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Bile-salt induction of apoptosis in human lymphoidand colonic epithelial cells: Relevance to colon cancer.
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Authors Samaha, Hanan El-Sayed Taha.
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BILE SALT-INDUCTION OF APOPTOSIS
IN HUMAN LYMPHOID AND COLONIC EPITHELIAL CELLS:
RELEVANCE TO COLON CANCER.
by
HANAN EL-SAYED TARA SAMAHA
A Dissertation Submitted to the faculty of the
. DEPARTMENT OF NUTRITIONAL SCIENCES
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 9 5
OMI Number: 9603380
OMI Microform 9603380 Copyright 1995, by OMI Company. All rights reserved.
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UMI 300 North Zeeb Road Ann Arbor, HI 48103
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have
read the dissertation prepared by Hanan E1-Sayed Taha Samaha
entitled Bile Salt-Induction of Apoptosis in Human Lymphoid
and Colonic Epithelial Cells: Relevance to Colon Cancer
and recommend that it be accepted as fulfilling the dissertation
requirement for the Degree of Doctor of Philosophy
/i. \ R I Dr. Harris Bernstein iC.~ t;.::;:>?~""I-'Q .................
Dr. Claire Payne 'YlAc P Date -< / / (Q; If fs-
Dr. tvanda Howe 11 Date
Dr. Charles Weber 6 /1 /16-Date
Date
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
2
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation
requirement. I I ~ Dr. Harris Bernstein ~ ~ Dissertation Director Date 7 I
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of a Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Request for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instance, however, permission must be obtained from the author.
SIGNED:
4
ACKNOWLEDGEMENTS
I thank God who surrounded me with people who made this work-possible.
I give an enormous thank-you, from the very bottom of my heart, to Dr. Harris Bernstein for all the time, encouragement, support, and guidance which he freely gave throughout the course of this research. I thank him very much for everything he did with me throughout my graduate studies.
I also thank Dr. Claire Payne who taught me much about this research. I acknowledge Dr. Payne for her time, for her flexibility, and for stimulating me always to reach my goals.
I specially thank Dr. Carol Bernstein for her helpful discussions, for her encouragement and for her support.
Also I thank very much Dr. Eileen Asher for teaching me, and for encouraging me throughout my graduate studies.
I thank Dr. Bobby Reid for his unfailing encouragement. Also I thank very much Dr. Wanda Howell and Dr. Charles Weber for their consistent support.
I am deeply grateful to my husband, Dr. Sherif EI Kholy, a person who cared about me, and put me infront of himself always during a difficult period of my life, and for his continued support and encouragement throughout my graduate studies.
A very special thank-you goes to the most wonderful children who gave me love and support, my two handsome boys Mohamed and Amro.
I thank my family who have always been there for me, my father and mother, the best I could ever have, my sisters Dr. Gihane Samaha and Samar Samaha who, despite the miles between us, sent their prayers, support, and love for which I am extremely grateful.
I thank my uncle Saad Samaha and his family, my aunt Fatma EI Miligi, my uncle Kamal, my mother in law Aida, Hend, Hoda, Nahla, Heba, Mona, Maha, Nesrine, Amira, Reem, Reham, Dalhia, Aisha, Hanan Abd EIKadar, and Enas and to all my other family members and friends without whom this work would not exist.
5
DEDICATION
This thesis is dedicated to the best mother (Fadia El
Okda) and the best father (El Sayed Taha Samaha) in whole
world. I really miss them very much and I hope God will
reunite us in paradise. I would also like to dedicate this
thesis to my sisters, Gihan and Samar. This thesis is also
dedicated to my husband Sherif and my two beautiful children
Mohamed and Amro.
6
TABLE OF CONTENTS
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . 12
LIST OF TABLES. 14
ABSTRACT
I . BACKGROUND
A. International incidence of colon cancer
B. Incidence of colon cancer in the u. S.
C. Mutations in colon carcinogenesis
1) RAS
2) p53 mutations and other genetic
alterations
D. Diet and colon cancer
1) Dietary fat
2) Fecal bile acids . 3) Bile acids in humans
4) Dietary fiber
5) Calcium
E. The nature of apoptosis and necrosis
1) Apoptosis
a) Structural changes in the apoptotic
cell
15
17
17
17
17
18
18
19
20
20
22
24
26
27
27
29
b) Nuclear collapse 29
c) Phagocytosis of apoptotic cells 30
d) Biochemical changes during apoptosis 30
TABLE OF CONTElqTS-Continued
e) Significance of apoptosis in relation
to cancer
2) Necrosis . . . . F. Role of the immune system in colon cancer . G. Bile salts/acids effects in colon
carcinogenesis
1) Role of goblet cells in protecting the
colon epithelium .
2) Proliferative activity and risk of colon
cancer ..
3) Mechanism of bile salt/acid action
4) Effects of bile salts/acids on the immune
system .
H. Neoplastic transformation
1) Crypt anatomy
2) Benign epithelial tumors and polyps
3) Adenoma-carcinoma sequence
4) Histological types .
5) Malignant epithelial tumors
6) Symptom Evaluation .
7) Anatomic distribution
8) Findings from the present study
II MATERIALS AND METHODS
7
32
33
34
35
35
36
37
38
38
38
40
41
43
44
44
45
45
47
TABLE OF CONTENTS-Continued
A. Cell line and tissue culture conditions
B. Bile salt treatment
C. Cell counts ..
D. Quantitation of apoptosis
E. One micron epoxy section
1) Prefixation with 3% glutaraldehyde
2) Fixation with 1% osmium tetroxide
3) Dehydration in a graded series of alcohol
solutions
4) Infiltration
5) Embedding in Spurr's epoxy resin
6) Thick section
7) Staining of tissue section
F. Electron microscopy · · G. Bile salt-induction of apoptosis in normal
human mononuclear cells · · . . . . . H. Bile salt-induction of apoptosis in colonic
epithelial cells . . . · · . . .
8
47
47
48
48
48
49
49
50
51
52
53
54
54
55
55
I. The second part of the study on human subjects 57
1) Patient recruitment 57
2) Tissue procurement . 58
3) Bile salt treatment 58
4) "Induced-apoptosis" bioassay 59
9
TABLE OF CONTENTS-Continued
III. RESULTS 61
A. NaDOC induction of apoptosis in a transformed
lymphocyte cell line ... ...... 61
B. Induction of apoptosis in normal human mononuclear
cells . . . . . . . . . . . . . . . . . . .. 74
C. NaDOC induction of apoptosis in colonic epithelial
cells . . . . . . . . . . . . 76
1) Preliminary Experiments with a normal
individual, a polyp patient and a cancer
patient at University Medical Center 76
a) Determination of assay conditions and
methods of measurement
b) Distribution of apoptotic cells and
type of enterocyte affected .
c) Effect of incubation with different
NaDOC concentrations on tissue
samples ..
d) Evaluation of NaDOC-induced apoptosis
in patients with different risks
for colon cancer
i. Normal subject
ii. Colon cancer patient
iii. Polyp patients ...
76
77
77
78
78
78
83
10
TABLE OF CONTENTS-Continued
iv. Conclusions on Preliminary
Experiments . . . . . .. 84
2) Study of bile salt induction of apoptosis in
colonic goblet cells from patients at the
Veterans Hospital
a) Determination of assay conditions and
methods of measurement . . .
b) Experimental parameters of the
apoptosis bioassay: Effect of
incubation in tissue culture media
on cells
i. Norcal patients
ii. Cancer patients
iii. Patients with non-malignant
84
84
87
87
92
polyps . . . . . . . . 92
C) Intraobservational Variation
IV. DISCUSSION ..
A. Bile salt induction of apoptosis in human
lymphoid cells .......... .
B. Bile salt induction of apoptosis in human
colonic epithelial cells
C. possible role of p53 in NaDOC-induced apoptosis
and colon carcinogenesis
96
100
100
103
107
11
TABLE OF CONTENTS-Continued
D. Hypothetical mechanisms 'for bile salt induction
of apoptosis .. .109
1) Elevation of calcium 109
2) Cytoskeletal changes 110
3) Endonuclease activation 112
E. Prospects for colorectal cancer prevention 113
V REFERENCES . . . . . . . . . . . . . 114
12
LIST OF FIGURES
FIGURE 1, Growth curves of NC-37 cells in the presence of a range of concentrations of NaDOC 62
FIGURE 2, Membrane integrity of NC-37 cells in the presence of a range of concentrations of NaDOC . . . . . . .. ..... 63
FIGURE 3, Percent apoptotic cells in the presence of a range of concentrations of NaDOC in NC-37 cells . . . . . . .. ....... 65
FIGURE 4, Comparison of the morphology of NC-37 cells, in the presence and absence of NaDOC . . 66
FIGURE 5, Representive electron micrograph showing the ultrastructure of control cells . . . . 68
FIGURE 6, Representive electron micrograph showing the ultrastructure of cells briefly reacted with NaDOC . . . . . . . . . . . . . . . . .. 69
FIGURE 7, Representive electron micrograph of NC-37 cells showing some of the characteristic features of an apoptotic cell .... 70
FIGURE 8, Representive electron micrograph of NC-37 cells showing some of the characteristic features of an apoptotic cell ... 71
FIGURE 9, Representive electron micrograph of an apoptotic cell showing unusual shapes 72
FIGURE la, Representive electron micrograph of apoptotic cell showing unusual shapes 73
FIGURE 11, Percent apoptotic cells in the presence of a range of concentrations of NaDOC in normal human mononuclear cells.. ...... 75
FIGURE 12, Dose-response curve for goblet cells of the normal colon epithelial tissue from a normal subject . . . . . . . . . . . . . . 79
FIGURE 13, Dose-response curve for goblet cells of the tumorous tissues from a colon cancer patient . . . . . " ...... 80
LIST OF FIGURES-Continued
FIGURE 14, Dose-response curve for goblet cells of the non-tumorous tissue from a colon cancer
13
patient . . . . . . . . . . . . 81
FIGURE 15, Light micrographs of biopsies of the normal mucosa of a non-colon cancer patient with adenomatous polyps (low risk group) 86
FIGURE 16, Electron micrograph of the normal colonic mucosa incubated in the absence of NaDOC . 88
FIGURE 17, Composite electron micrograph of the normal colonic mucosa of normal, neoplasia-free patients undergoing colonoscopy 89
FIGURE 18, Dose-response curve for goblet cells of the normal colonic mucosa derived from normal subj ects . . . . . . . . . . . . . . . . 91
FIGURE 19, Dose-response curve for goblet cells of the normal colonic mucosa derived from colon cancer patients . . . . . . . 93
FIGURE 20, Dose-response curve for goblet cells of the normal colonic mucosa derived from patients with polyps . . . . . . . . . . . . . .. 95
FIGURE 21, Plot of first score of percentage apoptosis versus second score for each patient . . 98
FIGURE 22, Plot of average first score of percentage apoptosis versus average second score for each patient . . . . . . . . . . . . . . 99
LIST OF TABLES
TABLE 1, NaDOC-induction of apoptosis in adenomatous and normal appearing mucosa parts of the same
14
patient. . . . . . . . . . . . . . . . . . .. 83
15
ABSTRACT
Death from cancer of the colon is the third leading
cause of cancer death in the United States. Epidemiologic
evidence indicates that life style factors and particula~ly
a high fat western diet play an important role in colon
cancer. Cheah (1990) concluded that bile salts present in
the feces as the result of a high fat diet are the most
important etiologic agent in colon cancer. The mechanism of
action of bile salts, however, is largely unknown. Bile
salts have been shown to cause DNA damage in some mammalian
cell types. As a first line of defence, the cell ordinarily
attempts to repair the DNA damage. However, failure to
repair damage can lead the cell to commit suicide
(apoptosis). Apoptosis is an active physiologic mode of
cell death.
In this study, apoptosis was detected using Wright
stained cytospins for lymphoid cells and one micron,
plastic-embedded sections for colonic epithelial cells using
the light microscope. I examined the effect of the bile salt
sodium deoxycholate (NaDOC) on three types of cells:
transformed human lymphocytes, normal human mononuclear
cells and colonic epithelial cells. I found that NaDOC
induces apoptosis in lymphoid cells. This might decrease
the number of lymphocytes available to defend against mutant
16
colonic epithelial cells that develop into tumors. Also I
found that NaDOC induces apoptosis in colonic goblet cells.
This induction of apoptosis was strong in colon epithelial
cells from normal individuals or polyp patients at low risk
for colon cancer. However, the goblet cells of the
apparently normal mucosa from colon cancer patients (and
polyp patients at high risk for colon cancer) were found to
be relatively resistant to the apoptosis-inducing effect of
NaDOC.
To explain these results the following model was
proposed. Under conditions of a high fat diet, bile salts,
may select for a population of apoptosis-resistant cells.
This may lead to repopulation of the colon with cells that
do not respond to DNA damage by entering the apoptosis
pathway. Rather, these cells may replicate and undergo
mutation at the sites of the DNA damage. These mutations
may contribute to colon carcinogenesis.
17
I. BACKGROUND
A. International incidence of colon cancer
Colorectal cancer is the third-ranking cancer in the
world, accounting for about 9 percent of the estimated 6.35
million invasive cancers occuring each year. The
international variation in incidence rates and the change
occuring after migration indicates the importance of
enviromental factors (Schottenfeld, 1995).
B. Incidence of colon cancer in the U. s.
Colon cancer is the third most common cause of cancer
mortality for men (after lung and prostate cancer) and the
third most common for women (after lung and breast cancer)
in the United States (Boring et al., 1993). A striking
variation in the frequency of colorectal cancer is seen in
different geographical areas. The highest incidence of
colon cancer is in the United States and Canada, while the
lowest incidence is in Asia, Africa and South America (Boyle
et al., 1985~. There are approximatly 160,000 new cases of
colon cancer in the United States every year (Rustgi and
Podolsky, 1992).
C. Mutations in colon carcinogenesis
Colon cancer is a multistep process involving both
mutational activation of oncogenes and mutational
18
inactivation of tumor suppressor genes [Rustgi and Podolsky,
1992; Fearon and Vogelstein, 1990 and Hamilton, 1992).
Development of colon cancer requires at least mutation of
four or five genes, while development of a benign tumor
requires fewer mutations (Rustgi and Podolsky, 1992).
l)"RAS
The RAS gene is considered to be the most common
oncogene in colon cancers, since it is found in at least 50%
of colon tumors and adenomas greater than 1 cm in size
(Rustgi and Podolsky, 1992; Fearon and Vogelstein, 1990). A
RAS gene mutation may be the initiating event in colorectal
tumor development (Fearon and Vogelstein, 1990). RAS gene
mutations are infrequent in histologically normal mucosa
adjacent to neoplastic tissue (Burmer and Loeb, 1989).
2) p53 mutations and other genetic alterations
Loss of specific chromosomal loci in any tumor
indicates that the involved regions may contain tumor
suppressor genes (Knudson, 1985). In colon tumors allelic
losses are very common in chromosomes 5q, 17p, and 18q.
They are found in about 70% of colon cancers (Rustgi and
Podolsky, 1992). There is evidence that these allelic
losses include tumor suppressor genes whose products
regulate normal growth and differntiation in a negative
fashion (Fearon and Vogelstein, 1990). Fearon and
19
Vogelstein, 1990 have reported a high frequency of allelic
loss of chromosome 17p in colorectal tumors. The common
region of chromosomal loss of chromosome 17p includes the
p53 gene (McBride et al., 1986; Fearon and Vogelstein,
1990). The p53 gene was reported to be defective in 75-80%
of colon tumors (Levine et al., 1991). Thus RAS appears to
be the most common oncogene and p53 the most common tumor
suppressor gene in colon tumors. Mutations and deletions of
the tumor-suppressor gene p53 are associated with the
development of late-stage, large, dysplastic polyps and
invasive carcinomas (Fearon and Vogelstein, 1990). The
total accumulation of genetic alterations rather than their
sequence of occurrence seems to be the most important for
colorectal cancer development.
D. Diet and colon cancer
The incidence of colorectal cancer is low in Japan and
high in the U. S. The rapid equalization of the incidence of
colon cancer between Japanese immigrants to the United
States and the general U. S. population indicates that it is
very likely that life style plays a major role in colon
cancer. Colon cancer is considered to have one of the
strongest relationships to diet of all diet related cancers
(Hill, 1986; Wynder, 1987; Bruce, 1987).
20
1) Dietary fat
There is a close relationship between per capita fat
intake and colorectal cancer incidence in various
populations (Armstrong and Doll, 1975). A raised levels of
dietary fat can lead to an increase in the proliferation of
colon epithelial cells of experimental animals (Stadler et
al., 1988; Lipkin, 1988), but the mechanism of this
proliferation is not known. Some studies suggested that it
might represent a compensatory proliferation after loss or
damage to surface epithelial cells (Bird et al., 1985).
Hyperproliferation of the colonic epithelium has been
regarded as a significant biomarker of the risk of colon
cancer (Lipkin, 1988; Cohen et al., 1990). High consumption
of animal fat might account for the high fecal bile acid
concentrations detected in populations at high risk for
colon cancer (Wynder, 1975).
