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l LIBRARY

Mlchlgan State
University

 

 

This is to certify that the

thesis entitled
Tyrosine Phosphorylation Status of Proteins in

Rat Colon Mucosa and in the Colon Cancer
Cell Lines HT-29 and HCT 116
presented by

Mridvika

has been accepted towards fulfillment
of the requirements for

Masters degree in Human Nutrition

Major professor

Date October 17, 1996

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this chockom from your record.
TO AVOID FINES return on or before date duo.

 

DATE DUE DATE DUE DATE DUE

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MSU lsAn Affirmative Action/Equal Opportunity Intuition
WM1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TYROSINE PHOSPHORYLATION STATUS OF PROTEINS IN RAT
COLONIMUCOSA.AND IN THE COLON CANCER CELL LINES
HT-29 AND HCT 116

BY

Mridvika

1k IflflESIIB

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MBSTER OF SCIENCE
Department of Food Science and Human Nutrition

1996

lfliSTTUUET

TYROSINE PHOSPHORYLATION STATUS OF PROTEINS IN RAT
COLONIMUCOSA.AND IN THE COLON CANCER CELL LINES
HT-29 AND HCT 116

BY

lflRIIHflEKA.

A study was conducted to determine protein tyrosine
phosphorylation status in colon mucosal cells of rats
treated with 1,2-dimethylhydrazine and in HT—29 and HCT 116
colon cancer cells at three points in the cell cycle.
Synchronized colon cancer cells were harvested at O, 0.5,
and 8 hrs after being induced to enter the cell cycle.
Extracted proteins were resolved with 2-dimensional
electrophoresis and tyrosine phosphorylated proteins were
detected using specific antibodies. In HT-29 colon cancer
cells four proteins (MW's 50, 56, 56 and 77 KD) had changed
tyrosine phosphorylation status at different points in the
cell cycle and in HCT 116 cells, ten such proteins (MW's 28
to 92KD) were detected. Two proteins of MWs 50 and 57KB were
common to both cell lines. In rat colon mucosa seven
proteins in molecular weight range 36-67 KD were found to be
tyrosine phosphorylated to a greater or lesser extent on the

third and seventh day after DMH treatment.

To my grandmother, mother and sister; three generations
of influential women whose mentoring I enjoy.

iii

ACKNOWLEDGMENTS

I would like to sincerely thank my major professor, Dr.
Maurice Bennink for his guidance, understanding, moral
support and constant encouragement. Thanks are also extended
to members of my committee, Dr. Dale Romsos and Dr. William
Helferich for their guidance and patience throughout the
program.

I would like to thank all my colleagues, and especially
Dr. Leslie Bourquin for his valuable input in this project.
Many thanks to my friends who have always been there for me.

Finally, I would also like to extend heartfelt thanks
to my dear family whose love and support have always been my

inspiration to achieve and excel.

iv

THUBLE1(IF CKNWTERTPS

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION

REVIEW OF LITERATURE

A.

B.

EPIDEMIOLOGICAL REVIEW

A.l Incidence of Colon Cancer

A.2 Distribution of Colon Cancer

A.3 Diet and Colon Cancer

CELL PROLIFERATION AND DIFFERENTIATION IN COLON

CANCER

EXPERIMENTAL MODELS IN CANCER RESEARCH

C.l Animal Models

C.2 Cell Culture and Cancer Research

GENETIC BASIS OF COLON CANCER

D.l Mutations in Colon Cancer

D.2 Alternative Modes of Regulation of Cell
Number

PROTEIN TYROSINE PHOSPHORYLATION

E.l Tyrosine kinases and Cancer

.....

E.2 Cell Cycle and Cancer
E.3 Cell Cycle and Tyrosine Kinases
E.4 Protein Tyrosine Kinases and Colon Cancer

F. JUSTIFICATION
G. OBJECTIVE
H. NULL HYPOTHESIS
MATERIALS AND METHODS

EXPERIMENTAL DESIGN

ANIMALS AND HOUSING

CHEMICALS AND REAGENTS
METHODS

1,2-Dimethylhydrazine Administration to Rats
Tissue Collection and Preparation

Cell Culture

Polyacrylamide Gel Electrophoresis
2-Dimensional Electrophoresis

Western Blotting

Visualization of Phospho-tyrosine Containing
Proteins

Analysis of Gels and Autoradiographs
RESULTS AND DISCUSSION

Study 1.

Study 2.

Study 3.

HT—29

vi

.....

.....

.....

HCT 116 52

.....

Study 4. ..... 59
SUMMARY AND CONCLUSIONS ..... 72
RECOMMENDATIONS FOR FUTURE RESEARCH ..... 75
BIBLIOGRAPHY ..... 77

vii

Table

Table

Table

Table

Table

LIST OF TABLES

Dietary composition of the modified

AIN-94 diet fed to rats in studies 1,

2 and 4. These quantities are for
10 Kg of diet.

Phosphotyrosine containing proteins
in HT-29 cells. The three symbols
within in each replicate represent
three time points, 0, 0.5 and 8
hours after addition of PBS.

Phosphotyrosine containing protein
spots in HCT 116 cells. The three
symbols in each replicate represent
three time points, 0, 0.5 and 8
hours after addition of FBS.

Phosphotyrosine containing protein
spots in DMH treated and control
rats. The three/four symbols in each
group represent three/four rats in
that group.

Phosphotyrosine containing proteins
common to HT-29 and HCT 116.

viii

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Proteins from colonic mucosal homogenates
from control rat (no DMH treatment) and
DMH treated rats resolved on 12%

acrylamide SDS—PAGE gels. The proteins were

stained with coomassie blue.

Proteins from colonic mucosal homogenates
from control rat (no DMH treatment), DMH
treated rats sacrificed on day 3 after
treatment and DMH treated rats sacrificed
on day 7 treatment resolved on 12%

acrylamide SDS-PAGE gels. The proteins were

stained with coomassie blue.

HT-29 cells 0 hrs after stimulation by the
addition of PBS to the media, left:
phosphotyrosine containing proteins
visualized by anti-phospho-tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

HT-29 cells 0.5 hrs after stimulation by
the addition of PBS to the media, left:
phosphotyrosine containing proteins
visualized by anti-phospho-tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

HT-29 cells 8 hrs after stimulation by the
addition of FBS to the media, left:
phosphotyrosine containing proteins
visualized by anti-phospho-tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

HCT 116 cells 0 hrs after stimulation by

ix

4O

42

48

49

50

.53

Figure 7.

Figure

Figure

Figure

Figure

8.

10.

11

the addition of FBS to the media, left:
phosphotyrosine containing proteins
visualized by anti-phospho—tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

HCT 116 cells 0.5 hrs after stimulation by
the addition of FBS to the media, left:
phosphotyrosine containing proteins
visualized by anti-phospho-tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

HCT 116 cells 8 hrs after stimulation by
the addition of FBS to the media, left:
phosphotyrosine containing proteins
visualized by anti—phospho-tyrosine mAb in
2-dimensional gels, right: proteins
resolved by 2-dimensional electrophoresis
and visualized by silver staining.

Colonic mucosal proteins from a control

(no DMH treatment) rat, resolved by

2-dimensional electrophoresis, and western
blotted for phosphotyrosine.

Colonic mucosal proteins from a DMH
treated rat (sacrificed day 3 after
administration), resolved by 2-
dimensional electrophoresis, and western
blotted for phosphotyrosine.

.Colonic mucosal proteins from a DMH

treated rat (sacrificed day 3 after
administration), resolved by 2-
dimensional electrophoresis, and western
blotted for phosphotyrosine.

54

55

61

62

63

INTRODUCTION

Colon cancer claims the lives of nearly 51,000 people
in the United States every year. The incidence of colon
cancer is related to the consumption of a western type diet
which is high in fat and low in fiber. A number of studies
have reported positive correlations between consumption of
fat (Stemmerman, Nomura and Heilbrun, 1984), meat or animal
protein (Enstrom, 1975) and colon cancer incidence (Thun,
1992; Howe, 1992) strongly suggesting a link between diet
and colon cancer.

Genetically, colon cancer is the combined result of
several mutations in the cells of the colon mucosa. These
mutations can result in either activation of oncogenes or
inactivation of tumor suppressor genes (Fearon and
Vogelstein, 1990). It has been suggested that the
chronological order in which these mutations come into
effect is not relevant to the assessment of the risk for
developing colon cancer. The severity of the risk for colon
cancer is related to the number of accumulated mutations

(Fearon and Jones, 1992). People with one or two mutations

2
might have a nOrmal appearing mucosa and still be at risk
for developing the disease. It is this group of people that
are likely to benefit most from dietary intervention of some
kind. To assess the risk for colon cancer in a clinical
setting, bio—markers have been developed.

