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Oncogene (2007) 26, 4817–4824

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ORIGINAL ARTICLE

Transgenic expression of human thymidylate synthase accelerates the development of hyperplasia and tumors in the endocrine pancreas M Chen1,5, L Rahman1,5,6, D Voeller1, E Kastanos1,7, SX Yang2, L Feigenbaum3, C Allegra1,8, FJ Kaye4, P Steeg1 and M Zajac-Kaye1 1 Molecular Therapeutics Program, Center for Cancer Research, Bethesda, MD, USA; 2National Clinical Target Validation Laboratory, Division of Cancer Treatment and Diagnosis, Bethesda, MD, USA; 3Laboratory of Animal Sciences, NCI-Frederic Cancer Research, Frederick, MD, USA and 4Genetic Branch, National Cancer Institute, Bethesda, MD, USA

Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and repair and elevated levels of TS have been identified as an important prognostic biomarker for colorectal cancer and several other common human malignancies. In addition, TS gene expression has been linked with cell-cycle regulation and cell proliferation through the ability of retinoblastoma protein to repress the transcriptional activation of E2F target genes such as TS. Therefore, overproduction of TS could participate in the progression to a neoplastic phenotype. Consistent with this model, a recent study has suggested that ectopic TS expression can induce a transformed phenotype in mammalian cells. To investigate the role of deregulated TS activity in tumor development, we generated transgenic mice that express high levels of catalytically active human TS (hTS) exclusively in the pancreas and low levels of hTS in multiple other tissues. Analyses of pancreatic tissue in TS transgenic mice revealed abnormalities within the endocrine pancreas, ranging from pancreatic islet hyperplasia to the detection of islet cell tumors. Overexpression of hTS in murine islets provides a new model to study genetic alterations associated with the progression from normal cells to hyperplasia to islet cell tumors, and suggests that this mouse model may be useful for regulating TS activity in vivo for development of cancer prevention and new therapies. Oncogene (2007) 26, 4817–4824; doi:10.1038/sj.onc.1210273; published online 12 February 2007 Keywords: thymidylate synthase; transgenic model; islet cell adenoma; endocrine pancreas

Correspondence: Dr M Zajac-Kaye, National Cancer Institute, NIH, Bldg 37 RM 1122, 37 Convent Drive, Bethesda, MD 20892, USA. E-mail: [email protected] 5 These two authors contributed equally to this work. 6 Current address: Center for Scientific Review, NIH, Bethesda, MD 20892, USA. 7 Current address: Department of ECE, University of Cyprus, Nicosia 1678, Cyprus. 8 Current address: Network for Medical Communication and Research, Bethesda, MD, USA. Received 3 August 2006; revised 3 November 2006; accepted 1 December 2006; published online 12 February 2007

Introduction Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and repair and has been extensively validated as a target for cancer therapeutics (Bertino and Banerjee, 2003). TS catalyses the reductive methylation of 20 -deoxyuridine 5-monophosphate (dUMP) to deoxythymidine-50 -monophosphate (dTMP), which undergoes further phosphorylation to deoxythymidine50 triphosphate (dTTP), a critical precursor for DNA synthesis. It has been shown that TS is required for cellular proliferation and, consequently, inhibition of TS results in cell growth arrest (Navalgund et al., 1980). Since the development and clinical success of 5fluorouracil, there has been intense interest in further defining the role of TS and designing a new generation of TS inhibitors that include pemetrexed, raltitrexed and capecitabine (Takimoto and Diggikar, 2005). Clinical studies have suggested that high TS levels are correlated with poor clinical outcome and resistance to TS inhibitors in different cancer types (Johnston et al., 1995; Chu and Allegra, 1996). Several studies have also shown that the level of TS expression can predict overall survival for patients with colon, breast and several other human cancers (Johnston et al., 1997; Pestalozzi et al., 1997; Allegra et al., 2003). These clinical data may be partly explained by the observation that tumors with elevated TS levels are also associated with increased tissue invasiveness and increased metastasis (Edler et al., 2000), although the mechanism underlying these findings is still unknown. In addition, tight control of TS activity is essential for normal DNA replication and division, and thus, dysregulation of this enzyme may disrupt homeostasis and potentially induce genetic abnormalities. Alterations in TS levels may also affect chromosomal stability and DNA repair by disrupting steady-state levels of nucleotide pools. For example, retinoblastoma (RB)-mediated cell-cycle arrest, that results in decreased TS expression, was shown to control the level and ratio of intracellular nucleotide pools (Angus et al., 2002). In addition, recent data have demonstrated that the ectopic overexpression of TS may play a role in mammalian tumorigenesis (Voeller et al., 2002; Rahman et al., 2004), although the mechanism of TS-induced neoplastic transformation remains to be

