Sprague–Dawley (Indianapolis, IN) at 4 weeks of age and were housed in polycarbonate cages (5 per cage). The animals were kept in a room lit for 12 h each day and maintained at 22 ± 0.5°C. 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 8 weeks of age mice received the first of 12 weekly gavage treatments with OH-BBN (TCI America, Portland, OR) in all three studies (the first week of treatment was designated week 1). Each 7.5 mg dose was dissolved in 0.1 ml of ethanol:water (25:75). The mice were weighed weekly and palpated for bladder masses twice per week. 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 (week –1) or beginning 1 week following the final treatment with OH-BBN (week 13) (Table I). Indomethacin treatment continued until the end of the study. Mice (unless killed early because of a large palpable bladder mass) were killed 32 weeks following the first OH-BBN treatment (medium length of time to death for control group was 28 weeks). 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, weeks 13–24, Group 1) or for the duration of the study (30 weeks, weeks 13–42, Group 2). The study was terminated 42 weeks after the initial OH-BBN administration (medium time to death of mice in control group was 35 weeks). In Experiment III (Table III) mice were administered indomethacin beginning either 1 week (weeks 13–61, Group 1) or 20 weeks (weeks 32–61, Group 2) following the final treatment with OH-BBN. In the latter group ~5% of OH-BBN-only treated mice (Group 3) had a palpable bladder mass. Indomethacin treatment continued until termination of the study. Mice were killed 61 weeks following the first OH-BBN administration (medium time length to death for control groups was 37 weeks). The Kaplan–Meier test was used to analyze survival data. Urinary bladder cancer multiplicity (average number of cancers per mouse) was analyzed by the Wilcoxon rank sum test.


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Table I. Effect of time of administration of indomethacin on OH-BBN-induced urinary bladder lesions in male B6D2F1 mice (Experiment I)

 

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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)

 

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Table III. Effect of indomethacin on OH-BBN-induced urinary bladder lesions when initiated 1 week after the carcinogen or when 5% of the male B6D2F1 mice had a palpapable bladder mass (Experiment III)

 
At necropsy urinary bladders were inflated with formalin, removed and, after fixation, observed under a high intensity light for gross lesions. Each lesion was then removed, processed for routine paraffin embedding within 24 h, cut into 5 µm sections and mounted for hematoxylin and eosin (H&E) staining or immunohistochemistry.

FHIT immunohistochemistry
Following deparaffinization and rehydration in graded alcohol dilutions, sections were heated in citrate buffer (0.01 M, pH 6.0) in a microwave oven (85–90°C, 3–5 min) before non-specific binding sites were blocked in goat/rabbit serum. Sections were incubated overnight at 37°C 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 conjugate. 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 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 NOVUS with the infiltrating small lymphocyte population and by the inhibition of NOVUS staining following preabsorption of the antibody with saturating concentrations of purified recombinant survivin.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Experiment I mice were administered indomethacin (20 mg/kg diet) beginning either 1 week prior to the carcinogen treatment or one week following the last treatment with OH-BBN (Table I and Figure 1). Mice were killed when they developed a large palpable mass or at 32 weeks following the initial dose of carcinogen. Of the mice treated only with OH-BBN, 22 of 68 developed palpable urinary bladder masses (which were subsequently diagnosed as cancers) and had to be killed. In contrast, of the mice treated early or late with indomethacin, 2 of 70 and 4 of 68, respectively, developed palpable masses. The average number of urinary bladder cancers per mouse (palpable and microscopic) was 0.53 in the OH-BBN-only treated mice, but only 0.09 and 0.06 in mice treated with indomethacin beginning prior to or after OH-BBN, respectively. This was an ~80% decrease in bladder cancers by either treatment regimen of indomethacin. The percentage of mice with benign 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.



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Fig. 1. Effect of time of administration of indomethacin on survival of B6D2F1 mice that received OH-BBN (Experiment I). The study was terminated 32 weeks after initial OH-BBN treatment. The groups were: •, indomethacin (20 mg/kg diet, 1 week prior to OH-BBN); {blacksquare}, indomethacin (20 mg/kg diet, 1 week after OH-BBN); {triangleup}, diet only.

 
In Experiment II (Table II and Figure 2) mice were administered OH-BBN alone or administered OH-BBN and indomethacin (20 mg/kg diet) beginning 1 week following the last dose of carcinogen (week 13). Mice received indomethacin either for a limited time (weeks 13–24, Group 1) or throughout the experiment (weeks 13–42, Group 2). Mice were killed when they developed a large bladder tumor or at 42 weeks after the initial treatment with OH-BBN. Of the mice treated with OH-BBN only, 31% developed palpable urinary bladder masses and had to be killed (Figure 2). In contrast, of the mice treated continually or for a limited time with indomethacin, 7 and 16%, respectively, developed palpable bladder masses. 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), which used a similar protocol. Thus, the data demonstrated the reproducibility of this urinary bladder cancer model.



