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Molecular Cancer Research 2:504-513 (2004)
© 2004 American Association for Cancer Research

Model Organisms

Mice Expressing SV40 T Antigen Directed by the Intestinal Trefoil Factor Promoter Develop Tumors Resembling Human Small Cell Carcinoma of the Colon1

James R. Gum, Jr.1,2, James W. Hicks1, Suzanne C. Crawley1, Stacey C. Yang1, Alexander D. Borowsky5, Christine M. Dahl1, Sanjay Kakar1,3, Dong Hoon Kim1, Robert D. Cardiff5 and Young S. Kim1,3,4

1 Department of Veterans Affairs Medical Center, San Francisco, California; Departments of 2 Anatomy, 3 Pathology, and 4 Medicine, School of Medicine, University of California, San Francisco, California; and 5 Department of Medical Pathology and Center for Comparative Medicine, University of California, Davis, California

Requests for reprints: James R. Gum Jr., GI Research Laboratory (151M2), Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. Phone: 415-750-2095; Fax: 415-750-6972. E-mail: jgum{at}maelstrom.ucsf.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The colonic epithelium contains three major types of mature cells, namely, absorptive, goblet, and enteroendocrine cells. These cells are maintained by a complex process of cell renewal involving progenitor and stem cells, and colon cancers develop when this process goes awry. Much is known about the genetic and epigenetic changes that occur in cancer; however, little is known as to the specific cell types involved in carcinogenesis. In this study, we expressed the SV40 Tag oncogene in the intestinal epithelium under the control of an intestinal trefoil factor (ITF) promoter. This caused tumor formation in the proximal colon with remarkable efficiency. ITFTag tumors were rapidly growing, multifocal, and invasive. ITFTag tumor cells express synaptophysin and contain dense core secretory granules, markers of neuroendocrine differentiation. The cell type involved in the early steps of ITFTag tumorigenesis was studied by examining partially transformed crypts that contained populations of both normal and dysplastic cells. The dysplastic cell population always expressed both Tag and synaptophysin. Cells expressing Tag alone were never observed; however, normal enteroendocrine cells expressing synaptophysin but not Tag were readily visualized. This suggests that ITFTag tumor cells originate from the enteroendocrine cell lineage following a transforming event that results in Tag expression. ITFTag tumors closely resemble human small cell carcinomas of the colon, suggesting the possibility that these tumors might be derived from the enteroendocrine cell lineage as well.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The colonic epithelium is composed of highly differentiated cells that are maintained throughout life by a complex process involving cell renewal, maturation, and death (1-4). The stem and progenitor cells that participate in this process have been the subject of considerable attention, but much still remains unknown about their characteristics and their patterns of growth and differentiation. Colon carcinomas arise from the cells that populate the colonic epithelium, driven by a stochastic series of genetic and epigenetic alterations (5-7). Many genes are altered in this process, either activated or inactivated, and several specific pathways have been proposed. Little is known, however, about the specific cell types involved. For example, it is not known whether stem cells, progenitor cells, maturing cells, or even differentiated cells are the precursors of colonic tumors.

Colon cancers comprise several histologic types. A major criterion for classification is mucin secretion. Non-mucin-secreting adenocarcinomas are further classified as well, moderately, or poorly differentiated depending on the degree of glandular differentiation they retain (8, 9). Colon cancers are classified as mucinous adenocarcinoma if >50% of the tumor are composed of mucin (9, 10). Some colon cancers exhibit an undifferentiated pattern of growth, retaining none of the glandular architecture of normal colon (11-13). These different histologic types of cancer have different growth characteristics and modes of dissemination and develop via characteristic genetic and epigenetic pathways (8-13). Given the varied histologic and pathologic features of individual human colonic tumors, it is likely that several cell types are capable of malignant transformation.

We have focused much attention on the roles of mucin in normal colon and colon cancer (14-16). The MUC2 mucin is abundantly and specifically expressed in small intestinal and colonic goblet cells and is variably expressed in colorectal cancers (15, 17, 18). Thus, the factors that control the regulation of this gene may reflect processes and signaling pathways important both for normal small intestinal and colonic differentiation and for the development of cancer (19-22). In mucinous colorectal cancers, MUC2 is expressed and secreted in abundance (23). Thus, these cancers possess this landmark feature of normal goblet cells.

