Glucagonlike Peptide 2 Analogue Teduglutide Stimulation of Proliferation but Reduction of Differentiation in Human Caco-2 Intestinal Epithelial Cells

Research

Original Investigation | ASSOCIATION OF VA SURGEONS Glucagonlike Peptide 2 Analogue Teduglutide Stimulation of Proliferation but Reduction of Differentiation in Human Caco-2 Intestinal Epithelial Cells

Lakshmi S. Chaturvedi, PhD; Marc D. Basson, MD, PhD, MBA

IMPORTANCE Short bowel syndrome occurs when a shortened intestine cannot absorb sufficient nutrients or fluids. Teduglutide is a recombinant analogue of human glucagonlike peptide 2 that reduces dependence on parenteral nutrition in patients with short bowel syndrome by promoting enterocytic proliferation, increasing the absorptive surface area. However, enterocyte function depends not only on the number of cells that are present but also on differentiated features that facilitate nutrient absorption and digestion.

OBJECTIVE To test the hypothesis that teduglutide impairs human intestinal epithelial differentiation.

DESIGN AND SETTING We investigated the effects of teduglutide in the modulation of proliferation and differentiation in human Caco-2 intestinal epithelial cells at a basic science laboratory. This was an in vitro study using Caco-2 cells, a human-derived intestinal epithelial cell line commonly used to model enterocytic biology.

EXPOSURE Cells were exposed to teduglutide or vehicle control.

MAIN OUTCOMES AND MEASURES We analyzed the cell cycle by bromodeoxyuridine incorporation or propidium iodide staining and flow cytometry and measured cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay. We used quantitative reverse transcription–polymerase chain reaction to assay the expression of the enterocytic differentiation markers villin, sucrase-isomaltase, glucose transporter 2 (GLUT2), and dipeptidyl peptidase 4 (DPP-4), as well as that of the putative differentiation signals schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2). Villin promoter activity was measured by a luciferase-based assay.

RESULTS The MTS assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated controls at a maximal 500nM (n = 6, P < .05). Teduglutide increased bromodeoxyuridine-positive cells vs untreated controls by a mean (SD) of 19.4% (2.3%) vs 12.0% (0.8%) (n = 6, P < .05) and increased the S-phase fraction by flow cytometric analysis. Teduglutide reduced the mean (SD) expression of villin by 29% (6%), Cdx2 by 31% (10%), DPP-4 by 15% (6%), GLUT2 by 40% (11%), SLFN12 by 61% (14%), and sucrase-isomaltase by 28% (8%) (n = 6, P < .05 for all). Author Affiliations: Department of Surgery, College of Human Medicine, Michigan State University, East CONCLUSIONS AND RELEVANCE Teduglutide increased Caco-2 proliferation but tended to Lansing (Chaturvedi, Basson); inhibit intestinal epithelial differentiation. The effects of mitogenic stimulation with Research Service, John D. Dingell Veterans Affairs Medical Center, teduglutide in patients with short bowel syndrome might be greater if the more numerous Detroit, Michigan (Chaturvedi, teduglutide-treated cells could be stimulated toward a more fully differentiated phenotype. Basson); Department of Anesthesiology, Wayne State University, Detroit, Michigan (Chaturvedi). Corresponding Author: Marc D. Basson, MD, PhD, MBA, Department of Surgery, College of Human Medicine, Michigan State University, 1200 E Michigan Ave, Ste 655, JAMA Surg. 2013;148(11):1037-1042. doi:10.1001/jamasurg.2013.3731 Lansing, MI 48912 (marc.basson Published online September 25, 2013. @hc.msu.edu).