Studies have shown that hyperproliferation of the
normal appearing colon is probably the earliest detectable
abnormality observed in people pre-disposed to colon cancer
(Lipkin, 1974; Deschner, 1978). Hyperproliferation is the
condition in which the dividing cells of the colon fail to
differentiate as they normally would into mature cells and
remain in a proliferating mode and is considered an early
bio—marker for colon cancer, measured by radio-labeling or
immuno-labeling in histological sections. These techniques
are time and labor intensive and require specific technical
training. Therefore, a protein marker for hyperproliferation
would offer an advantage in detection of colon cancer risk.

The objective of my study was to identify proteins
where tyrosine phosphorylation reflected the proliferation
state of the colon mucosal cells. This was studied in HT—29
and HCT 116 colon cancer cells and 1,2—dimethylhydrazine
treated animals. The proteins were studied using two
dimensional electrophoresis and immuno-detection with

monoclonal antibodies.

REVIEW OF LITERATURE

A . EPIDEMIOLOGICAL REVIEW
.A.1. Incidence of Colon Cancer

The past hundred years have seen a marked decline
in deaths caused by infectious diseases and an increase
in expected life span. As a result, cancer and
cardiovascular disease, traditionally "diseases of the
old age" incidence continues to increase in a population
that is rapidly aging. Whereas, the downward trends in
cardiovascular disease have been encouraging, after
decades of intensive research, cancer still continues to
be a major cause of mortality in the US with colon
cancer being among the deadliest of cancers. A 1996
publication of the American Cancer Society reported
133,500 new cases in 1996 of colon cancer and rectum
cancer combined, making it the fourth most incident
cancer (after breast, lung and prostrate) and the second

most common cause of cancer death (after lung) in the US

4

(Cancer Facts and Figures, 1996).

A.2. Distribution of Colon Cancer

Epidemiological studies have shown that colon cancer
incidence varies about twenty-fold across countries of the
world. The first indication of such a variation came from
Segi's compilations of cancer mortality statistics for the
years 1950-1957 (Segi, 1960). At the time, it was suspected
that these intercountry differences were inflated as a
result of differences in diagnostic and treatment facilities
and death certification practices across countries.
Subsequent studies have refuted these doubts and confirmed
Segi's findings (Muir et al., 1987; Parkin et al., 1992).
The highest rates of CC incidence are found in the developed
world - North America, Western Europe and Australia- with
age adjusted incidence rates of 25—35 cases per 100,000 in
the late 1980's. In contrast, much lower rates occur in
countries of Asia, Africa and southern Europe with the
lowest rates found in India, that is, 1-3 per 100,000 people

(Cancer Facts and Figures, 1992).

A13. Diet and Colon Cancer
Following the observation of the disparate rates of

colon cancer, differences in diet patterns across countries

5
were suggested as a possible explanation. Burkitt
hypothesized that a low-bulk, high starch diet might be
involved in the causation of colon cancer (Burkitt, 1969).
Since then, many epidemiological studies have examined
correlations between the consumption of certain nutrients
and non-nutrients and the incidence of CC. The focus of most
of these studies has been fat, meat, animal protein, fiber
or cereal intakes. A number of studies have reported
positive correlations between consumption of fat
(Stemmerman, Nomura and Heilbrun, 1984), meat or animal
protein (Enstrom, 1975) and colon cancer incidence (Thun,
1992; Howe, 1992). Some research has examined the
possibility that the high energy intake associated with such
diets might be the risk factor for colon cancer incidence
(Lyon et al., 1987). Extensive work also has been conducted
to ascertain the link between lifestyle patterns and colon
cancer incidence. Seventh-Day Adventists abstain from
smoking and drinking and most of them consume an ovo—lacto-
vegetarian diet, a diet including eggs and milk but
excluding meat. A reduced risk for colon cancer has been
reported in this population relative to the rest of the
population (Phillips and Snowdon, 1985; Phillips, 1975). A
lower colon cancer risk also has been associated with
consumption of diets with a high fiber content (Burkitt,

1969 ;Howe, 1992). In most of these studies, comparisons of

6
incidence and mortality from colorectal cancer and diet
across populations are based on food disappearance data
obtained from the Food and Agricultural Organization (FAQ).
All these studies indicate that diet plays a significant
role in colon cancer.

The incrimination of environmental factors rather than
genetic factors as important risk determinants for colon
cancer was further corroborated by studies of migrant
populations. It was observed that migrants moving from a low
risk area to a high risk area acquired, within their
lifetime, the high risk characteristic of the host
population. For example, Japanese migrating to the US
exhibit the high risk rates for colon cancer typical of US
whites rather than the low risk rates typical of Japan

(Haenzel and Kurihara, 1968).

B. CELL PROLIFERATION AND DIFFERENTIATION IN COLON CANCER
The high mortality rates due to colon cancer have led
to enormous interest in early detection and treatment of the
disease. Also, since the disease has a major dietary
component, detection of predisposition to colon cancer can
make dietary intervention possible at a stage early enough
to make a difference. To make early detection possible, the

proliferation and differentiation characteristics of colon

7
mucosal cells have been studied extensively. Many studies
have shown that epithelial cell proliferation and
differentiation is modified in conditions predisposing to
colon cancer.

Normal colon cells are columnar, mucous secreting, or
enteroendocrine and are arranged in crypts. In a normal
colonic crypt most of the DNA synthesis activity is confined
to the lower one-third of the crypt. Cell proliferation is
almost always completed in the lower two-thirds of the crypt
(Lipkin, 1988). The region of the crypt where cell division
occurs is termed the proliferative zone. The dividing cells
at the bottom of the crypt differentiate and mature as they
migrate up the crypt toward the luminal surface. By the time
these cells reach the luminal surface they are mature,
differentiated, non—proliferating cells (Maskens and
Deschner, 1977). Rarely does cell division occur at the
luminal surface of normal mucosa. This scenario is radically
altered in certain diseased states. An expansion of the
proliferative zone (also referred to as hyper-
proliferation), expansion of the major zone of DNA
synthesis, and high levels of DNA synthesis within colonic
crypts are some of the changes observed in the mucosa of
patients with gastrointestinal disturbances (Lipkin and
Higgins, 1988). Hyper-proliferation usually means an

increase in the mitotic index, which is a ratio of the

8
number of dividing cells in a crypt to the total number of
cells in the crypt.

Studies of proliferation and differentiation of colonic
mucosal cells have helped identify the stages of abnormal
development associated with an increased susceptibility to
colon cancer (Deschner, 1978; Lipkin, Venara and Winawer,
1985). Studies of normal appearing colon mucosa of people at
greater risk for developing colon cancer have shown that
hyperproliferation of the colonic mucosa is among the first
abnormalities detected. This hyperproliferation has been
observed in subjects with familial polyposis, sporadic
adenomas, or previous histories of familial or non—familial
colon cancers (Lipkin, 1974). Hyperproliferation leading
toward an expansion of the proliferative zone toward the
luminal surface corresponds to stage 1 in the development of
adenomas. In stage 1, the major zone of DNA synthesis,
however, continues to be the lower one-third of colonic
crypts. This stage has been observed in the colonic mucosa
of people with familial polyposis,‘isolated adenomas, colon
cancer and relatives of individuals who have familial
polyposis (Deschner and Lipkin, 1975). The next stage in
adenoma formation, stage 2, is characterized by a shift in
the principal zone of DNA synthesis from the lower one-third
toward the middle and upper thirds of the crypt (Maskens and

Deschner, 1977). This stage was first identified in the

9
colonic mucosa of people with a previous history of colon
cancer and was later observed in other high risk groups,
such as, familial polyposis, non—symptomatic relatives of
polyposis patients, people with sporadic polyps and colon
cancer patients and has never been observed in people with
no history of gastrointestinal disease (Deschner, 1980).
Stage 3 of adenoma development has been described as the
presence of isolated crypts with high levels of DNA
synthesis activity (Deschner and Maskens, 1981). The
labeling index (LI, the percentage of dividing cells in a
crypt) of these crypts was reported to be greater than
15%. Only one out of 13 control patients (no gastro—
intestinal abnormality) had such crypts, but 17 out of 26
patients with a history of colon cancer or isolated adenomas
were positive for the presence of these hyper-proliferative
crypts.

Hyper-proliferation and an upward shift in the
proliferative zone result in appearance of mitotic cells at
the mouth of the colonic crypts and in the flat mucosa. It
is believed that this leads to an accumulation of cells near
the mouth of the crypt in an otherwise flat mucosa. The
accumulated cells eventually form polyps which initially are
small and benign. With further changes over a period of

time, some of these polyps grow and develop into adenomas.