We have sought to create mouse models to better understand the development and characteristics of mucinous colorectal cancers. Earlier efforts used the MUC2 mucin gene promoter to drive the SV40 Tag oncogene (24). This hybrid oncogene, expressed in the small intestine, led to the continuation of goblet cells in the cell cycle even after their transit to the villi and ultimately to apoptosis as opposed to tumorigenesis. In the present study, we used the intestinal trefoil factor (ITF) promoter in an attempt to achieve Tag expression in mouse colonic goblet cells. Mice carrying this hybrid oncogene did develop tumors of the proximal colon but surprisingly not mucinous cancers. Instead, the tumors that developed resembled small cell carcinomas of the colon, a rare but deadly form of human colon carcinoma (11-13). Here, we describe the characteristics and development of tumors in these mice, which serendipitously constitutes an animal model for this lethal cancer.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Development of Hybrid Oncogene-Containing Lines
The ITFTag hybrid oncogene consists of –1,190 to +37 bp of the ITF gene promoter fused into the 5'-untranslated region of the SV40 Tag structural gene. A total of nine mice were obtained that incorporated at least one copy of this chimeric gene into their genome (Fig. 1A). Attempts were made to breed these mice by crossing them to C57BL/6 breeders, which were successful with founders 3, 4, 6, 7, and 8. During the process of establishing lines, it became apparent that, with one exception, the mice that incorporated the hybrid oncogene became emaciated and moribund at an early age (Fig. 1A). These mice were killed and, on postmortem examination, were found to have intestinal blockage. In most cases, this could be seen to be caused by a large tumor of the proximal colon, an example of which is shown in Fig. 1B. To determine if there was a relationship between Tag expression in the proximal colon and development of symptoms, we isolated RNA from the proximal colon of 5- to 7-week-old offspring mice and analyzed it for Tag message expression (Fig. 2). Northern analysis indicated that high-level Tag expression was found in ITFTag lines 7 and 8, lines in which the founders became moribund in 12 and 10 weeks, respectively (Fig. 2A). Line 6, in which the founder became moribund in 18 weeks, exhibited a faint, nearly imperceptible band, whereas Tag levels were undetectable using this technique in lines 3 and 4. To further examine this, we used reverse transcription-PCR (RT-PCR), a more sensitive technique, as a test for message levels (Fig. 2B). Here, the presence of Tag message was detected in lines 6, 7, and 8 but not in lines 3 and 4 or the nontransgenic control. Thus, good correlation between Tag expression in the proximal colon and development of symptoms is apparent. Because ITFTag lines 7 and 8 developed tumors at an early age with high efficiency, these lines were selected for further study.



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  		FIGURE 1. Incorporation of the ITFTag hybrid oncogene and tumor development. A. Southern blot analysis of tail DNA in nine founder mice and tail DNA from a nontransgenic control (NTG). Bottom, age at which the mice became moribund. B. Toluidine blue–stained section from the proximal colon of a 13.5-week-old F4 ITFTag line 8 male mouse showing tumor development. Bar, 500 µm.

 


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  		FIGURE 2. Tag expression in the proximal colon of asymptomatic mice. RNA was extracted from 2 cm segments of proximal colon (initiating immediately after the cecum) of F1-F3 mice from the indicated lines for Northern analysis. Mice were 5-7 weeks old and asymptomatic at time of killing. A. Northern analysis. The blot was subsequently stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to show that RNA was loaded in each lane. B. RT-PCR analysis, conducted as described in Materials and Methods.

 
Because frank tumor development occurred only in the proximal colon, we sought to determine expression levels for Tag in the tissues of these mice. RNA was isolated from various tissues of 7-week-old mice and Northern analysis was conducted (Fig. 3A). High-level Tag expression is apparent in the colon of both ITFTag lines 7 and 8 mice. In contrast, no expression was detected in the colons of nontransgenic animals. High-level Tag expression was also found in duodenum of ITFTag line 7 mice and trace expression was noted in stomach of both lines. Tag expression was highest in the proximal portion of the colon and diminished in the middle and distal portions, consistent with tumor development in the proximal location (Fig. 3B). Figure 3A also shows robust levels of Tag expression in three separate tumors isolated from each line. These tumors also had decreased levels of ITF message expression as compared with normal colon. Note that endogenous ITF is abundantly expressed in all segments of the small intestine and colon (Fig. 3A). Thus, the segment of the ITF promoter used to construct the ITFTag hybrid oncogene does not fully recapitulate the tissue-specific pattern of expression exhibited by the native ITF gene.