Pancreatic abnormalities in TS transgene model M Chen et al

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neoplastic transformation in rodent cells (Rahman et al., 2004), and (iii) is overexpressed in a variety of cancers (Pestalozzi et al., 1997; Shintani et al., 2003), we studied the phenotypic effect of expressing hTS in transgenic mice and have shown alteration of pancreatic growth in vivo that resulted in the progression of normal islet cells to hyperplasia and b-cell adenomas. Results

TS-T2

TS-T3

TS-T4

TS-c7

NIH3T3

a

TS-T1

Generation and characterization of human TS þ / transgenic mice Since ectopic human TS (hTS) overexpression can transform immortalized mammalian cells in vitro (Rahman et al., 2004), we generated hTS transgenic mice to study the effect of deregulated TS in an animal model system. When we originally designed our experiments to create TS transgenic mice, we selected the cytomegalovirus (CMV) promoter to express human TS in several different tissues. For example, it has been shown that the CMV promoter can be active in tissues such as the kidney, stomach, spleen and pancreas (Hoeflich et al., 1999; Zhan et al., 2000). Four TS-positive transgenic lines, designated TS-T1, TS-T2, TS-T3 and TS-T4, were established. DNA and protein blots of tail clippings were performed using a full-length TS cDNA probe and anti-TS antibody, respectively (Figure 1a; data not shown). The levels of TS expression in the tail clippings

