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[Cancer Research 66, 1929-1939, February 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Pten Deficiency Activates Distinct Downstream Signaling Pathways in a Tissue-Specific Manner

Lina I. Yoo1, David W. Liu1, Sandrine Le Vu1, Roderick T. Bronson2, Hong Wu3 and Junying Yuan1

Departments of 1 Cell Biology and 2 Pathology, Harvard Medical School, Boston, Massachusetts and 3 Department of Molecular and Medical Pharmacology, University of California Los Angeles School of Medicine, Los Angeles, California

Requests for reprints: Junying Yuan, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Phone: 617-432-4170; Fax: 617-432-4177; E-mail: junying_yuan{at}hms.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTEN deficiency predisposes to a subset of human cancers, but the mechanism that underlies such selectivity is unknown. We have generated a mouse line that conditionally deletes Pten in urogenital epithelium. These mice develop carcinomas at high frequency in the prostate but at relatively low frequency in the bladder, despite early and complete penetrance of hyperplasia in both organs. Cell proliferation is initially high in the bladder of newborn Pten-deficient mice but within days is inhibited by p21 induction. In contrast, proliferation remains elevated in Pten-deficient prostate, where p21 is never induced, suggesting that p21 induction is a bladder-specific compensatory mechanism to inhibit proliferation caused by Pten deletion. Furthermore, the AKT/mammalian target of rapamycin growth pathway, which is highly activated in Pten-deficient prostate, is not activated in bladder epithelium. Our results reveal alternative downstream signaling pathways activated by Pten deficiency that lead to tissue-specific susceptibilities to tumorigenesis. (Cancer Res 2006; 66(4): 1929-39)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor gene is one of the most frequently mutated/deleted genes in human cancer (1–4). Germ line PTEN mutations have been found to occur in 80% of patients with classic Cowden syndrome, a heritable multiple hamartoma syndrome characterized by a high risk of breast, thyroid, and endometrial carcinoma and occasional incidence of other types of cancers, including bladder and renal cell carcinoma. The types of cancer arising in Cowden syndrome patients exhibit strong tissue specificity.

Homozygosity for the null mutation (Pten–/–) in mouse models results in early embryonic lethality, indicating that the function of PTEN is critical for normal development (5–7). Pten heterozygous mice (Pten+/–) develop a spectrum of neoplasias in organs, such as the breast, thyroid, endometrium, and prostate, which closely resembles the situation in humans with PTEN mutation (5, 8–10). Somatic PTEN mutations in humans occur with a wide distribution of frequencies in sporadic primary tumors, with the highest frequencies in endometrial carcinomas and glioblastoma (11). PTEN mutation is among the most common genetic alterations reported in human prostate cancers, with loss of heterozygosity (LOH) of PTEN found in 30% of primary prostate cancers (12–14) and 63% of metastatic prostate cancer tissue samples (6). In contrast, the frequency of PTEN mutation in bladder cancer is considerably lower than that of prostate cancer (15, 16). The mechanism of tissue specificity in tumor predisposition as a result of PTEN loss is not clear.

A major substrate of PTEN is phosphatidylinositol 3,4,5-trisphosphate (PIP3), a lipid second messenger produced by phosphatidylinositol 3-kinase (PI3K; ref. 17). PTEN dephosphorylates PIP3 at the D3 position of the inositol ring to produce phosphatidylinositol 4,5-bisphosphate (PIP2). In the absence of PTEN activity, PIP3 concentrations are increased, leading to enhanced phosphorylation and activation of the survival-promoting factor AKT/PKB, whose activity is stimulated by PIP3 (18–21). Interaction of AKT with PIP3 results in an activating conformational change of AKT and its translocation from the cytoplasm to the plasma membrane, where it is further activated through phosphorylation. Activation of AKT has been shown to regulate a vast array of cellular responses, including cell proliferation, adhesion, growth, and survival (17, 22). AKT substrates include FOXO3a, TSC2 (Tuberin), and GSK-3ß, among others (23). AKT has been shown to phosphorylate FOXO3a, thereby preventing the nuclear translocation of FOXO3a. The phosphorylated FOXO3a transcription factor is thus unable to up-regulate proapoptotic target genes, and thus, cell survival is promoted (24). On the other hand, the phosphorylation of TSC2 by AKT has been shown to be important for regulation of cellular metabolism and growth. Specifically, phosphorylation of TSC2 by AKT disrupts complex formation of TSC2 with TSC1 (Hamartin), preventing the complex from inhibiting the small G protein Rheb, which in turn activates the mammalian target of rapamycin (mTOR). Thus, mTOR activates the 70-kDa ribosomal S6 kinase (p70S6K) and inhibits elongation-initiation factor 4E binding protein-1 (4E-BP1), both events that result in increased translation (25). Increased cell growth and cell size are direct consequences of abnormal AKT activation due to Pten deficiency (26).

There have also been reports that AKT can affect cell proliferation and survival by regulating protein levels and subcellular localization of the cyclin-dependent kinase (cdk) inhibitor (CKI) p21. Studies have shown that AKT can increase p21 levels by enhancing protein stability, either through direct phosphorylation of p21 (27) or through inhibition of GSK-3ß, which can phosphorylate p21 and increase its degradation (28). AKT has also been shown to induce cytoplasmic localization of p21 in cells overexpressing HER-2/neu, thereby blocking the cell growth inhibitory activity of nuclear p21 (29). In the nucleus, p21 binds to cyclin E/cdk2 complexes, inhibiting kinase activity, but p21 facilitates cyclin D association with cdk4/6 (30); thus, its effect on cell cycle progression may be dependent on the ratio of the cyclins, cdks, and p21 present in the cell. p21 can inhibit DNA replication by binding to and inhibiting the proliferating cell nuclear antigen (31). p21 also protects cells from apoptosis through multiple mechanisms, including inhibition of caspase-3 activation (7).