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  		FIGURE 3. Northern analysis of various tissues of ITFTag lines 7 and 8 mice. A. RNA was extracted from tissues of 7-week-old ITFTag lines 7 and 8 mice. Abbreviations: Duo, duodenum; Jej, jejunum; Ile, ileum; Col, entire colon; Sto, stomach; Liv, liver; Kid, kidney; Hea, heart; Bra, brain; NT, colon from nontransgenic mice; Tum 1, Tum 2, and Tum 3, tumors from the proximal colons of three mice (age 10-15.5 weeks) of each line. Blots were probed first for Tag and subsequently for ITF and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B. RNA was extracted from 3 cm segments of the proximal (Prox), middle (Mid), and distal (Dist) portions of the colon of two 5.5-week-old male ITFTag line 7 littermates and Northern analysis was conducted.

 
Cellular Structure and Characteristics of ITFTag Tumors
The tumors that developed in the colons of ITFTag mice were carcinomas, formed from solid sheets of cells lacking glandular architecture (Fig. 4). The tumors had an overlying layer of normal epithelial cells, and enveloped the entire colonic wall, invading into the muscularis propria (Fig. 4A and B). The tumor cells grew very aggressively, exhibiting abundant mitotic figures (Fig. 4C), with essentially all cells exhibiting proliferating cell nuclear antigen positivity (Fig. 4D). In contrast, in normal colon, only the cells near the base of the crypts exhibited proliferating cell nuclear antigen positivity (Fig. 4E). The average lifetime was 14.4 ± 1.1 weeks (n = 32) for ITFTag line 7 mice and 12.4 ± 1.7 weeks (n = 18) for ITFTag line 8 mice. ITFTag carcinoma cells expressed Tag, apparent both by Northern analysis (Fig. 3) and by immunohistochemistry (Fig. 4F). Tag has known anti-Rb and anti-p53 activities (25-27) and would be expected to be a major contributor to the uncontrolled growth of these cells.



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  		FIGURE 4. Morphology and gene expression in ITFTag tumors. A-D. ITFTag line 7 tumors. E. Nontransgenic mouse. F-H. ITFTag line 8 tumors. Similar results were obtained with both lines in all cases. A-C. H&E stained. Arrows, overlying normal epithelium in A, invasion of the muscularis propria in B, and mitotic figures in C. D. Proliferating cell nuclear antigen expression in tumor cells, although most cells of the overlying normal epithelium lack expression. E. Proliferating cell nuclear antigen expression in cells at the base of the crypts in normal colon. F. Tag expression in ITFTag tumor cells. G and H. Sections immunostained for multi-cytokeratins and synaptophysin, respectively. Sections were fixed with Bouin's solution (A, D, and E), methacarn (B and C), and formalin (F-H). Bars, 100 µm.

 
Our initial hypothesis was that expression of the ITFTag hybrid oncogene in mouse intestine may lead to the development of mucinous cancers, as ITF is highly expressed in goblet cells. ITFTag tumors do not, however, resemble mucinous colorectal cancers. They do not form a glandular architecture, and neither intracellular mucus nor pools of extracellular mucus are visible in H&E-stained sections (Fig. 4) or Alcian blue/periodic acid Schiff-stained sections (data not shown). Furthermore, as will be described below, Tag expression was not detected in goblet cells on immunohistochemical examination. The rapid growth and histopathologic and cytologic morphology of the tumors suggested the possibility of a neuroendocrine origin for ITFTag carcinomas. The epithelial origin of the cells was confirmed by their uniform staining with anti-cytokeratin antibody (Fig. 4G). Moreover, the cells stained strongly and uniformly positive for the panendocrine marker synaptophysin (Fig. 4H). Thus, ITFTag carcinoma cells seem to be derived from neuroendocrine cells, quite possibly from the enteroendocrine cell population of the colonic epithelium.