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mTS

hTS

Thymus Testis

Liver Salivary gland

Pancreas Muscle Brain Bone morrow Colon

Heart Intestine

Lung

Kidney

b

Lymph node

Actin

TS-c7 3T3 Spleen Somach

mTS hTS

FVB

8 9 10 11 12 13 14 15 16 17 18 TS-T4

7

TS-T3

6 TS-T2

c

3 4 5 TS-T1

1 2

TS-c7

elucidated. To pursue this latter observation, we have generated transgenic mice that express elevated levels of human TS protein predominantly in the mouse pancreas and have observed the phenotype of hyperplasia and adenoma that appears to be restricted to endocrine pancreatic tissue. Pancreatic endocrine tumors arise in the islet cells of Langerhans, which consist of four major cell types, a, b, g and PP that synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. b-Cell tumors are the most common tumor subtype which usually arise sporadically; however, a small proportion arise owing to loss of the multiple endocrine neoplasia type 1 (MEN1) gene (Wang et al., 1998). Activation of oncogenes such as c-myc and ras has also been observed during disease progression of sporadic endocrine tumors, but their role in tumor initiation remains unknown (Pavelic et al., 1995; Sato et al., 2000). The best-characterized murine model for pancreatic islet cell cancer is the targeted transgene expression of SV40 large-T antigen under the control of the rat insulin promoter (Hanahan, 1985). In this model, mice develop solid tumors in a multistep process starting with the normal islet cells progressing first to b-cell hyperplasia, then to angiogenic islets, and finally to solid tumors (Hanahan, 1985). In addition, involvement of the c-myc oncogene in the development of endocrine pancreatic neoplasm has been demonstrated using transgenic animals with reversible c-Myc protein expression (Pelengaris et al., 2002) or with elastase-driven c-myc in a Ink4a/Arf-null background (Lewis et al., 2003). In contrast to the SV40 and c-myc mouse models, targeted inactivation of the murine MEN1 gene results in a spectrum of endocrine tumors similar to human patients with the MEN1 syndrome (Crabtree et al., 2001). Mice with a heterozygous MEN1 inactivation developed pancreatic islet adenomas with a long latency period of 9–22 months of age (Crabtree et al., 2001), whereas conditional homozygous MEN1 knockout mice showed an adenoma latency of 6–11 months of age (Crabtree et al., 2003). Moreover, simultaneous loss of murine cyclin-dependent kinase (CDK) inhibitors, p18 and p27, which are regulated by menin (Milne et al., 2005), resulted in a spectrum of tumors similar to that seen in human MEN1 and MEN2 syndrome (Franklin et al., 2000). In addition, double heterozygotes for Rb and p53 null alleles or transgenic animals with activated CDK4 (Rane et al., 2002) develop a spectrum of endocrine neoplasia, including islet cell tumors (Williams et al., 1994). These results suggest that cell-cycle regulators play an important tumor suppressor or activator role in pancreatic islets and that simultaneous genetic changes in several of these regulators may be required for tumor development. CDK-mediated phosphorylation of the RB tumor suppressor results in loss of RB function and activation of the E2F-1 transcription factor that regulates the expression of several DNA synthetic enzymes including TS (DeGregori et al., 1995; Banerjee et al., 1998). Since TS (i) is a downstream effector of E2F-1 (DeGregori et al., 1995; Banerjee et al., 1998), (ii) can induce

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hTS

Figure 1 Expression of hTS in transgenic mice. (a) Immunoblot analysis can distinguish hTS from mTS in four independent TS transgenic lines (TS-T1, TS-T2, TS-T3 and TS-T4). Protein lysates were obtained from tail clippings of 4-week-old transgenic mice (lanes 1–4). Lysates from hTS-transfected NIH3T3 (TS-c7) or the parental NIH3T3 cells were used as controls (lanes 5 and 6). (b) High levels of ectopic hTS expressed exclusively in pancreatic tissues. Immunoblot analysis of tissues isolated from 4-month-old TS transgenic mice (lanes 1–18). (c) Comparison of pancreatic tissues among four TS transgenic lines ranging in age from 14 to 24 months (lanes 2–5). Pancreatic lysates from 8-month-old FVB mouse (lane 6) and TS-c7 cells (lane 1) were used as negative and positive controls, respectively.

Pancreatic abnormalities in TS transgene model M Chen et al

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a FdUMP

TS-T4

FVB

-

-

+

TS-T2 +

-

+ hTS/FdUMP

hTS 1

b

40000

(Catalytic Activity (dpms)