2) Fecal bile acids
In early studies, Cook et al. (1940) found that
deoxycholic acid injected subcutaneously in rats in an oily
vehicle, gave rise to local tumours. However Salaman and
Roe (1956) showed that the oily vehicre itself contained a
carcinogen. This suggested that deoxycholic acid may be
involved in tumor promotion, but not necessarily tumor
initiation. When both deoxycholic and lithocholic acids
21
were introduced into the rat rectum as an aquous solution,
tumors did not arize suggesting that these bile acids do not
cause tumor initiation. When the rats had been treated
first with the carcinogen dimethylhydrazine (DMH) both
deoxycholic and lithocholic acids were "found to be good
tumor promotors. Silverman and Andrews (1977) reported that
both deoxycholic and lithocholic acids increased the weak
mutagenic effect of the potent colon carcinogen DMH in the
Salmonella mutagensis assay system.
Cheah (1990), in an extensive review of the literature,
concluded that bile acids are the most strongly implicated
factors in the etiology of Colon cancer. This conclusion
was based, in part, on population comparisons, in which
there is a strong correlation between fecal bile acid
concentration and incidence of colon cancer (Reddy et al.,
1983; Hill et al., 1982; Narisawa et al., 1974). The
amounts of secondary bile acids (deoxycholic and lithocholic
acids) were higher in colon cancer patients than in normal
controls (Reddy et al., 1977). High bile acid
concentrations are known to accompany a high fat and low
fiber Western-type diet (Reddy 1981a; 1981b). A diet
induced increase of bile acid concentration in fecal water
results in higher intestinal cell lysis activity and higher
colonic cell proliferation (Lapre and Van Der Meer, 1992),
and higher incidence of colorectal cancer (Stadler et al.,
1988). Bull et al. (1983) suggested that proliferation of
colonic epithelial cells is drastically stimulated in
rodents by bile acids because of their intrinsic lytic
properties. Bull et al. (1983) used high non-physiologic
concentrations of NaDOC and they tested for induced cell
lysis, but not apoptosis.
22
However, in case control studies, fecal bile acid
concentration did not always parallel incidence of colon
cancer (Hill, 1982). This contradiction might be explained
by the influence of genetic factors as major determinants of
susceptibility to colon cancer (Morson et al., 1990a). It
is likely that both diet and genetic factors are interwoven
in the etiology of colon cancer but that the precise
composition of events varies from one individual to another
(Morson et al., 1990a).
3) Bile acids in humans
Bile acids are physiologically important steroidal
surfactants that are secreted into the small intestine to
emulsify fat. In humans, the primary bile acids, cholic and
chenodeoxycholic acids are secreted by the liver as
conjugates with glycine or taurine. Their function is to
emulsify fat in the duodenum and enable its digestion and
absorption in the small intestine. Only 1-4% of bile acids
escape to the colon where they are metabolised by the gut
23
bacterial flora. The major bile acids produced by bacterial
action in the large intestine are deoxycholic and
lithocholic acids (Paumgartner, 1986). These two bile acids
are termed secondary bile acids and they are the result of
deconjugation and 7~dehydroxylation (Hill, 1983). About 30-
50% of the bile acids entering the colon are returned to the
liver via the blood stream in the form of primary bile acids
and deoxycholic acid. Lithocholic acid is insoluble in
aquous solution and therefore is very poorly absorbed into
the blood stream. The bile acids that are returned to the
liver are then resecreted as part of bile for a further
enterohepatic cycle. The bile pool undergoes 6-10 cycles
per day: The number of cycles increases with increasing
dietary fat and therefore the amount of bile acid substrate
entering the colon is affected by dietary fat (Hill, 1990).
In the steady state, the concentration of bile acids in
systemic blood (Van Berge Henegouwen and Hofmann, 1983) and
urine (Alme et al., 1977) is low. Fecal excretion, which
approximately equals daily synthesis, is the major route for
the elimination of bile acids from the body (Setchell et
al., 1986). Serum bile acid levels are maintained by the
balance between intestinal absorption and hepatic
elimination of bile acids (Paumgartner, 1986).
Bile salts act as detergents because their molecular
structure consists of an ionic portion which is hydrophilic,
24
and an uncharged steroid backbone which is hydrophobic. In
addition, at certain bile salt concentrations, bile salts
will self aggregate to form micelles. The concentration at
which this occurs is called the critical micellar
concentration (CMC). Bile salt micelles may perturb
membranes by solubilizing membrane components. Lytic
activity of bile acids is strongly dependent on their
structure and increases with increasing hydrophobicity (Van
Der Meer et al., 1991; Lapre et al., 1992). The secondary
unconjugated bile acids (colonic) are more hydrophobic than
the primary conjugated bile acids and their lytic activity
is higher than that of the primary conjugated bile acids
(duodenal) (Van Der Meer et al., 1991). Hydrophobic
characteristics may enable the bile salt to bind to or
become embedded in the plasma membrane, thereby perturbing
the membrane. It is known that bile acids are toxic to
colonic epithelial cells (Rafter et al., 1986).
4) Dietary fiber
Burkitt (1971) suggested that a high fiber diet might
protect humans from colorectal cancer by increasing the
speed of intestinal transit. This would reduce the exposure
of the gut to carcinogens. Also the larger bulk of stools
might dilute carcinogens. Fiber may also protect humans by
adsorbing carcinogens and mutagens (Smith-Barbaro et al.,
25
1981) and modifying fecal bile salt excretion (Reddy et al.,
1980). Jass (1985) suggested that the products of bacterial
fermentation of fiber and undigested starch in the right
colon (acetic acid, propionic acid and butyric acids) serve
as the principal sources of energy for the colorectal
epithelium. These short chain fatty acids cause
acidification of the stool. Several studies have shown an
association between decreased incidence of colorectal cancer
and low stool pH (Samelson et al., 1985).
Butyrate is preferentially taken up by the colonic
epithelium accounting for 70% of its total energy
consumption (Scheppach, 1994). McIntyre et al. (1993)
showed that insoluble dietary fiber, by elevating butyrate
production in the distal large bowel, significantly
suppressed tumour formation in a rat model. Hague et al.
(1995) demonstrated that butyrate can induce apoptosis in
adenoma and carcinoma cell lines. Such an induction of
apoptosis may be significant in patients with adenoma,
suggesting that increased intake of dietary wheat bran may
be a good dietary strategy for preventing the development of
adenoma into carcinoma.
Cheah and Bernstein (1990) showed that dietary fiber,
especially cellulose, inhibits the DNA damaging ability of
bile acids. This might reduce bile acid promotor activity.
5) Calcium
Calcium ions might reduce the toxic effects of fecal
bile acids through their conversion of the bile acids to
insoluble calcium soaps (Newmark et al., 1984). Results
concerning the in vitro precipitation of bile acids by
calcium have been controversial. Some authors found that
calcium precipitates bile acids or decreases cytotoxicity
(Buset et al., 1986; Rafter et al., 1986), whereas other
26
workers found a marked increase in cytotoxicity in the
presence of calcium (Child and Rafter, 1986; Van Munster et
al., 1993). Abnormal patterns of cellular proliferation in
high risk individuals for colorectal cancer disappeared when
their diets were supplemented with calcium carbonate (Lipkin
and Newmark, 1985). Sorenson et al. (1988) showed that
increased intake of dietary calcium has a protective effect
against colon cancer and suggested that calcium might act
directly on the intestinal mucosa. Buset et al. (1986)
showed that the usual amount of calcium in an ordinary diet
inhibited the growth of colonic epithelial cells. Sorenson
et al. (1988) found that epithelial proliferation in the
bowel was enhanced in patients at high risk for colon
cancer. The ability of calcium to influence colon
carcinogenesis may be affected by interacting with other
food constituents. Calcium can bind to fiber which might
affect the amount of calcium available to interact with gut
lipids to form intestinal soaps (Eastwood and Mitchell,
1976) .
Milk is the highest source of calcium in most diets.
27
The intake of milk is higher in the United States than in
Japan, although the incidence of colon cancer is higher in
the United States than Japan. Milk is a high source of fat
as well as of calcium. The effect of ingesting milk on
colon carcinogenesis may thus reflect a balance of the
benefit of calcium and the deleterious influence of fat
(Sorenson et al. 1988).
E. The nature of apoptosis and necrosis
1) Apoptosis
The term "apoptosis" was introduced by Kerr, Wyllie and
Currie (Kerr et al., 1972) to describe the phenomenon of
programmed cell death, a physiologic mode of cell death in
which the cell dies by a programmed process (Kerr et al.,
1972; Kerr et al., 1987; Kerr and Searle, 1980; Wyllie et
al., 1980; and Eastman, 1990). Apoptosis, in Greek, refers
to leaves falling off trees in the autumn. in cell biology
this term applies to a natural form of cell death that is
presumed to be adoptive to the organism (Touchette and
Fogle, 1991). When cell death occurs as a result of
physiological stimuli, it almost always takes the form of
apoptosis (Kerr and Searle, 1980). Apoptosis is an active
28
II suicide II process requiring an intact energy generating
system (functiona~ mitochondria) (Cotter et al., 1990).
Apoptosis occurs during embryogenesis (Bursch et al., 1992),
during tissue involution as in the post partum uterus, in
the breast after weaning and in the liver during starvation
(which may be a beneficial adapt ion for reducing energy
expenditure and prevention of overfunction). Apoptosis is
also involved in the elimination of interdigital webs during
fetal development (Saunders, 1966), differentiation of the
retina (Young, 1984), in the deletion of redundant
epithelium after fusion of the palatine processes, and in
the formation of intestinal villi. Apoptosis also occurs
during normal homeostasis (Kerr, 1971; Kerr et al., 1987;
Kerr and Searle, 1980; Kerr et al., 1972; Searle et al.,
1982; Wyllie et al., 1980) and can be triggered by normal
physiologic stimuli such as glucocorticoids (Wyllie, 1980;
Cohen and Duke, 1984) or hormone withdrawal (Wyllie et al.
1973; Kyprianou and Issacs, 1988). Dive and Hickman (1991)
suggested that haemopoietic stem cells might repair cellular
damage inefficiently and this may predispose these cells to
cell death. Apoptosis is the antithesis of mitosis. The
balance between mitosis and apoptosis determines either
regression or growth of organs (Bursch et al., 1992). If
mitosis exceeds apoptosis, it may result in uncontrolled
growth which might be a reasonable definition for neoplasia
29
(Sarraf and Bowen, 1988).
a) Structural changes in the apoptotic cell
Apoptosis is characterized by cytoplasmic condensation.
The apoptotic cell shrinks (apoptosis was first named
shrinkage necrosis). "Zeiosis" , or rapid blebbing of the
plasma membrane might be due to modifications of the
cytoskeleton (Sanderson, 1982). It is important to note
that the plasma membrane in the apoptotic cell still
excludes dyes indicating preservation of membrane integrity.
This is in contrast to the necrotic cell in which the cells
take up the vital dyes indicating loss of membrane
integrity.
b) Nuclear collapse
The hallmark of apoptosis is the collapse of the
nucleus with preservation of the other organelles (Kerr et
al., 1972; Wyllie, 1980). The apoptotic cell is
characterized by peripheral condensation of the nuclear
chromatin. This condensation often gives rise to a
crescent-shaped appearance of the chromatin. In some cell
types, this nuclear condensation is associated with the
activation of an endonuclease which cleaves the cell's DNA
into nucleosome size fragments or multiples thereof, and the
formation of roughly spherical or ovoid membrane-bounded
structures with well preserved organelles known as apoptotic
bodies (Searle et al., 1982; Kerr et al., 1987; Kerr and
Searle, 1980; Wyllie et al., 1980; Searle et al., 1975).
30
The breaking up of the cell may be facilitated by the
vigorous "boiling" action of zeiosis (Cohen and Duke, 1992).
c) Phagocytosis of apoptotic cells
Phagocytosis of apoptotic cells or bodies in solid
tissues presumably protects the viable neighbour cells from
the damaging effects of released intracellular contents.
Release of these intracellular contents (proteases, other
enzymes, and oxidizing molecules) into the extracellular
tissue increase the probability of an inflammatory response
(Bursch et al., 1992) by direct toxicity-and by generating
chemotactic factors from extracellular matrix molecules
(Cohen and Duke, 1992). The resulting activation of
inflammatory cells especially neutrophils might risk
destruction of normal cells (Cohen and Duke, 1992; Morris et
al., 1984). Phagocytosis of apoptotic cells or bodies
before they lyse also 'ensure that the normal function of the
tissue is well preserved.
d) Biochemical changes during apoptosis
Fesus (1992) reported that during apoptosis a Ca++
dependent endonuclease is activated. Apoptosis is also
associated with an increase in tissue transglutaminase
activity (Bursch et al., 1992). This enzyme crosslinks
31
lysine and glutamine protein residues and it was
hypothesized (Bursch et al., 1992) that this may tighten the
membranes of apoptotic bodies and prevent their lysis prior
to phagocytosis.
Apoptotic cells were originally defined by the
characteristic margination of the chromatin, nuclear
fragmentation and condensation of the nucleus and cytoplasm
as seen in the electron microscope (Kerr et al., 1972).
Cleavage of DNA at internucleosomal linker regions,
resulting in a "ladder" of DNA fragments of 180-200 base
pairs and mUltiples has been associated with some cell types
entering apoptosis (Arends et al., 1990; McConkey et al.,
1988; McConkey et al., 1989; Cohen and Duke, 1984). However
a "ladder" of DNA fragments is also associated with necrosis
in some cell types (Collins et al., 1992). Thus, the "gold
standard II for determination of apoptosis has been
observation of the characteristic morphological changes by
electron microscopy (Samaha et al., 1995; Payne and Cromey,
1991; Kerr et al., 1972; Collins et al., 1992; Payne et
al., 1992). Once apoptosis is established under a given set
of circumstances using the "gold standard", other less time
consuming methods that correlate with the ultrastructural
appearance of apoptosis can be used to quantitate the degree
of apoptosis occuring with the cells in question.
32
e) Significance of apoptosis in relation to cancer
Apoptosis is an active process that occurs rapidly. It
does not appear to occur at random in all tissues but
specifically helps in the elimination of damaged or
redundant cells during normal development (Thompson, 1995).
Apoptosis also occurs in regression of prostatic and mammary
tumors upon removal of androgen and estrogen respectively.
Tumor promotors are used in the induction of
experimental cancer. Most tumor promotors cannot induce
cell proliferation on their own. Tumor promotors such as
phorbol myristate acetate (PMA) can act as specific survival
factors for the cells in which they promote tumor
development suggesting that they may inhibit apoptosis and
that apoptosis is an important step in the development of
cancer.
Cells from wide variety of human cancers have a
decreased ability to undergo apoptosis in response to some
physiologic stimuli (Hoffman and Liebermann, 1994).
Apoptosis appears to have a major role in tumor prevention
(Cotter et al., 1990). Cell death in response to DNA damage
in most cases has been shown to result from apoptosis (Lowe
et al., 1993). Apoptosis may serve as a natural defense in
multicellular organisms against the retention of cells with
unrepaired DNA-damage. Deleting cells with unrepaired DNA
damage may benefit the overall survival of the organism
33
(Ucker, 1991), since replication of DNA with unrepaired DNA
damages, might lead to mutations, accumulation of which may
lead to cancer.
2) Necrosis
Necrosis is a mode of cell death substantially
different from apoptosis (Wyllie, 1981; Searle et al.,
1982). Necrosis synchronously affects groups of contiguous
cells in vivo and is referred to as traumatic or accidental
cell death (Wyllie, 1981; Searle et al., 1982). Necrosis
usually occurs as a result of noxious stimuli, ischemia or
severe trauma. High concentrations of noxic substances
cause a progressive loss in membrane integrity, a collapse
of cellular homeostasis and depletion of ATP levels (Wyllie
et al., 1980). An early depletion of ATP levels preceding a
fatal disruption of the ionic gradients will cause the cell
to swell, rupture and to spill out degradative lysosomal
enzymes which mediate an inflammatory response and damage
the surrounding tissues (Dive and Hickman, 1991). It is a
non-physiological process, characterized at the cellular
level by pyknosis, dilation of the endoplasmic reticulum,
marked swelling of the mitochondria and other cell
organelles followed by general cell swelling and cell lysis
(Wyllie, 1981; Searle et al., 1982).
34
F. Role of the immune system in colon cancer
The colon, like the rest of the gastrointestinal tract,
is an important lymphoid organ, whose functional
organization is probably comparable to that of other organs
in which mucosa and lymphoid tissue come into intimate
contact. The gut immune system is the body's first line of
defense against intraluminal toxins (Elitsur et al., 1990).
This protection is provided within the lumen by secretory
IgA and within the mucosa by lymphocytes and macrophages
(Lewin, 1988).
The colon is endowed with isolated lymphoid follicles.
Isolated lymphoid follicles are the most abundant lymphoid
structure of GALT (~ut 8ssociated 1ymphoid Tissue) (Keren,
1988), which consists of more activated lymphocytes than the
peripheral circulation (Peters et al., 1989). The lymphoid
tissue functions not only to protect the individual against
infectious enteric diseases, but also appears to prevent
colon cancer caused by oncogenic agents in the diet or
produced by the metabolism.of intestinal bacteria (Keren,
1988). There is evidence of association between immune
deficiency and lymphoreticular neoplasms (Hoerni and
Laporte, 1970).
G. Bile salts/acids effects in colon carcinogenesis
1) Role of goblet cells in protecting the colon
epithelium
35
The surface of the gastrointestinal tract is maintained
by the balance between cell proliferation and cell loss
resulting from aging or" toxic insult (Waller et al., 1988).
These authors concluded that bile salts at high
concentration (25 mM) induced surface desquamation in the
large intestine followed by epithelial restitution by a
similar, but slower, process than occurs in the stomach.