In this study, we address the tissue specificity of tumor formation caused by Pten deficiency in conditional Pten mutant mice induced by Fabpl-Cre–mediated deletion. Our results show that Pten deficiency in different tissues elicits drastically different downstream signaling pathways and cellular responses that may be an important basis for the differential propensities for tumor formation following Pten loss in different tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Mice homozygous for floxed Pten exon 5 (Ptenloxp/loxP; ref. 32) were crossed to mice transgenic for Fabpl-Cre (33). F1 Ptenloxp/+;Fabpl-Cre+ mice were crossed with Ptenloxp/loxP mice to obtain Ptenloxp/loxp;Fabpl-Cre+ mice. P21–/– mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and crossed to the Pten conditional knockout mice. Littermate controls lacking the Fabpl-Cre transgene were used in all experiments. When bladder epithelium was needed for protein or RNA analysis, the epithelial layer was peeled away from the muscle in isolated bladders using forceps under a dissecting microscope.

PCR analysis. Mice were genotyped by PCR as described (32, 33) or with the JAX protocol. For detection of exon 5 deletion, organs were harvested and incubated overnight in digestion buffer [100 mmol/L Tris (pH 8), 1 mmol/L EDTA, 0.2% SDS, 200 mmol/L NaCl, 100 µg/mL proteinase K]. DNA was phenol/chloroform extracted and ethanol precipitated. The same primers used to genotype the loxP mice were used to detect Cre-mediated recombination at the Pten locus.

Immunohistochemistry. Tissues were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C, 16 to 24 hours. The next day, tissues were rinsed in PBS and stored in either PBS or 70% ethanol until they were paraffin embedded, sectioned, and mounted on slides. Some slides were stained with H&E. For immunohistochemistry, slides were deparaffinized, immersed in 0.5% hydrogen peroxide in PBS, boiled in 10 mmol/L citrate buffer (pH 6) in a microwave oven for 10 minutes, and blocked in 3% donkey serum/0.2% Triton X-100/PBS for 1 hour. Primary antibodies were diluted in block as follows: E-cadherin (10 µg/mL; Zymed, South San Francisco, CA), phospho-AKT Ser473 (1:50; Cell Signaling Technology, Beverly, MA), p21 (1:25; BD Biosciences, San Jose, CA), Ki-67 (1:50; DAKO, Carpinteria, CA), phospho-histone 3 Ser10 (1:1,000; Upstate, Charlottesville, VA), pan-uroplakin (1:1,000; gift from Tung-Tien Sun), ß-catenin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), p63 (1:100; gift from Frank McKeon). Most slides were washed and incubated with biotinylated secondary antibodies, and signals were amplified with an avidin-biotin complex kit and detected with a 3,3'-diaminobenzidine kit (Vector Labs, Burlingame, CA). Slides were counterstained with hematoxylin, dehydrated, and mounted. Slides incubated with ß-catenin antibody were stained with Cy3-conjugated secondary antibodies (Jackson Immunochemicals, West Grove, PA) and counterstained with Hoechst dye.

Immunoblots. Tissues were flash frozen in liquid nitrogen and stored at –80°C until processing. To prepare protein lysates, tissues were dounced in cold lysis buffer [20 mmol/L HEPES (pH 7.2), 5 mmol/L EGTA, 2 mmol/L EDTA, 5 mmol/L NaF, 40 mmol/L ß-glycerophosphate (pH 7.2), 1 mmol/L sodium orthovanadate, 1x protease inhibitor cocktail (Roche, Indianapolis, IN), 0.5% NP40] and spun at 13,000 rpm for 10 minutes. The supernatant was collected, and an equal volume of 2x SDS-containing electrophoresis sample buffer was added. Samples were run on SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and blocked in 3% milk/TBST. Primary antibodies were diluted in either 3% milk/TBST [phospho-FKHRL1 (FOXO3a) Thr32, 1:1,000 (Upstate); glyceraldehyde-3-phosphate dehydrogenase, 1:1000 (US Biological, Swampscott, MA); p27, 1:1,000 (Santa Cruz Biotechnology); Akt, 1:1,000] or 5% bovine serum albumin/TBST [p21, 1:500 (BD Biosciences); the rest of the antibodies were from Cell Signaling Technologies and diluted 1:1,000: 4E-BP1, phospho-mTOR Ser2448, phospho-S6 Ser240/244, phospho-AKT Ser473, p70S6K, phospho-p70S6K Thr389] and added to the blots for 1 hour at room temperature. Secondary antibodies were horseradish peroxidase–conjugated goat antibodies (Southern Biotechnology Associates, Birmingham, AL). Signals were detected with enhanced chemiluminescence (Amersham Biosciences, Piscataway, CA) on scientific imaging film (Kodak, Rochester, NY).

Cell size measurement. Sections that were double-stained for ß-catenin/Hoechst were photographed at the Nikon Imaging Facility (HMS), and pictures were taken at 20x using Metamorph Orca 100 software. Average cell size was defined by dividing bladder epithelial cell area of a given region on a section by the total number of nuclei in the area.