varied among the four transgenic lines and did not correlate with the estimated DNA copy numbers (data not shown). The hTS was resolved from the endogenous mouse TS on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) owing to electrophoretic mobility differences between the mouse and human TS (Rahman et al., 2004). The slower migrating mouse TS (Figure 1a, lanes 1–5) exhibited a signal that co-migrated with mouse NIH3T3 cells (Figure 1a, lane 6). In contrast, the transgenic hTS (Figure 1a, lanes 1–4) co-migrated with hTS that was stably transfected into NIH3T3 cells (Figure 1a, lane 5) (Rahman et al., 2004). To determine the tissue distribution of the transgenic hTS, we analysed the transgenic lines for hTS expression in different tissue types. In all four transgenic lines hTS protein expression was found predominantly in the pancreas (Figure 1b), whereas the endogenous murine TS was barely detectable in the different tissues. In addition, on prolonged exposure, low levels of hTS protein were observed in the heart, spleen, bone marrow, lymph nodes, thymus, liver, lung, testis, skin, intestine, muscle, kidney and stomach tissues. To compare hTS levels in pancreatic tissue among the four transgenic lines, proteins were immunoblotted using an anti-TS antibody. We observed that the levels of hTS expression in the pancreas were similar in all of the transgenic lines (Figure 1c, lanes 2–5). The presence of hTS in the pancreas of transgenic mice was also confirmed by immunoblot analysis using anti-TS antibody that can detect TS as both a free enzyme and a ternary complex with FdUMP (Johnston et al., 1993). We incubated pancreatic tissue lysates from both the hTS and control mice with excess FdUMP and 5,10-methylene-tetrahydrofolate, followed by SDS– PAGE and immunoblotting with the anti-TS antibody. Since the migration of the TS ternary complex on SDS– PAGE is retarded as compared to free TS, we observed that in the presence of excess folate and FdUMP, a 38.5 kDa species representing the TS ternary complex was detected along with the native TS band at 36 kDa (Figure 2a, lanes 2 and 6). In contrast, only the unbound native 36 kDa TS was detected when FdUMP and folate were omitted (Figure 2a, lanes 1 and 5). As expected, no hTS was detected in either the FVB control or treated sample (Figure 2a, lanes 3 and 4). The endogenous mouse TS could only be detected with a prolonged exposure that prevents the resolution of the free versus bound TS (data not shown). These results confirmed that the 36 kDa band observed in the pancreatic tissue by immunoblot analysis represents hTS. We also tested whether the ectopically expressed hTS in the pancreatic tissue from the transgenic mice retained catalytic activity in vivo. Using protein lysate isolated from both transgenic and wild-type FVB pancreas, we measured the conversion of FdUMP into dTMP by a tritium release assay (Johnston et al., 1993; Rahman et al., 2004). We found that hTS retained catalytic activity in the pancreas with up to a tenfold increase in TS catalytic activity in the hTS transgenic animal tissue as compared to the wild-type FVB mice (Figure 2b).

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Figure 2 Detection of hTS in TS transgenic mice. (a) Ectopic hTS retained its ability to form a ternary complex with FdUMP. Immunoblot analysis was performed with proteins isolated from pancreatic tissues of wild type or transgenic mice either treated or untreated with FdUMP and 5,10-methylene-tetrahydrofolate. The lysates from hTS transgenic lines, TS-T2 (lanes 5 and 6) and TS-T4 (lanes 1 and 2) and a wild-type FVB mouse (lanes 3 and 4) were immunoblotted with TS106 antibody. (b) TS catalytic activity was measured in TS-T1 (10–19-month-old) and TS-T2 transgenic mice (3-month-old) mice. Results shown from the transgenic line TS-T1 are represented by the mean7s.d. from five mice, each performed in triplicate. Results shown from the transgenic line TST2 are represented by the mean7s.d. from one mouse performed in triplicate.

Induction of pancreatic islet adenoma in TS transgenic mice To determine whether TS overexpression results in an age-dependent phenotype, a cohort of hTS mice (TS-T1 line) and age-matched hTS-negative littermates were euthanized every 2 months for up to 24 months of age. Hematoxylin and eosin staining of pancreatic tissue section from hTS animals revealed an increase in the number and size of islets (hyperplasia), which progressed to coalescence of two or more islets, and finally to islet adenoma (Figure 3a–d). At 3–8 months, four out of 21 (19%) of TS-T1 mice displayed an increase in the number and size of the islets, whereas none of 25 hTSnegative littermates showed this phenotype (Table 1). At 9–24 months, the TS-T1 mice displayed lesions ranging from islet hyperplasia to islet adenoma (Figure 3 and Table 1). Ten out of 171 mice (6%) of the TS-T1 transgenic line developed islet adenoma, a phenotype that was observed in 0% of the 136 hTS-negative littermates. One hundred and nine out of 171 (63%) TST1 mice examined at 9–24 months had islet hyperplasia as compared to 32 out of 136 (23%) mice examined Oncogene