10
It has been shown that most, if not all carcinomas are
formed from pre-existing adenomas.

Abnormal cell proliferation and differentiation and
pre-neoplastic gene expression are considered to be
biomarkers of an increased risk of colon cancer (Lipkin,
1988). Measurements of cell proliferation and
differentiation also aid research concerned with cancer
prevention through dietary intervention. These measurements
have aided the development and application of intermediate
biomarkers to studies of cancer prevention, thus precluding

the use of actual neoplasia as the endpoint.

C. EXPERIMENTAL MODELS IN CANCER RESEARCH
C.1. Animal models

The major advantage afforded by the use of animals in
cancer research is that it allows researchers to study the
various factors in diet which might individually play a role
in cancer promotion or prevention. Most of the animal models
are based on the administration of certain chemicals either
orally, subcutaneously or intravenously. These chemicals
cause mutations leading to increased cell division in target

tissues that results in the formation of tumors which are

ll
benign to begin with, and ultimately become cancerous as
additional mutations occur.

The use of natural cycasin or synthetic chemicals to
induce colon cancer in rats has been utilized for many years
(Laqueur and Spatz, 1968). Currently, the most commonly used
chemicals for inducing colon cancer in the rat are 1,2—
dimethylhydrazine (DMH), methylazoxy-methanol (MAM) or MAM
acetate (MAMAc). Since the introduction of symmetrical DMH
as a specific carcinogen for the colon by Druckery and his
associates, it has been used widely for investigating colon
carcinogenesis (Druckery et al., 1975). DMH induces hyper-
proliferation and other manifestations of abnormal cell
division and differentiation found in human colon mucosa
progressing towards malignancy. It is postulated to do so by
inducing aberrations in the normal sequence of DNA
replication and repair. In addition, it is also extremely
cytotoxic, so 1—3 days after administration,
hyperproliferation occurs to replace the dead cells. The
detoxifying enzymes in the liver convert DMH into the
activated methylating agent, methyl-azoxy-methanol and the
oxidative demethylation of the activated MAM results in the
methylation of the purine base guanine in both RNA and DNA.
Guanine methylation results in the introduction of changes
in the-DNA sequence (mutations) during replication. The

introduction of these changes in a rapidly proliferating

12
tissue such as the colonic epithelium leads to the formation
of tumors over a period of time.

In a number of studies Deschner and co-workers have
demonstrated the proliferative defects induced by 1,2-
dimethylhydrazine. In 1973, Thurnherr and Deschner reported
an increased number of proliferating cells close to the
luminal surface of colonic mucosa of mice in response to
seven weekly injections of DMH when compared to controls
which did not receive any DMH, even though the histology was
normal and the labeling index was unchanged (Thurnherr et
al., 1973). After 87 days, the colonic mucosal labeling
index of DMH-treated mice was 16.2%, whereas control mice
had a labeling index of 6.2%. At this time, the morphology
of colonic crypts from DMH-treated mice was also deranged.
In a similar study (Deschner, 1978), histological changes in
mice were observed in response to six weekly injections of
DMH. Labeling index peaked on the second and third days
after carcinogen administration to about 19% (compared to
6.6-10.7% for untreated controls), dropped to values close
to those of the controls, but rose again at week 7 after
carcinogen administration. In 1982, Deschner and Maskens
reported colonic mucosal cell proliferation in a group of
cancer patients and in DMH-treated mice (Deschner and
Maskens, 1982). The colonic mucosa of patients with sporadic

polyps was characterized by the presence of isolated crypts

13
with labeling index greater than 20%; such crypts were
absent in control subjects that had no gastrointestinal
disease. The distribution of proliferating cells in crypts
with labeling index greater than 15% was compared to crypts
with labeling index between 6-8%. Crypts having both high
and low labeling index demonstrated the Stage 1 and Stage 2
abnormalities described previously, but they were more
emphatically expressed in crypts with labeling index greater
than 15% (Deschner and Maskens, 1982). Isolated crypts of
DMH-treated mice had labeling indices between 21 to 38%,
whereas labeling index in crypts from untreated control mice
never approached 21%. In the control mice, proliferating
cells were located exclusively in the lower two-thirds of
colonic crypts. Crypts of the DMH treated mice showed an
extension of the proliferative compartment closer to the
lumen and an upward shift in the major zone of DNA synthesis

(Deschner and Maskens, 1982).

C.2. Cell Culture and Cancer Research

Animal models have contributed a great deal to our
understanding of colon cancer and the influence of nutrition
on cancer development, but these studies are often limited
by their long duration and high cost. In recent years

several colon cancer cell lines derived from human tumors

14
become commercially available. Since it is possible to
control for factors that can influence tumor development in
cell culture, these techniques have made a large
contribution to our understanding of colon cancer. Much
attention has been focused on the development of dependable,
early neoplastic markers in cell culture. Mutations which
initialize early steps in cancer cause changes in proteins.
There is potential to tap into one of these changes and use

as a marker.

D. GENETIC BASIS OF COLON CANCER
D.1. Mutations in Colon Cancer

The changes in the proliferation of cells of the colon
mucosa are caused by alterations in cell cycle control such
as delayed onset of normal terminal differentiation. Colon
cancer appears to be the cumulative result of mutational
activation of oncogenes and inactivation of tumor suppressor
genes (Fearon and Vogelstein, 1990). Some of the molecular
events underlying the initiation and progression of the
multistep process of tumorigenesis described have been
identified (Fearon and Vogelstein, 1990). Three stages of
adenoma development have been described. Early stage
adenomas are defined as adenomas less than one cm in size.

Intermediate stage adenomas are greater than one cm in size,

15
but do not contain foci of carcinoma. Late stage adenomas
are greater than one cm in size and contain foci of
carcinoma (Fearon and Jones, 1992). The genetic changes
associated with the progression of an early adenoma to
carcinoma include mutations of the K-ras gene, loss of
heterozygosity (LOH) at certain loci, and allelic deletions
in chromosomes 17p and 18q (Fearon et al., 1990). The gene
deleted in chromosome 18q, called DCC (Deleted in Colon
Cancer), is missing in 50% of late adenomas and 70% of
carcinomas. This gene encodes for a protein with significant
homology to the cell adhesion family of molecules. Absence
of the protein product of this gene probably alters cell
adhesion, causing diminution of growth restraining signals
associated with such adhesion. The 17p protein product, p53,
is a tumor suppressor protein whose deletion often marks the
conversion of adenomas into carcinomas (Fearon and Jones,
1992).

A genetic model for the development of colon cancer
proposed by Fearon and Jones in 1992 relates the timing of
the genetic events to the development of colon cancer
(Fearon and Jones, 1992). People with familial polyposis
have a germ-line mutation in the APC gene, that results in
hyperproliferation of the colonic epithelium. Somatic.APC
(adenomatous polyposis coli) gene mutations (chromosome 5q

LOH) can result in similar changes in patients without

l6
familial polyposis. Hyperproliferation of the colonic
epithelium precedes the formation of adenomas. The
transformation of early adenomas to intermediate adenomas is
often associated with K-ras gene mutation. Additional events
such as chromosome 18q LOH (the DCC mutation), and
chromosome 17p LOH (p53 mutation) often mark the transition

of a late adenoma to a carcinoma.

D.2. Alternative Modes of Regulation of Cell Number

The underlying cause of cancer is often thought to be
the loss of control on cell proliferation. Recently this
idea has undergone a change. With the realization that cell
number is a result of opposing effects of cell proliferation
and cell death, the latter has taken on greater
significance. Since recent studies have suggested that cell
death, like cell proliferation, is subject to molecular
control, thus cancer can result from loss of regulation of
either cell proliferation or cell death or the combination
of two (Green et al., 1996).

Apoptosis, or programmed cell death, is of particular
importance in this regard. One of the observations that
support this claim is that most physical and chemical agents
that have antitumor activity act to induce apoptosis

(Lennon, Martin and Cotter, 1991; Martin and Green, 1994;

17

Martin and Green, 1995). In addition, it has been noted that
frequency of apoptotic cells in tumor correlates very well
with the outcome. That is, tumors with low apoptotic indices
tend to be more aggressive than those displaying higher
incidence of apoptotic cell death (Arends, McGregor and
Wyllie, 1994). The observation that links apoptosis to
protein phosphorylation is that the process of apoptosis is
regulated by a number of oncogenes and anti-tumor oncogenes
(Martin and Green, 1994; Martin and Green, 1995; Bissonnette
et al., 1994), many of which are found to be mutated en
route to cancer.

The proteins involved in apoptosis can be grouped into
two categories - the ones that promote apoptosis and the
ones that inhibit apoptosis. Interestingly the effects of
these two classes of proteins on transformation are opposing
to their effect on apoptosis, which is consistent with
expected relationships between these activities. Bc1-2
(Korsemeyer, 1992; Reed, 1994) and Abl (McGohan, Cotter and
Green, 1994) promote transformation and block apoptosis,
while p53 acts to interfere with oncogenesis and promotes
some forms of apoptosis (Yonish-Rouach et al., 1991; Shaw et
al., 1992; Lowe et al., 1993). In view of these
observations, the activities of Myc and Ras are perhaps

surprising. Ras and Myc are two proto-oncogenes that

18
function in normal cells to promote proliferation but are
found to promote apoptosis under some conditions
(Bissonnette et al., 1994; Wyllie et al., 1987; Askew et
al., 1991; Evan et al., 1992; Tanaka et al., 1994).