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Fig. 2. Effect of indomethacin treatment for either 12 or 30 weeks beginning 1 week after OH-BBN (weeks 13–24 or 13–42, respectively) on survival of B6D2F1 mice (Experiment II). The study was terminated 42 weeks after initial OH-BBN treatment. The groups were: •, indomethacin (20 mg/kg diet for 12 weeks); {blacksquare}, indomethacin (20 mg/kg diet for 30 weeks); {triangleup}, diet only.

 
Experiment III was performed to determine the long-term effects of treatment with indomethacin on urinary bladder cancer formation (Table III and Figure 3). Mice were observed for a period of 61 weeks 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 beginning either 1 week following the last dose of OH-BBN (weeks 13–61) or when ~5% of the mice had palpable masses (weeks 32–61). In the latter group urinary bladder masses continued to develop at a similar rate to that of the OH-BBN-only treated mice for ~4 weeks after initiating treatment. After that time the rate of bladder mass formation was similar to the mice administered indomethacin early (i.e. beginning at week 13). At 61 weeks 66% of OH-BBN-only treated mice had developed a palpable bladder mass. In contrast, 26% of mice given indomethacin beginning early and 42% of mice given indomethacin late 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-only treatment group. The average number of cancers (palpable and microscopic) in the various groups at the end of the study were: OH-BBN-only treatment, 0.83; indomethacin weeks 13–61, 0.31; indomethacin weeks 32–61, 0.51.



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Fig. 3. Effect of indomethacin treatment beginning either 1 week after OH-BBN or when ~5% of mice had palpable urinary bladder masses (weeks 13–61 or 32–61, respectively) on survival of B6D2F1 mice (Experiment III). The study was terminated 61 weeks after initial OH-BBN treatment. The groups were: •, indomethacin (20 mg/kg diet one week after OH-BBN); {blacksquare}, indomethacin (20 mg/kg diet when 5% of mice had palpable masses); {triangleup}, diet only.

 
Urinary bladder cancers were collected from OH-BBN-only treated mice as well as from those mice treated with OH-BBN and indomethacin. The cancers were examined by immunohistochemistry for expression of FHIT and survivin. As can readily be seen in Figure 4, FHIT was highly expressed in normal bladder epithelium, but was not stained in the cancers. Interestingly, this gene was also underexpressed in benign lesions (e.g. papillomas). Survivin, an anti-apoptotic protein, is not routinely expressed in most normal adult tissues, including urinary bladder epithelium. Expression of survivin was observed in all of the bladder cancers induced in this study (Figure 5A–F). One should note, however, that this expression was quite variable, being relatively high in certain cancers but weakly expressed in others. 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-only treated mice were not observed. Thus, FHIT expression was low and survivin expression remained higher (but somewhat variable) in urinary bladder cancers.



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Fig. 4. (A) Normal mouse urinary bladder. The epithelium consists of three layers of cells (H&E, x200). (B) Adjacent tissue section showing strong cytoplasmic FHIT staining (DAB, dark brown) in the epithelium (x200). (C) Mouse bladder carcinoma (H&E, x100). (D) Adjacent tissue section showing reduced staining of FHIT in the tumor areas but still strong FHIT expression in the outer epithelial cells (x100). (E) Bladder carcinoma from mouse treated with indomethacin also showing reduced FHIT staining (x100). (F) Mouse bladder papilloma showing limited staining of FHIT (x100).

 


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Fig. 5. Immunohistochemical detection of survivin in mouse urinary bladder tumors. Sections of formalin-fixed, paraffin-embedded bladder specimens (5 µm) were stained with non-immune rabbit IgG (A, C, E and G) or an antibody to survivin (B, D, F and H) using DAB as a chromogen. (A and B) Normal bladder urothelium negative for survivin expression. Differential expression of survivin in the individual bladder tumors involved both cytoplasmic and nuclear staining. Magnification x100.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (7,8) showed that mutations in Ras were uncommon (<10%), while mutations in P53 were frequent (>50%). These results corresponded with 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. Recently we have employed microarray analysis to compare mouse bladder cancers with normal bladder epithelium to identify gene expression changes associated with the tumorigenic process (10). In order to further examine the relevance of the OH-BBN-induced mouse model of carcinogenesis, we examined two specific genes whose expression is altered in many human cancers, including those of the urinary bladder (1013). The two specific genes products were FHIT and survivin. FHIT is underexpressed in a wide variety of tumors, including lung, head and neck, esophagus, kidney and urinary bladder. This gene was initially examined because it fell within a region of chromosome 3 in humans that undergoes 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 ~60% of bladder tumors in humans, as assessed by immunohistochemistry. 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 decreased expression of FHIT, however, this alteration was observed much earlier during tumor progression, with a clear loss in small cancers and apparent loss in papillomas. Decreased expression of FHIT is often associated with methylation of the promoter region in many cancers (15). Preliminary studies employing cancers derived from this model showed altered methylation in the promoter regions of large 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 overexpressed in a wide range of human cancers, including breast, lung, esophagus and urinary bladder (16). As shown in the present study (Figure 5), survivin was virtually unexpressed 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. The profound role of survivin in bladder carcinogenesis is confirmed by our recent finding that mice overexpressing survivin in the bladder are highly sensitive to OH-BBN-induced bladder cancer (data not shown). Furthermore, the more limited number of cancers which did grow out in the presence of extended indomethacin treatment (breakthrough cancers) demonstrated similar expression of FHIT and survivin to bladder cancers from OH-BBN-only treated mice. This observation was also confirmed when RNA expression of additional tumor-related genes (e.g. c-Jun, cyclin B, aurora protein kinase and cyclin kinase inhibitor 2) was compared (10). The unchanged expression of FHIT and survivin might be expected, since these alterations may be necessary for the tumor process itself. Thus, two of the genes which are commonly associated with bladder cancer in humans, FHIT and survivin, were also shown to be 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 proteins. The data also suggest that breakthrough cancers are unlikely to be the ideal tissue for determining biomarker modulation by effective agents.