RNA preparation and reverse transcription-PCR. Tissues were processed as directed in the RNeasy Midiprep protocol (Qiagen, Valencia, CA). RNA was quantitated on a spectrophotometer. RNA (6 µg) was put in a reverse transcriptase reaction with 0.4 mmol/L deoxynucleotide triphosphates (dNTP), Rnasin, and Moloney murine leukemia virus-RT (Promega, Madison, WI) in reaction buffer. Reactions were incubated 1 hour at 37°C, then 1 minute at 95°C to inactivate enzyme. PCR reactions with the cDNA samples were done with 0.2 mmol/L dNTPs and with Ex Taq (Takara, Shiga, Japan). Primer concentrations were 0.5 µmol/L each for the p21 primers and 1 µmol/L each for the ß-actin primers. Primer sequences were p21Fwd (5'-CGGTGGAACTTTGACTTCGT-3'), p21Rev (5'-TCTGCGCTTGGAGTGATAGA-3'), ß-actinFwd (5'-TCATGAAGTGTGACGTTGACATCCGT-3'), and ß-actinRev (5'-CCTAGAAGCATTTGCGGTGCACGATG-3'). Cycling conditions for the p21 PCR were 94°C for 3 minutes, then 25 cycles of 94°C for 1 minute, 55°C for 1 minute, then 72°C for 1 minute followed by a final 10 minutes at 72°C. Cycling conditions for the ß-actin PCR were 94°C for 3 minutes, then 20 cycles of 94°C for 1 minute, 60°C for 1 minute, then 72°C for 1 minute followed by a final 10 minutes at 72°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of tissue-specific Pten-deficient mice by Fabpl-Cre. Mice that were homozygous for a floxed exon 5 encoding the catalytic domain of the Pten gene (32) were bred with mice carrying the Fabpl-Cre transgene (generously provided by Jeffrey Gordon). It has previously been shown that in this line of Fabpl-Cre transgenic mice, Cre is expressed in intestinal epithelium, and in urothelium of the bladder, kidney, and ureter (33). To determine the efficiency of the Fabpl-Cre–mediated Pten deletion in different tissues, we harvested various tissues from Ptenloxp/loxp; Fabpl-Cre+ mice and prepared DNA to test for the presence of Cre-mediated recombination at the Pten locus by PCR (Fig. 1A). The results showed that Cre-mediated deletion of Pten exon 5 was not limited to the organs previously described (33) but in fact occurred in a wide variety of urogenital organs. Recombination could be detected in the colon, kidney, ureter, bladder, prostate, seminal vesicle, and vagina at 14 days of age (Fig. 1A) and also in the penis (data not shown) when examined at later ages. In contrast, we found that there was no detectable recombination in other organs, such as the heart, spleen, liver, brain, lung, and uterus. Pten is therefore specifically deleted in a subset of the lower abdominal reproductive and excretory organs of Ptenloxp/loxp; Fabpl-Cre+ mice.


Figure 1
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  		Figure 1. Tissue-specific deletion of Pten and tumor development in Ptenloxp/loxp;Fabpl-Cre mice. A, DNA was harvested from various organs from 14-day-old Ptenloxp/loxp;Fabpl-Cre+ mice. Samples were assayed by PCR using primers in the Pten locus to detect Cre-mediated recombined alleles (0.35-kb band) and nonrecombined alleles (1.1-kb band). B, protein lysate from bladder epithelium from 8- and 28-day-old Ptenloxp/loxp;Fabpl-Cre+ and Ptenloxp/loxp mice was run on SDS-PAGE and blotted with an antibody for PTEN. C, survival of male and female Ptenloxp/loxp;Fabpl-Cre+ and Ptenloxp/loxp mice was monitored over 13.5 months. A significant difference between survival curves of male Ptenloxp/loxp;Fabpl-Cre+ mice and male Ptenloxp/loxp mice was found (P = 0.0002) by log-rank test. There was also a significant difference between male Ptenloxp/loxp;Fabpl-Cre+ mice and female Ptenloxp/loxp;Fabpl-Cre+ mice (P = 0.0018). There was no significant difference between female Ptenloxp/loxp;Fabpl-Cre+ mice and Ptenloxp/loxp mice. D, tumor types and frequencies were identified in Ptenloxp/loxp;Fabpl-Cre+ mice over a 13.5-month study. The number of mice examined for each organ (n) is indicated. Frequency of anterior prostate tumor-bearing mice was calculated as a percentage of male mice examined, and the frequency of vaginal tumor-bearing mice was a percentage of examined female mice only.

 
To verify that PTEN protein levels were diminished in the bladder and other tissues that underwent Cre-mediated deletion of Pten, we did immunoblots against PTEN using tissue protein lysates. We found a marked decrease in PTEN in bladder epithelium from 8- and 28-day-old Ptenloxp/loxp; Fabpl-Cre+ mice (Fig. 1B).

Fabpl-Cre–mediated Pten deletion leads to tumor development in urothelial cells. It was soon apparent that the Ptenloxp/loxp; Fabpl-Cre+ mice died at an accelerated rate compared with their Ptenloxp/loxp littermates and were specifically developing benign and malignant neoplasias in the same organs where Pten was deleted. We monitored the survival of the mice over a period of 13.5 months and found significantly increased mortality in male Ptenloxp/loxp; Fabpl-Cre+ mice compared with that of male Ptenloxp/loxp mice (Fig. 1C). The survival of Ptenloxp/loxp; Fabpl-Cre+ mice was sex influenced: 75% of female Ptenloxp/loxp; Fabpl-Cre+ mice survived by 13.5 months of age, but only 45% of the male Ptenloxp/loxp; Fabpl-Cre+ mice were still alive by the same age. With respect to the male Ptenloxp/loxp; Fabpl-Cre+ mice, one third of the deaths was attributed to inability to urinate due to urinary tract blockage, with accompanying hydronephrosis of the kidney. Such mice seemed largely asymptomatic until ~1 day before the sudden onset of death due to failure to urinate. Some of these mice had urethral cancers, but excessive hyperplasia in the urinary tract or abnormal prostate growth may also have been responsible for blocking urine flow in other cases. Two thirds of the deaths of the male Ptenloxp/loxp; Fabpl-Cre+ mice were due to other types of tumor growth, as described below. The female Ptenloxp/loxp; Fabpl-Cre+ mice also developed tumors in all the organs they possessed which underwent Cre-mediated Pten deletion, but they never lost the ability to urinate. Because some of the male mice that died due to urine blockage may have developed tumors that would have killed them later in life, it is not feasible to compare death rates due to tumors alone in males compared with females.

A spectrum of tumors as well as hyperplasia were detected in the Ptenloxp/loxp; Fabpl-Cre+ mice in organs that underwent Cre-mediated deletion of Pten. These tumors included urothelial carcinoma of the bladder, ureter, and kidney; squamous cell carcinoma of the vagina and rectum; adenocarcinoma of the colon; and carcinomas of the prostate, seminal vesicles, and urethra (Fig. 1D and Fig. 2). Within the first year of life, we frequently found tumors of the anterior prostate, with 97% of the males developing this type of tumor (Fig. 1D and Fig. 2B). In male Ptenloxp/loxp; Fabpl-Cre+ mice a year of age or older, the tumor had often taken over the entire prostate and seminal vesicle in a bilateral fashion. This is in contrast to Pten+/– mice, where only mild neoplasia in the coagulating gland was detected by 1 year of age. This illustrated a dose-dependent effect of Pten deletion on tumorigenesis in the coagulating gland. Urethral tumors were also quite common, occurring in 48% of the Ptenloxp/loxp;Fabpl-Cre+ mice by 13.5 months of age. Additionally, 41% of the Ptenloxp/loxp;Fabpl-Cre+ females developed vaginal squamous cell carcinoma (Fig. 2D). Carcinomas of the kidney, bladder, ureter, and colon (Fig. 2F) were less common but were also well represented (Fig. 1D).