The Bcl-2 family of proteins are a family of unique
proteins. The protein encoded by the BCL-2 gene, for
example, bears no significant amino acid homology with other
proteins whose biochemical mechanism of action is known.
From the analysis of the amino acid sequence, it appears
that Bcl-2 is an integral membrane protein whose
transmembrane domain and C terminal region are essential for
optimal function (Cazals-Hatem et al., 1992; Krajewski et
al., 1993; Tanaka et al., 1993; Borner et al., 1994). At
present, six mammalian homologs of Bcl-2 have been reported,
including Bax, Bcl-X, Mcl-l, A1, Bad and Bak (Oltvai et al.,
1993; Boise et al., 1993; Kozopas et al., 1993; Lin et al.,
1993a; Yang et al., 1995; Chittenden et al., 1995; Kiefer et
al., 1995 ; Farrow et al., 1995). Of these, the Bax, Bad and
Bak proteins function as promoters of cell death.
Conversely, the Mcl-l and A1 proteins appear to be
suppressors of cell death, along with Bcl-2.

Although the BCL-2 gene was first discovered because of
it's role in t(14;18) translocations found in non-Hodgkin's
lymphoma, high levels of BCL-Z gene expression have been

found in a variety of human cancers including 90% of

l9
colorectal cancers (Reed, 1994; Reed, 1995). Moreover, BCL-2
gene expression has been reported to be suppressed by p53,
through direct or indirect mechanisms. In experiments where
p53 function was conditionally restored to a p53—deficient
murine leukemia line, p53 was shown to induce marked
decreases in bcl-2 gene expression followed by apoptotic
cell death (Miyashata et al., 1994). The p53 gene located on
chromosome 17p is mutated in greater than 70% of human

colorectal cancers.

E. PROTEIN TYROSINE PHOSPHORYLATION
E.1. Tyrosine Kinases and Cancer

Phosphorylation and dephosphorylation of cellular
proteins at their tyrosine, serine or threonine residues is
a predominant method for regulating enzyme activity. Many of
these proteins have a role in regulating the cell cycle.
Protein tyrosine kinases (PTKs), therefore, were an obvious
choice for researchers in their quest to better understand
the molecular mechanisms underlining the cellular changes
associated with cancer (Hunter and Cooper, 1985).

The highly conserved sequences of the genes encoding
PTKs in species ranging from humans to simple eukaryotes
suggest the importance of tyrosine phosphorylation in

cellular physiology (Hunter and Cooper, 1985). Today we

20
recognize two major classes of PTKs - (1) those whose genes
have become a part of retroviruses, and (ii) those
associated with growth factor receptors.

The genes for the type of PTK's that become a part of
the retroviruses are present in the normal eukaryotic genome
as proto-oncogenes. The protein products of these proto—
oncogenes are membrane PTK's. These proteins have an
exoplasmic ligand binding site, a trans-membrane part and a
cytoplasmic kinase site. Under normal conditions, the
binding of a specific ligand on the ligand-binding site of
the PTK activates the kinase to phosphorylate cellular
proteins. This can trigger a cascade of events that
culminates in a response by the cell, cell division being
one such response. The capture of receptor tyrosine kinases
(RTK) into the genomes of acutely transforming retroviruses
has been one of the predominant mechanisms by which these
molecules become overexpressed or inappropriately expressed.
For example, the RTK’s erbB (chicken) (Ulrich et al., 1984),
sea (chicken) (Smith et al., 1989), kit (feline) (Besmer et
al., 1986), and fms (feline) (Hampe et al., 1982) were first
uncovered in this fashion. Both overexpression and mutation
appear to be features of the RTK sequences captured by
retroviruses. N-terminal truncations usually remove a

significant portion of the extracellular ligand binding

21
domain and are found in v-kit (Besmer et al., 1986), v—erbB
(Ulrich et al., 1984) and v-sea (Smith et al., 1989). Thus,
a mutation resulting from the action of a carcinogen can
lead to the conversion of a proto—oncogene to an oncogene
that codes for a PTK with the exoplasmic ligand binding site
either missing or dysfunctional. The activation of the
kinase activity of this protein thus becomes independent of
ligand binding (Hunter and Cooper, 1985). Such a cell
receives signal to divide even in the absence of external
stimulus, throwing the cell into a hyper-proliferative
state.

A good example of this would be the overexpression or
aberrant expression of epidermal growth factor(EGF)
receptors that have been reported in cell lines derived from
gastric cancer (Yasui et a1, 1988) and squamous cancer of
various origins such as skin, tongue, esophagus, gingiva and
vulva (Yamamoto et al, 1986). On the other hand, activated
pp600‘m',a known cytoplasmic kinase, has been observed to
be activated in colon carcinoma cells (Cartright, Meisler

and Eckhart, 1990).

E.2. Cell Cycle and Cancer
Accumulating evidence suggests that derangements in the

cell cycle machinery may contribute to the uncontrolled cell

22
growth characteristic of a tumor. Over the past few years,
cell biologists have made remarkable progress in
understanding of the control of the cell cycle and
identification of the proteins and factors that drive it.

The eukaryotic cell cycle consists of a phase of
chromosomal DNA replication (S phase) followed by
segregation of the replicated chromosomes into two daughter
cells during M phase. In most animal cells, gap phases G1
and 62 occur between M and S phases and between S and M
phases, respectively. During these gap phases, information
is integrated by the cell to determine the readiness of the
cell to enter either S or M phase. Thus, the Gl/S and G2/M
boundaries serve as major checkpoints for control of
progression of the cell through the cell cycle (Norbury and
Nurse, 1992).

Research on cell cycle control in several distinct
experimental systems has recently converged. Today, much is
understood about the proteins comprising the machinery which
drives the eukaryotic cell cycle. The key regulators of cell
cycle progression are proteins of the cdc2 protein kinase
family (p34wd), a highly conserved family of proteins in
eukaryotic cells. The kinase activity of these proteins is
dependent upon the association with another highly conserved
class of proteins called cyclins. Thus the p34“” family of

proteins are often referred to as cdks (cyclin—dependent

23

kinases) (Motokura and Arnold, 1992). Cyclins fluctuate in
level during cell cycle progression. Individual cyclins
typically peak in concentration during the stage of cell
cycle in which they are required and are degraded once that

point has passed (Doree and Galas, 1994).

E.3. Cell Cycle and Tyrosine Kinases

Different combinations of cdks and cyclin subunits form
kinase complexes which act at different points in the cell
cycle (Norbury and Nurse, 1992). In addition to association
with the different cyclin subunits at different stages of
the cell cycle, cdk activity is regulated by a multiple
phosphorylation events also. The best characterized example
is an inhibitory tyrosine phosphorylation Tyr-15 of p34“d
in the ATP-binding domain. The state of tyrosine
phosphorylation of p34““, and thus the entry into mitosis,
is determined by a balance of kinase and phosphatase
activities. A family of protein kinases, plond
phosphorylate Tyr-15 of p34““'s inhibiting it's kinase
activity, thereby preventing the onset of mitosis (Russel
and Nurse, 1987). A family of p34‘3dcz specific tyrosine
phosphatases (p80““”) dephosphorylate the same site,
activating p34wd and permitting entry into M phase.

Phosphorylation has also been implicated in regulating

24
the abundance of Ga cyclins in the cell. In yeast
phosphorylation of Cln 2, a<3_cyclin that binds Cdc 28
provides the signal that is necessary for it’s rapid
degradation. It is speculated that Cdc 28 is the primary
kinase that phosphorylates Cln 2. Targeting Cln 2 for
degradation by means of phosphorylation it's cognate CDK
provides a mechanism by which activation of the Cdc 28 CDK
by Cln 2 could target that cyclin for degradation, thus
rendering it self-limiting (Lanker et al., 1996).