They suggested that the slower restitution in the large
intestine than in the stomach is due to the more rapid
formation and more efficient maintainance of the mucoid
layer in the stomach following insults. This indicates the
importance of the goblet cells which produce the mucoid
layer. Goblet cells synthesize and secrete high molecular
weight glycoproteins called mucin. Within the mucus gel,
other components such as water, electrolytes, sloughed
epithelial cells and secreted immunoglobulins are found.
This produces a physical and chemical barrier that protects
the epithelium from luminal agents.
Mucins in adenomas and normal colorectal epithelium may
differ both in quantity and quality (Morson et al., 1990a).
In normal colorectal goblet cell's mucins, the peripheral
36
sugars are mainly represented by sialic acid 'and two neutral
sugars: N-acetyl galactosamine and (in the right colon)
fucose (Morson et al., 1990a). The acidity of the large
bowel mucosa is related to both sulphate groups and sialic
acid (Morson et al., 1990). In the majority of individuals,
normal large bowel sialic acid occurs in an O-acetylated
form in mucin causing resistance of the large bowel mucosa
to digestion by neuraminidase (Jass et al., 1988). Changes
in sialic acid structure, especially loss of O-acetyl group
have been reported in adenomas (Culling et al., 1977). High
acidity in the colon may stimulate intestinal peristalsis
and colonic mucus production which may reduce the contact of
carcinogens and cocarcinogens with colonocytes. Low pH
decreases the solubility of bile acids (Roberton, 1993).
Also, low pH may inhibit. the bacterial conversion of primary
bile acids into cocarcinogenic secondary bile acids
(lithocholic and deoxycholic acid) (Thornton, 1981).
2) Proliferative activity and risk of colon cancer
There is much evidence that factors which stimulate
colonic cell proliferation enhance tumor development and
factors that inhibit colonic cell proliferation inhibit
tumor development (Jacobs, 1988). Low proliferative
activity is considered a protective factor against colon
cancer development as documented by the studies on 7th Day
37
Adventist vegetarians, a group with low mortality from colon
cancer (Lipkin et al., 1985). On the other hand,
proliferative index is not predictive of colon cancer risk
on an individual basis (Biasco et al., 1992).
3) Mechanism of bile salt/acid action in relation to
colon cancer
Bile salts have been known for some time to be
cytotoxic to cells. Previous studies evaluating the
cytotoxicity of bile salts, however, generally employed high
concentrations that caused cell lysis (Velardi et al., 1991;
Van der Meer et al., 1991 and Lapre et al., 1992). Such
studies primarily measure necrosis, a passive form of cell
death which is trauma induced (Wyllie, 1981). These studies
did not evaluate the more regulated form of cell death,
apoptosis, in response to bile salt treatment.
Exposure to bile salts could increase the formation of
reactive oxygen (Craven et al., 1986). Bile salts induce
phospholipid breakdown with release of arachidonic or other
membrane lipid moieties and this generates reactive oxygen
species. These cause cellular damage and death and promote
cell turnover. Thus bile salts stimulate compensatory
proliferation of surviving colonic epithelial cells (Craven
et al., 1986). Others suggest that bile salts stimulate
colonic cell proliferation through increased production of
38
lipoxygenase products (DeRubertis et al., 1984). Numerous
authors have suggested that increases in cell proliferation
precede the development of colon cancer both in humans and
experimental animals (Spagnesi et al., 1994).
Bile salts potentiate superoxide anion release from
activated rat peritoneal neutrophils which might perturb the
plasma membrane (Dahm et al., 1988). Bile salts have also
been shown to inhibit glutathione S-transferase (Schneider,
1992), effectively raising intracellular free radicals which
are known to damage DNA (Imlay and Linn, 1988). Bile acids
cause DNA damage in eukaryotic cells (Kandell and Bernstein,
1991). Unrepaired DNA damage may then lead to apoptosis
(Corcoran and Ray, 1992; Carson et al., 1986).
4) Effects of bile salts/acids on the immune system
The problem of impairment of lymphocyte function by
bile acids and its impact on colon cancer has been studied
by Keane et al. (1984), Elitsur et al. (1990) and Gianni et
al. (1980). However those studies did not clarify the
mechanism by which bile acids impair lymphocyte function.
It is possible that there was cell loss through apoptosis in
their systems, which could account for the overall decrease
in lymphocyte function. In the present study, I examined
the effect of sodium deoxycholate (NaDOC), in concentrations
similar to those found in the fecal water (Rafter et al.,
39
1987}, on induction of apoptosis in lymphoid cells. As will
be reported in the Results, I found that NaDOC, at high
physiologic concentrations can induce apoptosis in an
Epstein-Barr virus' transformed human lymphoid cell line and
in normal (untransformed) mononuclear cells.
H. Neoplastic transfor.mation
1) Crypt anatomy
The normal crypt is the basic functional unit of the
large bowel. It is shaped like a test tube, with a closed
and an open end. Three main cell types are represented
within the crypt and surface epithelium. The most numerous
are the columnar cells, goblet cells and endocrine cells.
The epithelial lining of the intestine acts as barrier that
restricts many threatening noxious elements to the
intestinal lumen (Madara et al., 1988). Most cells within
the intestinal crypts are transitory with a limited life
expectancy and a limited capacity to divide. Cell turnover
times vary according to cell type, with goblet cell turnover
being about 5 to 6 days and columnar cell turnover about 2
to 3 days (Bjerknes and Cheng, 1981). The three types of
cells are derived from a common stem cell at or near the
crypt base. The early stages of neoplastic transformation
are not accompanied by obvious morphological changes but can
be distinguished by cell kinetic studies (Sunter, 1984).
40
The earliest changes are usually abnormal proliferation; the
proliferative zone gradually increases in size with
hyperplasia and there is a shift of the proliferative
compartment to the upper part of the crypt.
2) Benign epithelial tumors and polyps
Polyps are usually restricted to mucosal growths
projecting into the bowel lumen. Polyps can be small or
large, benign or malignant and may exist singly, in small
numbers or in large numbers up to hundreds. Inflammatory
masses and tumors arising from the submucosa or the muscle
layers of the colon are referred as polypoid lesions (Morson
et al., 1990a). The term polyp does not indicate the nature
of underlying lesion.
Various pathological processes may lead to formation of
epithelial polyps. A polyp may result from neoplasia,
inflammation, malformation and dysmaturation (Morson et al.,
1990a). The major types of polyps occuring in the colon are
the following:
a) Benign neoplastic polyps termed adenomas.
b) Inflammatory polyps arising in chronic inflammatory
bowel diseases.
c) Hamartomas polyps arising in normal colonic
epithelium but haphazardly arranged or malformed
(Morson et al., 1990a).
41
d) Hyperplastic polyps which have arisen by epithelial
dysmaturation (Morson et al., 1990a).
The histologic type of polyp has a relationship with
presence or absence of carcinoma. Hyperplastic polyps are
more common in geographical areas in which the population is
at high risk areas for colorectal cancer (Morson et al.,
1990a). Hyperplastic polyps and adenomas differ at the
ultrastructural level (Kaye et al., 1973). Cooper et al.
(1979) and Franzin and Novelli (1982) have concluded that
conversion of a hyperplastic polyp to a carcinoma is rare.
Morson et al. (1990a) suggested that hyperplastic polyps can
be regarded as a "paraneoplastic" rather than a
preneoplastic lesion. However, Rokkas et al. (1993)
concluded that all patients determined to have small polyps,
either hyperplastic or adenomatous, during flexible
sigmoidoscopy should be examined by total colonoscopy.
3) Adenoma-carcinoma sequence
Population screening programmes show that adenomas
occur in the large intestine at an earlier age of onset than
carcinomas (Bussey, 1968). Several studies noted that most
colonic carcinomas originate in previously benign neoplastic
polyps (Jass, 1989; Correa et al., 1977; Vamosi-Nagy and
Koves, 1993). Adenomatous polyps have a 2.7-9.4% chance of
containing invasive carcinomas (Cranley et al., 1986).
42
Development of an adenoma is thought to reflect the stepwise
accumulation of mutations within progenitor cells (Morson et
al., 1990a). Adenomas show abnormal cytology, disturbance
of growth and differentiation which is expressed in the term
dysplasia (Konishi and Morson, 1982). Adenomas reveal a
spectrum of slight deviation from normal to severe dysplasia
or carcinoma in situ (Morson et al., 1990a). The number of
adenomas can be considered an important risk factor for
development of more adenomas and adenocarcinomas (Konishi
and Morson, 1982). The other important morphological
features that determine the malignant potential of an
adenoma are its size, growth pattern and grade of dysplasia
(Konishi and Morson, 1982). Animal studies have shown that
bile salts increase colonic ornithine decarboxylase (Takano
et al., 1981; DeRubertis et al., 1984). Induction of
ornithine decarboxylase has been suggested to be involved in
the final common pathway of most tumor promotors (Morson et
al., 1990a). Adenomas and carcinomas of the human large
intestine have high level of ornithine decarboxylase three
times more than the normal large intestine (LaMuraglia et
al., 1986).
It is widely accepted that patients with adenomatous
polyps have an increased potential for malignant change
compared to the normal population (Morson et al., 1990b;
Buntain et al., 1972), and that development of colon cancer
43
proceeds through an adenoma-carcinoma sequence (Hill et al.,
1978) .
4) Histological types
Adenomas are classified according to their microscopic
architecture as tubular, tubulovillous and villous (Morson
et al., 1990a). A change in the configuration of the
lesion, as followed radiographically, from a smooth, rounded
outline to an irregular shape indicates a worse prognosis
and that malignancy may present. A villous element
increases the risk of an adenoma developing into carcinoma
(Morson et al., 1990a). Several studies have shown adenomas
to be more prevelant in populations at high risk for
colorectal cancer (Correa et al., 1977; Rickert et al.,
1979). Hill (1990) concluded from his studies and other
studies that secondary bile acids might have a role in
colorectal cancer, in the progression from adenoma to
carcinoma but not in the formation of adenomas. Secondary
fecal bile acids may increase the size of adenomas (Hill,
1983 and 1990). Patients with colorectal adenomas have
increased serum bile acids and significantly increased
concentrations of deoxycholic acid compared with patients
without these benign precursors of colorectal cancer
(Bayerdorffer et al., 1993 and Bayerdorffer et al., 1994).
It is still unclear how microadenomas are formed and
44
subsequently how they develop into carcinoma.
5) Malignant epithelial tumors
The 5-year survival rates of colon cancer in whites for
the per~od 1960 to 1963 was 43%, while for the period of
1977 to 1983 it was 53% (Silverberg, 1987). This
improvement in survival rates suggests the importance of
screening tests, early diagnosis and monitoring patients at
high risk. Patients at high risk for colon cancer include
patients with polyposis coli, patients with a past history
of colorectal cancer or adenomas, patients with a family
history of colorectal cancer or adenomas, and patients with
inflammatory bowel disease (Bond, 1993). The majority of
colorectal cancers are adenocarcinomas in which tubular
architecture is related to the grading of the colon cancer
(Morson et al., 1990b).
6) Symptom Evaluation
According to current practice patients with signs or
symptoms of colorectal cancer should be offered a complete
examination of their entire large bowel. Any persistent
change in the bowel movement pattern should raise suspicion
of colorectal cancer until proven otherwise, especially in
persons greater than 40 years of age. Newly developed
diarrhea, fatigue, constipation, rectal bleeding or cramping
abdominal discomfort may be signs of a colorectal tumor
(Bond, 1993). The passage of bright red blood, with or
without clots, usually at the time of defecation, is very
often seen with cancers of the rectum or sigmoid (Kurtz,
1995). At present, however no reliable biomarker is
available for early detection of colon cancer
susceptibility.
7) Anatomic distribution
45
More than half of all colonic cancers occur either in
the sigmoid colon (35 percent) or the cecum (22 percent) .
The distribution throughout the remaining colon is as
follows: ascending colon (12 percent), transverse colon (10
percent), and descending colon (7 percent) (Schottenfeld,
1995) .
8) Findings from the present study
In addition to studying bile salt induction of
apoptosis in lymphocytes, I have also investigated bile
salt-induction of apoptosis in the normal appearing colonic
mucosa of normal subjects, adenomatous patients and cancer
patients. I found that NaDOC, at high physiological
concentrations, induces a high level of apoptosis in normal
colonic goblet cells from normal subjects. However the
normal appearing portion of the colon in colon cancer
patients and high risk polyp patients was relatively
resistant to NaDOC-induction of apoptosis. These findings
46
have implications both for the basic mechanism of colon
carcinogenesis and the development of a biomarker for early
detection of colon cancer susceptibility.
47
II MATERIALS AND METHODS
A. Cell line and tissue culture conditions
An Epstein-Barr virus-transformed cell line (NC-37) was
obtained from the American Type Culture Collection. These
cells were derived from a donor whose serum was positive for
Epstein-Barr virus (EBV) antibodies. Cells were incubated
at 37°C in a humidified CO2 incubator in Dulbecco's minimal
Eagle media (DMEM) (pH 7.3), containing 10% fetal bovine
serum. The fetal bovine serum was heat-inactivated at 56°C
for 30 minutes. Penicillin (100 units/ml), streptomycin
(100 ~g/ml), and glutamine (20 mM) were added and the media
was buffered with 20 mM Hepes. The medium was changed every
5 days. Cells were seeded into fresh media at 4 x 104
cells/ml to maintain growth in logarithmic phase.
Experiments were initiated with DMEM containing 5 x 105
cells/mI.
B. Bile salt treatment
Sodium deoxycholate (ICN biochemicals) (NaDOC) was made
as a 0.1 M stock solution in sterile deionized water and
added in specified amounts to the growth media at the time
of treatment. The pH remained constant (pH 7.3) over the
entire range of concentrations of NaDOC used in the
experiments. All incubations were performed at 37°C in a
humidified CO2 incubator.
48
C. Cell counts
Counts were determined in duplicate by light microscopy
using a Spencer Counter. Membrane integrity was determined
using the eosin Y dye exclusion assay described by Payne et
al. (1992).
D. Quantitation of apoptosis
Apoptotic cells were distinguished from normal and
necrotic cells by their morphology using light microscopy of
Wright's-stained cytospin preparations (Payne et al., 1992).
This procedure was used to determine the proportion of
apoptotic cells among total visible cells. Approximately
500 cells were scored for each determination. The
morphologic criteria used to identify apoptotic cells were:
condensation of the nuclear chromatin, fragmentation of the
nucleus, increased cytoplasmic vacuolization, tinctorial
changes in the cytoplasm from blue to purple or gray and the
formation of apoptotic bodies.
E. One micron epoxy sections
Fixation is the first step in preparation of any
biological material: it preserves the structure of cells
with the minimum alteration from the living state, protects
the tissue against disruption during embedding and
sectioning, and prepares the tissue for staining and
exposure to an electron beam. An ideal fixative should kill
the tissue quickly with minimal shrinkage or swelling.
1) Prefixation with 3% glutaraldehyde
Cells were prefixed with 3% glutaraldehyde.
49
Prefixation with glutaraldehyde toughens soft tissue due to
both intra-and intermolecular crosslinkage in protein
molecules and prevents leakage of the cell's protein
molecules to the outside. However prefixationwith
glutaraldehyde doesn't impart electron scattering ability.
Thus osmium tetroxide is used as a post- fixative to impart
some contrast and to allow lipid retention in the tissue.
The slow penetration of glutaraldehyde is balanced by its
high reactivity. The recommended concentration range is 2-
6%. Higher glutaraldehyde concentrations result in
excessive shrinkage of tissues and lower concentrations lead
to extraction of proteins.
2) Fixation with 1% osmium tetroxide
Osmium tetroxide (OS04) acts not only as a fixative of
cytoplasmic detail, but also as a stain. The deposition of
osmium in the tissues is an important factor in the
enhancement of contrast. Its high atomic weight ensures
that any structure in which osmium is deposited during
fixation appears dense in the electron image (Carr and
Toner, 1982). It has a poor penetration power and is
expensive. Since osmium tetroxide is a slow penetrant, the
50
tissue is usually cut up into sections of less than 1.0 mm
in thickness and then transferred to vials containing OS04
to ensure rapid and complete penetration of the fixative
into the centre of each small piece. It reacts at a
moderate rate with proteins but rapidly with phospholipids
and unsaturated fatty acids. As OS04 is reduced, the
specimen will turn black. Overfixation results in oxidation
of membranes and leaching out of various cell components.
Following fixation in OS04' all the tissue samples are
rinsed in a buffer to remove any free OS04 (not reduced)
which might precipitate in the cells causing secondary
blackening.
3) Dehydration in a graded series of alcohol solutions
For making alcohol dilutions, 95% ethanol was used.
30% ethanol was prepared by taking 30 ml of 95% ethanol and
adding enough distilled water to equal a total volume of 95
mI. 50%, 70% and 90% ethanol solutions were prepared in a
similar way. Dehydration of samples was carried out by
passing each sample through a graded series of alcohol
solutions at increasing concentrations. Each dehydration
step lasted for 15 minutes. The final step, exposure to
100% ethanol, was done twice to ensure that the tissue was
completely anhydrous. Dehydration was done at room
temperature.
51
4) Infiltration
The plastic mixture (Spurr's epoxy) was infiltrated
into the specimen following dehydration in absolute ethanol.