Figure 2
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  		Figure 2. Tumors in the Ptenloxp/loxp;Fabpl-Cre+ mice. A and B, H&E sections of anterior prostate from 4.5-month-old male mice. C and D, vaginal tissue from female mice. E and F, sections of H&E-stained colons from 13.5-month-old females. A, C, and E, normal tissue from Ptenloxp/loxp mice. B, D, and F, prostatic carcinoma, vaginal squamous cell carcinoma, and colon adenocarcinoma, respectively, from Ptenloxp/loxp;Fabpl-Cre+ mice. Magnification, x200 (A-D) and x100 (E-F).

 
We examined young Ptenloxp/loxp; Fabpl-Cre+ mice for abnormal cell growth and evidence of early tumor development. We found that epithelial hypertrophy and nuclear atypia were evident in bladders of Ptenloxp/loxp; Fabpl-Cre+ mice at birth (Fig. 3A and B), and both hypertrophy and hyperplasia were present with 100% penetrance by 6 weeks of age in bladders of Ptenloxp/loxp;Fabpl-Cre+ mice (Fig. 3C). We also observed characteristic vacuoles containing dead cells and enlarged and abnormally shaped nuclei in the hyperplastic bladder epithelium. Measurements of cell size on bladder sections of control Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice stained for ß-catenin expression and counterstained with Hoechst dye revealed a ~50% increase in cell size in 8-day-old Ptenloxp/loxp; Fabpl-Cre+ mice compared with that of their Ptenloxp/loxp littermates (Fig. 4A).


Figure 3
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  		Figure 3. Urothelial hyperplasia and carcinoma in Ptenloxp/loxp;Fabpl-Cre+ mice. A and B, bladders from Ptenloxp/loxp (A) and Ptenloxp/loxp;Fabpl-Cre+ (B) mice less than 24 hours old. C, hyperplastic bladder from 6-week-old Ptenloxp/loxp;Fabpl-Cre+ mouse. D, high-grade invasive bladder urothelial carcinoma (low magnification). E and F, developing prostate from 8-day-old Ptenloxp/loxp (E) and Ptenloxp/loxp;Fabpl-Cre+ (F) mice. Magnification: x400 (A-C and E-F) and x100 (D).

 

Figure 4
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  		Figure 4. Cell size and proliferation in Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice. A, sections of bladder epithelium from 8-day-old Ptenloxp/loxp (p47 and p50) and Ptenloxp/loxp;Fabpl-Cre+ (p51) mice were stained for ß-catenin/Hoechst with Cy3-tagged secondary antibodies and photographed. Images were quantitated for cell area divided by number of nuclei in that area. More than 180 nuclei were counted for each genotype from multiple fields. B, bladder sections from Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice at various ages were immunostained for Ki-67. The frequency of Ki-67+ cells in the urothelium was counted. Over 700 cells from one to four mice were counted for each genotype at each time point. A statistical difference between age-matched Cre+ and Cre– mice was only found in 3-day old, 4-day-old, and 30-day-old mice (P < 0.0005) by {chi}2 test. C, prostate sections from Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice at 4, 10, and 16 weeks of age were immunostained for Ki-67 and counted. More than 900 cells from one mouse were counted for each genotype at each time point. A statistical difference between Cre+ and Cre– mice was found at each time point (P = 0.0018 for 4-week-old mice and P < 0.0001 for 10- and 16-week-old mice) by {chi}2 test.

 
Over time, a fraction of the hyperplastic and hypertrophic bladders in Ptenloxp/loxp; Fabpl-Cre+ mice progressed to high-grade urothelial carcinoma (Fig. 3D). Bladder cancer was positively identified in 22% of the Ptenloxp/loxp; Fabpl-Cre+ mice examined by 13.5 months of age. Although the frequency of bladder tumor formation is low in Ptenloxp/loxp; Fabpl-Cre+ mice, the Pten-deficient bladder carcinomas, once they are formed, progress to late-stage cancer, including invasion and metastasis. Examination of histopathology slides of bladder carcinomas revealed that the tumors in some cases invaded through the muscle wall (Fig. 3D). Furthermore, using an antibody specific for pan-uroplakin, a marker for differentiated transitional epithelium, we were able to show by immunohistochemical staining the lung metastasis of urothelial carcinomas in multiple animals, indicating the aggressiveness of the tumors that developed.

Given the full penetrance of the hyperplasia in the Pten-deficient bladder epithelium from a very early age, it is striking that the frequency of bladder cancer development is so low compared with that observed in other tissues. For example, in the anterior prostate, hyperplasia was consistently observed in the Ptenloxp/loxp;Fabpl-Cre+ mice at 8 days of age (Fig. 3E and F) and always progressed to carcinoma (Fig. 1D). Because the frequency of PTEN LOH in human bladder cancer is also considerably lower than some of the other tissues, such as prostate (15, 16, 34, 35), we investigated the mechanism that decreases the efficiency of tumor formation in the bladder after Pten loss. We focused on early events in the hyperplastic bladder because the urothelial carcinomas we observed were highly aggressive, suggesting that the inefficiency of bladder epithelial transformation as a result of Pten loss is not due to a block in the final steps of tumor formation.

Mechanism of hyperplasia in Ptenloxp/loxp; Fabpl-Cre+ mice. The hyperplasia evident in the Pten-deleted tissues prompted us to investigate whether we could detect increased cell proliferation in those sites. We stained bladder sections from 3-day-old to 16-week-old Ptenloxp/loxp;Fabpl-Cre+ and Ptenloxp/loxp mice for Ki-67, a marker of cycling cells (Fig. 4B). Although there was a large increase in frequency of Ki-67+ cells in Pten-deficient mice at 3 days of age, there was a decrease in Ki-67+ cells in bladders from the Ptenloxp/loxp;Fabpl-Cre+ mice 1 week of age and older. This was surprising, given the epithelial hypercellularity in the bladders of 4-week-old Ptenloxp/loxp;Fabpl-Cre+ mice and the previously reported increase in cell proliferation in the skin, mammary gland, and brain of Pten-deficient mice (26, 36, 37).