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mall intestine failure occurs when the mucosa becomes Cell Culture atrophic after prolonged starvation or when 1 or more We studied Caco-2 brush border enzyme intestinal epithelial S bowel resections decrease the amount of small intestinal cells, a subclone of the original Caco-2 cell line, selected for their surface area available to interact with luminal nutrients. Criti- ability to differentiate in culture toward an enterocytic pheno- cally ill patients who have not received enteral feeding may ex- type as indicated by the formation of an apical brush border and hibit mucosal barrier failure early, leading to bacterial translo- the expression of brush border enzymes.12 We maintained these cation and a septic response.1 After recovery from their acute cells at 37°C with 8% carbon dioxide in Dulbecco modified Eagle events, such patients may have difficulty readapting to enteral medium with 4500 mg/L of D-glucose, 4mM glutamine, 1mM nutrition because the atrophied mucosa is unable to handle the sodium pyruvate, 100 U/mL of penicillin, 100 μg/mL of strep- digestive load. After massive small-bowel resection, patients ex- tomycin, 10 μg/mL of transferrin, 10mM 4-(2-hydroxyethyl)-1- perience more pronounced difficulties. Although adaptation to piperazineethanesulfonic acid (pH 7.4), and 3.7 g/L of sodium short bowel syndrome (SBS) occurs,2 its capability is limited, and bicarbonate, supplemented with 10% fetal bovine serum. All many patients require permanent total parenteral nutrition or studies were performed on the cells within 15 passages. small-bowel transplantation, each of which has well-recognized attendant morbidities and a substantial long-term mortality Proliferation rate.3,4 Proliferation was assessed using a colorimetric MTS assay as We attempt to palliate such patients by manipulating their described by the manufacturer (Promega Corporation). Briefly, diets in composition and frequency to make it more readily di- Caco-2 cells (10 000 cells per 96-well plate) were plated in cell gestible, adding antiperistaltic agents to slow motility and in- culture medium for 24 hours. On the next day, the cells were crease the dwell time of the nutrients within the gut, as well as incubated with 0 to 1000nM teduglutide in serum-free me- treating bacterial overgrowth where appropriate. However, the dium for 72 hours. The experiment was terminated by adding main modalities of treatment for SBS focus on the stimulation 20 μL of 1 mg/mL of MTS solution to each of 96 wells. After 30 of proliferation. Supplementation of growth hormone, glutamine, minutes of incubation in the dark at 37°C, the absorbance of and enteral nutrition has been effective in promoting intestinal each well was measured at 490 nm using a microplate reader adaptation in selected patients with SBS.5-7 It has also been re- (Molecular Devices, LLC). ported that glutamine acts better in combination with growth hormone in animal investigations.8 Recently, glucagonlike pep- Bromodeoxyuridine Incorporation Assay tide 2 (GLP-2) and the GLP-2 analogue teduglutide (ALX-0600) Bromodeoxyuridine (BrdU) incorporation was assessed using have been reported to promote intestinal growth in patients with a fluorescein isothiocyanate conjugated BrdU flow kit as de- SBS.9,10 Teduglutide is believed to be more biologically active than scribed by the manufacturer (BD Biosciences). Briefly, Caco-2 native GLP-2 in stimulating intestinal epithelial proliferation be- cells (10 000 cells per 96-well plate) were plated in cell cul- cause teduglutide is resistant to GLP-2 degradation by dipepti- ture medium for 24 hours. On the next day, the cells were in- dyl peptidase 4 (DPP-4),9,11 but its effect on the regulation of incubated with 500nM teduglutide and human epidermal growth testinal epithelial differentiation has not been studied. factor in serum-free medium for 72 hours. Before termina- In the present study, we investigated the effects of teduglu- tion of experiments, the cells were incubated with 10μM BrdU tide in the modulation of proliferation and differentiation in hu- for 6 hours before flow analysis. man Caco-2 intestinal epithelial cells. We hypothesized that teduglutide impairs human intestinal epithelial differentiation. To Cell Cycle Analysis test this hypothesis, we compared the expression of villin, DPP- Caco-2 cells (10 000 cells per 96-well plate) were plated in cell 4, sucrose-isomaltase, glucose transporter 2 (GLUT2, also known culture medium for 24 hours. The next day, the cells were in- as SLC2A2), and caudal-related homeobox intestine-specific tran- cubated with 500nM teduglutide in serum-free medium for 72 scription factor (Cdx2), as well as the putative differentiation hours. The cells were harvested and washed twice with ice- marker schlafen 12 (SLFN12), in the presence or absence of tedu- cold phosphate-buffered saline containing 50% fetal bovine se- glutide. We also assessed the effects of teduglutide on Caco-2 cell rum and then fixed with 70% ethanol for 1 hour and incu- proliferation to verify biological activity in this model. bated in 0.35 mL of phosphate-buffered saline containing 50 μL/mL of propidium iodide (Sigma-Aldrich), 66 U/mL of RNase (Invitrogen), and 10% Triton X-100 (Sigma-Aldrich) on ice for Methods 60 minutes. The DNA content analysis was performed by a FAC- Scan with CellQuest software (BD Biosciences). Materials Dulbecco modified Eagle medium was obtained from Sigma- RNA Isolation and Quantitative Reverse Aldrich. Penicillin-streptomycin and 0.5% trypsin–EDTA were Transcription–Polymerase Chain Reaction from Gibco. The 3-(4,5-dimethylthiazol-2-yl)-5-(3- The RNA was extracted using the RNeasy kit (Qiagen). The carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium complementary DNA (cDNA) generated was amplified using (MTS) inner salt was purchased from Promega Corporation. Te- SYBR Green Real-time PCR Master Mix on an Applied Biosys- duglutide (CPZ1435 HD-33) was obtained from Creative Pep- tems 7500 Real-time PCR System (Invitrogen). Expression lev- tides Inc. Human epidermal growth factor and other com- els were determined from the threshold cycle values using the mon laboratory reagents were obtained from Sigma-Aldrich. method of 2−ΔΔCt with 18S expression as the reference control