Electron microscopic analysis of ITFTag tumors revealed the presence of cells with large, pleiomorphic nuclei and scant cytoplasm (Fig. 5A). Many cells can be seen to contain dense core secretory vesicles (Fig. 5B and C), characteristic of neuroendocrine cells (12). Thus, electron microscopic examination confirms a neuroendocrine phenotype for these cells.



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  		FIGURE 5. A-C. Electron microscopy of ITFTag tumors. Arrows, dense core secretory granules. Bars, 2 µm.

 
Biogenesis of ITFTag Tumors
The development of ITFTag tumors is of interest given their aggressive growth characteristics and unexpected neuroendocrine character. Microscopic analysis of the proximal colons and tumors of ITFTag mice revealed the presence of crypts that seemed to be partially transformed (i.e., which contained both normal and dysplastic colonocytes). This was investigated further first by staining sections of developing ITFTag tumors with antibodies to Tag and synaptophysin (Fig. 6A and B). Here, normal-appearing crypts are apparent in which neither goblet cells nor other cell types exhibit demonstrable Tag expression. Crypts could be seen, however, which contained, in addition to normal cells lacking Tag expression, populations of dysplastic Tag-expressing cells (Fig. 6A). The Tag-expressing cells often seemed to be tightly bunched and spatially disorganized, having nuclei lying above their normal basolateral location. Immunostaining of serial sections revealed that the portions of a crypt that contained Tag-expressing cells also contained synaptophysin-expressing cells (Fig. 6B). This observation suggests two possibilities for the initiation of tumorigenesis: that the Tag-expressing cells are derived from neuroendocrine cells or that non-neuroendocrine cells transdifferentiate after the onset of Tag expression into neuroendocrine cells. To examine this process more precisely, we used double-label fluorescent immunostaining so that the expression of both antigens could be visualized in the same cell. Tag-expressing cells were imaged with green fluorescent dye (Alexa488)and synaptophysin-expressing cells were visualized with red fluorescent dye (Alexa594). The merged image (Fig. 6C) reveals the colocalization of Tag and synaptophysin in the same cells of partially transformed crypts. We used this technique to investigate the development of ITFTag tumors. In normal-appearing crypts, cells expressing detectable levels of Tag are not apparent, although cells expressing synaptophysin are (Fig. 6D and E). These cells are presumably enteroendocrine cells, which express this marker but not Tag. Partially transformed crypts can be seen that contain small groups of cells that express both Tag and synaptophysin (Fig. 6C-F). Importantly, cells that express Tag but not synaptophysin are never visualized. Note that, in Fig. 6E, a synaptophysin-expressing, apparently normal enteroendocrine cell (without Tag expression) can be seen immediately adjacent to a cell expressing both markers. In more advanced regions of ITFTag tumors, cells proliferating between normal-appearing crypts are apparent (Fig. 6F and G) as are solid sheets of tumor (Fig. 6G).



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  		FIGURE 6. Analysis of tumorigenesis in ITFTag mouse proximal colon. A and B. Serial sections showing immunohistochemical detection of crypts containing groups of cells expressing Tag (A) and synaptophysin (B). Arrows, partially transformed crypts containing significant populations of cells expressing these markers. Bars, 25 µm. C. Partially transformed crypts containing populations of cells expressing both Tag (green) and synaptophysin (red) by double-label immunofluorescence microscopy. The merged image confirms coexpression of both markers in the same cells. Arrows, partially transformed crypts. D and E. Sequential steps in tumorigenesis. Arrowheads, enteroendocrine cells expressing synaptophysin but not Tag; short arrows, partially transformed crypts; long arrows, crypts containing a large proportion of transformed cells. Note the presence of a cell expressing both Tag and synaptophysin immediately adjacent to the enteroendocrine cell in E. F. Transformed cells spreading between crypts. G. A region of tumor has replaced nearly all normal crypts.