As a comparison, we determined the labeling ratio of Ki-67+ cells in the prostate of Ptenloxp/loxp; Fabpl-Cre+ mice, which also exhibited epithelial hyperplasia (Fig. 4C). Interestingly, we detected a clear increase in the Ki-67+ labeling ratio in the prostate of 4- to 16-week-old Ptenloxp/loxp; Fabpl-Cre+ mice when compared with that of Ptenloxp/loxp mice. This result is consistent with other Ki-67 studies in the prostates of Pten-deficient or heterozygous mice (5, 10, 38, 39). The comparison of Pten-deficient bladder and prostate suggests a possible tissue-specific divergence in the signaling pathways downstream of PTEN and could address the low tumorigenesis rates in the bladder.

We also examined the possibility that a reduction in epithelial cell apoptosis contributed to the hyperplasia we observed in the bladder. We did immunohistochemical staining for terminal deoxynucleotidyl transferase–mediated nick-end labeling–positive cells and activated caspase-3–positive cells in bladder epithelium. By both methods, the rate of apoptotic death in the urothelium was too low to be measured (data not shown). This strongly suggested that the increase in cell number in the Ptenloxp/loxp; Fabpl-Cre+ bladders was attributable to an increase in proliferation during the animals' first week of life (Fig. 4B).

Differential activation of PTEN downstream targets. To determine the molecular mechanisms that lead to hyperplasia and hypertrophy in Pten-deficient bladder epithelium, we examined the phosphorylation levels of known downstream effectors of the AKT signaling pathway, such as FOXO3a, mTOR, p70S6K, and S6. We isolated bladder urothelium by cleanly dissecting away the muscularis propria and made protein lysate for immunoblotting. Surprisingly, we found minimal increase in phosphorylation levels of AKT (Fig. 5A), and no difference in the phosphorylation levels of FOXO3a, mTOR, p70S6K, or S6, the predicted downstream targets of AKT, in the bladder samples from Ptenloxp/loxp; Fabpl-Cre+ mice ranging from 5-day-old to 3-month-old mice when compared with that of age-matched Ptenloxp/loxp mice (Fig. 5A; data not shown). It was particularly noteworthy that there was no difference in S6 phosphorylation in bladders from 2-day-old mice (Fig. 5A), an age when there was a marked increase in proliferation and cell size in the Pten-deficient bladders. This was an unexpected result because we had established that there was a significant increase in epithelial cell size in the Ptenloxp/loxp; Fabpl-Cre+ bladders (Fig. 4A). These results suggest that the hypertrophy of bladder epithelial cells induced by the Pten deficiency is unlikely to be mediated through the activation of the mTOR/p70S6K pathway, a known mechanism by which the activation of AKT leads to increases in cell size (40–42).


Figure 5
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  		Figure 5. Immunoblots for Akt/p70S6K signaling pathway activation in Pten-deficient epithelium. A, protein lysates were made from bladder epithelium dissected away from the muscle, from Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice of various ages. Blots were probed with various antibodies as indicated. B, prostate lysates from 35-day-old and 6-month-old animals were probed with similar antibodies. C, lysates from bladder and prostate were compared for Akt expression.

 
Like the bladders in Ptenloxp/loxp;Fabpl-Cre+ mice, the prostates consistently became hyperplastic from a very early age. Immunoblots of prostate samples revealed that there was a clear up-regulation in the phosphorylation of AKT, FOXO3a, mTOR, p70S6K, and S6 in Ptenloxp/loxp;Fabpl-Cre+ mice compared with that of Ptenloxp/loxp mice (Fig. 5B). Furthermore, this constitutive activation of the AKT pathway was maintained over a period of months. This is in stark contrast to the immunoblot data from the bladder samples, where no difference was detectable. The lack of Akt activation in the bladder was not due to low levels of Akt expression, as we have compared endogenous levels of total Akt in bladder and prostate and found comparable but slightly higher levels of Akt in the bladder compared with the prostate (Fig. 5C).

Normal urothelial differentiation marker expression. Because the mTOR pathway was not activated in Pten-deficient hypertrophic bladders, we asked whether the increased cell size in the Pten-deficient urothelium is due to a higher representation of terminally differentiated cells in the bladder. Tissue sections from Ptenloxp/loxp and Ptenloxp/loxp; Fabpl-Cre+ mice were stained with antibodies against pan-uroplakin and p63 to examine differentiation in the urothelium. We found staining of pan-uroplakin in the Pten-deficient bladders to be present in intermediate and terminally differentiated cells, just as in the normal bladders (Supplementary Fig. S1A-B). In addition, p63 was present in the nuclei of intermediate and basal urothelium of both the Pten -deficient and wild-type bladders, with superficial epithelial cells staining negative for p63 in both cases (Supplementary Fig. S1C-D). A count of p63-positive nuclei in Pten-deficient (90.4%) and wild-type bladders (90.3%) from 14-day-old mice revealed that the frequency of p63-positive cells was virtually identical. An immunoblot for pan-uroplakin in bladder epithelial lysates from Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice showed no difference in protein levels of this differentiation marker (Supplementary Fig. S1E). Therefore, a normal frequency of urothelial cells seems to develop a terminally differentiated phenotype in the Pten-deficient bladders. This suggested to us that the increase in cell size in the Pten-deficient bladders may not be due to a defect in differentiation.