Thus, the regulation of cell entry into mitosis can be
affected by the balance of competing kinases and
phosphatases. Each of these protein families are further
regulated by a multitude of phosphorylation events. The
activity state of any of these crucial proteins can be
affected by upstream events triggered by extracellular
signals. Many extracellular signaling pathways converge on a
protein kinase family termed the mitogen-activated protein
(MAP) kinases (Pelech et al., 1993). After stimulation, MAP
kinases phosphorylate a broad spectrum of substrates
involved in different cellular functions and events
reflecting the central position of these protein kinases in
the cellular phosphorylation network (Mordet, 1993).
Activation of the MAP kinase pathway regulates translation

through rsk (Blenis, 1993) and protamine kinase (Reddy et

25
al., 1993) which phosphorylate eukaryotic protein synthesis
initiation factor eIF4-E. MAP kinases also regulate
transcription by phosphorylating nuclear transcription
factors (c-jun, c-myc, c-tall, etc)(Cheng et al., 1993;
Pulverer et al., 1991; Pulverer et al., 1993; Seth et al.,
1991). The activities of MAP kinases also include the
regulation of microtubule dynamics (Gotoh et al., 1991),
metabolism (Merral et al., 1993; Pelech et al., 1991), and
release of second messengers such as arachidonic acid (Lin
et al., 1993). MAP kinases are also postulated to be
involved in cell cycle control and tumor proliferation
though the interaction between MAP kinases and the
cdk/cyclin complexes are not yet fully understood (Ahn,

1993).

E.4. Protein Tyrosine Kinases in Colon Cancer

PTK activity in human colon carcinoma has been found to
be altered in comparison to surrounding normal tissue
(Sakanoue et al., 1991). Examination of PTK activity in
cytosolic and particulate fractions of homogenates from twenty
human colon carcinomas revealed an increase in the PTK
activity in the particulate fraction and decrease in the PTK
activity in the cytosolic fraction when compared to normal

adjacent tissue (Sakanoue et al., 1991). In addition, a marked

26

increase in tyrosine kinase activity was observed to accompany
a rise in ornithine decarboxylase activity in rectal mucosa
from patients with adenomatous or hyperplastic polyps
(Colarian et al., 1991). A similar phenomenon was observed in
the colonic mucosa of rats in response to the carcinogen
azoxymethane (AOM), that is, an increase in the tyrosine
kinase activity was observed as the ornithine decarboxylase
activity increased (Arlow et afluq 1989). In another study
maximal increase in the tyrosine kinase was reported to occur
five days after the ADM administration and was 70% over the
control. This rise in tyrosine activity could be partially
attributed. to increased tyrosine kinase activity' of the
epidermal growth factor receptor (EGF-R). Interestingly, the
increase in the tyrosine kinase activity of the EGF-R is
caused by a 250% increase in the tyrosine phosphorylation of
the receptor itself (Relan et al., 1995). The exposure of
human colon cancer cell lines to differentiation promoting
factors like sodium butyrate significantly attenuated tyrosine
protein kinase activity. This increase concomitant with an
increase in the activity alkaline phosphatase, an enzymatic
marker“ of intestinal differentiation indicating that the
induction of differentiated phenotype in colon cancer is
associated with a marked decrease in tyrosine protein kinase

activity and tyrosine phosphorylation (Schwartz et al., 1995).

27

F. JUSTIFICATION

To briefly summarize the preceding review, colon cancer
is accompanied by marked changes in the proliferation state of
the colon mucosa. Most of the information about the
proliferation in colon mucosa has been provided by
histological studies. At present labeling indices and
proliferation patterns are the most dependable markers
available for assessment of colon cancer risk. Unfortunately,
the determination of these assessment markers is a time and
labor intensive process. It usually involves multiple steps,
namely, fixing, embedding and cutting sections of the tissue
followed by a staining process involving either a radioactive
labeling technique or immunodetection. The justification for
the search of a protein marker is that it is far more
convenient and accessible to test for a protein in a clinical
or a diagnostic setting. A technique that would enable us to
detect changes in proliferation state without doing histology
would be of tremendous advantage in diagnosis and early
detection. A detection technique that is easier and faster to
conduct would make possible for more people to undergo the
screening process. If the detection of colon cancer is early,
the intervention has a much higher probability of being

successful.

28
G. OBJECTIVE

My objective was to determine if cellular proteins change
in detectable levels in relation to the proliferative state of
the tissue. Furthermore, in View of the evidence for the role
of tyrosine phosphorylation in cell division, changes in
protein tyrosine phosphorylation might be expected to reflect
changes in proliferation state of the colon mucosa. Following
this, my second objective was to detect changes, if any, in
tyrosine phosphorylation of proteins that occur in a
hyperproliferative state. Serine and threonine phosphorylated
proteins, which are also crucial in cellular proliferation,
were not a part of this study, being too wide a field to fit
the scope of my study.

The experimental approach undertaken IX) achieve these
objectives was to first measure changes in cellular proteins
in colon mucosa of rats treated with dimethylhydrazine. This
was followed by measuring changes in protein tyrosine
phosphorylation. Since the colon mucosa have cells in various
stages of the cell cycle at any given time, changes in protein
tyrosine phosphorylation, that are very subtle to begin with,
might have been difficult to detect. Therefore, changes in
protein tyrosine phosphorylation in response to different
phases of time cell cycle were examined in aa cell culture

system where it is possible to synchronize the cells to enter

29

the cell cycle simultaneously. .After development of the
technique in cell culture, changes in protein tyrosine

phosphorylation in rat colon mucosa were determined.

H. NULL HYPOTHESIS
The null hypothesis to be tested in this study is that
the amount of tyrosine phosphorylation in colon. mucosal

proteins is the same in DMH treated and non-DMH treated rats.

MATERIALS AND METHODS

EXPERIMENTAL DESIGN

Study' 1. Study' 1 examined changes in colon. mucosa
proteins following a single dose of DMH. The study used four
rats, two received a single dose of DMH (the dosage of DMH in
all the rat studies was 20mg/kg body weight) and the other two
rats were given saline with no DMH (controls). All four rats
were sacrificed two days following the treatment and the colon
mucosal proteins were extracted.

Study 2. Study 2 was conducted to determine changes in
colon mucosal proteins three and five days after DMH
administration. This study utilized five Sprague-Dawley rats;
four were given a single dose of DMH and the fifth rat was
given saline. The control and two DMH treated rats were
sacrificed on the third day after treatment and the other two
rats were sacrificed on fifth day after the treatment. The
mucosal proteins were extracted and analysed.

Study 3. A cell culture study was conducted to determine
changes in protein tyrosine phosphorylation by two dimensional

electrophoresis (2-DE). Cells were induced tx> cycle

30

31
simultaneously and were harvested at times 0, 0.5 and 8 hours
after the stimulus had been provided. The cell lysates were
analysed for protein tyrosine phosphorylation using 2—DE and
western blotting.

Study 4. Ten rats which were randomly assigned to one of
three treatment groups: 1) Saline administration with no DMH
to be sacrificed on the first day after injections, this group
had four rats 2) single dose of DMH and sacrificed om the
third day after the injections, this group had three rats and,
3) a single dose of DMH and sacrificed on seventh day after
the injections, this group had three rats. The colon mucosal

protein were extracted from all of these rats.

ANIMALS AND HOUSING

Seven to eight. ‘week old Sprague-Dawley rats were
purchased from Harlan Sprague-Dawley (Indianapolis, IN). The
rats were housed individually in stainless steel hanging wire
cages. The animal room was temperature (68°-75°F) and humidity
(40-70%) controlled and a twelve hour light-dark cycle was
maintained. The rats were fed a modified AIN-93(G) diet (Table
1.). All the protocols for animal housing and injections were
approved by the All University Committee on Animal Use and

Care.

32

Table 1. Dietary composition of the modified AIM-94 diet fed
to rats in studies 1,2 and 4. These quantities are
for 10 Kgs of diet.

 

 

Ingredients Quantity
(Kg)
Corn Starch 3.49
Casein 2.0
Sucrose 1.0
Corn Oil 1.5
Fiber 0.5
Mineral Mix (AIN-93) 0.35 .
Vitamin Mix (AIN-93) 0.1
L-Cystiene 0.03
Choline Chloride 0.025

 

 

 

 

33

CHEMICALS AND REAGENTS

Dietary components were obtained from Teklad Test Diets
(Madison, WI). Sym-l,2-dimethyl-hydrazine was purchased from
Sigma Chemical Company (St. Louis, MO). The polyvinylidene
diflouride (PVDFj membrane and the Rennaissance
Chemiluminescence used for the detection of proteins were
obtained from DuPont NEN Research Products (Boston, MA). Anti-
phosphOtyrosine—horseradish peroxidase conjugate, goat anti-
mouse IgG-horseradish peroxidase conjugate and anti-PCNA
monoclonal antibody were all purchased from Oncogene Science
(Uniondale, NY). Bicinchoninic acid was obtained from Pierce
Chemical Company (Rockford, IL). Cell lines HT-29 and HCT 116
were purchased from American Type Cell Culture Association
(Bethesda, MD). Fetal bovine serum was obtained from Idfe
Technologies (Giethersburg, MD). Penicillin/streptomycin stock
and all cmher Chemicals were obtained from Sigma Chemical

Company.