 
The observation that the duodenum of ITFTag line 7 mice also expressed Tag (Fig. 3) prompted us to examine Tag expression in this tissue. Here, we found that Tag expression was detected in a population of villi epithelial cells (Fig. 7A and C-E). These cells did not express synaptophysin (Fig. 7B-E), nor were they goblet cells as delineated by morphologic criteria. The villi in duodenum were enlarged and dysplastic cells were evident. Tumor formation, however, never occurred. Thus, the ITFTag hybrid oncogene causes expansion of a different population of cells in the small intestine of these animals than it does in the colon.



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  		FIGURE 7. Tag expression in ITFTag line 7 proximal small intestine. A and B. Immunohistochemical detection of Tag (A) and synaptophysin (B) in ITFTag line 7 small intestine. Note that there is a population of epithelial cells that expresses Tag but not synaptophysin. Bars, 100 µm. C-E. Merged images showing Tag expression (green) and synaptophysin (red). Note that epithelial cells expressing Tag do not also express synaptophysin. Arrowheads, enteroendocrine cells that expresses synaptophysin but not Tag.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The transgenic lines described here develop tumors of the proximal colon with remarkable efficiency within their first 3 months of life, with tumors being observed in 32 of 32 ITFTag line 7 mice and 18 of 18 ITFTag line 8 mice. These tumors contain rapidly dividing, aggressive cells and seem to be multifocal in origin. This conclusion is supported by both the large size and efficient development of tumors that envelop the proximal colon and the numbers of partially transformed crypts that are present in the colons of these mice. Metastases to the liver, brain, lungs, or other organs were not apparent on gross examination of these mice. In addition, sections of livers of four ITFTag line 7 mice and two ITFTag line 8 mice were examined for metastases using Tag and synaptophysin immunohistochemistry. No evidence for metastases was found in this study (data not shown). Three samples of lung and two samples of brain were also examined in this way with similar negative results. This may be due to the aggressive and multifocal nature of the primary tumor causing death early in the disease process, before metastases can develop. Alternatively, ITFTag tumors may be inherently nonmetastatic. These tumors express phenotypic markers for epithelial cytokeratins and synaptophysin.

The efficiency with which the ITFTag lines develop tumors enabled elucidation of the early steps of colonic tumorigenesis in these mice. Synaptophysin-staining, apparently normal enteroendocrine cells are readily detected in the proximal colonic epithelium of ITFTag mice. Conversely, cells that express Tag but not synaptophysin cannot be detected (Fig. 6). Crypts containing populations of synaptophysin-expressing and Tag-expressing cells are, however, relatively abundant. A reasonable interpretation of these observations is that cells of the enteroendocrine cell lineage undergo an initial transforming event that results in the activation of the transgene and therefore in Tag expression. This seems to result in a clonal expansion of transformed cells. The clones seem to expand rapidly, which results in crypts containing a large proportion of transformed cells. Examples of such crypts can be seen in Fig. 6D and E. These cells seem to overgrow the normal boundary of their crypts, proliferating into the lamina propria and occupying the space between crypts (Fig. 6F). Finally, the carcinoma cells outgrow the normal crypts, forming solid sheets of undifferentiated tumor (Fig. 6G). The nature of the initial transforming event is not known; however, one possibility is demethylation of the transgene promoter. Previous work has shown a possible role for methylation in ITF promoter regulation (28).