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gene. Human 18S primers used were 5′-CGCCGGTC- Figure 1. Effect of Teduglutide on Human Caco-2 Intestinal Epithelial Cell CAAGAATTTCACCTCT-3′ (upstream) and 5′-CCCTCGATGCTCT- Proliferation TAGCTGAGTGT-3′ (downstream). Human villin primers used

were 5′-TGCTATCTATGGTGTGGGAAGG-3′ (upstream) and 5′- A a a TCCTGTAGTCTCTTGGTGTTGG-3′ (downstream). Other mark- 120 a ers used were the following: SLFN12 (forward 5′-ATCTGGGCT- TGCAAGAGAAC-3′ and reverse 5′-TTTTTGCCAGCTTCTGCTTT- 100 3′), Cdx2 (forward 5′-CAACCTGGACTTCCTGTCAT-3′ and reverse 5′-CACAGACCAACAACCCAAAC-3′), DPP-4 (forward 5′- 80 TTTGGGGCTGGTCATATGGAGGG-3′ and reverse 5′- ACTCCCACCGGGATACAGGGG-3′), GLUT2 (forward 5′- 60 AGCTGCATTCAGCAATTGGACCTG-3′ and reverse 5′- ATGTGAACAGGGTAAAGGCCAGGA-3′), and sucrase- 40

isomaltase (forward 5′-AAACCTACATGTGGGTGGTGGTCA-3′ % of Control Cell Proliferation and reverse 5′-AACAGAGAACCCTGTGCCATCTGA-3′). The cycle 20 conditions for the polymerase chain reaction were 1 cycle of 5 0 minutes at 95°C and 40 cycles of 15 seconds at 95°C, 30 sec- 062 125 250 500 1000 onds at the annealing temperature (60°C), and 30 seconds at Teduglutide, nM B 60°C for extension. a 30 Statistical Analysis 25 All experiments were performed independently at least 3 times, a

with similar results. Data sets were analyzed using paired or 20 unpaired t tests with Bonferroni correction as appropriate. Sta- tistical significance was set at P < .05. 15

10 % of BrdU-Positive Cells % of BrdU-Positive

Results 5 Teduglutide Increases Caco-2 Intestinal 0 Epithelial Cell Proliferation Teduglutide (500nM) – + – Teduglutide significantly increased cell numbers at higher con- EGF (50 ng/mL) – – + centrations (250-1000nM) compared with cell numbers in untreated control cell populations (Figure 1A). We further con- The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- firmed the effect of 500nM teduglutide on cell proliferation sulfophenyl)-2H-tetrazolium (MTS) dye assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated control cells with BrdU incorporation, followed by flow analysis. The cells at a maximal 500nM (n = 6, P < .05). A, Teduglutide (250-1000nM) incubated with 500nM teduglutide exhibited a greater mean significantly increased cell numbers compared with untreated control cells as (SD) proportion of BrdU-positive cells than untreated control measured by the MTS assay. B, Teduglutide (500nM) and human epidermal cells (21.1% [1.5%] vs 12.0% [0.8%], n = 6, P < .05) (Figure 1B). growth factor (EGF) (50 ng/mL), used as a positive control, significantly increased the proportion of bromodeoxyuridine (BrdU)–positive cells vs The cells incubated with a positive control epidermal growth untreated control cell populations. factor (50 ng/mL) also exhibited a greater mean (SD) propor- aP < .05. Gray bar indicates treated cells. The plus sign and minus sign indicate tion of BrdU-positive cells than untreated control cells (26.7% whether the cells received or did not receive what is written on the side in the figure. [3.7%] vs 12.0% [0.8%], n = 6, P < .05) (Figure 1B).