Activation of p21 in the bladder epithelial cells of Ptenloxp/loxp;Fabpl-Cre+ mice. AKT activation has been found to induce the up-regulation of p21 expression in 293 and MCF7 cells (27) and to down-regulate expression of p27 in embryonic stem cells (43). To determine if the bladder epithelium of Ptenloxp/loxp;Fabpl-Cre+ mice has altered levels of CKIs, we compared p21 and p27 expression in Ptenloxp/loxp;Fabpl-Cre+ and Ptenloxp/loxp mice by immunoblots. We detected reduced levels of p27 (Fig. 6A) in the bladders of Ptenloxp/loxp;Fabpl-Cre+ mice compared with Ptenloxp/loxp mice even at 2 days of age, suggesting that lower p27 levels may contribute to increased cell proliferation in newborn mouse bladders. More strikingly, we detected increased levels of p21 in Pten-deficient bladders (Fig. 6B). This induction in p21 first occurred around 5 days of age and was maintained over at least the first 3 months of life. This was an intriguing result because the increase in p21 protein could explain the unexpected decrease in Ki-67+ cycling cells in the bladders of 1-week-old Ptenloxp/loxp;Fabpl-Cre+ mice, because an elevated level of p21 is known to inhibit cell proliferation (44–46). The timing of p21 induction exactly corresponded to the decrease in proliferation rates observed by Ki-67 staining in the bladder.


Figure 6
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  		Figure 6. Altered p21 expression in bladders from Ptenloxp/loxp;Fabpl-Cre+ mice inhibits cell proliferation. A and B, bladder urothelium from Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice was blotted for p27 (A) and p21 (B) expression. C, quantitation of frequency of p21-positive cells in bladder epithelium in mice of different ages. More than 1,000 cells were counted from each genotype at each time point from one to two mice. Frequency of p21-positive cells in age-matched Cre+ versus Cre– mice was significantly different (P < 0.0001, {chi}2) at all ages examined. D and E, bladder sections were stained for p21 expression in 4-week-old female Ptenloxp/loxp (D) and Ptenloxp/loxp;Fabpl-Cre+ (E) mice. Note nuclear expression of p21 in superficial bladder epithelium. Few epithelial cells in the superficial layer of normal bladder are positive for p21, whereas Pten-deficient superficial epithelial cells are all positive for p21. F, mice which were p21+/– and p21–/– were bred to the Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mice. Bladders from mice of various resulting genotypes as shown were harvested, sectioned, and stained for phospho-Histone 3 Ser10. Frequency of the positive nuclei was counted. More than 900 cells were counted from one to three mice for the H3-stained sections. Data from some pairs of genotypes were tested for statistical significant difference by {chi}2 test. **, P < 0.0001; *, P < 0.01. G, lysates from 28-day-old Ptenloxp/loxp and Ptenloxp/loxp;Fabpl-Cre+ mouse bladders and a 12-month old Ptenloxp/loxp;Fabpl-Cre+ mouse bladder carcinoma were immunoblotted for antibodies as indicated. H, lysates from a 12-month-old Ptenloxp/loxp mouse anterior prostate and three Ptenloxp/loxp;Fabpl-Cre+ mouse anterior prostate carcinomas were immunoblotted for antibodies as indicated.

 
To examine if the levels of p21 transcripts were altered in the bladders of Ptenloxp/loxp; Fabpl-Cre+ mice, we analyzed bladder RNA samples from 3-month-old mice by reverse transcription-PCR (RT-PCR) for p21 transcript level. We detected higher levels of p21 transcripts in the RNA from the bladders of Ptenloxp/loxp; Fabpl-Cre+ mice than that of Ptenloxp/loxp mice (Supplementary Fig. S2A), which suggested that an alteration in the p21 expression as a result of Pten deficiency in bladder epithelium was at least in part due to either an increase of p21 transcription or a stabilization of p21 transcripts. AKT activation may also result in stabilization of p21 protein, as suggested by other studies (27, 28); but that cannot be the only mechanism in this case, because the RT-PCR result showed an increase in p21 transcripts.

In contrast to Ptenloxp/loxp; Fabpl-Cre+ bladders, Ptenloxp/loxp;Fabpl-Cre+ prostates showed no change in p27 or p21 levels (Supplementary Fig. S2B) while showing a clear up-regulation in phosphorylation of FOXO3a, mTOR, and S6 (Fig. 5B). These results showed a stringent tissue specificity in the activation of AKT downstream targets as a result of Pten deficiency and provides an important possible explanation for the different propensities for tumor formation in different tissues.

We did immunohistochemistry to examine the subcellular localization of p21 in bladder cells. There was a significantly increased frequency of p21-positive cells in the hyperplastic bladder epithelium of Ptenloxp/loxp;Fabpl-Cre+ mice (Fig. 6C-E). In Ptenloxp/loxp bladder epithelium, p21 expression was restricted to a fraction of the large, terminally differentiated cells localized in the superficial layers. In contrast, in Ptenloxp/loxp;Fabpl-Cre+ mice the p21-positive cell population is greatly expanded in both the superficial and intermediate layers of urothelium. Interestingly, the p21 expression in both Ptenloxp/loxp mice and Ptenloxp/loxp;Fabpl-Cre+ mice was limited to the nucleus.

Increased cell proliferation in p21–/–; Ptenloxp/loxp; Fabpl-Cre bladder urothelium. We were interested whether p21 induction plays a role in limiting cell proliferation in the Pten-deficient bladders at 1 week of age. To address this question, we bred p21–/– mice to the Ptenloxp/loxp; Fabpl-Cre+ mice and generated mice with four genotypes: (a) p21+/+;Ptenloxp/loxp, (b) p21–/–; Ptenloxp/loxp, (c) p21+/+; Ptenloxp/loxp; Fabpl-Cre+, (d) p21–/–;Ptenloxp/loxp; Fabpl-Cre+. In mice 1 and 4 weeks of age, we isolated bladders and examined the mitotic cell marker phospho-histone 3 Ser10 (Fig. 6F) and Ki-67 (Supplementary Fig. S2C) by immunohistochemistry. Intriguingly, there was a distinct increase in mitotic cell frequency in p21–/–;Ptenloxp/loxp; Fabpl-Cre+ mice compared with mice of all other genotypes in both assays. This showed that p21 induction in the Ptenloxp/loxp; Fabpl-Cre+ bladder inhibits urothelial cell proliferation in mice 1 week of age and older, but when p21 is also deleted in addition to Pten as in the p21–/–; Ptenloxp/loxp; Fabpl-Cre+ mice, the urothelial cells continue to proliferate at higher rates. The induction of p21 in response to Pten deletion may therefore be a type of bladder-specific tumor suppression mechanism. Experiments are in progress to determine if the bladder tumorigenesis rate in p21–/–;Ptenloxp/loxp;Fabpl-Cre+ mice is increased compared with that of Ptenloxp/loxp;Fabpl-Cre+ mice. We will note here that lysate from a bladder carcinoma arising from a p21–/–;Ptenloxp/loxp;Fabpl-Cre+ mouse was compared with normal and hyperplastic bladder samples for AKT and FOXO3a phosphorylation by immunoblotting (Fig. 6G), and we found no increase in phosphorylation in the carcinoma. This suggests that the lack of AKT activation in the Pten-deficient bladders is maintained in tumors. Similarly, lysates from anterior prostate carcinomas from three Ptenloxp/loxp;Fabpl-Cre+ mice were immunoblotted for phospho-AKT and FOXO3a (Fig. 6H), and we found that the increased levels of phosphorylation were maintained in the tumors.

