Gastroenterology

Gastroenterology

Volume 115, Issue 5, November 1998, Pages 1144-1153
Gastroenterology

Alimentary Tract
Oncogenic ras induces gastrin gene expression in colon cancer,☆☆

https://doi.org/10.1016/S0016-5085(98)70085-XGet rights and content

Abstract

Background & Aims: The expression of gastrin, as a tumor growth factor, is significantly increased in some colon cancers compared with the low levels found in normal mucosa. The aim of this study was to elucidate the transcriptional mechanisms of gastrin induction in colon cancer. Methods: Gastrin messenger (mRNA) levels and K-ras genotype were determined in colon cancer cell lines and surgical specimens. Colon cancer cells were transfected with oncogenic ras expression vectors, and transcriptional activity was assayed with gastrin-luciferase reporter genes. Results: Colon cancer cell lines and tissues with K-ras mutations all had significantly higher gastrin mRNA levels than those that were ras wild type. Treatment of several ras mutant cell lines with PD98059, an inhibitor of mitogen-activated protein kinase kinase, resulted in a decrease in endogenous gastrin mRNA levels. The effects of ras on gastrin expression appeared to be mediated through the gastrin promoter because transfection of oncogenic ras and activated raf expression vectors both induced gastrin-promoter, luciferase-reporter genes. The inductive effects of oncogenic ras could be blocked by the coexpression of dominant negative forms of raf and extracellular regulated kinase. Conclusions: Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway.

GASTROENTEROLOGY 1998;115:1144-1153

Section snippets

Cell lines and tissues

All cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in the following media: RPMI 1640 (Colo320HSR and Colo205), Dulbecco's modified Eagle medium with high glucose (HT29, Hct116, Caco-2, and LoVo), and Leibovitz's L-15 (SW480), each supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) and 2% penicillin/streptomycin (10,000 U/mL). Surgical specimens were snap-frozen on resection and stored at −80°C. Treatment of cells

Gastrin mRNA levels in colon cancer

A sensitive RPA was developed for the detection and quantification of gastrin mRNA levels in colon cancer cell lines and tissue samples obtained from surgical resection. Antisense riboprobes derived from human gastrin (exon 2) and cyclophilin gene templates were used in each assay. Cyclophilin mRNA, transcribed from a ubiquitous housekeeping gene, served as an internal control for mRNA integrity and reaction conditions. Total RNA samples from seven colon cancer cell lines and two pairs of human

Discussion

Several studies have noted that colon cancer tumors often have increased levels of gastrin peptides, particularly incompletely processed forms.14, 15, 18 Normal gastrin expression in the colon appears to be much higher during fetal development than after maturity.17 Hence, the elevated expression of gastrin in colon cancer likely occurs through oncofetal transcriptional mechanisms, which to date have not been well characterized. The results of this study establish that oncogenic activation of

Acknowledgements

The authors thank Drs. Channing Der (kinase expression vectors and p(Py)2Luc), Richard Rippe (pGL Col3 Luc), and Timothy Wang (pGem gastrin exon 2) for providing plasmids; Drs. David Brenner, Cyndi Bradham, Laura Licato, and Martin McMahon for helpful discussions; Dr. Shrikant Bangdiwala for advice about statistical analysis; and Dr. Kevin Behrns (University of North Carolina) and Tissue Procurement facilities at the Lineberger Cancer Center (University of North Carolina) and Massachusetts

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      Citation Excerpt :

      It is, therefore, interesting to notice that the new ligand of F1-ATPase that we have identified in the present study is also overexpressed in these tumor cells. Indeed, the gene coding for gastrin is a target of oncogenic pathways frequently activated in colon cancer such as APC/β-catenin or Ras pathways (38–41). Therefore, high concentrations of gastrin precursors, including G-gly have been observed in colon tumors and in blood of patients with colorectal cancer (42–45).

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    Address requests for reprints to: Loyal G. Tillotson, M.D., Ph.D., CB 7038, Room 148, Glaxo Building, University of North Carolina, Chapel Hill, North Carolina 27599-7038, e-mail: [email protected]; fax: (919) 966- 7468.

    ☆☆

    Supported by grant R29 DK49860 from the National Institutes of Health, a grant from the Glaxo Institute of Digestive Health Basic Research, and grant P30DK34987 from the Center for Gastrointestinal Biology and Disease.

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