The composition of Spurr's epoxy Mixture is: 10 gm ERL-4206
(vinyl cyclohexene dioxide) (primary epoxy resin), 6 gm DER
736 (diglycidyl ether polypropylene glycol) (flexibilizer,
epoxy resin), 26 gm NSA (nonenyl succinic anhydride)
(anhydride hardener), and 0.4 gm DMAE (dimethylamino
ethanol) (accelerator). This Spurr's kit was obtained from
Electron Microscopy Sciences. Since viscous materials are
more accurately measured by weight than by volume, all of
the above components were added by weight. Each component
of the mixture was added in turn to a glass bottle for
weighing on a trip balance scale at room temperature. A
pipette with its tip broken off to allow easy flow was used
to dispense the required amount of each component. All
materials used that had been in contact with an epoxy resin
were either kept in a hood (i.e. pencils and forceps), or
were discarded in appropriate receptacles.
Infiltration was started by adding the embedding media
(Spurr's epoxy) mixed with an equal quantity (1:1) of 100%
ethanol to the vials containing the tissue specimens. The
tissues were exposed at room temperature for 2 to 4 hours.
Then the tissues were removed with a forceps and put into
new clean vials and the embedding media (Spurr's Epoxy'
52
Mixture) added. The duration of infiltration must be
sufficient to insure good penetration. When the specimens
are in 100% Spurr's Epoxy Mixture, the vials must be shaken
gently to expose all surfaces of the tissue specimens to the
liquid epoxy resin. Infiltration is very critical since
poorly preserved tissue will result if the pure resin
mixture doesn't permeate the specimen completely.
5) Embedding in Spurr's epoxy resin
Embedding in freshly made Spurr's epoxy was done as
previously described (Payne et al., 1992; Payne and Cromey,
1991). Flat embedding is a faster process than capsular
embedding, since with flat embedding a number of good sized
tissue specimens can be embedded in the same dish at the
same time. In capsular embedding one piece of tissues is
embedded in every capsule. The tissue is placed, along with
its identification code number, in a small rubber mould
filled with liquid embedding medium. Polymerization takes
8 hours at 70°C, but the samples can be left in an oven at
70°C overnight. The polymerized form of the embedding
medium is hard and durable. The tissues are surrounded and
infiltrated by this supporting media (liquid embedding
medium) forming a convenient block ready for sectioning.
53
6) Thick sectioning
Sectioning of the tissue was done using an
ultramicrotome. An ultramicrotome is made up of a knife
holder, specimen holder, advance system, observation system
and illumination system. The knife holder is clamped in the
microtome and the block is fixed in a movable chuck. The
block must advance towards the knife at each step by only 1
~m in order to cut sections of this thickness.
An initial trim of the specimen to form a pyramid shape
was done using a sharp razor blade. The specimen was
trimmed so that the tissue was in the center and at the tip
of the pyramid. Triming of the block is a very critical
step since a large block face will cause chatter in the
sections and a rapid dulling of the glass knife edge used in
the following step.
Glass knives were made by mechanically breaking glass
strips into squares and then breaking each square into two
triangular shaped pieces using a glas~ knife-maker. The
knife was provided with a trough filled with distilled water
acting as a fluid reservoir to lie behind the cutting edge.
The trough was made of tape firmly attached to the shoulder
of the knife. So that sections, once cut, will float on the
surface. When viewed in reflected light, Their interference
colours serve as approximate guide to section thickness
through a dissecting microscope: yellow is thicker, gray is
54
thinner. Sections were removed from the trough filled with
distilled water using an eyelash hair. Fixation of the
section was done by mounting the section in a drop of
distilled water on the center of a clean slide on a hot
plate for about 3 minutes.
7) Staining of tissue section
The tissue sections that were placed on a slide were
next stained with toluidine blue. A drop of toluidine blue
was put on the fixed section on a hot plate until a green
rim formed all around the drop. Then the slide was removed
from the hot plate washed with distilled water and left to
dry. This staining is useful for identifying apoptotic
cells. Light stain or heavy stain compromise the
identification of apoptotic cells. Apoptotic cells are
characteristically more heavily stained than normal non
apoptotic cells.
F. Electron microscopy
Electron microscopy is considered one of the gold
standards for defining apoptosis (Payne et al., 1992).
Apoptosis was confirmed by Dr. Claire Payne, using electron
microscopy.
G. Bile salt-induction of apoptosis in nor.mal human
mononuclear cells
55
To validate the physiological relevance of bile salt
induction of apoptosis in transformed lymphocytes, I also
studied the induction of apoptosis in normal human
mononuclear cells using the same concentrations of NaDOC as
used with the transformed lymphocytes. Whole human blood
was obtained from healthy donors by venipuncture. The
normal mononuclear fraction (lymphocytes and monocytes) was
prepared by Ficoll/Hypaque gradient separation as described
by Payne and Glasser (1981). The mononuclear preparation
was incubated with the same concentrations used above with
NC-37 cells for 6 and 12 hours. Frequencies of apoptotic
cells were measured by light microscopy using whole mount
cytospin preparations.
H. Bile salt-induction of apoptosis in colonic epithelial
cells
A preliminary study of bile salt induction of apoptosis
was carried out in University Medical Center. NaDOC
induction of apoptosis was studied in normal appearing
mucosa from normal subjects, polyp patients and colon cancer
patients. The procedure used was as follows: Tissue was
removed from a patient in the G.I. clinic at University
Medical Center as part of a medically indicated procedure.
56
Tissue from this specimen was cut into 2 mm pieces with a
sharp scalpel on a hard dental wax surface and 4 pieces
placed into each of seven vials containing Dulbecco's
minimal Eagle media plus either 0, 0.02, 0.05, 0.1, 0.19,
0.39, 0.77, 0.97, 2.9 or 4.8 roM NaDOC. The tissues were
incubated at 37°C in a humidified CO2 incubator for four
hours. At the end of the incubation period, the media was
replaced with 3% glutaraldehyde made up in 0.1 M phosphate
buffer (pH 7.2), and fixed for 2 hours at 4°C. After an
overnight buffer rinse, the tissues were post-osmicated with
osmium tetroxide, dehydrated in a graded series of ethanol
solutions, and embedded in Spurr's epoxy resin. At least 3
blocks of every sample were thick sectioned, and stained
with toluidine blue. All the epithelial cells were counted
and the percentage "dark cells" determined using light
microscopy. "Dark cells" are characterized by margination
or condensation of nuclear chromatin and have a dense
cytoplasm. Such "dark cells" were identified as apoptotic
under the electron microscope by Dr. Payne.
Although this'preliminary study involved tissues from
only a few patients it gave significant information for
designing the second part of the study. On the basis of the
results of the first experiment, I determined the doses of
NaDOC, times of exposure and epithelial cell type to focus
on in the second experiment.
57
I. The second part of the study on human subjects
1) Patient recruitment
The second part of the study on human subjects was
conducted on 15 patients. Colon biopsies were evaluated for
bile acid-induced apoptosis. The subjects were healthy
volunteers. All the subjects were above the age of 45.
These subjects entered the study in the G.I. clinic at the
Veterans Affairs Hospital, where they had a sigmoidoscopy
procedure performed. Their diagnosis was recorded in their
clinical charts. The patients were classified into 3
clinical groups according to the colonoscopy procedure
performed. Group I was composed of 4 normal sUbjects. We
excluded from this group patients with any inflammatory
bowel disease, diverticulosis, or with a history of colon
cancer or polyp formation. Also, the normal subjects did
not have a family history of colonic genetic lesion or
history of ulcerative colitis or colon cancer. Group II was
composed of 6 patients. These patients either had a past
history of polyp removal, or had a'polypectomy on the same
day as the procedure used to obtain our biopsy samples. The
polyps were evaluated according to their size, their
numbers, their histological type and their degree of
dysplasia. Group III was composed of 6 patients. These
patients had a past history of colon cancer.
58
2) Tissue procurement
For all subjects, biopsy specimens were taken 20 cm
from the anal verge with a flexible sigmoidoscope. For each
subject, seven separate biopsies were obtained and treated
as described below.
3) Bile salt treatment
The biopsies were taken immediatly, placed in sterile vials,
containing 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 mM sodium
deoxycholate (NaDOC) made up in complete Eagle's minimum
essential media (MEM). Complete MEM consists of 500 ml of
sterile Eagle minimum essential medium (MEM) alpha
modification (Sigma), 5 ml non-essential amino acids
(Sigma), 5 ml penn/strep (Penicillin-streptomycin solution)
(Gibco BRL); penicillin G 10,000 units/ml, streptomycin
sulfate 10,000 mcg/ml in normal saline, 2.5 ml hepes buffer
(1M) (USB) and 50 ml fetal bovine serum (Gibco). The vials
were kept on ice until the start of the incubation period.
All seven vials were then placed, with the caps loosened, in
a humidified CO2 -incubator and incubated at 37°C for 3
hours. The vials were pre-incubated for 45 minutes, before
adding the tissue samples in a humidified CO2 -incubator to
allow for pH equilibrium until the start of the incubation
period. At the end of the incubation period, the vials were
removed from the incubator and the tissue culture media were
59
replaced with cold 3% glutaraldehyde made up in 0.1 M
phosphate buffer (ph 7.2). The tissues were then fixed for
3 hours at 4°C in the glutaraldehyde fixative.
Subsequently, the tissue was stored in 0.1 M phosphate
buffer (containing 10% sucrose) in the refrigerator. Then
the tissue was processed for routine electron microscopy as
described by Payne et al. (1984). The tissue was
postosmicated in 1% osmium tetroxide, dehydrated in a graded
series of alcohol solutions of increasing alcohol content
and embedded in fresh Spurr's epoxy resin. One micron-epoxy
sections were then prepared using glass knives. The
sections were stained with toluidine blue and the percentage
of "darkly-stained" (apoptotic) goblet cells was determined
by light microscopy.
4) "Induced-apoptosis" bioassay
For each subject, at least two tissue specimens were
examined at each dose of NaDOC. A total of at least 200
cells were scored for each specimen. The percentage of
apoptotic goblet cells at each dose was calculated as the
average obtained from two to four specimens. The average
percentage of apoptosis versus concentration of NaDOC was
then plotted for each subject.
Then 35 slides of the tissues treated with 1 mM NaDOC
were coded by another investigator and their original
60
designations covered up. These coded slides were scored
blindly by me to determine intraobservational variation as
measured by the correlation coefficient between the primary
and secondary measurements.
61
III. RESULTS
A. NaDOC induction of apoptosis in a transfor.med lymphocyte
cell line
A cell suspension of the transformed lymphocyte cell
line NC-37 was divided into 8 aliquots; NaDOC was added to
the aliquots to yield final concentrations of 0.00, 0.05,
0.10, 0.19, 0.39, 0.77, 1.54 and 3.1 rnM. The cell count of
the untreated control increased threefold over the 24 hours
of incubation. Cell growth was inhibited in the presence of
0.05, 0.1, 0.19 and 0.39 rnM NaDOC in a dose-dependent manner
(Figure 1). The higher concentrations of 0.77, 1.54 and 3.1
rnM were toxic (data not shown), as evidenced by significant
lysis of the cells within 20 minutes. At a concentration of
0.39 rnM, there was a 50% decrease in the number of cells at
20 minutes. This rapid decrease was not observed at the
lower doses of NaDOC. After 24 hours in 0.39 rnM NaDOC,
there were about one-fifth as many cells as at time zero.
Dye exclusion assays, performed at 12 hours and 24
hours of incubation, showed that membrane integrity (cells
able to exclude dye) was approximately the same for cells
grown in the presence of 0.00, 0.05 or 0.1 rnM NaDOC, either
at 12 hours or 24 hours of incubation (Figure 2). However,
membrane integrity was markedly decreased after growth in
the presence of 0.19 and 0.39 rnM of NaDOCi"only 67% of the
62
1.6~-----------------,
1.4
1.2
'eO 1.0 .-l
~
r-I 0.8 e: .......... III r-I r-I 0.6 . Q) U
0.4 0.19 roM NaDOC
0.2 a . 39 roM NaDOC
0.0 ~---------~------~ o 5 10 lS 20 2S
Time of incubation (hrs)
Figure 1:· Growth curves of NC-37 cells in the presence of a
range of concentrations of NaDOC.
100 .---------------------------~
QJ
~ QJ 90 '0 ::s
r-I 0 ~ QJ
0 +J
QJ 80 r-I
.0 (II
U)
r-I r-I OJ 0
QJ 0'1 70 (II +J ~ QJ 0 f..I OJ III
0
0.0 0.1 0.2 0.3 0.4
mM NaDOC
Figure 2: Membrane integrity of NC-37 cells, measured by
ability to exclude eosin Y dye, after 12 and 24 hours
incubation in the presence of a range of concentrations of
NaDOC.
63
remaining cells could exclude dye after incubation in 0.39
mM NaDOC for 24 hours.
64
Apoptotic cells were identified and quantitated by
light microscopy using Wright's-stained cytospins (Payne et
al., 1992). The following criteria were used to identify
apoptotic cells in whole mount cytospin preparations stained
with Wright's stain: margination and condensation of the
nuclear chromatin often with the formation of crescents,
deeply stained cytoplasm, increased cytoplasmic
vacuolization, nuclear fragmentation and formation of
apoptotic bodies. The percent apoptotic NC-37 cells was
found to increase with increasing concentrations of NaDOC
after 12 hours incubation at 37°C. Apoptosis increased in a
dose-dependent manner with a threshold value around 0.1 mM
(Figure 3) .
Light micrographs (whole mount cytospin preparations)
of NC-37 cells incubated in the presence and absence of
NaDOC are shown in Figure 4. The control cells are
characterized by a dispersed chromatin pattern and a
basophilic cytoplasm (Figure 4A). NC-37 cells treated with
0.39 mM NaDOC for 12 hours show many typical apoptotic cells
(Figure 4B). The cell shrinkage, increased vacuolization,
deeply stained cytoplasm, condensed chromatin and
fragmentation of the cell into discrete bodies are
characteristic of apoptotic cells (Wyllie et ale 1980;
65
35 ,----------------,
30
25 . !J)
.,..f !J)
0 +J
20 0. 0 0. I1S
Q.I tn 15 . ro +J C Q.I {} J..4 10 . Q.I 01
5
o ~-------------~ 0.0 0.1 0.2 0.3 0.4
mM NaDOC
Figure 3: Percent apoptotic cells measured after 12 hours
incubation in the presence of a range of concentrations of
NaDOC in NC37 cells.
66
B
Figure 4: Comparison of the morphology of NC-37 cells, in
the presence and absence of NaDOC (X 2,900 Whole mount
cytospin preparations, Wright stain)
A) Control cells incubated without NaDOC. Cells
are round to oval in shape and the nucleus (N) contains
finely dispersed chromatin.
B) Cells incubated for 12 hours in the presence of
0.39 mM NaDOC. Many cells are condensed, have pyknotic
nuclei and are irregular in shape. Some cells were captured
in the process of forming apoptotic bodies (arrow).
67
Searle et al. 1975; Payne et al. 1992; Payne and Cromey,
1991). Many of the nuclei were fragmented into small round
bodies. After 24 hours incubation at the same concentration
of NaDOC (0.39 mM), secondary necrosis of apoptotic cells
become apparent.
Cell morphology after bile salt treatment was studied
using electron microscopy (Figures 5-10). Within 20
minutes of treatment with 0.39 mM NaDOC, significant surface
blebbing became apparent (Figure 6). This surface blebbing
occurred before apoptosis was apparent in the treated cell
population; the nuclear features in the cells showing
blebbing remained similar to that of the untreated controls
(Figure 5). Classic apoptotic cells exhibiting margination
of chromatin and vacuolization (Figure 7) and apoptotic body
formation (Figure 8) were identified after 12 hours
treatment with NaDOC. Increased cytoplasmic density,
another typical feature of apoptosis, is seen in both of
these cells. Some NaDOC-treated cells exhibited increased
cellular rigidity. This was evidenced by elliptical shapes,
or rigid long protuberances (Figures 9 and 10). These
unusual rigid forms were not seen when apoptosis was induced
by tritium decay or natural killer cells using the same
cell line (data not shown) .
. I~.: .,. ~ . '-. ,", ,., ~ " . :
:~.,-; "
, ..
...
Figure 5: Representative electron micrograph showing the
ultrastructure of control cells (uranyl acetate; lead
citrate). Control cells display a smooth cellular profile.
68
Nucleus (N) shows dispersed chromatin pattern with an intact
nucleolus (arrow) (X 10,100) (m= mitochondrial profile, er=
endoplasmic reticulum) (photograph courtesy Dr C. Payne).
69
~;:-:-.~~~------ '--
Figure 6: Representative electron micrograph showing the
ultrastructure of cells briefly reacted with NaDOC (uranyl
acetate; lead citrate). Cells treated for 20 minutes with
0.39 mM sodium deoxycholate. Note abundant surface blebbing
(arrows). The nucleus (N) appears normal as evidenced by
the dispersed chromatin and an intact nucleolus (X 10,100)
(photograph courtesy Dr C. Payne).
70
v
Figure 7: Representative electron micrograph of NC-37 cells
showing some of the characteristic features of an apoptotic
cell. (12 hours incubation with 0.39 mM NaDOC) (uranyl
acetate, lead citrate). This cell displays an increase in
cytoplasmic density, multiple, large (V) and small (v)
cytoplasmic vacuoles, and margination and condensation of
nuclear chromatin with crescent formation (arrow) (X 10,900)
(photograph courtesy Dr C. Payne).
71
."fo,;" .' ..