Teduglutide Increases S-phase Cells in the Cell Cycle lutide reduced the mean (SD) expression of differentiation of Intestinal Epithelial Cells marker transcripts: villin was reduced by 29% (6%), DPP-4 by We further performed cell cycle analysis using propidium io- 15% (6%), sucrase-isomaltase by 28% (8%), and GLUT2 by 40% dide staining, followed by flow analyses. Teduglutide also in- (11%) (n = 6, P < .05 for all) (Figure 3). creased the proportion of diploid cells in S phase compared with the S-phase fraction of untreated control cell populations Teduglutide Decreases the Expression of Intracellular (Figure 2A and B). Signal Proteins Human Caco-2 intestinal epithelial cells were treated with Teduglutide Decreases the Expression of Enterocytic 500nM teduglutide in serum-free medium for 72 hours, and Differentiation Markers quantitative reverse transcription–polymerase chain reac- Human Caco-2 intestinal epithelial cells were treated with tion was performed with cDNA amplified from RNA. We mea- 500nM teduglutide in serum-free medium for 72 hours, and sured the effect of teduglutide on the expression of 2 intracel- quantitative reverse transcription–polymerase chain reac- lular signals (Cdx2 and SLFN12) that might influence enterocytic tion was performed with cDNA amplified from RNA. Tedug- proliferation and differentiation. Teduglutide reduced the

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Figure 2. Effect of Teduglutide on the Human Caco-2 Intestinal Epithelial Figure 3. Effect of Teduglutide on the Expression of Differentiation Cell Cycle Marker Transcripts in Human Caco-2 Intestinal Epithelial Cells

A Control Debris Villin 600 Aggregates DPP-IV 1.0 Dip G1 a SI

500 Dip G2 rRNA a GLUT2 0.8 a

Dip S 18S a 400 0.6

300 0.4 No.

0.2 200

Relative Expression of mRNA/ Expression Relative 0 100 ––– –+++ + Teduglutide (500nM)

0 Teduglutide reduced the expression of villin, dipeptidyl peptidase 4 (DPP-4), 0 50 100 150 200 250 sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) differentiation Channels (PI-A) markers compared with untreated control cells as measured by quantitative reverse transcription–polymerase chain reaction (n = 6, P < .05 for all). mRNA Teduglutide (500nM) indicates messenger RNA; rRNA, ribosomal RNA. 600 aP < .05. The plus sign and minus sign indicate whether the cells received or did not receive what is written on the side in the figure.

500

mean (SD) expression of the Cdx2 by 31% (10%) and of SLFN12 400 by 61% (14%) (n = 6, P < .05 for both) (Figure 4).