Since the original work of Hanahan (29) using the rat insulin II gene promoter to drive Tag expression in pancreatic ß cells, Tag has been used for the construction of many hybrid oncogenes, yielding cancer models in transgenic mice. Cells of neuroendocrine lineage may be especially susceptible to transformation by SV40. Garabedian et al. (30) unexpectedly obtained transformation of prostate neuroendocrine cells when the cryptdin-2 gene promoter, normally active in intestinal Paneth cells, was used to drive Tag expression in transgenic mice. Other models of prostate cancer employing Tag also result in tumors expressing neuroendocrine markers with varied frequency (31, 32). Moreover, Lee et al. (33) obtained neuroendocrine tumors of the colon and pancreas using the glucagon gene promoter. In work especially relevant to this study, Syder et al. (34) recently described a transgenic model using transcriptional regulatory elements from the B-subunit of the H+,K+-ATPase gene to drive Tag expression in gastric preparietal cells. Here, Tag expression occurred first in a hyperplastic population of preparietal cells that coexpressed the endogenous B-subunit, a marker of this cell type. These cells seemed to transdifferentiate into cancer cells that strongly express neuroendocrine markers and that exhibit weaker staining of Tag and a lack of staining for the B-subunit of the ATPase. Thus, a transdifferentiation step is evident in this model. In the ITFTag model, cells expressing Tag but not synaptophysin were not observed; thus, no evidence for transdifferentiation was found. It should be noted, however, that the data presented cannot formally rule out the possibility of a short-lived, Tag-expressing precursor cell initiating transformation and transdifferentiating into a neuroendocrine cancer cell. Along these lines, Moser et al. (35) noted that adenomas forming in ApcMin/+ mice produced patches of cells of all lineages normally found in the intestine. This suggests that tumorigenesis, at least in this model, may initiate in pluripotent stem cells or from their immediate daughters. Other work indicates the importance of Wnt signaling in maintaining the stem cell compartment in normal intestine as well as for the development of colonic tumors (36, 37). These data suggest that cancer cells may not be capable of arising directly from fully differentiated colonic epithelial cells. Thus, the possibility that ITFTag cancer cells arise from progenitor cells committed to the enteroendocrine cell lineage may be more likely than their being derived from fully differentiated enteroendocrine cells.

Two major roles for Tag in tumorigenesis have been described. Tag binds to and inactivates both Rb and p53 (25-27). By inactivating Rb, Tag removes the normal G1-phase to S-phase cell cycle checkpoint and therefore facilitates uncontrolled cycling (25, 26). By inactivating p53, Tag ablates p53-dependent apoptosis (38, 39). These dual activities certainly account for much of the oncogenic potential of Tag. Other activities have been attributed to Tag as well, which may also contribute to its oncogenic potential. The small splice variant of Tag (small t) transactivates the transcription of some genes and inhibits phosphatase PP2A, which induces cytoskeletal disorganization and tight junction defects (40-42). PP2A is also probably necessary for transformation in some cell types (43). Previous studies have noted the secretion of growth-enhancing substances from SV40-transformed cells (44, 45). All of these activities could contribute to the growth-inducing activities in ITFTag tumors.

ITFTag tumors closely resemble human small cell carcinomas of the colon. These tumors express synaptophysin, indicating neuroendocrine differentiation. Small cell carcinomas grow rapidly, exhibiting many mitotic figures and an undifferentiated pattern of growth (11-13). Carcinoid tumors of the colon also express panendocrine markers such as synaptophysin, but these tumors are relatively slow growing and are well differentiated (46). Also expressing panendocrine markers are the large cell neuroendocrine carcinomas, but these tumors have differentiated growth patterns (46). Like ITFTag tumors, small cell carcinomas of the colon are usually found in the proximal colon. They are morphologically similar to small cell carcinomas of the lung (9). They are often associated with adenomas or adenocarcinomas, suggesting the possibility that they represent clonal outgrowths from these lesions. Unlike ITFTag mice, human patients with small cell carcinoma of the colon usually present with metastases and their prognosis is poor. These human tumors are rare, representing ~1% of all colon cancers (12), but some may be inappropriately classified as poorly differentiated adenocarcinomas; thus, their frequency may be underestimated.

ITF is normally expressed in goblet cells of the small intestine and colon (47). Because the ITFTag hybrid oncogene is expressed in cells of the enteroendocrine cell lineage, the ITF promoter used (–1,190 to +37 bp) is not active in the same cell type as is the endogenous ITF promoter. Because several founder mice exhibited essentially the same pattern of expression, it is doubtful that the site of integration plays a role in governing cell type–specific expression. Rather, the explanation for the difference in expression pattern between the transgene and endogenous ITF probably lies in the length of the promoter used for transgene construction. Podolsky et al. did a detailed series of studies characterizing the ITF promoter (48-50). These studies indicate that goblet cell–specific ITF gene expression is controlled by several positive and negative regulatory elements found in the ITF promoter between –2,216 bp and the start of transcription. A silencer inhibitor element was identified between –2,216 and –2,204 bp that seems to be important for permitting expression in goblet cell–like cell lines (50). Silencer elements were noted near –1,900 and –200 bp and positive regulatory elements were noted near –1,590, –180, and –135 bp (48, 49). It is not clear how these elements would function in vivo in normal tissues. It is certainly likely, however, that goblet cell–specific expression requires a complex series of promoter elements and the segment of the 5'-flanking sequence used to construct the ITFTag hybrid oncogene may not contain all elements required for specificity. The complexity of this regulation is underscored by the observation that, in the small intestine, the transgene is expressed in a different cell lineage than it is in the colon (Figs. 6 and 7).