METHODS

Dimethylhydrazine Administration to Rats: Colonic
epithelial cell proliferation was induced by a single
subcutaneous injection of DMH. DMH was administered in a 0.15M
saline, 1 mM EDTA solution with the concentration adjusted

such that 0.1 ml contained 20 mg DMH/ kg body weight. The pH

34
of the solution was adjusted to 7.4 with sodium carbonate
prior to injecting the animals.

Tissue Collection and Preparation: Rats were sacrificed
by carbon dioxide asphyxiation followed by exsanguination. The
entire colon was removed, cut open longitudinally, and rinsed
briefly with cold running water followed by PBS (ice-cold) to
remove fecal debris. Mucosal cells were scraped off with a
glass microscope slide and homogenized immediately in
homogenization buffer [20mM 3-[N-morpholino]propanesulphonic
acid, 150mM NaCl, 1% triton X-100 (v/v), 1% deoxycholate(v/v),
0.1% sodium dodecyl sulphate(v/V), 1mM ethylenediamine-
tetraacetic acid.Na2, 100uM sodium orthovanadate, 250uM
phenylmethylsulphonyl flouride, 10ug/ml leupeptin, lug/ml
pepstatin] for studies 1 and 2, and lysis buffer [9.5 M urea,
2% nonidet P-40 detergent, 2% ampholytes(pH 3-10), 5% B-
mercaptoethanol] for study 4. Since the ampholytes and the B-
mercapto-ethanol can interfere with the protein estimation,
these were added after protein estimation. The homogenates
were centrifuged at 14,000 rpm for ten minutes to obtain a
clear protein extract which was used for 2-DE. The protein
content of homogenates was determined using the bicinchoninic
acid (BCA) assay (Smith et al., 1989).

Cell Culture: For each cell line, HT-29 and HCT 116,

approximately 1x1061cells were seeded into each of ten, 100 nmi

35

diameter polystyrene tissue culture plates. Cells were seeded
in McCoy's 5A medium supplemented with 10% FBS plus
antibiotics. After 48 hours of culture, the medium was removed
from plates, plates were rinsed once with PBS, and fresh
McCoy's 5A nedium containing 0.1% FBS and antibiotics was
added. Cells were maintained in this low serum medium for 48
hours, at which time the majority of cells would be expected
to be in.C%/G¢jphase of the cell cycle. The serum deficient
medium was sufficient to maintain cell survival, but not
sufficient to stimulate cell proliferation. After 48 hours in
the serum deficient medium, cells from two plates for each
cell line were harvested in lysis bUffer. These cell lysates
formed the 0 hour samples. The medium in the remaining eight
plates for each cell line was switched to McCoy's 5A medium
containing 10% FBS and antibiotics. Cells from two plates per
cell line were harvested in lysis buffer at 0.5 and 8 hours
after switching to medium containing 10% FBS. This experiment
was replicated thrice.

Cell lysates were combined for each pair of duplicate
plates at each time point. The lysates were allowed to stand
on ice for thirty minutes and were then transferred to 1.5 ml
microcentrifuge tubes and centrifuged for 10 minutes at 14,000
rpm to obtain a clear supernate that was used for further
analysis.

Polyacrylamide Gel Electrophoresis (PAGE): The protein

36

homogenates collected from study 1 and 2 were combined 1:1
with SDS sample buffer and separated on 12% or 10% accrylamide
gels. The gels were stained with a half hour immersion in 0.2%
solution of brilliant coommassie blue followed by several
changes of destaining solution. High and low molecular weight
markers were also run on the same gels.

2-Dimensional Electrophoresis (2-DE): Changes in
phosphorylation of cellular proteins were studied using 2-DE
followed by western blotting. 2-DE was conducted as described
by Dunn and Burghes (1983) and in accordance with the
instructions provided by the electrophoresis equipment
supplier (Hoefer Scientific instruments; San Fransisco, CA).
The first dimension was an isoelectric focusing (IEF) tube gel
run in 7.5 cm long glass tubes (ID-1.5mm) using a pH gradient
of 3-10. The samples (30 ug of protein) were focussed for 4
hours at a constant voltage of 500 Volts. At this time the
tube gels were removed from the glass tubes and equilibrated
for fifteen minutes in equilibration buffer (10% glycerol, 4.9
mM DTT, 2% SDS, trace of bromophenol blue, 0.125 M tris base,
pH 6.8). The second. dimensional separation(SDS-PAGE) was
carried out in 8x10 cm mini-gels of 1.5 mm thickness (10%
total acrylamide, 2.7% bis). The tube gels were annealed to
the slab gels with agarose (1% solution in tank buffer). Mouse

IgG Fc fragments were loaded in a well adjacent to the tube

37
gel and served as a marker for the electrophoresis,
electrophoretic transfer and western blotting. Electrophoretic
separation was carried out at a constant current of 80 mA.

Western Blotting: After the 2nd dimension
electrophoresis, gels were equilibrated in Towbin's transfer
buffer [25 mM tris, 192 mM glycine, 20% methanol] for 15
minutes at 25 C. Electrophoretic transfer was carried out in
wet conditions using a Hoefer transfer unit. Transfers were
conducted overnight in Towbin's transfer buffer at a constant
current of 100 mA. The transfer unit was cooled by circulating
cold water.

Visualization of phospho-tyrosine containing proteins
.After electrophoretic transfer, the PVDF membranes were
allowed to air-dry to facilitate protein adherance to the
membranes. The membranes were rewetted with 100% nethanol
followed by distilled water before incubating in blocking
buffer [3% BSA. in wash buffer [0.1% tween—20 in PBS]]
overnight at 4°C. This was followed by a two hour incubation
with anti-phosphotyrosine monoclonal antibody (dilution
1:1000) in blocking buffer. After three washes of ten minutes
each in wash buffer, the membranes were incubated with goat
anti-mouse IgG-horseradish peroxidase conjugate for one hour.
Membranes then were washed once for fifteen minutes and four

times for five minutes each in wash buffer. Phosphotyrosine

38

containing proteins were detcted using Enhanced
Chemiluminescence (Renaissance Western Blot Reagent; DuPont
NEN Research Products). Emitted signal was captured on
Reflection autoradiograghy film (DuPont NEN).

.Analysis of Gels and Autoradiographs: The densitometric
analysis was a densitometric program called ImageQuant. The
analysis was done by a combination of this program and careful

visual examination.

RESULTS AND DISCUSSION

Study 1. The hyperproliferative changes produced in the rat
colon after administration of a colon carcinogen like DMH or
azoxymethane mimic the early stages of development of colon
cancer (Deschner and Maskens, 1982). The purpose of this study
was to identify proteins whose level of expression increased
or decreased in response to DMH, to be used as markers for the
risk for colon cancer. For this the rats were administered
DMH. Control rats were not given DMH. The colon mucosal
proteins were extracted from each of the two treatments and
analyzed by SDS-PAGE and visualized by coomassie blue stain.
Approximately forty bands were visualized in this manner. The
protein bands that changed in intensity between the two
treatments, namely, DMH and no DMH administration had
molecular weights in the range of 30 to 45 KDa (Figure 1.).
These changes in the protein levels, however, were
inconsistent between animals. Therefore, we concluded that
another study had to be conducted to look at changes in colon

mucosal protein levels resulted from DMH administration.

39

40

24 kDa_ Z--
I
20.1 kDa -
l42ldhL_ .-.,-

 

Figure 1. Proteins from colonic mucosal homogenates from
control rat (no pun treatment) and DMH treated
rats resolved on 12% acrylamide SDS-PAGE gels.
The proteins were stained with coomassie blue.

41

Study 2. From the review of the literature regarding
induction of colon cancer by DMH administration it appears
that the proliferation state of colon mucosa, expressed as
labeling index (LI), changes over a period of several days
after the carcinogen administration (Deschner, 1978). There is
an initial increase in proliferative activity in response to
the widespread cytotoxicity, a result of chemical
administration. This is reflected by the increase in labelling
index on days immediately following the carcinogen
administration. The labelling index has been reported to
decrease to a value closer to the baseline value after a few
days (Deschner, 1978). This study was conducted to determine
if the change in level proteins suggested in study 1 were
transient or sustained over a few days. Five rats were used
for this study, four of which were administered DMH. The
control rat was not given. DMH. In this study the rats
receiving DMH were sacrificed on the third and fifth day after
carcinogen administration. The mucosal proteins were extracted
and analyzed as they were in study 1. Each lane of the SDS-
PAGE contained equal amounts of protein. Figure 2 shows a gel
from this study in which the proteins have been stained with
coommassie blue. It was observed that the amount of proteins
with molecular weights in 30 KDa to 45 KDa range changed

relative to control on the third and the fifth day after DMH

42

Figure 2. Proteins from colonic mucosal homogenates from
control rat (no DMH treatment), DMH treated
rats sacrificed on day 3 after treatment and DMH
treated rats sacrificed on day 7 treatment
resolved on 12% acrylamide SDS-PAGE gels.
The proteins were stained with coomassie blue.