Pancreatic abnormalities in TS transgene model M Chen et al

4820 Normal

Hyperplasia

a

b

c

d

Coalescence

Adenoma

Figure 3 Pancreatic lesions in TS transgenic mice. (a) TS overexpression leads to progression from normal pancreatic islets to (b) islets hyperplasia, (c) coalescence of hyperplastic foci as depicted by the arrow and (d) adenoma. Hematoxylin and eosin (H&E)-stained slides of paraffin-embedded fixed pancreas from (a) FVB mice and from (b–d) TS transgenic mice, TS-T1. Magnification at  32. Photographs were obtained from (a) 8-, (b) 23-, (c) 19- and (d) 17-month-old mice.

Table 1

Summary of pancreatic lesions in TS-T1 transgenic mice and in the age-matched negative littermates

Age (months)

Islet phenotype

Transgenic mice +/

/

Transgenic lines

hTS

(%)

hTS

(%) 0 0

3–8

Hyperplasia Adenoma

4/21 0/21

19 0

0/25 0/25

9–24

Hyperplasia Adenoma

109/171 10/171

64 6

32/136 0/136

Table 2 Summary of pancreatic lesions analysed in three TS transgenic mice and in the age-matched negative littermatesa

23 0

Islet phenotype

Transgenic mice hTS

TS-T2 TS-T3 TS-T4 Total

Adenoma Adenoma Adenoma Adenoma

(+/)

1/30 2/17 2/22 5/69

(%) 3 12 9 7

hTS

(/)

0/14 0/12 0/8 0/34

(%) 0 0 0 0

Abbreviations: TS, thymidylate synthase; hTS, human TS.

Abbreviations: TS, thymidylate synthase; hTS, human TS. aAnimals range from 18 to 24 month of age.

from the same age group of hTS-negative littermates (Table 1). We also examined the pancreas from three additional hTS transgenic lines TS-T2, TS-T3 and TST4 at 18–24 months of age. We found that 5 out of 69 (7.2%) of total mice examined had adenomas, whereas this phenotype was observed in 0% out of 34 of these older age-matched hTS-negative littermates (Table 2). We also observed a twofold increase in the phenotype of coalescing hyperplastic islets in the TS transgenic animal as compared to the TS-negative littermates. The degree of hyperplasia and adenoma was quantitated using the Automated Cellular Imaging System (ACIS) (San Juan Capistrano, CA, USA), see Materials and methods for methodological details (Yang et al., 2005). As expected,

we found that the islet area is much larger in the hTS transgenic mice than in the normal littermate (64.3 vs 8.6%, respectively). To quantify the insulin content in the pancreas of normal and hTS transgenic mice, the average insulin intensity was generated by ACIS. Again as expected, we found that the total insulin content in the hTS transgenic mice was approximately three-fold higher than the normal littermate. To determine the localization of human TS in the pancreatic tissue, we isolated both the endocrine islets and the exocrine cells from several TS transgenic and wild-type FVB mice. Total protein extract isolated from islet and exocrine cells were resolved on SDS–PAGE and analysed by immunoblot using an antibody against

Oncogene

Pancreatic abnormalities in TS transgene model M Chen et al

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TS. We found that hTS was present in both islets and exocrine cells (Figure 4, lanes 4–6), but was absent in the control FVB mice (Figure 4, lanes 1–3). We also performed immunohistochemical analysis of pancreatic sections from hTS mice using pancreatic endocrine markers to determine the presence of islet cell-specific peptides (Figure 5). We observed that the increase in volume of pancreatic islets relative to the exocrine pancreas was a result of an increase in b-cells expressing insulin, but not the a-cell (glucagon) or g-cell (somatostatin) (Figure 5). In addition, we measured insulin levels in blood samples obtained from TS transgenic animals with documented islet cell hyperplasia and islet cell adenoma and from several age-matched control animals (Table 3). We found that the insulin blood levels from TS transgenic mice diagnosed with islet cell adenoma