In a previous study, we observed that Tag expression in small intestinal goblet cells driven by the MUC2 promoter resulted in apoptosis as the cells transited from the crypts to the villi (24). The efficiency of this apoptosis was high enough to cause ablation of almost all villi goblet cells. In the present study, we noted the existence of a population of Tag-expressing cells in ITFTag line 7 small intestinal villi (Fig. 7). Examination of H&E-stained sections as well as TUNEL-stained (51) sections (data not shown) indicated that this Tag-expressing cell population was not apoptotic. Thus, although goblet cells may be shunted into an apoptotic pathway if they continue in the cell cycle after their migration to the villi, other small intestinal epithelial cells apparently do not share this fate. This conclusion is supported by other studies in which Tag was expressed in other villi epithelial cell types (52). This propensity to undergo apoptosis suggests a possible antitumor mechanism especially important in goblet cells.

In summary, the ITFTag transgenic lines described here form aggressive, neuroendocrine cell tumors in the proximal colon of transgenic mice. As such, they constitute an animal model for small cell carcinoma of the colon, a human cancer with an especially poor prognosis. ITFTag tumors seem to be derived from the enteroendocrine cell lineage, suggesting the possibility of this origin for human small cell carcinomas as well. Future studies will focus on the genes expressed by these tumors that enable their rapid initiation and aggressive, invasive growth.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Preparation of the ITF Promoter/Tag Hybrid Oncogene and Development of Transgenic Mice
A bacterial artificial chromosome clone containing the mouse ITF gene (clone RP23-172D20) was purchased from Research Genetics (Huntsville, AL). A segment of the 5'-flanking region of this gene initiating at –1,190 bp and terminating at +59 bp was synthesized by PCR using this clone as target DNA. The forward primer for this reaction was 5'-GGACGCACTGAACTAACTAATGGC and the reverse primer was 5'-TAGCCAGAGGGCTCTGGTATCGATGGCAGCAGGC, with the underlined bases changed from the gene sequence to introduce a ClaI site. The 1,249-bp amplicon was cloned into pCR-4TOPO vector (Invitrogen, Carlsbad, CA), retrieved with EcoRI, given blunt ends with Klenow polymerase, and digested with ClaI. pBluescript (SK–) containing the SV40 early region (24) was digested with SalI, treated with Klenow polymerase, and digested with ClaI. The PCR amplicon was cloned into this construct. The ITFTag hybrid oncogene generated in this procedure initiated at –1,190 bp of the ITF gene promoter and transitioned into the Tag structural gene at +37 bp of the 5'-untranslated region. Following its retrieval by digestion with XhoI and BamHI, transgenic mice were produced at the Transgenic Mouse Facility, University of California (Irvine, CA) by microinjection into C57BL/6 x BALB/c F2 hybrids. Southern blots were used to identify founders, which were mated to C57BL/6 mice obtained from Charles Rivers (Wilmington, MA) to produce transgenic offspring for analysis. The offspring mice used in this study were from F1 to F7 for ITFTag line 7 and from F1 to F5 for ITFTag line 8.

