Combination of aging and dimethylhydrazine treatment causes an increase in cancer—stem cell population of rat colonic crypts

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Abstract

Aging is associated with increased incidence of colon cancers. It is also becoming evident that cancer stem cells (CSC) play a vital role in the pathogenesis and prognosis of colon cancer. Recently, we reported the presence of colon cancer stem-like cells in macroscopically normal mucosa in patients with adenomatous polyps and that they increase with aging, suggesting that aging may predispose the colon to carcinogenesis. In the current study we have examined the combined effects of aging and carcinogen exposure on the status of colon CSCs in an experimental model. We used young (4–6 months) and aged (22–24 months) rats and exposed them to the carcinogen, dimethylhydroxide (DMH). We investigated the expression of colon cancer stem cell markers, CD44, CD166, EpCam, and ALDH1 as well as EGFR expression in normal colonic crypt epithelium following carcinogen treatment. Our results demonstrate that aging per se or carcinogen treatment alone causes an increase in the number of colon cancer stems cells, as evidenced by increased immunoreactive-CSC-markers positive cells in the colonic mucosa. In aged rats, carcinogen exposure results in a more pronounced increase in colon cancer stem cells. Our study shows that in aging colon the effects of carcinogens are more pronounced, and an increase in colon CSCs is one of the earliest changes preceding tumor development. Moreover, the current investigation of the use of a panel of immunohistochemical markers of colon CSC can potentially serve as a prognostic marker during screening for colon cancer.

Introduction

A growing body of evidence supports the contention that epithelial cancers including the colorectal cancer are diseases driven by a small set of self renewing cells, termed cancer stem cells (CSC) or cancer-initiating cells, that are distinct from the bulk of the cells in the tumor [1]. CSCs are widely believed to arise from the normal stem cells or progenitor cells upon mutation(s) [1], [2], [3]. Two recent transgenic animal studies have demonstrated that crypt stem cells are the cell of origin of intestinal cancers [4], [5]. CSC possess not only the ability to self-renew but also differentiate and can give rise to heterogeneous tumors [1], [2], [3], [4], [5]. Currently, CSCs are identified by specific surface epitopes. Several stem cell markers have been proposed that highlight the stem cell or stem cell-like populations in several tissues including breast, colon and pancreas [6], [7]. Most commonly used markers for detection of colonic stem cells are CD44, EpCAM, CD133, CD166, Lgr5, etc. Dalerba et al. demonstrated that only a subpopulation of CD44 high/EpCAM high cells were able to form tumors in xenografts [6]. More recently aldehyde dehydrogenase 1 (ALDH-1) has also been shown to be a stem cell marker for colon [8].

Aging is associated with an increased risk for colon cancer [9], [10], [11]. The occurrence of benign and malignant tumors increases with age in the colon [12]. It has also been demonstrated that during aging, colonic crypt proliferative activity increases and apoptosis decreases [13]. The mechanisms related to the increased incidence of colorectal cancer with advancing age have been a subject of intense investigations. Currently there is no definitive explanation for the changes observed in the colonic crypts, such as increased proliferation and decreased apoptosis, as well as increased expression of EGFR, preceding overt neoplasia. Since the stem cells are considered to be the origin of neoplastic transformation in solid tumors as well as intestinal neoplasias, we decided to investigate the status of stem cells in the normal appearing colonic crypts in aging. Recently, we reported the presence of colon cancer stem-like cells in macroscopically normal mucosa in patients with adenomatous polyps and that they increase with aging [14]. These changes are paralleled by increases in the expression of EGFR [11], [15].

In the current study we examined the combined effects of aging and carcinogen exposure on the stem cell population of colonic crypts of rats. We focused on the expression of stem cell markers in normal appearing colonic mucosa of rats exposed to the carcinogen dimethylhydrazine (DMH) in young and aged rats.

Section snippets

Animals

Male Fischer-344 rats, aged 4–6 (young) or 22–24 month (old) were purchased from the National Institute on Aging (Bethesda, MD). All procedures were performed according to the standards for use of laboratory animals established by the Institute of Laboratory Animal Resources, National academy of Sciences, and were approved by the Animal Investigation Committee at Wayne State University School of Medicine. The details of animal handling have been previously published [11].

Carcinogen treatment

Groups (n = 6) of 4–6 and

Results

For each tissue sample, the colonic crypts with longitudinal orientation and complete section from base to lumen, were analyzed. The expression of markers in the colonic crypts was quantitated by counting the stained cells in the crypt base under high power field of the microscope. Twenty colonic crypts were counted and an average number of stained cells per crypt was recorded. The same procedure was applied to all the markers tested. The differences in expression levels were tested by

Discussion

In this study we demonstrate that the number of cancer stem-like cells in colonic crypts of rats increase significantly due to aging and in response to carcinogen treatment. Moreover, these effects are additive. The increase in stem cell population is maximal in aged and carcinogen treated animals.

These results are very similar to our recent observation in normal appearing colonic mucosa from patients with adenomatous polyps, which are considered to be precursors of colorectal cancer [14]. In

Acknowledgments

This work was supported by grants (A.P.N.M.) from the NIH/NIA (AG014343) and the Department of Veterans Affairs.

References (16)

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