300 No. Discussion 200 Small intestinal mucosal function is likely to be determined

100 by the total absorptive surface area available to interact with luminal contents and by the phenotype of the enterocytes that line the lumen. Although some nutrient transport is passive, 0 0 50 100 150 200 250 most digestive functions require active transport proteins or Channels (PI-A) digestive enzymes that are increasingly expressed as the enterocyte matures. This study suggests that mitogenic stimuli a B such as teduglutide, although increasing the number of en- 20 terocytes available and the absorptive surface area, may not proportionately increase the amount of mucosal protein avail- 15 able for these energy-dependent critical digestive tasks. There are many different markers of the mature entero- 10 cytic phenotype. Enzymes of the brush border membrane and transport proteins are characteristic features of the differen- 5 tiated enterocytes.13 For this study, we selected villin, DPP-4, % of S-phase Cell Cycle sucrose-isomaltase, and GLUT2. Villin is a calcium-regulated 0 –+actin-binding protein of the microvillus core of the brush bor- Teduglutide (500nM) der, expressed in crypts and villi of the small and large intestines.13,14 Expressed in the intestinal brush border of ma- Teduglutide increased bromodeoxyuridine-positive cells vs untreated control ture enterocytes, DPP-4 and its expression are controlled by cells by a mean (SD) of 19.4% (2.3%) vs 12.0% (0.8%) (n = 6, P < .05) and 15,16 increased the S-phase fraction by flow cytometric analysis. Teduglutide diet composition. Sucrose-isomaltase is a glucosidase en- (500nM) significantly increased the diploid (Dip) S-phase fraction compared zyme expressed in the brush border of mature enterocytes and with untreated control cells as measured by flow cytometry. A, Typical cell cycle is increasingly expressed during developmental maturation of distribution. B, Our results. PI-A indicates propidium iodide A. 17,18 aP < .05. Black bar indicates treated cells. The plus sign and minus sign indicate the intestine. The levels of the fructose and glucose trans- whether the cells received or did not receive what is written on the side in the porter GLUT2 are regulated by components of diet such as fruc- figure. tose and fat,19 and increased expression of GLUT2 and the hexose transporter sucrose-isomaltase has been reported after

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tunately, the pharmaceutical company that manufactures Figure 4. Effect of Teduglutide on the Expression of 2 Intracellular Signal Proteins That May Influence Differentiation in Human Caco-2 Intestinal teduglutide declined to make human biopsy samples from pre- Epithelial Cells clinical testing available for external study. Nevertheless, in- vestigators have commonly used human Caco-2 intestinal epithelial cells,12,25-28 so these findings suggest the possibility that

Cdx2 overdriving cell proliferation may result in a population of en- 1.0 SLFN12 terocytes that is on average less mature and less differentiated than at baseline. These results do not mean that tedug- a rRNA 0.8 lutide or other mitogenic agents, such as growth hormone, 18S should not be used in SBS. Indeed, some evidence suggests that 0.6 these agents can help to facilitate weaning from total paren- a teral nutrition in selected subgroups of patients with SBS. It

0.4 has been reported that treatment with teduglutide reduced de- pendency on parenteral nutrition for some patients with SBS 29-31 0.2 and intestinal failure. However, many of the patients who benefited by demonstrating reduced parenteral fluid depen- Relative Expression of mRNA/ Expression Relative dency in these studies still required some parenteral fluids and 0 –++– total parenteral nutrition, while mitogenic stimuli alone seem Teduglutide (500nM) insufficient to benefit patients with extreme short gut syn- dromes. In addition, the effects of growth hormone or tedu- Teduglutide reduced the expression of schlafen 12 (SLFN12) and caudal-related glutide regress rapidly if the drugs are stopped because of the homeobox intestine-specific transcription factor (Cdx2) compared with untreated control cells as measured by quantitative reverse transcription– rapid progression of migration from crypt to villous tip and then polymerase chain reaction (n = 6, P < .05 for both). mRNA indicates messenger cell loss in vivo as mature enterocytes are shed into the lu- RNA; rRNA, ribosomal RNA. men. Therefore, others have raised concerns about the possi- aP < .05. The plus sign and minus sign indicate whether the cells received or did bility of neoplastic transformation in response to such long- not receive what is written on the side in the figure. term mitogenic stimuli.32 These results suggest that it may be useful to focus at least some future efforts on the promotion polyamine-induced differentiation in vivo.20 Both GLUT2 and of enterocytic differentiation as an alternative to or a syner- sucrose-isomaltase are commonly used markers of entero- gistic treatment with mitogenic interventions. Several stimuli cytic differentiation.13-16,20 have been reported to promote such differentiation, includ- The mechanism of the apparent inhibition of differentia- ing transforming growth factor β,33,34 gastrin-releasing pep- 35 36 37 tion by teduglutide is not clear. It is possible that more rapid pro- tide and its receptor, bombesin, calcium and vitamin D3, liferation does not allow adequate time for differentiation. Al- Notch pathway,38 and sodium butyrate.39 ternatively, GLP-2 stimulation not only may initiate mitogenic It seems possible that the physical stimulation of repeti- signals but also might directly inhibit some of the intracellular tive deformation that occurs during peristalsis or villous signals that trigger differentiation. A member of the caudal- motility may facilitate enterocyte differentiation.23,40,41 related homeobox gene family of transcription factors, Cdx2 is Early low-level feeding, which stimulates such mechanical involved in enterocyte lineage specification. The function of activity by the gut, may be trophic for the gut mucosa even Cdx2 within the cell is to induce differentiation and inhibit pro- in the presence of adequate parenteral nutrition. This is well liferation at the level of gene transcription.21 It activates many established in the critical care literature but is perhaps not intestine-specific genes such as sucrase-isomaltase and lactase- applied universally. phlorizin hydrolase.22 Schlafen 12 is a member of the schlafen There are other phenotypic characteristics of effective superfamily that is expressed in humans. Rat schlafen 3 is or- enterocytic differentiation that we have not studied, and in thologous to human SLFN12 and has recently been implicated vivo investigations might yield different results, particularly in intestinal epithelial differentiation.23,24 Therefore, the tedu- in the aberrant neuroendocrine environment that is likely to glutide-induced decreases in Cdx2 or SLFN12 could contribute characterize patients with SBS. Nevertheless, these results to diminished differentiation in teduglutide-treated intestinal raise a caution that purely mitogenic stimuli may have sub- epithelial cells. This question awaits further study. optimal effects on the small intestinal epithelium. Combin- It would be important to confirm these studies in vivo be- ing a mitogenic stimuli, such as teduglutide, with a differen- cause there are obvious differences between cell lines in cul- tiating agent might improve weaning from total parenteral ture and the intestinal epithelium in an intact human. Unfor- nutrition in patients with extreme SBS.