Finally, to determine if p21 induction plays a role in suppressing activation of the AKT pathway, we made bladder epithelial lysates from p21–/–;Ptenloxp/loxp and p21–/–;Ptenloxp/loxp;Fabpl-Cre+ mice and immunoblotted for phospho-AKT, mTOR, and S6 (Supplementary Fig. S2D). As with the p21+/+ mice, we found no difference in activation of the AKT/mTOR pathway, showing that p21 induction in Pten-deleted urothelium does not suppress activation of the AKT pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Loss of Pten expression has been strongly linked to the development of multiple types of human cancer, such as glioblastoma, breast cancer, and prostate cancer, but its importance in suppressing the development of other types of malignancies, such as bladder cancer, has been controversial (15, 16, 47, 48). An analysis of tumor predisposition in Cowden syndrome patients (PTEN+/–) also revealed biases towards the development of tumors in certain organs, such as breast and thyroid (11), although genitourinary cancers are also common. The molecular basis for such differential tumor predispositions is not understood. To address this issue, we generated mice that were conditionally deficient for Pten in urothelium of the bladder, ureter, and kidney and in epithelium of the urethra, vagina, prostate, colon, and seminal vesicle. These mice developed a spectrum of epithelial tumors in the same organs that underwent Cre-mediated deletion at the Pten locus. Ptenloxp/loxp;Fabpl-Cre+ mice died at an accelerated rate compared with Ptenloxp/loxp mice or wild-type mice, due to tumors and obstructed urine flow. The tumors were characteristically aggressive and in a few cases metastasized to the lung. The aggressiveness of the tumors is consistent with the known effects of PTEN deficiency on cell motility and angiogenesis, in addition to proliferation and survival (49, 50). In future studies, the Ptenloxp/loxp;Fabpl-Cre+ mouse line may serve as a unique, valuable model for high-grade urothelial carcinoma, prostate adenocarcinoma, and vaginal squamous cell carcinoma.

Hyperplasia was apparent in the bladders of Ptenloxp/loxp;Fabpl-Cre+ mice before 6 weeks of age and increased epithelial cell size was evident even at birth, suggesting that loss of Pten exerts an early effect on the epithelial cells. Surprisingly, however, despite the increased number of epithelial cells in the Pten-deficient bladders, cell proliferation rates were elevated only during the first week of life, after which they dropped to normal. We also examined apoptosis rates and found very low levels of apoptosis in the bladders. This led us to conclude that Pten deletion in the bladder was causing increased proliferation during a 1-week time span in newborn mice, leading to a hyperplasia that was maintained during the life of the animal.

A general survey of protein phosphorylation in the bladder was done with immunoblots to determine which signaling pathways had been activated by Pten deficiency. In the Pten-deficient bladder, we found that very few downstream targets of PI3K were phosphorylated or changed as described in other tissues or cell types. We did find that the protein levels of p27 were decreased from 2 days of age through at least 3 months of age. Previous studies had shown that this might be mediated through AKT inhibition of FOXO3a activation, because FOXO3a can induce p27 expression (51), or through dysregulation of SCFSKP2, which causes degradation of p27 (52). FOXO3a is unlikely to play a role in decreased p27 levels in the Ptenloxp/loxp;Fabpl-Cre+ mice because we could not detect a change in phosphorylation of FOXO3a. Decreased levels of p27 were found to be associated with increased tumor grade in bladder cancers (53, 54), suggesting that the down-regulation of p27 in the Pten-deficient bladders may contribute to tumorigenesis. The low p27 levels may even be responsible for the initial burst in proliferative rates during the first week of life in the Pten-deficient animals. It will be interesting to identify the mechanism by which Pten deficiency leads to p27 reduction, because it could be an AKT-independent pathway downstream of PTEN that leads to cancer in the bladder. We did not find any change in subcellular localization of p27 in the Pten-deficient bladder tissue (data not shown), in contrast to recent reports that AKT activation leads to relocalization of p27 from the nucleus to the cytoplasm in human breast cancer (55–57).

We found that the levels of p21 in the bladder epithelium of Ptenloxp/loxp; Fabpl-Cre+ mice were elevated between 2 and 5 days of age, and this increase was maintained over time. Previous studies have suggested that AKT can regulate p21 levels by altering protein stability, either through direct phosphorylation of p21 (27) or through inhibition of GSK-3, which can phosphorylate p21 and regulate its degradation (28). However, we believe the increase in p21 is not due to GSK-3 inhibition because we did not detect any alteration in GSK-3 phosphorylation (data not shown). Furthermore, because we can detect an increase in p21 mRNA, a change in p21 protein stability cannot be the only mechanism to explain its up-regulation. The increase in the p21 mRNA in the Pten-deficient bladder may be mediated through an increase in p21 transcription or a reduction in the degradation of p21 mRNA in intermediate and superficial cells, or alternatively due to a differentiation defect (e.g., by an abnormal expansion in the population of superficial epithelial cells that normally express p21). The latter explanation seems less likely because differentiation still occurs normally according to some variables, such as uroplakin and p63 expression. However, given the fact that the frequency of p21-positive cells is increased in Pten-deficient bladders, we must still consider the possibility that there is a differentiation defect.