43
administration. However, any definite conclusions about the
identity of protein bands of interest could not be drawn from
this study. This was partly because of the large number of
bands observed III the coomassie stained gfifl. that caused
overlapping of protein bands because of insufficient
resolution.

In the course of these experiments, the significance of
kinases and phosphatases in cell growth and division became
more apparent in View of many of the oncogenic proteins being
found to have kinase activity. It was realized that noticeable
changes in response to mutagenic mutations might occur in the
extent of phosphorylation of certain key proteins and not in
the level of the proteins which we had earlier attempted to
detect by coommassie staining. In View of this information, we
decided.tx> look at changes in tyrosine phosphorylation of
colon mucosal proteins as ea result of DMH administration.
Therefore, the next two experiments focussed on studying
tyrosine phosphorylation of proteins using western blotting.

The problem of poor resolution using SDS-PAGE was solved
by switching to 2-dimensional electrophoresis (2—DE). 2-DE
separates proteins according to their molecular weights and
their isoelectric points which allows for a higher degree of
resolution. In the first dimension the proteins are

isoelectrically focussed in a tube gel. The second dimension

44
is run perpendicular to the first and achieves separation
according to molecular weights. 2—DE has never been used to
detect total changes in protein tyrosine phosphorylation in

the proteins of the colonic mucosa.

Study 3. A cell culture study was conducted for this
purpose. HT-29 and HCT 116 cells were grown in culture. The
cells were serum deprived for 48 hours which arrests the cells
in GO because the serum deprived media will support cell
survival but does not have enough growth factors for the cells
to continue dividing. The 0 hour collection was made at this
point. The media was then replaced by serum replenished media
to provide necessary growth factors and nutrients. At this
point the cells proceed through the next mitotic cycle by
intensively synthesizing DNA and proteins. The signal for the
onset of DNA and protein synthesis travels from the plasma
membrane to the nucleus, the site of nucleic acid synthesis.
The binding of the growth factors to their respective
receptors activate the kinases associated with the receptors
or the membrane. This initiates a cascade of kinase and
phosphatase activities. These events occur rapidly, therefore,
the next sample was taken 0.5 hr after the media was switched.

It was assumed that the phosphorylation and dephosphorylation

45

that occurred in the first half hour would be seen in this
sample. The next sample was taken eight hours after serum
replacement. The cells were all collected in lysis buffer.
Equal amounts of protein as determined by BCA protein
estimation assay were analyzed with 2-DE. The level of
expression of proteins was determined by silver staining the
gels. The tyrosine phosphorylation of proteins was determined
by western blotting followed by inmmno—probing with anti-
phospho-tyrosine monoclonal antibody. The silver stained gels
and phospho-tyrosine autoradiographs were analyzed using a
densitometer. The density of the spot in the silver stained
gels and pmospho-tyrosine autoradiographs was taken to be
indicative of the level of expression of the protein and the
level of tyrosine phosphorylation of the protein respectively.

Approximately thirty to thirty-five phospho—tyrosine
containing proteins were detected by this method in HT-29 and
HCT 116 cells. The total number of proteins detected were
about seventy. These were visualized by silver staining. The
phosphotyrosine containing proteins that were observed to
increase or decrease in the autoradiographs were carefully
examined in the silver stained gels. This was to assure that
the change observed. was due to change in the tyrosine
phosphorylation state of the protein and not due to change in
the level of expression of the protein. Since the experiment

was conducted in triplicate, there are three sets of values

46

for each cell line. The variation in the intensity of the
protein spots between triplicates was quite large. Only the
spots that changed in a consistent manner between the
replicates are reported in Tables 2 and 3. The results are
reported as intensity of protein spots as compared to the
intensity of the particular spot in control (time, 0
hour)(Table 2 and 3). These results have been obtained by a
careful visual examination of the protein spots. An accurate
densitometeric evaluation could not be obtained due to the
varying levels of background found in the different parts of
the gels which cannot be excluded from the spot when
demarcating the area.

The number and clarity of the protein spots observed
with 2-DE was much greater than that obtained with PAGE and
clearly justified the use of 2—DE, which is more time and
labor intensive than PAGE. The approximate molecular weights
and approximate isoelectric points were calculated from the
relative distance traveled by a certain protein in each
perpendicular direction and are also reported in the Tables 2
and 4. The only spots reported here are the ones that did not
change in intensity in the silver stained gels. Therefore, the
changes observed are due to changes in protein tyrosine
phosphorylation and not due to the difference in the level of

the protein present in cell.

47

HI;22 The 50 kDa protein of approximate isoelectric point
6.4 was not detected in the first replicate. In the second
replicate of the experiment, it was detected only in the half
hour cells. In the third replicate, however, it was detected
at all three time points. The intensity of the spot in the
phosphotyrosine autoradiograph for one half hour cells was
greater than the 0 hour and the eight hour cells. This pattern
suggests that the 50 kDa protein is transiently phosphorylated
at the onset of the mitotic cell cycle after which it reverts
to it's baseline level of tyrosine phosphorylation.

The 56 kDa protein of approximate isoelectric point 5.7
was observed to decrease at the half hour time point in all
three replicates compared to the controls at 0 hour. At eight
hours this phospho-tyrosine spot increased to either the
baseline control level or even higher. This protein appears to
be dephosphorylated for a short time at the beginning of the
cell cycle.

Another 56 kDa protein with an isoelectric point of 5.5 was
observed to be tyrosine phosphorylated to a greater extent at
the half hour time point compared to the 0 hour control. In
the first and the second replicate the phosphorylation seemed
to return to the baseline control level, but stayed elevated
in the third replicate. This could indicate a protein which

stays at the higher level of tyrosine phosphoryaltion for

48

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51

The three symbols within
three time points, 0 min,
addition of PBS.

Phosphotyrosine containing proteins in HT-29 cells.
each replicate represent
30 min and 8 hrs after

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MWkDa pI 1st Replicate 2nd Replicate 3rd Replicate
(SD) (SD)
0 30 8 0 30 8 0 30 8
min min hr min min hrs min min hrs
48.9(2.3) 6.4(0.0) - P - P > P
56.8(0.7) 5.7(0.2)
P < P P < > P < P
56.8(0.7) 5.5(0.l)
77.6(2.0) 3.8(0.l) P > P P > P P > >
- P P - P P
Key : - Not present at detectable level

P Present at detectable level
> Present at level greater than control
< Present at level lesser than control

 

52

longer period than the two proteins described earlier.

The 75 kDa protein with isoelectric point 3.8 was only
detected in the second and third replicates. In the second and
third replicates, this spot was not detected at 0 hour time
point. At the half hour and eight hours time points the level
of tyrosine phosphorylation appears to remain constant. This
protein apparently is not phosphorylated at it's tyrosine
residues when the cell is in the non-dividing state. But once
the cell starts cycling, it stays phosphorylated for a long

period of time.

HCI_11§ The proteins discussed here for HCT 116 cells are
tabulated in Table 3. The 40 kDa protein with isoelectric
point 6.1 appears to increase in intensity at half hour time
point compared to 0 hour control in phospho-tyrosine
autoradiographs. It was observed to stay elevated in the first
and the third replicates, but seemed to return to baseline
eight hour level in the second replicate.

fume 42 kDa protein with isoelectric point 6.6 was not
detected in the first replicate. In the second and third
replicate, the intensity of the spot in the phospho-tyrosine
autoradiographs was observed to be increased at the half hour
time point as compared to the 0 hour control. At the eight

hour time point, the level of phosphorylation seemed to return

53

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54

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Table 3. Phosphotyrosine containing proteins in HCT 116
cells. The three symbols within each replicate

56

represent three time points, 0 min, 30 min and 8
hrs after addition of FBS.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P Present at detectable level

> Present at level greater than control

.< Present at level lesser than control

MW kDa pI lst 2nd 3rd
(SD) (SD) Replicate Replicate Replicate

o 30 s o 30 s 0 30 a
min min hrs min min hrs min min hrs

28.7 (9.9) 5.4 (0.2) P P > - P - -

41.1 (4.1) 6.1 (0.1) P > > P > P -

42.1 (3.8) 6.6 (0.0) P ' > P -

42.9 (5.6) 4.9(0.1) - P - - P -

51.0 (4.4) 6.5 (0.1) P > P P > P -

58.7 (7.0) 5.5(0.1) - P - P > - - P

59.9 (11.1) 5.0 (0.0) - P P - P P

60.2 (4.0) 4.7 (0.3) - P - P > P - P

70.0 (4.7) 7.5 (0.0) - P < -

92.1 (3.8) 4.4 (0.1) - P - P > P -

Key : - Not present at detectable level

 

57
to baseline in replicate II, but seemed to stay elevated in
replicate III.