ARTICLE INFORMATION Author Contributions: Drs Chaturvedi and Basson Analysis and interpretation of data: All authors. Accepted for Publication: May 13, 2013. had full access to all the data in the study and take Drafting of the manuscript: All authors. responsibility for the integrity of the data and the Critical revision of the manuscript for important Published Online: September 25, 2013. accuracy of the data analysis. intellectual content: All authors. doi:10.1001/jamasurg.2013.3731. Study concept and design: Basson. Statistical analysis: Chaturvedi. Acquisition of data: All authors.

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Administrative, technical, and material support: colon carcinoma cells. Cancer Res. 29. Jeppesen PB, Gilroy R, Pertkiewicz M, Allard JP, Chaturvedi. 1988;48(7):1936-1942. Messing B, O’Keefe SJ. Randomised Study supervision: All authors. 14. Kato M, Kusumi T, Tsuchida S, Tanaka M, Sasaki placebo-controlled trial of teduglutide in reducing Conflict of Interest Disclosures: None reported. M, Kudo H. Induction of differentiation and parenteral nutrition and/or intravenous fluid peroxisome proliferator–activated receptor γ requirements in patients with short bowel Funding/Support: This work was supported in part syndrome. Gut. 2011;60(7):902-914. by grant 1 R56 DK096137-01 from the National expression in colon cancer cell lines by troglitazone. Institutes of Health (Dr Basson). J Cancer Res Clin Oncol. 2004;130(2):73-79. 30. Jeppesen PB, Pertkiewicz M, Messing B, et al. Teduglutide reduces need for parenteral support Role of the Sponsor: The National Institutes of 15. Darmoul D, Lacasa M, Baricault L, et al. Dipeptidyl peptidase IV (CD 26) gene expression in among patients with short bowel syndrome with Health had no role in the design and conduct of the intestinal failure. Gastroenterology. study; collection, management, analysis, and enterocyte-like colon cancer cell lines HT-29 and Caco-2. J Biol Chem. 1992;267(7):4824-4833. 2012;143(6):1473-1481.e3. www.gastrojournal.org interpretation of the data; and preparation, review, /article/S0016-5085(12)01316-9/abstract?referrer or approval of the manuscript; and decision to 16. Basson MD, Emenaker NJ, Rashid Z. Effects of =http://www.ncbi.nlm.nih.gov/pubmed/?term submit the manuscript for publication. modulation of tyrosine phosphorylation on brush =Gastroenterology.%202012;143(6):1473. Previous Presentation: Presented at the 37th border enzyme activity in human Caco-2 intestinal Accessed August 10, 2013. Annual Surgical Symposium of the Association of epithelial cells. 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