Abstract
Bile acids have been shown to be
important in the etiology of colorectal cancer and are known to
cause DNA damage in mammalian cells. We previously found
that in vitro exposure of colonic mucosal biopsies to bile acids
at concentrations comparable to those found in fecal water after
high fat meals induces apoptosis in goblet cells. At the
light microscopic level, the apoptotic cells appeared to be
darkly stained using either toluidine blue or a polychrome
stain. Since transmission electron microscopy (TEM) is the
"gold standard" for the identification of apoptotic
cells, samples from various patient groups were studied by
TEM. The characteristic features of apoptotic cells were
identified in the darkly stained goblet cells, and included
chromatin condensation and margination, segregation of the
nucleolus, an increase in electron density and the appearance of
cytoplasmic vacuoles.
Colonic epithelial cells with DNA damage
are often removed by apoptosis. If a colonic mucosal cell
population should arise which was apoptosis resistant, then
DNA-damaged cells would not be removed, and, for proliferating
cells, this could result in DNA replication past damaged sites,
an increased probability of mutation, and therefore an increased
cancer risk. Previous reports, based on a small number of
individuals, indicated that patients with a history of colorectal
cancer or having familial adenomatous polyposis (FAP) often have
a high proportion of cells resistant to induction of apoptosis in
the normal-appearing portion of their colonic epithelium.
In the present study, mucosal
biopsies from 72 patients were examined. Biopsies were
taken at 20 cm from the anal verge, from the cecum and from the
descending colon. The patients included 17 individuals with
a history of colorectal cancer, 37 with adenomas, 14 who were
neoplasia free, and 4 with localized or extensive ulcerative
colitis. For biopsies taken 20 cm from the anal verge, the
mean bile salt-induced apoptotic index (AI) among normal
individuals, with its standard error was 57.6+/-3.47 whereas the
mean (AI) of individuals with a history of colon cancer was
36.41+/-3.12. The mean AIs of the normal and cancer groups
differ significantly at the level of p<0.05 by Tukey's test.
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Confocal images of Jurkat cells reacted with a polyclonal antibody against poly(ADP-ribose) polymers. Upper left: cells treated with 0.4 mM N-methyl-N'-nitro-N-nitrosoguanidine for 30 min; upper right: cells treated with 0.5 mM sodium deoxycholate (NaDOC) for 30 min; lower left: untreated control cells; lower right: control for the immunostaining reaction where the same staining procedure was followed as for the other three, except that the primary antibody was omitted. For experimental details see reference below. |
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Confocal images of NF-kB (a bile salt-induced stress response protein) and lkB-gamma (a NF-kB inhibitory protein) localization in the nucleus and cytoplasm of HCT-116 colon cells. Cells were reacted with poyclonal antibody against lkB-gamma (red) followed by monoclonal antibody against activated p65 subunit of NF-kb (green), using an immunofluorescent biotin/avidin sandwich technique. Areas of colocalization appear orange to yellow. Left: untreated control cells; right: cells treated with 0.5 mM NaDOC for 4 h at 37 degrees Celsius. |
Variability in resistance to apoptosis
was assessed using samples taken at different locations within
the colon from patients in three different clinical groups: 1)
normal subjects (no evidence of polyps or colon cancer), 2)
colonic adenomas (no evidence of colon cancer) and 3) patients
with resected colon cancer or ulcerative colitis. Three
possible sources of variation was evaluated: variation between
subjects, variation based on location and variation between
biopsies. The greatest source of variability in the normal
group was between subjects; the greatest source of variability in
the adenoma group was based on location and the greatest source
of variability for patients with resected colon cancers or
ulcerative colitis was between biopsies. The variability
between biopsies that characterized the high risk group of
subjects is the basis for a "patchiness" in
apoptosis resistance. It is possible that these
"resistant patches" are "hot spots" for
mutations that result in cellular transformation, neoplasia and
malignancy. Issues of variability are also being addressed
using fresh mucosa obtained during surgical resection of portions
of the colon taken from patients with colon cancer, large
adenomas or diverticulitis.
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Immunohistochemical staining of activated NF-kB in the normal mucosa of patients with colon cancer obtained during surgical resection of a portion of the colon. Paraffin sections were prepared from two distinct areas of the mucosa (distant from the tumor), one showing resistance to NaDOC-induced apoptosis (A) and the other showing senstivity to NaDOC-induced apoptosis (B). (A) Immunostained with a monoclonal antibody against the activated form of NF-kB. Note the brown staining (DAB reaction) in the nucleus and cytoplasm at the base of the crypt. B. In apoptotic resistant mucosal regions, note the lack of brown staining in the crypt. |
Future studies will be directed at
understanding the causes of the resistance to bile salt-induced
apoptosis. Immunohistochemical studies at both the light
and electron microscopic level will be critical to understanding
which stress proteins, anti-oxidant genes, transcription factors
or oncogene products and their crypt position and/or sub-cellular
location are altered in the pre-cancerous mucosa.
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Model indicating the fate of cells after interaction with high physiologic concentration of bile salts. A cell that has missing or inadequate stress response proteins may undergo apoptosis. Cells with adequate stress response proteins likely repair any damage and survive as normal cells. Surviving cells with a genetic or epigenetic changes causing high constitutive levels of stress-response proteins in vivo, could produce clones of resistant cells that become prone to further mutations, some of which lead to cellular transformation and neoplasia. |
REFERENCE: Payne C, Crowley C, Washo-Stultz D, Briehl M, Bernstein H, Bernstein C, Beard S, Holubec H, Warneke J: The stress-response proteins poly(ADP-ribose) polymerase and NF-kB protect against bile salt-induced apoptosis. Cell Death and Differentiation 5:623-636; 1998..