I · (~ i:i .S$~~ .-.-." .f. ~~1
• - ~~I
.. • •. • ...... ~ I(
". , .... ~.~; . .,'., ~ .' :.:~ " ~" .... ~1In ........ : ..
.:,:." • ...;.'_~ ....... f '.: ~ '~' • • " • -:
Figure 8: Representative electron micrograph of NC-37 cells
showing some of the characteristic features of an apoptotic
cell. (12 hours incubation with 0.39 mM NaDOC) (uranyl
acetate, lead citrate). This cell displays increased
cytoplasmic density, numerous cytoplasmic vacuoles (v),
fragmentation of the nucleus and apoptotic body formation
(arrow) (X 9,600) (photograph courtesy Dr C. Payne).
:a
Figure 9: Representative electron micrograph of apoptotic
cell showing unusual shapes (12 hours incubation with 0.39
mM NaDOC) (uranyl acetate, lead citrate). Comparison of a
72
normal cell with normal nucleus (N) (left) and an electron-
dense apoptotic cell (Right). The apoptotic cell shows
increased cytoplasmic density, numerous cytoplasmic
vacuoles, and an elliptical shape (arrow) (X 7,600)
(photograph courtesy Dr C. Payne).
73
.. • \
'I' .. . ,
Figure 10: Representative electron micrograph of apoptotic
cell showing unusual shapes (12 hours incubation with 0.39
mM NaDOC) (uranyl acetate, lead citrate). An apoptotic cell
with increased cytoplasmic density, cytoplasmic vacuoles (v)
and a rigid-appearing long protuberance (X 10,700)
(photograph courtesy Dr C. Payne).
74
B. Induction of apoptosis in nor.mal human mononuclear cells
Since induction of apoptosis was demonstrated with
cultured transformed lymphocytes it was decided to determine
if normal human mononuclear cells also underwent induction
of apoptosis in response to NaDOC. Mononuclear cells were
obtained and treated as indicated in Materials and Methods.
After 6 hours incubation, with NaDOC apoptosis was induced
(Figure 11). The percentage of cells undergoing apoptosis
after 6 hours incubation with NaDOC rose from 0.17% at 0.00
mM to 35% at 0.78 mM. After 12 hours incubation, the
percentage of apoptosis rose with increasing concentration
to a maximum of 14.26% at 0.39 mM (data not shown). After
this, there was a decrease in the percentage of apoptosis
at 0.78 mM NaDOC. This decrease may have been due to
development of secondary necrosis. Complete lysis was
observed after 6 hours and 12 hours at the highest
concentration (1.56 mM) NaDOC included in the study.
75
35 ~------------------------~
30
25 ~ .~
~ 0 ~ ~ 20 0 ~ ~
m 15 ~
~ ~ ~ m 0
10 ~ m ~
5
o ~------------------------~ 0.0 0.2 0.4 0.6 0.8
roM NaDOC
Figure 11: Percent apoptotic cells measured after 6 hours
incubation in the presence of a range of concentrations of
NaDOC in normal human mononuclear cells.
76
C. NaDOC induction of apoptosis in colonic epithelial cells
1) Preliminary Experiments with a normal individual, a
polyp patient and a cancer patient at University
Medical Center
a) Determination of assay conditions and methods of
measurement
NaDOC was chosen as the bile salt to be used in the
apoptosis bioassay because NaDOC is the most common bile
salt in human feces (Van Faassen et al., 1987). Thus biopsy
specimens were incubated in the absence and presence of
different concentrations of NaDOC as described in the
Materials and Methods.
One micron epoxy sections of the different tissue
samples were stained with Toluidine blue. Apoptotic and
non-apoptotic goblet cells were scored using the light
microscope. The criteria used to score apoptotic goblet
cells were condensation and/or margination of the chromatin
or increased density of cytoplasm surrounding the mucin
granules in profiles where the nucleus was out of plane of
section.
Some samples identified as having apoptotic goblet
cells at the light microscopy level were also checked at the
ultrastructural level by electron microscopy by Dr. Payne
and the presence of apoptosis confirmed.
b) Distribution of apoptotic cells and type of
enterocyte affected
77
Of the three distinct morphologic types of
differentiated enterocytes within the human distal colonic
mucosa (columnar, goblet and enterochromatin cells), only
the goblet cells were found to undergo apoptosis in response
to NaDOC at high physiological levels. Therefore goblet
cells, which have a distinctive morphology, were
specifically scored in this study. NaDOC induced apoptosis
in virtually all locations of goblet cells within the crypt,
representing both immature and mature mucous-secreting
cells. Although the goblet cells normally undergo
differentiation followed by senescence and death persumably
byapoptosis (Specian and Oliver, 1991), this uninduced
apoptosis is restricted to cells at the luminal surface
(Gavrieli et al., 1992). Intestinal cells have been
reported to undergo apoptosis in response to DNA-damaging
agents such as chemotherapeutic drugs and radiation (Ijiri
and Potten, 1983), but the specific cell type affected was
not mentioned in these studies.
c) Effect of incubation with different NaDoe
concentrations on tissue samples
Goblet cells in tissue specimens from a normal
individual that were incubated in the absence of bile salts
78
for up to four hours contained large numbers of mucin
granules and had normal nuclei with dispersed chromatin.
This indicates that in vitro incubation adequately preserved
the normal structure of colonic epithelial cells. However,
treatment with 1.0 mM NaDOC and higher concentrations of
NaDOC caused some degeneration after a four hour incubation
at 37°C in the tissues from the normal subject, a polyp
patient, and in the tissues from cancer patient.
d) Evaluation of NaDOC-induced apoptosis in patients
with different risks for colon cancer
i. Normal subject
The effect of treatment with increasing concentrations
of NaDOC on apoptosis in the colonic biopsies from one
normal subject is shown in Figure 12. After four hours
incubation, 30% of colonic goblet cells were apoptotic at
1.5 mM NaDOC. This contrasts with the low frequency of
spontaneously occuring apoptotic goblet cells (1.2%) found
in the zero dose controls. Higher concentrations of NaDOC
(i.e. 3.1 and 6.2 mM NaDOC) caused degranulation of some
goblet cells and all the nuclei were lysed (data not shown).
ii. Colon cancer patient
NaDOC induction of apoptosis was studied in the non
tumorous tissue and the tumorous tissue from a' colon cancer
79
35
w ~ ~ ~
30 0
~ ~
25 ~ ~ 0 ~
~ 20 0
w '" w 0 15 ~ ~ 0 ~ ~
~ 10
~ ~ ~ ~ ~ 5 0 ~ ~ ~
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
roM NaDOC
Figure 12: Dose-response curve (percent apoptosis vs NaDOC
,concentration) after 4 hours incubation for goblet cells of
normal colon epithelial tissue from a normal subject. Each
point is an average based on measurements made with two
different specimens having the same tratment.
80
35 ~---------------------------,
30
25
20
15
10
5
~ o \~ 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
mM NaDOC
Figure 13: Dose-response curve (percent apoptosis vs NaDOC
concentration) after 4 hours incubation for goblet cells of
the tumorous tissues from a colon cancer patient. Each
point is an average based on measurements made with two
different specimens having the same tratment.
81
35
Ul ..... ..... 30 OJ 0
+J OJ ..... 25 .0 0 tJI
!j.j 20 0
Ul
'" Ul 0 15 +J 0. 0 0. ro
10 OJ tJI ro +J c: 5 OJ 0 J..l OJ 14 .....
0
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2
mM NaDOC
Figure 14: Dose-response curve (percent apoptosis vs NaDOC
concentration) after 4 hours incubation for goblet cells of
the non-tumorous tissue from a colon cancer patient. Each
point is an average based on measurements made with two
different specimens having the same tratment.
82
patient. The tumorous tissue showed almost complete
resistance to NaDoe induction of apoptosis (Figure 13). The
non-tumorous tissues from the this patient also showed
little, if any, NaDoe induction of apoptosis (Figure 14) .
In contrast to the normal individual the non-tumorous
tissues of the cancer patient didn't show complete lysis at
3.1 mM. Moreover the tumorous tissues from the same patient
showed resistance to cell lysis not only at 3.1 roM, but up
to 6.2 mM.
83
iii. Polyp patients
In this preliminary investigation NaDOC-induction of
apoptosis in non-malignant adenomatous polyp tissue was also
examined. Table 1 shows NaDOC-induction of apoptosis in
non-adenomatous tissue and adenoma tissue after 2, 3 and 4
hours incubation with 1.0 mM NaDOC. This table shows that
NaDOC-induction of apoptosis in the non-adenomatous tissue
was higher than in the adenoma tissue after 2, 3 and 4 hours
incubation. These data suggest that the adenomatous tissue
may be more resistant to induction of apoptosis than the
normal appearing mucosa from the same patient.
Table 1 NaDOC-induction of apoptosis in adenomatous and
normal appearing mucosa parts of the same patient
Incubation period Percentage Percentage apoptosis in apoptosis in
1.0 mM NaDOC normal appearing adenomatous mucosa part
(hrs)
2 17.1 (1) • 6.2 (2)
3 14.0 (1) 11.1 (1 ) , 4 19.9 (2) 5.0 (1)
* The numbers in parenthesis indicate the number of
independent tissue samples measured from the same patient.
Where two samples were measured, the percentage apoptosis is
84
an average.
iv. Conclusions on Preliminary Experiments
In general this preliminary experiments were useful in
show~ng that goblet cells were the type of colon epithelial
cells most sensitive to bile acid induction of apoptosis.
These experiments also allowed me to determine the range of
NaDOC concentrations to which normal colonic tissue was
sensitive to induction of apoptosis and that 4 hours is a
sufficient period for incubation. In addition the results
showed that the tumorous tissue as well as the non-tumorous
tissue from one cancer patient was very resistant to NaDOC
induction of apoptosis and lysis. Furthermore I found that
the normal tissue of a polyp patient was more sensitive to
induction of apoptosis than the adenomatous tissue of the
same polyp patient.
2) Study of bile salt induction of apoptosis in colonic
goblet cells from patients at the Veterans Hospital
a) Determination of assay conditions and methods of
measurement
Based on the preliminary results described above, a
more extensive series of patients was examined for the
sensitivity of their colon epithelial cells to bile acid
induced apoptosis. As in the preliminary experiments, NaDOC
was used in the apoptosis bioassay because NaDOC is the most
85
common bile salt in human feces (Van Faassen et al., 1987).
I found that concentrations of NaDOe higher than 1.0 mM
(i.e. from 1.5 to 3.0 mM) lysed epithelial cells in a high
proportion of the cases from normal subjects, polyp patients
and patients with a history of colon cancer. Therefore,
data were analyzed only from the samples treated with 0, 0.5
and 1.0 mM NaDOe.
Representive one micron epoxy sections of human colonic
biopsies stained with toluidine blue are shown in Figure
(15A,B). Apoptotic goblet cells (treated with NaDOe) have
very dense stained nuclei or increased density of the
cytoplasm surrounding the mucin granules (Figure 15B)
compared with the nucleus and cytoplasm of untreated goblet
cells (Figure 15A). The criteria used to score apoptotic
goblet cells were condensation and/or margination of the
chromatin or increased density of the cytoplasm surrounding
the mucin granules in profiles where the nucleus was out of
the plane of section.
86
J .~ L
B
e' ..... · .J '!'.~ *. ~. ~ .....
< • '!'oI:"
. .. .. .,. -... ..". . ..
{
LP
Figure 15: Light micrographs of biopsies of the normal
mucosa of a non-colon cancer patient with adenomatous polyps
(low risk group) . (A) Tissue incubated for 3 hours at 37°C
in the absence of NaDOC (LP= lamina propria, M= mucin
granules and L= lumen of crypt). (B) Tissue incubated for 3
hours at 37°C in the presence of NaDOC. Note the dark
staining of the nucleus (n) and the cytoplasm (arrow) of the
goblet cells, which are identified by their abundant mucin
granules (M). (One micron epoxy sections, Toluidine blue 0
stain: X 2,800)
b) Experimental parameters of the apoptosis bioassay:
Effect of incubation in tissue culture media on
cells
87
Goblet cells from normal tissue specimens that were
incubated in the absence of bile salts, contained large
numbers of mucin granules and had normal nuclei with
dispersed chromatin (Figure 16 and 17A). This illustrates
that in vitro incubation without NaDOC adequately preserved
the normal structure of colonic epithelial cells in the
normal subjects and the low risk polyp patient. However,
treatment with 1.0 mM NaDOC caused some tissue degeneration
after a three hour incubation at 37°C (Figure 17B,C) (Samaha
et al., 1995b).
i. Normal patients
The dose-response curves obtained with colonic biopsies
from four normal subjects are shown in Figure 18. Each
point is an average of 2 to 4 measurements. The biopsies in
this group were similar in showing a strong induction of
apoptosis, reaching 45% to 63% apoptotic goblet cells at 1.0
mM NaDOC. This contrasts with the low frequency of
spontaneously occuring apoptotic goblet cells, 0% to 4%,
found in the zero dose controls (Samaha et al., 1995b).
88
Figure 16: Electron micrograph of the normal colonic mucosa
of a polyp patient incubated in the absence of NaDOC For 3
hours at 37°C in a CO2 -incubator. (M =mucin granules, N
=nucleusi Uranyl Acetate, Lead Citrate) (photograph courtesy
Dr C. Payne).
89
Figure 17: Composite electron micrograph of the normal
colonic mucosa of normal, neoplasia-free patients undergoing
colonoscopy. The biopsies were incubated in vitro in the
absence or presence of NaDOC For 3 hours at 37°C in a CO2 -
incubator. (M =mucin granules, N =nucleus; Uranyl Acetate,
Lead Citrate) (photograph courtesy Dr C. Payne).
(A) Incubation without NaDOC. Note the excellent
tissue preservation and lack of apoptosis (LP =lamina
propria; arrow is pointed to a basal lamina).
(B) Incubation in the presence of 1 mM NaDOC. The
goblet cell shows characteristic features of apoptosis,
including condensation and margination of chromatin and
increase in nuclear and cytoplasmic electron density.
(C) Incubation in the presence of 1 mM NaDOC. The
goblet cell has characteristic features of apoptosis,
including condensation and margination of chromatin,
increase in nuclear and cytoplasmic electron density and
loss of nucleolar structure (arrows).
90
~ l .fl .. 'I" >. t .. ..
" ~
•. "':\., ." " .', " . . I, .
' ... , ...... :.
91
70 ~-------------------------------,
m 60 ~ ~ ~ 0
~ 50 ~
~
~ ~
~ 40 0
m '" m 0 30 ~ ~ 0 ~ ro ~ 20 ~ ro ~ a ~ 0 10 ~ ~ ~
0
0.0 0.2 0.4 0.6 O.S 1.0 1.2
mM NaDOC
Figure 18: Dose-response curve (percentage apoptosis vs
NaDOC concentration) for goblet cells of the normal colonic
mucosa derived from normal subjects; after 3 hours
incubation with or without NaDOC~ Each point represents the
overall percentage apoptosis determined from the total
number of cells scored from 2 to 4 blocks at each dosage of
NaDOC.
92
ii. Cancer patients
Figure 19 shows the results obtained upon NaDOC
tratment of biopsies from the normal-appearing mucosa of the
colons of six patients with a history of colon cancer. Each
point is an average of 2 to 4 measurements. The
percentanges of apoptotic cells within these biopsies, after
treatment with 1.0 roM NaDOC, were substantially lower (1%-
23%) than those observed in biopsies from normal subjects
(Samaha et al., 1995b).
iii. Patients with non-malignant polyps
Figure 20 shows the results obtained from six
unselected polyp patients. The dose-response curves from
these cases separated into two patterns (low induction or
high induction of apoptosis). Goblet cells within biopsies
from two polyp patients showed strong induction of apoptosis
by 1.0 mM NaDOC (61%-63%) similar to the induction of
apoptosis shown by goblet cells from normal individuals at
1.0 mM NaDOC (Figure 18). Goblet cells from four polyp
patients showed weak induction of apoptosis (2%-21%) similar
to the induction of apoptosis shown by goblet cells from
cancer patients at 1.0 mM NaDOC (Figure 19). The two polyp
patients whose goblet cells showed strong induction of
apoptosis (and were therefore at low risk for colon cancer
by the bioassay) were also determined to be clinically at
93
70 ~-----------------------------,
60
so
40 -
30
20 -
10 -
0~~~~L-J 0.0 0.2 0.4 0.6 O.S 1.0 1.2
roM NaDOC
Figure 19: Dose-response curve (p~rcentage apoptosis vs
NaDOC concentration) for goblet cells of the normal colonic
mucosa derived from colon cancer patients after 3 hours
incubation with or without NaDOC. Each point represents the
overall percentage apoptosis determined from the total
number of cells scored from 2 to 4 blocks at each dosage of
NaDOC.
low risk (both had a past history of small polyps with· no
recurrence at the time the current biopsies were taken) .
94
The four patients whose goblet cells showed low induction of
apoptosis (and were therefore at high risk for colon cancer
by the bioassay) were considered to be at increased risk
clinically. One patient had an early onset of polyps at 48
years of age, a second had mUltiple polyps with one more
than 3 cm. in size (villous adenoma type), a third had 2, 5
and 3 polyps removed at 3 month intervals (the 5 polyps were
removed when the present biopsy was taken), and the fourth
had 5 tubular adenomatous polyps removed at the time of
biopsy; this same patient had a prior history of a
tubulovillous adenoma.