Ex oc r En ine do cr in e

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Ex oc ri En ne do cr in e To ta l To ta l

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hTS

Tubulin 1

2

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6

Figure 4 Detection of hTS protein expression in islets and exocrine pancreas. Pancreatic tissue was obtained from FVB control mice and the human TS transgenic line TS-T1. Immunoblot analysis of lysates prepared from total pancreas and isolated exocrine and endocrine islet cells from control FVB mice (lanes 1– 3) and TS-T1 (lanes 4–6). All animals were 8-month-old.

H&E

Insulin

were 122.8 ng/ml (n ¼ 6) as compared to a mean of 3.84 ng/ml (n ¼ 14) in TS-negative littermates and a mean of 4.38 ng/ml (n ¼ 19) in TS-positive mice with normal islets. A smaller increase in insulin blood levels of 8.33 ng/ml (n ¼ 18) was also detected in TS-positive mice diagnosed with islet cells hyperplasia (Table 3). In addition, fasting blood glucose levels were lower in TSpositive animals with hyperplastic islets (3.5770.46 mM/ l (n ¼ 5)) as compared to glucose levels in TS-positive animals having normal islets (5.171.17 mM/l (n ¼ 5)) having a P-value of 0.025.

Discussion Although TS has been studied extensively for the past 30 years, there have been no model systems that have examined the phenotypic effect of TS overexpression in vivo. Such a murine model is of potential interest since TS plays a critical role in DNA synthesis and repair, is required for normal cell proliferation, and elevated levels of TS have been identified as an important prognostic biomarker for colorectal cancer as well as for several other human malignancies (Pestalozzi et al., 1997; Shintani et al., 2003). In addition, we recently observed that elevated levels of ectopic TS can participate in the transformation in murine cells in vitro and that these TS-transformed cells give rise to xenograft tumors in nude mice (Rahman et al., 2004). Therefore, to study the role of deregulated TS in vivo, we developed a human TS vector using the strong eukaryotic CMV promoter and generated four independent human TS transgenic lines. We observed that the transgenic mice appeared healthy and lived up to

Glucagon

Somatostatin

FVB

hTS

Figure 5 Detection of insulin expression in TS-induced pancreatic adenoma. Immunohistochemical staining was performed on TS transgenic mice (hTS) (17-month-old) and normal FVB control with antibodies against insulin, glucagon and somatostatin (5-mm sections of formalin-fixed, paraffin-embedded mouse pancreas tissues at  32 magnification). Oncogene

Pancreatic abnormalities in TS transgene model M Chen et al

4822 Table 3 Insulin levels in TS transgenic mice and age-matched negative littermates Islet phenotype Normal Normal Hyperplasia Adenoma

Age (months)

hTS

Insulin (ng/ml)a

Number of animals

9–24 9–24 9–24 16–24

(/) (+/) (+/) (+/)

3.8471.04 4.3971.04 8.3371.68 122.80764.72

14 19 18 6

Abbreviations: TS, thymidylate synthase; s.d., standard deviation. a Numbers represent mean7s.d.

