Gastroenterology

Gastroenterology

Volume 124, Issue 5, May 2003, Pages 1348-1357
Gastroenterology

Basic-alimentary tract
Progastrin stimulates murine colonic epithelial mitosis after DNA damage

https://doi.org/10.1016/S0016-5085(03)00288-9Get rights and content

Abstract

Background & Aims:

Transgenic mice that overexpress progastrin are more susceptible than either wild-type mice or mice that overexpress amidated gastrin to chemical carcinogen-induced colonic adenomas. We have investigated whether alterations in the regulation of apoptosis or mitosis after DNA damage contribute to the effects of progastrin on murine colonic epithelium.

Methods:

Apoptosis and mitosis were assessed on a cell positional basis in murine intestinal epithelium after γ-irradiation. Mice analyzed were progastrin overexpressing, gastrin overexpressing, gastrin knockout, and their wild-type counterparts. The expression of cell cycle regulators was analyzed by gene array and Western blotting.

Results:

Apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice 4.5 hours after 8 Gy γ-radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy γ-radiation in wild-type, gastrin-knockout, and gastrin-overexpressing mice. However, significant colonic mitosis persisted in progastrin-overexpressing mice up to 24 hours after 8Gy γ-radiation. Increased levels of cdk4 and cyclin D1 proteins were found in the colonic epithelium of progastrin-overexpressing mice relative to wild-type animals after γ-radiation.

Conclusions:

After DNA damage by γ-radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin-overexpressing mice. Persistently elevated cdk4 and cyclin D1 in progastrin overexpressing mice accounts for the capacity of colon cells to continue with the cell cycle after DNA damage.

Section snippets

Animals

The following mouse strains were used: INS-GAS, hGAS, GAS-KO, FVB/N (wild-type counterparts of INS-GAS and hGAS), and C57BL/6 (wild-type counterparts of GAS-KO). hGAS mice are transgenic for 1.3 kb of the human gastrin promoter and the coding sequence of human preprogastrin, resulting in overexpression of the transgene in hepatocytes and elevated (1–100 nmol/L) serum levels of human progastrin.13 INS-GAS mice contain a gastrin transgene consisting of 0.4 kb of the insulin promoter upstream of

In hGAS mice, colonic mitosis persists after γ-irradiation

Mitotic figures were present in the colonic crypts of unirradiated FVB/N and hGAS mice (Figure 1A and C), and apoptotic bodies were seen at the base of colonic crypts from both strains after 8 Gy γ-irradiation (Figure 1B and D). However, hGAS mice additionally showed persistent mitotic figures in colonic crypts after 8Gy γ-irradiation (Figure 1D).

These findings have been confirmed by quantitative analysis of apoptotic and mitotic indices in the small intestinal and colonic crypts of hGAS and

Discussion

We have investigated the effects of expression of gastrin and its precursor peptides on spontaneous and γ-radiation–induced apoptosis and mitosis in murine intestinal epithelia. The most important observation was of higher levels of mitosis (P < 0.01) in the colonic crypts of hGAS mice compared with INS-GAS, wild-type (FVB/N), and GAS-KO animals at various times after 8 Gy γ-radiation.

There has been considerable recent interest in the role of gastrin and its precursors in colon cancer. Because

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      We therefore administered the IGF-1R antagonist AG1024 to hGAS and C57BL/6 mice and assessed distal colonic crypt length (Figure 6A), mitotic figures per hemi-crypt (Figure 6C), Ki67-positive cells per hemi-crypt (Figure 6D), DCAMKL-1–positive cells per hemi-crypt (Figure 6B), and relative expression of Lgr5 transcripts (Figure 7) 52 hours after drug administration.27 As previously described,13,15,23 cell number per hemi-crypt, mitotic figures per hemi-crypt, number of DCAMKL-1–positive cells per hemi-crypt, and Lgr5 expression were significantly increased in the colons of DMSO-treated hGAS mice compared with C57BL/6 mice (Figures 5–7 and Supplementary Figure 2). Administration of AG1024 to hGAS mice, however, resulted in significant reductions in the number of total cells, mitotic cells, DCAMKL-1–positive cells, and Lgr5 messenger RNA (mRNA) transcripts in the colon compared with DMSO-treated hGAS mice.

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    Supported by the North West Cancer Research Fund. Dr. Pritchard holds an Advanced Fellowship for Clinicians from the Wellcome Trust.

    1

    The authors thank Prof. R. Dimaline for useful discussions.

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