70 .-----------~----------------~
O'---~-----r----+---~----~--~
0.0 0.2 0.4 0.6 0.8 1.0 1.2
roM NaDOC Figure 20: Dose-response curve (percentage apoptosis vs
95
NaDOC concentration) for goblet cells of the normal colonic
mucosa derived from low-risk (top two curves) patients with
polyps and high-risk (lower four curves) patients with
polyps; after 3 hours incubation with or without NaDOC.
Each point represents the overall percentage apoptosis
determined from the total number of cells scored from 2 to 4
blocks at each dosage of NaDOC.
C) Intraobservational Variation
The scoring of percentage apoptosis on any slide is a
somewhat subjective procedure. Therefore to determine the
reproducibility of these scores intraobserveral variation
was measured.
96
Thirty-five slides containing colonic epithelial tissue
treated with 1 mM NaDOC and that had been previously scored
by me were chosen by another investigator and then coded. I
reexamined those slides individually and quantitated the
percentage of apoptosis. This was done blindly without
knowing the previous values obtained. The reading of those
slides were named second score. Three of the 35 slides from
one patient were wrongly substituted for by three other
slides from the same patient, so that comparisons involving
these slides are invalid.
The correlation coefficient was calculated for all the
data including the three mis-substituted slides. The value
obtained was 0.75 (Figure 21). When the data for the mis
substituted slides was removed, the correlation coefficient
were 0.83.
Two additional slides from one individual case showed a
poor correlation. Therefore I reexamined them, using
different light intensities, and concluded that differences
in light intensity may have affected my perception of the
percentage of apoptosis in this case.
97
Each point in Figures 18, 19 and 20 is the average of
2-4 determinations. Thus to estimate the reproducibility of
each plotted point we average the 2 or 3 measurements made
during the first scoring, and the 2 or 3 made during the
second scoring. The resulting plot of the data is shown in
Figure 22. The correlation coefficient between the first
score and the second score was 0.78 and with the mis
substituted set removed, it was 0.84.
These results indicate that the scoring of slides was
reasonably reproducible.
80
• 70
60
~ ~ 50 0 0 m ~ 40 C 0 0
30 ~ m
20
10
0
0 20 40 60 80
First score
Figure 21: The percentage apoptosis for each patient (at 1
mM NaDOC treatment), termed first scoring, is plotted
against the percentage apoptosis for each of these slides
determined blindly, (i.e. with the identity of the slide
covered up), termed second scoring. The points based on a
mis-substitution of the three slides for a particular
patient are indicated by the symbolo(r= 0.75 for all 35
slides; r= 0.83 with the mis-substituted data removed) .
98
99
70
60 • • • 50
~ ~ 0 0 40 00
~ ~ 0 30 0 ~
00
20
10
0
0 10 20 30 40 50 60 70
First score
Figure 22: The average percentage of apoptosis* for each
patient (at 1.0 mM NaDOC treatment), termed first scoring,
is plotted against the average percentage apoptosis for each
of these slides determined blindly, termed second scoring.
The point based on a mis-substitution of three slides for a
particular patient is indicated by the symbolc(r= 0.78 for
all points; r= 0.84 with mis-substituted data removed) .
(* determined by averaging the percentage apoptosis
determined using slides prepared from different specimens)
100
IV. DISCUSSION
A. Bile salt induction of apoptosis in human lymphoid cells
It has been well documented that many different kinds
of stimuli can trigger apoptosis (Lennon et al., 1990).
Agents that produce necrosis at high concentrations often
produce apoptosis at lower concentrations (Lennon et al.,
1991). I have shown that a bile salt, sodium deoxycholate
(NaDOC), induces apoptosis in an Epstein Barr virus
transformed human lymphoid cell line (NC-37).
This was the first demonstration that a bile salt can induce
apoptosis in any cell type. I started this research with
transformed lymphocytes, rather than normal lymphocytes, as
they were more convenient to obtain in large quantities.
The transformed lymphocytes can be grown in culture, thereby
precluding the need for human blood donors.
I found that at high concentrations of NaDOC the NC37
cells died by necrosis whereas at low concentrations the
cells died by apoptosis. Light microscopic examination
revealed that apoptosis was the predominant mode of NC-37
cell death at doses of NaDOC sO.39 roM after a 12 hours
incubation period. Necrosis occurred at doses greater than
0.39 roM and after a longer incubation period with sO.39 roM.
Bile acids at high concentrations have a strong detergent
action, which may cause membrane damage accompanied by
dissolution (Lapre et al., 1992; Rafter et al., 1986)
resulting in cell lysis ..
101
Since NaDOC induction of apoptosis was demonstrated
with cultured transformed lymphocytes it was decided to
determine if normal human mononuclear· cells also underwent
induction of apoptosis in response to NaDOC. The percentage
of apoptosis in these cells after 6 hours incubation with
NaDOC rose from 0.2% at 0 rnM to 35% at 0.78 rnM (Figure 8).
After 12 hours incubation, the percentage of apoptosis
decreased persumably due to development of secondary
necrosis.
As detailed in the Introduction, epidemiological
evidence suggests that bile acids have an etiologic role in
colon cancer. Bile acids are thought to promote colon cancer
but the mechanism is not known. Bile acids can cause DNA
damage in prokaryotic and eukaryotic cells (Zheng and
Bernstein, 1992; Kandell and Bernstein, 1991). DNA damage
induction by bile acids is probably indirect and may result
either from cytoskeleton-induced changes in conformation of
chromatin (Puck and Krystosek, 1992) making it more
susceptible to endonuclease attack, or from the generation
of free radicals (Craven et al., 1986; Schneider, 1992)
which are known to damage DNA (Imlay and Linn, 1988).
Unrepaired DNA damage can then lead to apoptosis (Corcoran
and Ray, 1992; Carson et al., 1986).
102
The present finding that NaDOC induces apoptosis in
lymphoid cells at high physiological concentrations may
provide a mechanism for a role of bile salts in colon cancer
through a general decrease in immunosurveillance. The
concentrations of NaDOC found to induce apoptosis (~0.39 mM)
are comparable to levels found in the gut since Rafter et
al. (1987) found that the concentration of deoxycholic acid
in the aqueous phase of human stool is 0.2 mM. Therefore,
the GALT (Qut Associated ~ymphoid Tissue), which consists of
more activated lymphocytes than the peripheral circulation
(Peters et al., 1989), may be adversely affected by these
levels of bile salts.
Apoptosis can be added to the known list of effects of
bile acids on lymphocytes (Keane et al., 1984; Gianni et
al., 1980). Bile salt-induced apoptosis of lymphoid cells
can clarify the mechanism of bile salt impaired lymphocyte
function described by Keane et al. (1984). They found no
diminution in cell viability by trypan blue exclusion after
incubation of the lymphocyte with NaDOC. Since apoptotic
cells can exclude trypan blue for sometime in culture, this
test does not rule out that apoptosis occured in their
system. Exposure of cells of the immunosurveillance system
to elevated bile acids in the lamina propria of the gut may
lead to their apoptotic death, lowering the number available
to carry out immune attack on mutant colonic epithelial
103
cells that had progressed along the pathway to colon cancer.
Our finding that bile salts induce apoptosis in one
cell type, raises the possibility that bile salts may induce
apoptosis in other cell types. Therefore, I next tested the
effect of NaDOC on colonic epithelial cells, since bile
salts are considered to be promotors of colon cancer
(Narisawa et al., 1974; Reddy et al., 1992).
B. Bile salt induction of apoptosis in human colonic
epithelial cells
I found, for the first time, that NaDOC, induces a high
level of apoptosis in the colonic goblet cells of normal
sUbjects. Also I found that the normal-appearing portion of
the colon of subjects with colon cancer, or at high risk for
colon cancer, is relatively resistant to NaDOC induction of
apoptosis (Figures 19 and 20). The goblet cells from the
tumorous tissue itself, from the one colon cancer patient
studied, were highly resistant to NaDOC-induced apoptosis
and lysis up to 6.24 mM (Figure 13). The sub-micellar
concentrations of NaDOC (Brito and Vaz, 1986) (sl.0 mM),
employed in this study, were within, or close to, the range
normally found in the colonic contents. Van Fassen et al.
(1987) showed that deoxycholic acid occurs at about 1.85 mM
in the feces of individuals with a mixed Western diet.
Rafter et al. (1987) showed that DCA is the main soluble
104
bile acid in fecal water occuring at about 0.2 mM. Thus the
concentrations of bile salt used in this study is probably
within the range that accompanies a high fat diet.
The very wide variation observed in the resistance to
NaDOC-induction of apoptosis among patients with non
malignant polyps suggests that this bioassay may prove
useful as a prognostic marker of the susceptibility of such
patients to colon cancer. That is, patients with polyps
that show resistance to bile salt-induced apoptosis (similar
to the colon cancer patients) may be at higher risk than
those whose induction of apoptosis is closer to that of
patients with normal colons.
DeRubertis et al. (1984) found in rats that 4 hours
after intracolonic instillation of 1mM NaDOC there was no
inflammatory reaction, although increasing the time of
exposure or increasing the dose of NaDOC resulted in an
inflammatory reaction and lysis of goblet cells. This
result is similar to the present study since absence of
inflammation is always associated with apoptosis, not
necrosis. However this study did not address the question
of whether apoptosis was occuring under these conditions.
Colonic cell proliferation increases with age in
individuals consuming a high fat diet (Stadler et al.,
1988). A high level of fat ingestion over several decades
exposes the colonic lumen to chronically high concentrations
105
of bile salts that'have been produced to aid in digestion of
fat. Bile salts have been shown to cause DNA damage in
mammalian cells (Kandell and Bernstein, 1991). Apoptosis
serves to protect humans from cancer by eliminating cells
with unrepaired DNA damage (Lane, 1992). The susceptibility
of the goblet cells to the induction of apoptosis by NaDOC
might be a selective advantage in avoiding colon
carcinogenesis. Apoptosis may be an efficient method of
preventing malignant transformation in colon epithelial
cells by removing cells with unrepaired DNA damage that
would otherwise tend to mutate upon replication. This view
is in accord with that of Cohen and Duke (1992), who have
noted "better dead than wrong" with respect to induction of
apoptosis in lymphocytes. The selective deletion of cells
with unrepaired DNA damage has also been reported in other
studies (Searle et al., 1975; Potten, 1977).
A high level of fat ingestion, causing a high
concentration of NaDOC in the colon may lead to a high
frequency of apoptosis in colonic goblet cells. Over time,
however, continuation of this process may select for
survival of mutant progenitor goblet cells which have become
relatively resistant to apoptosis induction. Prolonged
intake of a high fat diet may thus result in the
repopulation of colonic mucosa by mutant cells resistant to
induction of apoptosis. When these cells replicate their
DNA past these damages, there is a high probability of
further mutations occuring, some of which may lead to
carcinogenesis.
106
The above explanation for the increased resistance to
apoptosis found in the normal portion of the colon of colon
cancer and high risk patients is supported by recent
experiments of Magnuson et al. (1994). They showed that
when rats were chronically fed the bile acid cholic acid,
the cells within their colonic crypts developed resistance
to induction of apoptosis by the carcinogen azoxymethane.
Both cells within normal appearing colonic crypts and cells
within aberrant crypt foci (preneoplastic lesions of colon
cancer) were resistant to induction of apoptosis.
Hague et al. (1995) found low induction of apoptosis in
the carcinoma cell line PC/JW/F1 and that a colonic adenoma
cell line AA/C1 was relatively resistant to NaDOC induction
of apoptosis. These authors speculated that deoxycholate
acts as a tumor promotor in the large intestine through
selection for a sub-populations of cells resistant to
deoxycholate-induced apoptosis.
Many studies have focused on cellular kinetics in the
colonic mucosa aiming to discover early markers of
neoplastic transformation which could be used in devising
preventive strategies to reduce the risk of colon cancer
(Lipkin et al., 1983; Biasco et al., 1992; Rosen, 1992).
107
The approach reported here involving NaDOC-induction of
apoptosis in biopsy specimens of the colon epithelium may
prove useful as a possible biomarker for susceptibility to
colon cancer.
Relative bile salt-resistance to apoptosis in high risk
polyp patients and colon cancer patients might also suggest
changes in the mucosa surrounding the lesion and development
of IItransitional mucosa ll• Transitional mucosa is the
thickened mucosa that may extend for several centimeteres
beyond the edge of a colorectal cancer or large adenoma.
Transitional mucosa differs morphologically and functionally
from normalcolorectal mucosa. Several authors have
suggested a link between transitional mucosa and colorectal
cancer in an evolutionary sequence (Rognum et al., 1982).
C. Possible role of p53 in NaDOC-induced apoptosis and colon
carcinogenesis
The role of apoptosis in colon cancer can be clarified
by considering the action of the tumor suppressor gene pS3.
The normal function of the the pS3 gene is to act as a
policeman to allow the cell to deal with DNA damage (Lane,
1992). The pS3 protein is a transcriptional regulator
mediating G1 arrest following DNA damage, allowing time for
DNA repair (Lane, 1992). When there is a loss or mutation
of the pS3 gene, the cell cannot respond normally to DNA
108
damage (Lane, 1992). Kuerbitz et al. (1992) pointed out
that a CRC cell line possessing normal p53 function arrested
normally following DNA damage, while a transfected mutant
p53 acted in a dominant negative manner to prevent arrest.
p53 blocks cell division, so that the cell has chance to
repair its DNA, but if there is too much damage, p53
programs the cell to commit suicide (Lane, 1992).
Gene p53 is required for induction of apoptosis by
several DNA damaging agents (Lowe et al., 1993). However
Hague et al. (1995) found that NaDOC induces apoptosis in a
carcinoma cell line obtained from a patient with familial
polyposis syndrome in the absence of the p53 gene. This
implies that NaDOC-induced apoptosis may not depend on p53
function.
Allelic loss or mutation of the p53 gene occurs in the
late stage of development of colon cancer in 70-80% of colon
tumors (Lane, 1992). Therefore p53 mutation may account, in
part, for the high resistance to NaDOC-induced apoptosis of
colonic goblet cells from tumorous tissue.
D. Hypothetical mechanisms for bile salt induction of
apoptosis
109
There are at least three distinct mechanisms (alone or
together) by which bile salt might trigger apoptosis.
1) Elevation of calcium
Bile salts are known to cause release of calcium from
the endoplasmic reticulum (Combettes et al., 1988).
Elevated intracellular calcium appears to induce several
aspects of apoptosis. The earliest detectable change in
cells undergoing apoptosis is a rapid sustained increase in
cytosolic calcium concentration (Schwartzman and Cidlowski,
1993). Elevated intracellular calcium mediates cytoskeletal
alterations which appear to be part of apoptosis. Elevated
calcium also seems to activate a transglutaminase that
crosslinks proteins during apoptosis (Knight et al., 1991).
It also activates both a protease that may degrade the
cytoskelton (Carson and Ribeiro, 1993), and the Ca++ Mg++
dependent endonuclease present in most nuclei (Cohen and
Duke, 1984). Elevation of intracellular calcium appears to
be the rate limiting step in DNA fragmentation in ionophore
treated thymocytes (McConkey et al., 1989). A sustained
elevation of cytosolic calcium may be part of a general
physiologic pathway of apoptosis (Orrenius et al., 1989).
Also, calcium antagonists inhibit DNA fragmentation induced
110
by acetaminophen in mice (Ray et al., 1993). McConkeyet
al. (1988) concluded that increase in cytosolic calcium
concentration might be significant in tetrachlorodibenzo-P
dioxin (TCDD) induced apoptosis and in thymocyte DNA
fragmentation, since agents that inhibit this increase block
both DNA fragmentation and apoptosis. McConkey et al.
(1989) concluded that calcium-stimulated DNA fragmentation
is the apparant cause of apoptosis in rat thymocytes. The
role of elevated calcium in bile salt-induced apoptosis in
colonic goblet and lymphoid cells is currently unknown.
2) Cytoskeletal changes
I found that NaDOC causes surface blebbing early in
treatment (20 minutes exposure) in lymphoid cells. Surface
blebbing is a consistent marker for the disruption of the
cytoskeleton (Orrenius et al., 1989). Kaneko et al. (1993)
reported that bile acids may promote tumor formation by
disturbance of the cytoskeleton. Surface blebbing is also a
general phenomenon associated with the late stages of
apoptosis (Orrenius et al., 1989; Wyllie et al., 1980).
Surface blebbing can result from increased cytosolic
calcium, and bile salts have been shown to release calcium
from the endoplasmic reticulum (Combettes et al., 1988).
The association of the cytoskeletal network and the
interaction of different classes of cytoskeletal filaments
111
have been suggested to depend on the cytosolic calcium level
(Orrenius et al., 1989). This dependence was supported by
the finding that the calcium ionophore calcimycin (Nicotera
et al., 1986) and quinones (Jewell et al., 1982), which
raise calcium, causes surface blebbing.
There are at least two mechanisms by which increased
calcium may cause surface blebbing. First, increased calcium
causes the dissociation of actin from actinin, a protein
that serves as an intermediate in the association of
microfilaments with actin-binding protein in the plasma
membrane (Orrenius et al., 1989). The second mechanism by
which calcium may increase blebbing could involve activation
of a protease which removes the plasma membrane anchor for
the cytoskeleton, forming sites of weakness leading to
surface blebs (Orrenius et al., 1989).