24 months without overt tumor formation. On protein immunoblot analysis of adult mouse tissues, we detected a high steady-state level of ectopic TS in pancreatic tissues and low levels of human TS protein expression in 14 out of 18 other tissues examined. These data demonstrate that this CMV promoter shows tissue restriction with high-level expression in the pancreas as reported previously (Zhan et al., 2000). Scheduled necropsy of pancreatic tissues showed progression from normal islets to islet hyperplasia and finally to islet cell adenoma in the hTS transgenic mice. Hyperplastic islets first became apparent in the transgenic mice by 3–8 months; however, this phenotype was not seen in any of the TS-negative littermates at these ages. In the older mice, from 9 to 24 months, the frequency of hyperplastic islets was three-fold higher in the TS-positive mice as compared to the TS-negative littermates. Islet cell adenoma developed only in the TS transgenic mice and not in the TS-negative littermates. We also kept a separate cohort representing three independently derived TS transgenic lines and their negative littermates for a period of 18–24 months. We again found that islet adenoma was observed in all of the independent transgenic lines and not in the TS-negative littermates. Development of islet cell adenoma in these TS transgenic animals occurred at a modest frequency and required a latency period of approximately 11 months of age. This long latency period may not be unexpected given that other mouse models for islet cell neoplasia, such as the Rb þ / knockout, required the cooperating loss of p53 function to exhibit the phenotype (Williams et al., 1994). In addition, even the transgenic expression of the oncogenic SV40 large-T antigen showed a latency period, which was accelerated by cross-breeding mice with Rb and p53 null alleles (Casanovas et al., 2005). These latter data were interpreted to suggest that the large-T antigen might only partially abrogate RB suppressor function and that there is a sufficient amount of active protein in Rb þ / þ tissues to keep the cells in a state of proliferative latency (Casanovas et al., 2005). Although the ectopic overexpression of TS, which is a critical enzyme required for DNA synthesis and cell proliferation, may participate in b-cell hyperproliferation, it is unlikely to participate as a rate-limiting oncogenic event. In addition, TS is only one of several different proposed downstream mediators for inactivated RB/E2F function. Our data, therefore, suggest Oncogene

that elevated levels of TS may accelerate the deregulated proliferation of susceptible cells that have already initiated the oncogenic process, and these cells must still acquire additional genetic events for tumor development. The detection of islet cell adenomas exclusively in hTS mice, however, is further evidence that elevated TS levels in human cancers may not simply be a passive biomarker for cell proliferation. An explanation for why the pre-malignant phenotype was detected in endocrine b-cells, but not in pancreatic exocrine cells which also appeared to express elevated levels of hTS, is still undefined. Mouse and human cases of exocrine pancreatic cancer are dependent on aberrant K-ras/growth factor signaling pathways (Almoguera et al., 1988), whereas endocrine pancreatic islet cell cancer models show no incidence of K-ras mutations in humans and, in contrast, are associated with concomitant RB/p53 inactivation in the mouse model (Williams et al., 1994). Since the TS gene has been identified as a downstream mediator of the RB/E2F pathway, it is possible that murine TS deregulation may favor the induction of an islet cell phenotype.

Materials and methods Generation and screening of transgenic mice A 2540 bp NruI/NsiI fragment containing hTS cDNA driven by CMV promoter was excised from pcDNA3.1zeo-TS vector (Rahman et al., 2004) and used to generate an FVB transgenic mice. Founder mice were identified by Southern blot of BamH1-digested genomic DNA and by polymerase chain reaction (PCR) using primers specific for human TS-specific primers: forward, 50 -TTGCCCCCCGCCGCACAG-30 and reverse, 50 -CACAGCAACTCCTCC-AAAACACC-30 . Tissue extract preparation, antibodies and Western-blot analysis Tissues were homogenized using the Powergen 125 homogenizer following the manufacturer’s protocol (Fisher Scientific, Pittsburgh, PA, USA). The homogenate was lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 0.5 mM dithiothreitol, 1 mM phenyl-methylsulfonyl fluoride and 10 mg/ml each of leupeptin and aprotinin. Protein extract (100 mm) was resolved by SDS–PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-TS antibody (clone TS-106) in a 1:200 dilution (Alexandrova et al., 1995; Rahman et al., 2004). The a-tubulin (clone DM1A) and actin antibody were purchased from Sigma (St Louis, MO, USA) and used in 1:1000 dilution as loading controls. TS catalytic activity TS catalytic activity was determined using 100 mg of tissue extract, 105 M [5-3H]FdUMP, 200 pmol FdUMP, 200 pmol 5,10-methylene tetrahydrofolate and 20 mM b-mercaptoethanol in a 200 ml final volume as described previously (Rahman et al., 2004). Histology, immunohistochemistry and quantitative analysis of insulin content Sections of mouse pancreas were surgically excised, fixed for 24 h in 10% neutral-buffered formalin, transferred to 70% ethanol, processed and embedded in paraffin (Histoserv Inc., Germantown, MD, USA). Sections were cut at 5 mm and