It will be very interesting to determine the mechanism by which the large population of p21+ cells is generated. The most studied regulator of p21 transcription is p53. Activation of p53 has been shown to increase the levels of p21 transcription (58). p53, however, is unlikely to be responsible for p21 induction in Pten-deficient bladders for at least two reasons. First, we found that p53 protein levels were barely detectable in the bladder samples, and p53 was not up-regulated in the Pten-deficient bladders by immunoblot or by RT-PCR (data not shown). Second, several studies have suggested that Pten deletion would actually result in decreased levels of p53 due to MDM2 up-regulation (59–62). Immunohistochemical analysis of p21 staining patterns showed that p21 was only detectable in the nuclei of epithelial cells in all bladders, and a significantly higher percentage of p21+ cells was present in the Pten-deficient bladders. Pten loss therefore did not induce cytoplasmic localization of p21 in our mice (29).

The consequence of p21 induction in the bladder was inhibition of urothelial cell proliferation. The timing of p21 induction in the Ptenloxp/loxp;Fabpl-Cre+ mice at 5 days of age perfectly coincided with a precipitous drop in proliferation rate as measured by Ki-67 staining. Furthermore, Pten/p21 double-deficient bladder cells continued to proliferate at higher levels than cells from Pten or p21 single-deficient bladders after 1 week of age, showing that p21 was at least partially responsible for the reduction in proliferation. The induction of p21 may be a key bladder-specific tumor suppression mechanism to limit abnormal growth in the presence of constitutive cell proliferation signals, and drugs designed to induce p21 in p21-negative tumors may be an effective strategy for chemotherapy.

It is worth noting that whereas Wu et al. (50) found increased levels of activated AKT in 55% primary bladder tumors, we did not observe any increase in activated AKT in tissue from hyperplastic Pten-deleted bladders. We have verified that there are similar levels of AKT in the bladder compared with the prostate so the failure to detect an increase in AKT activation is not due to an absence of AKT in the bladder. It is likely that in the bladder, there is either a negative feedback loop that mitigates the effect of Pten deletion on AKT activation, or there is a partial redundancy in PTEN function. Regardless, our results suggest that AKT/mTOR pathway inhibitors, such as rapamycin, may not be useful for the treatment of Pten-negative bladder tumors. This is especially critical given the fact that rapamycin analogues are being tested as chemotherapeutic agents, and whereas they may be effective against certain tumor types, our study shows the necessity of recognizing tissue-specific differences when selecting drug therapy.

The striking tissue specificity exhibited by Pten-deficient bladder and prostate epithelial cells in activating p21 or p70S6K/FOXO3a suggests that this represents two distinct choices of cell fate. What are the critical determinants for differential regulation of p21 or p70S6K? One possible determinant is the levels of AKT activation. The lack of AKT activation in the bladders of the Ptenloxp/loxp;Fabpl-Cre+ mice may explain why the p70S6K pathway was not activated. Trotman et al. (39) have shown that fine-tuning levels of activated AKT in the prostate through the use of a series of PTEN hypomorphic mice results in dose-responsive tumor progression, illustrating the exquisite sensitivity of cancer development to levels of AKT activation. A certain level of AKT activity may be required to up-regulate the p70S6K signaling pathway. Alternatively, the endogenous levels of AKT signaling pathway targets may play an important role in determining cell fate. For instance, we found that Pten deficiency in the prostate resulted in increased absolute levels of p70S6K in addition to increased activation of p70S6K, whereas in the bladder, there was no effect of Pten deletion on either (data not shown). This suggests that there is some prostate-specific mechanism that results in increased p70S6K protein levels when PTEN is absent, which may be responsible for the differential activation of this pathway. Another interesting possibility is that exogenous factors secreted by stromal cells may enhance AKT signaling in the prostate compared with the bladder. For example, You et al. (63) have shown that conditioned medium from prostate stromal cells induces the PI3K pathway in primary prostate epithelial cells. As for an explanation why p21 is up-regulated only in bladder but not prostate tissue, it is possible that because the rates of proliferation are so high in Pten-deficient urothelium during the first week of life, there is increased induction of negative feedback mechanisms to block proliferation. However, the molecular mechanisms mediating this effect remain to be determined.

In conclusion, our study shows an interesting contrast that Pten deficiency in a tissue preferentially predisposed to cancer formation leads to the activation of FOXO3a and p70S6K pathways, whereas in the bladder which is less sensitive to tumor formation, Pten deficiency leads to decreased levels of p27, increased levels of p21, and a decrease in proliferation rates. We believe the key to the relatively low rate of tumorigenesis in the bladder lies in the fact that the cell population, which increases in response to Pten loss, is largely nonproliferative, p21 positive, and differentiated. We are currently in the process of determining if the p21–/–;Ptenloxp/loxp;Fabpl-Cre+ mice have increased rates of bladder cancer development compared with the Ptenloxp/loxp;Fabpl-Cre+ mice. Clearly, it will be important to study the expression and mutation of both the p21 and PTEN genes in human bladder cancer samples. Understanding the specificities of downstream signaling pathways will be key to the development of effective, tailored, and tissue-specific anticancer therapies.


   Acknowledgments
 
Grant support: Grants CA89434 and DAMD17-02-1-0403 (J. Yuan), NIH grant RO1CA107166 (H. Wu), Department of Defense grant PC031130 (H. Wu), and American Cancer Society postdoctoral fellowship (L.I. Yoo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Radha Verman, Ben Pu, and Ho Jung Yoo for technical assistance; Jeffrey Gordon (Department of Molecular Biology and Pharmacology, Washington University in St. Louis School of Medicine, St. Louis, MO) for the Fabpl-Cre+ mice; Tung-Tien Sun (Departments of Pharmacology, Urology, and Dermatology, New York University, New York, NY) and Frank McKeon (Department of Cell Biology, Harvard University, Boston, MA) for anti–pan-uroplakin and anti-p63 antibodies, respectively; and Michael Boyce, Roberto Olea-Sanchez, and Alexei Degterev for helpful comments on the article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/ 7/05. Revised 8/27/05. Accepted 11/ 8/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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