There is evidence that cytochalasin B causes surface
blebbing and participates in apoptosis via its action on
actin (Kolber et al., 1990). Cytoskeletal changes are known
to cause changes in the conformation of DNA (Puck and
Krystosek, 1992) making it more susceptible to intranuclear
endonuclease attack (Kolber et al., 1990). It is thus
possible that early cytoskeletal changes may trigger the
apoptotic process by a process involving DNA degradation.
112
3) Endonuclease activation
An early event in apoptosis is the activation of a
specific endonuclease which reduces chromosomal DNA to
oligonucleosomal fragments, usually preceding cell death
(Cohen and Duke, 1984; Arends et al., 1990). This enzyme is
activated by Ca++ and Mg++ but is inhibited by Zn++ (Cohen
and Duke, 1984). Arends et al. (1990) concluded that
selective activation of endonuclease was responsible for not
only DNA fragmentation but also for the major nuclear
morphologic changes. Cohen and Duke (1984) suggested that
activation of the endonuclease could be considered a general
mechanism for apoptosis. Numerous studies showed that
fragmentation of DNA into mUltiples of 180 base pair
subunits by cleavage in the linker region between
nucleosomes results from activation of the Mg++ Ca++
dependent endonuclease in rat and mouse thymocytes (Wyllie,
1980; Cohen and Duke, 1984; Arends et al., 1990; McConkeyet
al., 1988; Duke et al., 1983). Studies with cell types
other than thymocytes, however, indicate the lack of
involvement of an endonuclease during apoptosis (Bursch et
al., 1992; Collins et al., 1992; Samaha et al., 1995).
Whether this endonuclease has a role in the apoptotic
process induced specifically by bile acids is unknown.
113
E. Prospects for colorectal cancer prevention
It would appear worth while to further investigate the
phenomenon of bile acid-induced apoptosis in order to
evaluate the contribution of high fat diet to the etiology,
pathogenesis and progression of colon preneoplasia to colon
neoplasia. Bile acid-induced apoptosis may also prove
useful as a biomarker for detection of patients with
increased risk of colon cancer, but further investigation of
this potential is needed. Should this biomarker prove to be
reliable, individuals at risk could then be readily
identified and advised to take all possible steps towards
preventing colon cancer. Such steps would include a low
fat, high fiber diet, dietary supplements designed to
acidify the colon contents and frequent monitoring for high
risk lesions.
114
V REFERENCES
Alme, B., Bremmelgaard, A., Sjovall, J. and Thomassen, P. (1977) Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gasliquid chromatography-mass spectrometry. J. Lipid Res. 18, 339-362.
Arends, M. J., Morris, R. G. and Wyllie, A. H. (1990) Apoptosis. The role of the endonuclease. Amer. J. Pathol. 136, 593-608.
Armstrong, B. K. and Doll, R. (1975) Enviromental factors and cancer incidence and mortality in different countries, with special reference to dietary practices. Int. J. Cancer. 15, 616-631.
Bayerdorffer, E., Mannes, G. A., Ochsenkuhn, T., Dirschedl, P. and Paumgartner, G. (1994) Variation of serum bile acids in patients with colorectal adenomas during a one-year follow-up. Digestion 55, 121-129.
Bayerdorffer, E., Mannes, G. A., Richter, W.O., Ochsenkuhn, T., Wiebecke, B., Kopcke, W. and Paumgartner, G. (1993) Increased serum deoxycholic acids levels in men with colorectal adenomas. Gastroenterology 104, 145-151.
Biasco, G., Paganelli, G. M., Miglioli, M. and Barbara, L. (1992) Cell proliferation markers in the gastrointestinal tract. J. Cell Biochem., Supplement 16G, 73-78.
Bird, R. P., Medline, A., Furrer, R. and 'Bruce, W. R. (1985) Toxicity or orally administered fat to the colonic epithelium. Carcinogenesis 6, 1063-1066.
Bjerknes, M. and Cheng, H. (1981) The stem-cell zone of the small intestine epithelium. 1. Evidence from paneth cells in the adult mouse. Am. J. Anat. 160, 51-63. .
Bond, J. H. (1993) Screening and early detection In: Colorectal Cancer. Ed Harold J. Wanebo Mosby St. Louis, Baltimore, Boston, Chicago, London, Madrid Philadelphia, Sydney and Toronto. pp 149-157.
Boring, C. C., Squires, T. S. and Tong, T. (1993) Cancer Statistics. CA 43, 7-26.
Boyle, P., Zaridze, D. G. and Smans, M. (1985) Descriptive epidemiology of colorectal cancer. Int. J. Cancer 36, 9-18.
115
Brito, R. M. M. and Vaz, W. L. C. (1986) Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine. Analyt. Biochem. 152, 250-255.
Bruce, W. R. (1987) Recent hypotheses for the origin of colon cancer. Cancer Res. 47, 4237-4242.
Bull, A. W., Marnett, L. J., Dawe, E. J. and Nigro, N. D. (1983) Stimulation of deoxythymidine incorporation in the colon of rats treated intrarectally with bile acids and fats. Carcinogenesis 4, 207-210.
Buntain, W. L., ReMine, W. H. and Farrow, G. M. (1972) Premalignancy of polyps of the colon. Surgery, Gynecology and obstetrics 134 (3) 499-508.
Burkitt, D. P. (1971) Epidemiology of cancer of the colon and rectum. Cancer 28, 3-13.
Burmer, G. C. and Loeb, L. A. (1989) Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma. Proc. Natl. Acad. Sci. (U. S. A.), 86, 2403-2407.
Bursch, W., Oberhammer, F. and Schulte-Hermann, R. (1992) Cell death by apoptosis and its protective role against disease. TiPS 13, 245-251.
Buset, M., Lipkin, M., Winawer, S., Swaroop, S. and Freedman, E. (1986) Inhibition of human colonic epithelial cell proliferation in vivo and in vitro by calcium. Cancer Res. 46, 5426-5430.
Bussey, H. J. R. (1968) Age and cancer of the large intestine. In Cancer and aging. Thule International Symposia. Arthur Engel and Tage Larsson (eds) Nordiska Bokhandelhns Forlag, Stockholm 1. page 203-214.
Carr, K. E. and Toner, P. G. (1982) Techniques and applications In Cell structure: An introduction to biomedical electron microscopy. Churchill Livingstone. Edinburgh London Melbourne and New York. page 108-124.
Carson, D. A. and Ribeiro, J. M. (1993) Apoptosis and disease. Lancet 341, 1251-1254.
Carson, D. A., Seto, S., Wasson, D. B. and Carrera, C. J. (1986) DNA strand breaks, NAD metabolism, and progammed cell death. EXp. Cell Res. 164, 273-281.
Cheah, P. Y. (1990) Hypotheses for the etiology of colorectal cancer-an overview. Nutr. Cancer 14, 5-13.
116
Cheah, P. Y. and Bernstein, H. (1990) Modification of DNA by bile acids: a possible factor in the etiology of colon cancer. Cancer Letters 49, 207-210.
Child, P. and Rafter, J. (1986) Calcium enhances the hemolytic action of bile salts. Biochim. Biophys. Acta 855, 357-364.
Cohen, J. J. and Duke, R. C. (1984) Glucocorticoid activation of a calcium-dependent endonuclease leads to cell death. J. Immunol. 132, 38-42.
Cohen, J. J. and Duke, R. C. (1992) Apoptosis and programmed cell death in immunity. Annual Review of Immunol., 10, 267-293.
Cohen, S. M. and Ellwein, L. B. (1990) Cell proliferation in carcinogenesis. Science 249, 1007-1011.
Collins, R. J., Harmon, B. V., Gobe, G. C. and Kerr, J. F. R. (1992) Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int. J. Rad. BioI. 61, 451-453.
Combettes, L., Dumont, M., Berthon, B., Erlinger, S. and Claret, M. (1988) Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells. J. BioI. chern. 263, no 5, 2299-2303.
Cook, J. W., Kennaway, E. L. and Kennaway, N. W. (1940) Production of tumors in mice by deoxycholic acid. Nature (London) 145, 627.
Cooper, H. S., Patchefsky, A. S. and Marks, G. (1979) Adenomatous and carcinomatous changes within hyperplastic colonic epithelium. Dis. Colon Rectum, 22, 152-156.
Corcoran, G. and Ray, S. (1992) The role of the nucleus and other compartments in toxic cell death produced by alkylating hepatotoxicants. Toxicol. Appl. Pharmacol. 113, 167-183.
Correa, P., Strong, J. P., Reif, A. and Johnson, W. D. (1977) The epidemiology of colorectal polyps. Prevalence in New Orleans and international comparisons. Cancer, 39, 2258-2264.
117
Cotter, T. G., Lennon, S. V., Glynn, J. G. and Martin, S. J. (1990) Cell death via apoptosis and its relationship to growth. Development and differentiation of both tumour and normal cells. Anticancer Res. 10, 1153-1160.
Cranley, J. P., Petras, R. E., Carey, W. D., Paradis, K. and Sivac, M. V. (1986) When is endoscopic polypectomy adequate therapy for colonic polyps containing invasive carcinoma? Gastroenterology, 91, 419-427.
Craven, P. A., Pfanstiel, J. and DeRubertis, F. R. (1986) Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation. J. Clin. Invest. 77, 850-859.
Culling, C. F. A., Reid, P. E., Worth, A. and Dunn, W. L. (1977) A new histochemical technique of use in the interpretation and diagnosis of adenocarcinoma and villous lesions in the large intestine. J. Clin. Pathol. 30, 1056-1062.
Dahm, L. J., Hewett, J. A. and Roth, R. A. (1988) Bile and bile salts potentiate superoxide anion release from activated, rat peritoneal neutrophils. Toxicology and Applied Pharmacology 95, 82-92.
DeRubertis, F. R., Craven, P. A. and Seito, R. (1984) Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products. J. Clin. Invest. 74, 1614-1624.
Dive, C. and Hickman, J. A. (1991) Drug-target interactions: only the first step in the commitment to a programmed cell death? Review. Br. J. Cancer 64, 192-196.
Duke, R. C., Chervenak, R. and Cohen, J. J. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc. Natl. Acad. Sci. USA 80, 6361-6365.
Eastman, A. (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells 2, 275-280.
Eastwood, M. A. and Mitchell, W. D. (1976) Physical properties of fiber: A biological evaluation. In Fiber in human nutrition. ed. G. A. Spiller and R. J. Amen pp 109-129. New York: Plenum.
Elitsur, Y., Bull, A. W. and Luk, G. (1990) Modulation of human colonic lamina propria lymphocyte proliferation. Effect of bile acids and oxidized fatty acids. Digestive Diseases and Sciences 35 (2), 212-220.
118
Fearon, E. R. and Vogelstein, B. (1990) A genetic model for colorectal tumorigenesis. Cell 61, 759-767.
Fesus, L. Today 13,
(1992) Immunoregulation: (8), A16-A:t7.
Apoptosis. Immunology
Franzin, G. and Novelli, P. (1982) Adenocarcinoma occurring in a hyperplastic (metaplastic) polyp of the colon. Endoscopy, 14, 28-30.
Gavrieli, Y., Sherman, Y. and Ben-Sasson, S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell BioI. 119, 493-501.
Gianni, L., Di Padova, F., Zuin, M. and Podda, M. (1980) Bile acid-induced inhibition of the lymphoproliferative response to phytohemagglutinin and pokeweed mitogen: an in vitro study. Gastroenterol. 78, 231-235.
Hague, A., Elder, D. J. E., Hicks, D. J. and Paraskeva, C. (1995) Apoptosis in colorectal tumour cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate. Int. J. Cancer 60, 400-406.
Hamilton, S. R. (1992) Molecular genetics of colorectal carcinoma. Cancer 70, 1216-1221.
Hill, M. J. (1982) Genetic and enviromental factors in human colorectal cancer. In Colon Carcinogenesis. Malt R, Williams RCN (eds). MTP Press Ltd, Lancaster 73-79.
Hill, M. J. (1983) Bile, bacteria and bowel cancer. Gut, 24, 871-875.
Hill, M. J. (1990) Bile flow and colon cancer. Mut. Res. 238, 313-320.
Hill, M. J. (1986) Bile acids and colorectal cancer in humans. In Dietary Fiber, Basic and Clinical Aspects (G. V. Vahouny and D. Kritchevsky, eds) , New York: Plenum. 497-513.
119
Hill, M. J., Morson, B. C. and Bussey, H. J.R. (1978) The aetiology of the adenoma-carcinoma sequence. Lancet i, 245-247.
Hill, M. J., Taylor, A. J., Thompson, M. H. and Wait, R. (1982) Faecal steroids and urinary volatile phenols in fou~ Scandinavian populations. Nutr. Cancer 4, 67-73.
Hoerni, B. and Laporte, G. (1970) Immunological disorders in the aetiology of lymphoreticular neoplasms. Biomedicine 15, 841-850.
Hoffman, B. and Liebermann, D. A. (1994) Molecular controls of apoptosis: differentiation/growth arrest primary response genes, proto-oncogenes, and tumor suppressor genes as positive& negative modulators. Oncogene 9, 1807-1812.
Ijiri, K. and Potten, C. S. (1983) Response of intestinal cells of differing topographical and hierarchical status to ten cytotoxic drugs and five sources of radiation. Br. J. Cancer 47, 175-185.
Imlay, J. A. and Linn, S. (1988) DNA damage and oxygen radical toxicity. Science 240, 1302-1309.
Jacobs, L. R. (1988) Role of dietary factors in cell replication and colon cancer. Am. J. Clin. Nutr. 48, 775-779.
Jass, J. R. (1985) Diet, butyric acid and differentiation of gastrointestinal tract tumours. Med. Hypoth. 18, 113-118.
Jass, J. R. (1989) Do all colorectal carcinomas arise in preexisting adenomas? World J. Surg. 13, 45-51.
Jass, J. R., Sugihara, K. and Love, S. B. (1988) Basis of sialic acid heterogeneity in ulcerative colitis. J. Clin. Pathol. 41, 388-392.
Jewell, S. A., Bellomo, G., Thor, H. Orrenius, S. and Smith, M. (1982) Bleb formation in hepatocytes during drug metabolism is caused by disturbances in thiol and calcium ion homeostasis. Science 217, 1257-1259.
Kandell, R. L. and Bernstein, C. (1991) Bile salt/acid induction of DNA damage in bacterial and mammalian cells: implications for colon cancer. Nutr. Cancer 16, 227-238.
120
Kaneko, M., Kodama, M., Inoue, F. and Horikoshi, J. (1993) Lithocholic acid-sensitive mutant cells isolated from Chinese hamster ovary cells are cross-sensitive to cytoskeleton-disturbing agents. Toxicology letters 66, 211-219.
Kaye, G. I., Fenoglio, C. M., Pascal, R. R. and Lane, N. (1973) Comparative electron microscopic features of normal, hyperplastic and adenomatous human colonic epithelium. Gastroenterology 64, 926-945.
Keane, R. M., Gadacz, T. R., Munster, A. M., Birmingham, W. and Winchurch, R. A. (1983) Impairment of human lymphocyte function by bile salts. Surgery 95, 439-443.
Keren, D. F. (1988) Gastrointestinal immune system and its disorders. In Gastrointestinal Pathology. Harvey Goldman, Henry Appelman and Nathan Kaufman (editors). Williams and Wilkins London page 247-285.
Kerr, J. F. R. (1971) Shrinkage necrosis: a distinct mode of cellular death. J. Pathol. 105, 13-20.
Kerr, J., Searle, J., Harmon, B. and Bishop, C. (1987) Apoptosis. In: Perspectives on Mammalian Cell Death (C. S. Potten,eds), Oxford University Press, Oxford, New York. 93-128.
Kerr, J. F. R. and Searle, J. (1980) Apoptosis: its nature and kinetic role. In: Radiation Biology in Cancer Research (R. E. Meyn and H. R. Withers, eds) , Raven Press, New York. 367-384.
Kerr, J. F. R., Wyllie, A. H. and Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. cancer 26, 239-257.
Knight, C. R., Rees, R. C. and Griffin, M. (1991) Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumour progression. Biochim. Biophys. Acta 1096, 312-318.
Knudson, A. G. (1985) Hereditary cancer, oncogenes and antioncogenes. Cancer Res. 45, 1437-1443.
Kolber, M. A., Broschat, K. o. and Landa-Gonzalez, B. (1990) Cytochalasin B induces cellular DNA fragmentation. FASEB J.
4, 3021-3027.
121
Konishi, F. and Morson, B. C. (1982) Pathology of colorectal adenomas: a colonoscopic survey. J. Clin. Pathol. 35, 830-841.
Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V. and Kastan, M. B. (1992) Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. 89, 7491-7495.
Kurtz, R. c. (1995) Diagnostic approach to the symptomatic patient. In Cancer of the colon, rectum and anus. editors Alfred M. Cohen, Sidney J. Winawer, 'Michael A. Friedman and Leonard L. Gunderson. pp 371-375.
Kyprianou, N. and Issacs, J. T. (1988) Activation of programmed cell death in the rat ventral prostate after castration. Endocrinol. 122, 552-562.
LaMuralgia, G. M., Lacaine, F. and Malt, R. A. (1986) High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann. Surg. 204, 89-93.
Lane, D. P. (1992) p53, guardian of the genome. Nature, 358, 15-16.
Lapre, J. A., Termont, D. S. M. L., Groen, A. K. and Van Der Meer, R. (1992) Lytic effects of mixed micelles of fatty acids and bile acids. Am. J. Physiol. 263, G333-G337.
Lapre, J. A. and Van Der Meer, R. (1992) Diet-induced increase in colonic bile acids stimulate lytic activity of fecal water and proliferation of colonic cells. carcinogenesis 13, 41-44.