Expression of the COX-2 gene was examined and showed minimal expression in cancer cells (data not shown). This result confirmed 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). Despite the fact that COX-2 appears to be minimally expressed in bladder cancer cells, prior studies in our laboratory have shown that the COX-2 inhibitor celecoxib is a highly effective chemopreventive agent when administered beginning at the time of OH-BBN treatment of male B6D2F1 mice (1).

We have employed indomethacin to examine a number of questions. First, whether treatment starting with the first OH-BBN or following the last OH-BBN administration exhibited similar efficacy. At 32 weeks after the initial OH-BBN treatment (Experiment I) 32% of mice in the OH-BBN-only treated group developed palpable masses, but only 3 and 6% of mice given indomethacin beginning 1 week prior to or 1 week after OH-BBN, respectively, developed palpable masses. When all cancers (both palpable and microscopic) were examined, the average number of bladder cancers in the OH-BBN-only treated group was 0.53. 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. It should also be noted that the relative efficacy that we obtained with indomethacin was similar to that which we previously obtained with the COX-2 inhibitor celecoxib (1). Thus, indomethacin (a non-specific COX inhibitor) and celecoxib (which is preferentially a COX-2 inhibitor) appear to be strikingly effective. We obtained similar results (i.e. similar efficacy of indomethacin and celecoxib) in a UV model of skin carcinogenesis in which COX-2 overexpression was clearly associated with tumor progression (25).

In the second experiment (Experiment II) we examined whether limited treatment with indomethacin would be effective as a chemopreventive regimen. 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 (weeks 13–42) or for a limited time (weeks 13–24). Continual treatment resulted in ~80% decreases in both palpable urinary bladder masses or in total bladder cancers (palpable and microscopic). In contrast, while limited indomethacin treatment decreased palpable masses by ~50%, it had no effect on the total numbers of cancers, implying that the effects of indomethacin treatment were rapidly lost when administration was stopped. This agrees with our previous data with piroxicam in rat colon (26) and celecoxib in mouse skin (27), which similarly showed that the effects of NSAIDS were rapidly lost. These data also agree with studies in humans showing regrowth of adenomatous polyps following removal of NSAIDs (28).

Finally, we examined the effects of indomethacin in an extended study (Experiment III) to determine: (i) whether urinary bladder cancers continued to develop when mice were observed for a longer time (i.e. 61 weeks rather than 32 weeks); (ii) whether indomethacin would still be profoundly effective in preventing cancers when examined at this later time point; (iii) whether very late administration of indomethacin would be effective in fairly established bladder cancers. Indomethacin treatment was initiated either 1 week following the last dose of OH-BBN (week 13) or when ~5% of the mice had palpable masses (week 32). We observed that bladder cancers continued to occur in the OH-BBN-only treatment group throughout the experiment despite the fact that the carcinogenic stimulus was stopped ~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 1 week following the last OH-BBN treatment 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 ~40–50% of the lifetime of the animals. Delaying indomethacin treatment for 20 weeks (until 5% of mice had palpable masses) still reduced the final incidence of palpable bladder masses to 42%. In these animals palpable tumors continued to rapidly develop for ~4 weeks (Figure 3). However, following this time cancers developed at a rate similar to the mice given indomethacin early (week 13). 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 implies that NSAIDs 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 in addition to previously demonstrated alterations in the EGFr pathway, it exhibits alterations in expression of FHIT and survivin that parallel known changes in human bladder cancer. Furthermore, the data on the NSAID indomethacin present additional supporting evidence that: (i) NSAIDs are effective when administered beyond the time of tumor initiation; (ii) NSAIDs by themselves do not appear to be therapeutic; (iii) NSAIDs must be administered continually to have a striking chemopreventive effect; (iv) expression of a variety of potential tumor-related genes was similar in tumors derived from control mice and indomethacin-treated mice, arguing that ‘breakthrough’ tumors from a highly effective agent may not be optimal for identifying genes which are modulated by the agents.


    Acknowledgments
 
The authors wish to thank Ms Mary Jo Cagle and Ms Jeanne Hale for secretarial and editorial services and Ms Bonnie Mould, Mr Tom Morgan and Ms Julie Gray for technical assistance. The studies were supported in part by NCI contract no. N01-CN-05119-MAO and NCI grant no. R01 CA96131.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 27, 2004; revised September 21, 2004; accepted November 26, 2004.





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