Southern, Northern, and RT-PCR Analyses
The ITFTag transgene was detected in tail DNA following PstI digestion, electrophoresis, and blotting using a full-length Tag probe (24). Bands of 732 and 3,230 bp are predicted assuming complete digestion of concatemeric transgene. Total RNA was isolated from tissues using Tri reagent (Molecular Research Center, Cincinnati, OH) and blots were prepared and processed as described (24). Probes for Tag and glyceraldehyde-3-phosphate dehydrogenase were those used previously (24). A probe containing 63 to 238 bp of mouse ITF was prepared by RT-PCR from normal mouse colon RNA using procedures described previously (53). The forward primer was 5'-CTGTTGGTGGTCCTGGTTGC and the reverse primer was 5'-GGCAACATTTGGGATACTGGAGTC. The 175-bp product was cloned into pCR2.1 vector (Invitrogen) and retrieved with EcoRI for probe isolation. RT-PCR analysis was conducted using primers that span the sole intron excised to generate the large Tag message. The forward primer was 5'-AGGTCTTGAAAGGAGTGCCTGG and the reverse primer was 5-GAGTCAGCAGTAGCCTCATCATCAC. RT reactions were conducted using RNA (1.5 µg) and random primers with SuperScript II reverse transcriptase (Invitrogen) as described (54). PCR amplifications (54) used 30 cycles of 30 seconds at 94°C, 30 seconds at 63°C, and 30 seconds at 72°C. The 309-bp product was visualized by ethidium bromide staining following electrophoresis on 2% agarose gels.

Immunohistochemistry and Electron Microscopy
Tissue samples were fixed with formalin, methacarn, or Bouin's solution and embedded in paraffin by the San Francisco VA Cell Imaging Laboratory Core Facility (San Francisco, CA). Immunohistochemistry was conducted on 5 µm sections using Histostain-Plus kits (Zymed, South San Francisco, CA). Antigen unmasking was done in 10 mmol/L sodium citrate buffer (pH 6.0) by microwaving at high power for 2 minutes and at low power for 13 minutes followed by a 30-minute cooling period. Primary antibodies used were proliferating cell nuclear antigen (clone PC-10, 1:3,000 dilution, Sigma Chemical Co., St. Louis, MO), Tag (SC-148, 1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), multi-cytokeratin (NCL-AE1/AE3, 1:20 dilution, Novocastra, Newcastle upon Tyne, United Kingdom), and synaptophysin (SC-17750, 1:100 dilution, Santa Cruz Biotechnology). All antibody incubations were at 37°C for 1 hour with the exception of multi-cytokeratin, which was at room temperature for 75 minutes. Reaction with 3,3'-diaminobenzidine substrate was for 3 to 4 minutes with all antibodies, except Tag, which was for 12 minutes, followed by counterstaining with hematoxylin. Samples were processed for electron microscopy using uranyl acetate staining as described (24).

Double-Label Immunofluorescence Microscopy
Formalin-fixed paraffin sections were hydrated, treated for 10 minutes in 3% H2O2 in methanol, and subjected to antigen unmasking as described above. Following three rinses in PBS, the sections were treated for 1 hour in Histostain-Plus blocking solution. Sections were incubated 1 hour at 37°C with rabbit anti-Tag (SC-147, Santa Cruz Biotechnology) diluted 1:100 in 1% bovine serum albumin in TBS with 0.1% Tween 20. Following PBS rinsing, the sections were treated with the Histostain-Plus biotinylated secondary antibody solution. After rinsing, the sections were treated with a 1:1,000 dilution of streptavidin-Alexa488 (Molecular Probes, Eugene, OR) conjugate for 30 minutes. Sections were again blocked and incubated with 1:100 rabbit anti-synaptophysin (catalogue no. 18-0130, Zymed) for 1 hour at 37°C. Goat anti-rabbit-Alexa594 conjugate (Molecular Probes) was used at 1:1,000 dilution to detect the anti-synaptophysin. The slides were mounted with Prolong Antifade mounting medium (Molecular Probes). Slides were examined with a Zeiss Axioplan 2 fluorescent microscope (Zeiss, Thornwood, NY). Images were captured and merged with Open Lab software.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Sandra Huling and Juan Engel (San Francisco VA Cell Imaging Laboratory) for their aid with electron and fluorescence microscopy, respectively, and Tom Fielder (Transgenic Mouse Facility, University of California) for transgenic mouse production.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Department of Veterans Affairs Medical Research Service and the Theodora Betz Foundation and NIH grants RR14905 and CA84294 from the Comparative Medicine Program, National Center for Research Resources and the National Cancer Institute, NIH (A.D. Borowsky and R.D. Cardiff). Back

Received February 26, 2004; revised July 1, 2004; accepted July 16, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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