Lennon, S. V., Martin, S. J. and Cotter, T. G. (1990) Induction of apoptosis (programmed cell death) in tumour cell lines by widely diverging stimuli. Biochem. Soc. Trans. 18, 343-345.
Lennon, S. V., Martin, S. J. and Cotter, T. G. (1991) Dosedependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif. 24, 203-214.
Levine, A. J., Momand, J. and Finlay, C. A. (1991) The p53 suppressor gene. Nature (London), 351, 453-456.
Lewin, K. J. (1988) Enteric infections In Gastrointestinal Pathology edited by H. Goldman, H. Appelman and N. Kaufman. Williams and Wilkins. London. page 286-305.
122
Lipkin, M. (1988) Biomarkers of increased susceptibility to gastrointestinal cancer: new applications to studies of cancer prevention in human subjects. Cancer Res. 48, 235-245.
Lipkin, M., Blattner, W. E., Fraumeni Jr., J. F., Lynch, H. T., Deschner, E. and Winawer, S. (1983) Tritiated thymidine labeling distribution as a marker for hereditary predisposition to colon cancer. Cancer Res. 43, 1899-1904.
Lipkin, M. and Newmark, H. (1985) Effect of added dietary calcium on colonic epithelial proliferation in subjects at high risk for familial colonic cancer. N. Engl. J. Med. 313, 1381-1384.
Lipkin, M., Uehara, K., Winawer, S.,Sauchez, A., Bauer, C., Philips, R., Lynch, H. T., Blattner, W. and Fraumeni J. F. (1985) Seventh-day adventist vegetarians have a quiescent proliferative activity in colonic mucosa. Cancer Lett. 26, 139-144.
Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A. and Jacks, T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature, 362, 847-849.
Madara, J. L., Nash, S., Moore, R. and Atisook, K. (1988) Structure and function of the intestinal epithelial barrier in health and disease. In Gastrointestinal Pathology edited by H. Goldman, H. Appelman and N. Ka~fman. page 306-324.
Magnuson, B. A. and Bird, R. P. (1994) Resistance of aberrant crypt foci to apoptosis induced by azoxymethane in rats chronically fed cholic acid. Carcinogenesis 15, 1459-1462.
McBride, o. W., Merry, D. and Givol, D. (1986) The gene for human p53 cellular tumor antigen is located on chromosome 17 short arm (17p13). Proc. Natl. Acad. Sci. USA. 83, 130-134.
McConkey, D. J., Hartzell, P., Duddy, S. K., Hakansson, H. and Orrenius, S. (1988) 2, 3, 7, 8-tetrachlorodibenzo-pdioxin kills immature thymocytes by Ca++ mediated endonuclease activation. Science 242, 256-259.
McConkey, D. J., Hartzell, P., Nicotera, P. and Orren ius , S. (1989) Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J. 3, 1843-1849.
123
McIntyre, A., Gibson, P. R. and Young, G. P. (1993) Butyrate production from dietary fibre and protection against large bowel cancer in a rat model. Gut 34, 386-391.
Morris, R. G., Hargreaves, A. D., Duvall, E. and Wyllie, A. H. (1984) Hormone-induced cell death. Surface changes in thymocytes undergoing apoptosis. Am. J. Pathol., 115, 426-436.
Morson, B. C., Dawson, I. P., Day, D. W., Jass, J. R., Price, A. B. and Williams, G. T. (1990a) Benign epithelial tumors and polyps In Gastro-intestinal Pathology. Blackwell Scientific Publications. Oxford London. Edinburgh Boston Melbourn. page 563-596.
Morson, B. C., Dawson, I. P., Day, D. W., Jass, J. R., Price, A. B. and Williams, G. T. (1990b) Malignant epithelial tumors. In Gastro-intestinal Pathology. Blackwell Scientific Publications. Oxford London. Edinburgh Boston Melbourn. page 597-629.
Narisawa, T., Magadia, N. E., Weisburger, J. H. and Wynder, E. L. (1974) Promoting effect of bile acids on colon carcinogenesis after intrarectal instillation of N-methyl-Nnitro-N-nitrosoguanidine. J. Natl. Cancer Inst. 53, 1093-1097.
Newmark, H. L., Wargovich, M. J. and Bruce, W. R. (1984) Colon cancer and dietary fat, phosphate and calcium: a hypothesis. J. Natl Cancer Inst. 72, 1323-1325.
Nicotera, P., Hartzell, P., Davis, G. and Orrenius, S. (1986) The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2
+ is mediated by the activation of a non-lysosomal proteolytic system. FEBS Lett. 209, 139-144.
Orren ius , S., McConkey, D. J., Bellomo, G. and Nicotera, P. (1989) Role of Ca2
+ in toxic cell killing. TiPS 10, 281-285.
Paumgartner, G. (1986) Serum bile acids. Physiological determinants and results in liver disease. J. Hepatol. 2, 291-298.
Payne, C. M., Bjore, C. G. and Schultz, D. A. (1992) Change in the frequency of apoptosis after low- and high-dose xirradiation of human lymphocytes. J. Leuk. BioI. 52, 433-440.
124
Payne, C. M. and Cromey, D. W. (1991) Ultrastructural analysis of apoptotic and normal cells using digital imaging techniques. J. Comput-Assist. Micros. 3, 33-50.
Payne, C. M. and Glasser, L. (1981) Evaluation of surface markers on normal human lymphocytes containing parallel tubular arrays: a quantitative ultrastructural study. Blood 57, No.3, 567-573.
Payne, C. M., Nagle, R. B. and Lynch, P. J. (1984) Quantitative electron microscopy in the diagnosis of mycosis fungoides. A simple analysis of lymphocytic nuclear convolutions. Archives of Dermatology 120 (1) 63-75.
Peters, M. G., Secrist, H., Anders, K. R., Nash, G. S., Rich, S. R. and MacDermott, R. P. (1989) Normal human intestinal B lymphocytes. Increased activation compared with peripheral blood. J. Clin. Invest. 83, 1827-1833.
Potten, C. S. (1977) Extreme sensitivity of some intestinal crypt cells to X and Y irradiation. Nature, 269, 518-521.
Puck, T. T. and Krystosek, A. (1992) Role of the cytoskeleton in genome regulation and cancer. Int. Rev. Cytol. 132, 75-108.
Raff, M. c. (1992) Social controls on cell survival and cell death. Nature 356, 397-400.
Rafter, J. J., Child, P., Anderson, A., Alder, R., Eng, V., and Bruce, W. R. (1987) Cellular toxicity of fecal water depends on diet. Am. J. Clin. Nutr. 45, 559-563.
Rafter, J. J., Eng, V. W. S., Furrer, R., Medline, A. and Bruce, W. R. (1986) Effects of calcium and pH on the mucosal damage produced by deoxycholic acid in the rat colon. Gut 27, 1320-1329.
Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yorkin, R. D. and Corcoran, G. B. (1993) Ca2
+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. FASEB J. 7, 453-463.
Reddy, B. S. (1981a) Dietary fat and its relatioship to large bowel cancer. Cancer Res. 41, 3700-3705.
Reddy, B. S. (1981b) Diet and excretion of bile acids. Cancer Res. 41, 3766-3768.
125
Reddy,B. S., Ekelund, G., Bone, M., Engle, A. and Domellof, L. (1983) Metabolic epidemiology of colon cancer: dietary patterns and faecal sterol concentrations of three populations. Nutr. Cancer 5, 34-40.
Reddy, B. S., Engle, A., Simi, B. and Goldman, M. (1992) Effect of dietary fiber on colonic bacterial enzymes and bile acids in relation to colon cancer. Gastroenterology 102, 1475-1482.
Reddy, B. S., Mangat, S., Sheinfil, A., Weisburger, J. H. and Wynder, E. L. (1977) Effect of type and amount of dietary fat and l,2-dimethylhydrazine on biliary bile acids, and neutral sterols in rats. Cancer Research 37, 2132-2137.
Reddy, B. S., Watanabe, K. and Sheinfil, A. (1980) Effect of dietary wheat bran, alfalfa, pectin and carrageenan on plasma cholesterol and fecal bile acid and neutral sterols in rats. J. Nutr. 110, 1247-1254.
Reddy, B. S. and Wynder, E. L. (1973) Large bowel carcinogenesis: fecal constituents of populations with diverse incidence rates of colon cancer. J. Natl. Cancer Inst. 50, 1437-1442.
Rickert, R. R., Auerbach, a., Garfinkel, L., Hammond, E. C. and Frasca, J. M. (1979) Adenomatous lesions of the large bowel: an autopsy survey. Cancer 43, 1847-1857.
Roberton, A. M. (1993) Roles of endogenous substances and bacteria in colorectal cancer. Mutation Res. 290, 71-78.
Rognum, T. a., Elgio, K., Brandtzaeg, P., Orjasaeter, H. and Bergan, A. (1982) Plasma carcinoembryonic antigen concentrations and immunohistochemical patterns of epithelial marker antigens in patients with large bowel carcinoma. J. Clin. Pathol. 35, 922-933.
Rokkas, T., Karameris, A. and Mikou, G. (1993) Small polyps found at sigmoidoscopy: are they significant? HepatoGastroenterol. 40, 475-477.
Rosen, P. (1992) An evaluation of rectal epithelial proliferation measurement as a biomarker of risk for colorectal neoplasia and response in intervention studies. Eur. J. Cancer Prevent., 1, 215-224.
Rustgi, A. K. and Podolsky, D. K. (1992) The molecular basis of colon cancer. Annual Rev. Med. 43, 61-68.
126
Salaman, M. H. and Roe, F. J. C. (1956) Further tests for tumour initiating ability: N, N-Di-(2-Chloroethyl)-paminophenybutyric acid (CB1348) as initiators of skin tumour formation in the mouse. Brit. J. Cancer 10, 363-378.
Samaha, H., Asher, E., Bernstein, C., Payne, C. M. and Bernstein, H. (1995a) Evaluation of cell death in EBVtransformed lymphocytes using agarose gel electrophoresis, light microscopy and electron microscopy.- Induction of classic apoptosis by the bile salt, sodium deoxycholate. Leuk. Lymph. (in press) .
Samaha, H., Bernstein, C., Payne, C. M., Garewal, H. S., Sampliner, R. E. and Bernstein, H. (1995b) Bile salt induction of apoptosis in goblet cells of the normal human colonic mucosa: relevance to colon cancer. Acta microscopica (in press) .
Samelson, S. L., Nelson, R. L. and Nyhus, L. M. (1985) Protective role of faecal pH in experimental colon carcinogenesis. J. Roy. Soc. Med. 78, 230-233.
Sanderson, C. J. (1982) mediated cytotoxicity. cytotoxicity, ed. W. R. York: Plenum.
Morphological aspects of lymphocyte In Mechanisms of cell mediated Clark, P. Golstein, pp. 3-21. New
Sarraf, C. E. and Bowen, I. D. (1988) proportions of mitotic and apoptotic cells in a range of untreated experimental tumours. Cell Tissue Kinet. 21, 45-49.
Saunders, J. w. (1966) Death in embryonic systems. Science 154, 604-612.
Schneider, H. (1992) A factor in the increased risk of colorectal cancer due to ingestion of animal fat is inhibition of colon epithelial cell glutathione Stransferase, an enzyme that detoxifies mutagens. Medical Hypothesis 39, 119-122.
Schottenfeld, D. (1995) Epidemiology. In Cancer of the colon, rectum and anus. editors Alfred M. Cohen, Sidney J. Winawer, Michael A. Friedman and Leonard L. Gunderson. pp 11-24.
Schwartzman, R. A. and Cidlowski, J. A. (1993) Apoptosis: The biochemistry and molecular biology of programmed cell death. Endocrine Rev. 14, 2, 133-151.
127
Searle, J., Lawson, T. A., Abbott, P. J., Harmon, B. and Kerr, J. F. R. (1975) An electron-microscope study of the mode of cell death induced by cancer chemotherapeutic agents in populations of proliferating normal and neoplastic cells. J. Pathol. 116, 129-138.
Searle, J., Kerr, J. F. R. and Bishop, C. J. (1982) Necrosis and apoptosis: Distinct modes of cell death with fundamentally different significance. Pathol. Annual. 17, 229-259.
Setchell, K. D. R., Street, J. M. and Sjovall, J. (1986) Fecal bile acids In: The bile acids: chemistry, physiology, and Metabolism. Volume 4: Methods and Applications. eds K. D. R., Setchell, D. Kritchevsky and P. P. Nair. Page 441-570.
Silverberg, E. (1987) Cancer Statistics. CA Cancer J. Clin. 37, 2-19.
Silverman, S. J. and Andrews, A. W. (1977) Bile acids: Comutagenic activity on the Salmonella-mammalian microsome mutagenicity test. J. Natl Cancer lnst. 59, 1557-1560.
Smith-Barbaro, P., Hanson, D. and Reddy, B. S. (1981) carcinogen binding to various types of dietary fiber. J. Natl. Cancer lnst. 67, 495-497.
Sorenson, A. W., Slattery, M. L. and Ford, M. H. (1988) Calcium and colon cancer: a review, Nutr. Cancer 11, 135-145.
Specian, R. D. and Oliver, M. G. (1991) Functional biology of intestinal goblet cells. Am J. Phsiol 260, C183-C193.
Spagnesi, M. T., Tonelli, F., Dolara, P., Caderni, G., Valanzano, R., Anastasi, A. and Bianchini, F. (1994) Rectal proliferation and polyp occurrence in patients with familial adenomatous polyposis after sulindac treatment. Gastroenterology 106, 362-366.
Stadler, J., Stern, H. S. and Yeung, K. A. S. (1988) Effect of high fat consumption on cell proliferation activity of colorectal mucosa and on soluble faecal bile acids. Gut 29, 1326-1331.
Sunter, J. P. (1984) Cell proliferation in gastrointestinal carcinogenesis. Scand. J. Gastroenterol. 19, (suppl104), 45-55.
128
Takano, S., Matsushima, M., Erturk, E. and Bryan, G. T. (1981) Early induction of rat colonic epithelial ornithine and S-adenosyl-L-methionine decarboxylase activities by Nmethyl-N-nitro-N-nitrosoguanidine by bile salts. Cancer Res. 41, 624-628.
Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456-1462.
Thornton, J. R. (1981) High colonic pH promotes colorectal cancer. Lancet i, 1081-1083.
Touchette, N. and Fogle, S. (1991) Apoptosis: it chimes with mitosis. J. NIH Res. 3, 75-78.
Ucker, D. S. (1991) Death by suicide: one way to go in mammalian cellular development? The New BioI. 3, 103-109.
Vamosi-Nagi, I. and Koves, I. (1993) Correlation between colon adenoma and cancer. Eur. J. Surg. Oncol. 19, Suppl. 1, 619-624.
Van Berge Henegouwen, G. P. and Hofmann, A. F. (1983) Systemic spill-over of bile acids. Eur. J. Clin. Invest. 13, 433-437.
Van der Meer, R., Termont, D. S. M. L. and De Vries, H. T. (1991) Differential effects of calcium ions and calcium phosphate 9n cytotoxicity of bile acids. Am. J. Physiol. 260, G142-G147.
Van Faassen, A., Bol, J., Van Dokkum, W., Pikkar, N. A., Ockhuizen, T. and Hermus, R. J. J. (1987) Bile acids, neutral steroids and bacteria in feces as affected by a mixed, a lacto-ovovegetarian, and a vegan diet. Amer. J. Clin. Nutr. 46, 962-967.
Van Munster, I. P., Tangerman, A. De Hann, A. F. J. and Nagengast, F. M. (1993) A new method for the determination of the cytotoxicity of bile acids and aqueous phase of stool: the effect of calcium. European J. Clin Invest. 23, 773-777.
Velardi, A. L. M., Groen, A. K., Oude Elferink, R. P. J., Van der Meer, R., Palasciano, G. and Tytgat, G. N. J. (1991) Cell type-dependent effect of phospholipid and cholesterol on bile salt cytotoxicity. Gastroenterol. 101, 457-464.
Waller, D. A., Thomas, N. W. and Self, T. J. (1988) Epithelial restitution in the large intestine of the rat following insult with bile salts. Virchows Archiv. A Pathol. Anat. 414, 77-81.
Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556.
129
Wyllie, A. H. (1981) Cell death: a new classification separating apoptosis from necrosis. In: Cell death in biology and pathology, edited by Bowen, I. D. and Lockshin, R. A. New York, New York: Chapman and Hall. p. 9-34.
Wyllie, A. H., Kerr, J. F. R. and Currie, A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306.
Wyllie, A. H., Kerr, J. F. R., Macaskill, I. A. M. and Currie, A. R. (1973) Adrenocortical cell deletion: the role of ACTH. J. Pathol. 111, 85-94.
Wyllie, A. H., Morris, R. G., Smith, A. L. and Dunlop, D. (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67-77.
Wynder, E. L. (1975) The epidemiology of large bowel cancer. Cancer Res. 35, 3388-3394.
Wynder, E. L. (1987) Amount and type of fat/fiber in nutritional carcinogenesis. Prevo Med. 16, 451-459.
Young, R. W. (1984) Cell death during differentiation of the retina in the mouse. J. Compo Neurol. 229, 362-373.
Zheng, Z-Y. and Bernstein, C. (1992) Bile salt/acid induction of DNA damage in bacterial cells: effect of taurine conjugation. Nutr. Cancer 18, 157-164.