Pancreatic abnormalities in TS transgene model M Chen et al

4823 mounted on salinated slides for hematoxylin and eosin staining. Slides were read by a certified mouse pathologist (Taconic Anmed, Rockville, MD, USA). Following the pathologist’s diagnosis of hyperplastic islets, we reexamined the slides under light microscopy. Individual islet area was measured using a  4 microscope objective lens and an ocular micrometer. Mice were diagnosed as hyperplasic when in an area of at least four islets, each individual islet measured 0.375 mm or greater. For immunohistochemistry, sections were cut by the NCI-Frederick Pathology Lab (PHL, Frederick, MD, USA). Sections were incubated for 30 min with the primary antibodies against insulin (1:500, DAKO, Carpinteria, CA, USA), glucagon (1:500, DAKO) and somatostatin (1:1000, DAKO). Antigen–antibody complexes were detected by the peroxidase ABC kits (Vector Labs, Burlingame, CA, USA) according to the manufacturer’s instructions. Immunohistochemical staining signal of insulin was analysed quantitatively using the ACIS (ChromaVision Medical Systems, Inc., San Juan Capistrano, CA, USA) as described previously (Yang et al., 2005). The areas of the whole pancreas and each of the islets were measured by ACIS and the ratio was calculated by dividing the combined area of all the islets by the area of the pancreas. To quantify the insulin content in the pancreas of normal and hTS transgenic mice, five staining areas were scored with a  40 tissue-scoring tool in ACIS. The insulin content in the pancreas was determined by multiplying the area of islets by the average insulin intensity as generated by ACIS. Insulin enzyme-linked immunosorbent assay (ELISA) and glucose levels Serum insulin was measured using an ultrasensitive mouse insulin ELISA kit according to the manufacturer’s protocol (ALPCO Diagnostics, Salem, NH, USA). Duplicate serum samples were used for all measurements and the Mercodia internal control of mouse insulin was used as recommended by the manufacturer (ALPCO Diagnostics). For blood glucose

levels, fasting TS-positive and age-matched negative control animals were chosen before knowing the phenotype of pancreatic islets at the time of the test. Mice tails were clipped (1–2 mm) with a sharp scalpel and a drop of blood was placed onto a glucometer test strip (Bayer, IN, USA) and the result recorded using a Glucometer Elite XL (Bayer, IN, USA). These animals were killed at a later time point and the islet cell phenotype was determined. Isolation of islets from exocrine pancreas Pancreatic islets were isolated from four TS þ / transgenic mice and four wild-type FVB mice as described previously (Martinez et al., 2001). Each mouse was perfused through the heart with Hank’s balanced salt solution (HBSS, Sigma) to remove blood from the pancreatic vessels. A cannula was inserted into the common biliary duct and collagenase (0.4 mg/ ml; 20–25 ml) was pumped through the cannula until the pancreas was fully inflated. The pancreas was excised and incubated at 371C for 20 min for collagenase digestion. The solution was replaced with cold HBSS and the pancreas was dissociated by agitation. The digested pancreas tissues were pooled and passed through a 500 mm mesh (Fisher Scientific). The crude islet mix was then washed three times by centrifugation at 200 g. The islets were collected under a dissecting microscope and the freshly isolated islets were resuspended in ice-cold RIPA buffer.

Acknowledgements We thank Alfredo Martinez for help with islet cells isolation, Steve Jay for help with the animals and Philip Martin for helpful discussions. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and has complied with all relevant federal guidelines and NIH policies.

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