The 43 kDa protein with isoelectric point 4.9 was not
detected 1J1 replicate III. 131 the first anui the second
replicate, this protein was only detected at the half hour
time point. It appears to be a protein that is only
transiently tyrosine phosphorylated at the onset of the cell
division.

The 51 kDa protein with isoelectric point of 6.5 was
observed to have increased tyrosine phosphorylation at half
hour time point compared to 0 hour time point in all three
replicates. At eight hours the level of tyrosine
phosphorylation seemed to decrease to baseline levels in the
first and the second replicates, but seemed to stay elevated
in replicate III.

The 58 kDa protein with approximate isoelectric point 5.5
was phosphorylated at tyrosine residues to a greater extent
compared to 0 hour control. At eight hours, though, the level
of phosphorylation differed in all three replicates. In the
first replicate, the intensity of the spOt seemed to return to
the baseline level, which was undetectable in this replicate.
In the second replicate, the level of tyrosine phosphorylation
decreased lower than detectable levels at eight hours. In the

third replicate the level of tyrosine phosphorylation remained

58
constant at the half hour and the eight hour period.

The 59 kDa protein with approximate isoelectric point 5.0
was not detected in the third replicate. In the first and
second replicate, however, the tyrosine phosphorylation spot
increased in intensity at the half hour time point and stayed
elevated at the eight hours time point.

The 60 kDa protein has an approximate iso-electric point
of 4.7. In all replicates the level of tyrosine
phosphorylation increased at the half hour time point compared
to 0 hour control. In the first and the second replicate the
level of tyrosine phosphorylation seemed to decrease to the
level of the 0 hour control at the 8 hr time point, except in
replicate III where the tyrosine phosphorylation appeared to
stay constant over the eight hour period.

The 70 kDa protein with iso-electric point of 7.5 was not
detected in the first replicate. In the second and the third
replicate the level of tyrosine phosphorylation increased from
undetectable levels to detectable levels at the half hour time
point. At eight hours it stayed detectable in both replicates
II and III, however, the level though stayed constant in the
third replicate and decreased slightly in the second
replicate.

The 92 kDa protein with iso-electric point 4.4 was

tyrosine phosphorylated at increased levels at half hour time

59
periods compared to 0 hour control in the three replicates. At
eight hours the levels of tyrosine phosphorylation seemed to
come down to the baseline levels in replicates I and II.
However, the level of tyrosine phosphorylation appeared to

remain constant at the eight hour period in replicate III.

Study 4. The next study was conducted with ten rats. Of
these, four were controls with no DMH treatment, three were
sacrificed on day three after a single DMH injection and other
three were sacrificed a week after the DMH injections (Table
4). Approximately ninety protein spots were detected in the
phospho-tyrosine autoradiographs. The approximate molecular
weights and isoelectric points of these proteins were
calculated using the Rf values and comparing them to those for
the markers. There was a large variation in the patterns of
the appearance and disappearance of phospho-tyrosine spots.

Tflua 36 KD protein with approximate iso-electric point
4.35 was present at a higher level in the three rats at day
three after the DMH injection. But on day seven, it had
decreased to control levels.

The 37 kDa protein with iso-electric point 4.4 followed
the same pattern as described for the 36 kDa protein. The
protein showed a much darker spot in all third day rats

compared to the control and the seventh day rats.

60

Table 4. Phosphotyrosine containing protein spots in DMH
treated and control rats. The three/four symbols in
each group represent three/four rats in that
treatment group.

 

 

MW (kDa) pI Control DMH-day DMH-day 7
36 4.3 P P - > > > P P P P
37 444 P 1' - => > => P 1P 1? P
42 5.3 - P - > > > - .. .. ..
44 54 - 1’ - => >’ > - - 4- -
54 5.0 - P - > > > > > > P
67 6:5 P I' P IP <: < 4< 4<
67 6.2 P P - - - - - < <

 

 

 

 

 

 

 

Key : - Not present at detectable level
P Present at detectable level
> Present at level greater than control
< Present at level lesser than control

61

Isa-electric focussing #1

 

EDVJ'SGS

 

 

Figure 9. Colonic mucosal proteins from a control(no DMH
administration) rat, resolved by 2-dimensionanl
electrophoresis, and western blotted for
phosphotyrosine.

62

[so-electric focussing
-.

7

 

    

 

HOVJ‘SGS

A
Yf

 

Figure 10. Colonic mucosal proteins from a DMH treated rat
(sacrificed day 3 after administration), resolved
by 2-dimensional electrophoresis, and western
blotted for phosphotyrosine.

63

Isa-electric focussing
’L

 

 

ESVESGS

 

 

Figure 11. Colonic mucosal proteins from a DMH treated rat
(sacrificed day 7 after administration), resolved
by Z-dimensional electrophoresis, and western
blotted for phosphotyrosine.

64

Table 5. Phospho-tyrosine containing proteins common to
HT-29 and HCT 116.

 

 

MW (kDa) pl Level of Phospho-Tyrosine
0 min 30 min 8 hrs

50 6.4 P > P

57 55 P > P

 

 

 

 

 

 

 

Key : P Present at detectable levels
> Present at levels higher than control

 

65

The 43 kDa protein had an iso-electric point of 5.4 was
present in undetectable levels in two of the three control
rats. On the third day after DMH treatment the level of the
tyrosine phosphorylation of the protein was elevated in all
three rats. The protein was not detected in phospho-tyrosine
autoradiographs in seventh day rats, presumably because the
tyrosine phosphorylation was below detectable levels.

The 44 kDa protein with iso-electric point 5.4 was
detected only in the third day rats, with the exception of one
rat in the control group. Seemingly this protein was tyrosine
phosphorylated in response to carcinogen administration but
did not remain so for a very long time.

The 54 kDa protein of iso-electric point 5.0 was the only
protein in this study that exhibited elevated tyrosine
phosphorylation on day three and stayed elevated on day seven
of the experiment.

The 67 kDa protein with iso-electric point 6.5 was
present at a constant level of tyrosine phosphorylation in
control and third day rats. The tyrosine phosphorylation
seemed to decrease on day seven of the experiment in all four
rats in this group.

The 67 kDa protein with iso-electric point 6.2 was not
detected in phospho-tyrosine autoradiographs for third day
rats. In the seven day rats it was detected only in two of the

four rats where it was present at a lower density than the

 

66

control rats :Ui the phospho-tyrosine autoradiographs. This
protein, therefore, appears to be dephosphorylated and stay

dephosphorylated, as a result of carcinogen administration.
The large variation in tyrosine phosphorylation of
proteins can be explained on the basis that the cells of the
colon nmcosa ixira live animal, unlike the cells grown in
culture, are a mixture of cycling cells and are not
synchronized in terms of cell cycle. In addition to that,
there is also a diurnal effect in terms of proliferation,
i.e., some cells are always dividing in the tissue, but the
fraction of cells diving at certain times of the day are
higher than the others. Therefore, at any given time, the
mucosal cells are in various phases of the cell cycle. The
extent of tyrosine phosphorylation of a protein probably
depends on the fraction of total cells present in the phase of
cell cycle in which that protein is tyrosine phosphorylated.
The pattern of increase or decrease in phosphorylation of
mucosal proteins seen over a period of several days can be
important in assessing the validity of that protein as a
marker for hyperproliferation. The proteins that are
phosphorylated for a short period of time, almost transiently,
suggests a regulatory role for these proteins in the cell
cycle. These proteins are probably placed at crucial branch
points of the cell regulatory pathways and are only required

to be phosphorylated or dephosphorylated transiently. During

67
the short interval of it’s being active, this protein probably
activates other downstream proteins. The proteins that are
phosphorylated or dephosphorylated for longer periods of time
probably have a functional role, for which they are required
to be in the modified or active form for the duration of the
time the cell takes to divide.

The proteins that are phosphorylated or dephosphorylated
for longer periods of time show a greater potential for being
used as a marker for dividing cells as there is a greater
possibility of these being detected. On the other hand,
though, the changes in phosphorylation of these proteins may
just be downstream event of mutations in the regulatory
proteins and thus might be seen later than regulatory
proteins. Changes :hi phosphorylation cu? the proteins
regulatory may be, therefore, a more sensitive marker for
proliferation.

From a careful examination of the molecular weights of
the proteins identified in this study, some suggestions can be
made regarding the identity of these proteins. These
suggestions are based on the comparisons between the molecular
weights of the proteins. Only the proteins that have been
implicated in the regulation of the proliferation of cells or
cell division have been considered. Most of these proteins are
either known to be tyrosine phosphorylated under physiological

conditions or a strong possibility of such post-translational

68
modification exists.

Bcl-2, a 26 kDa protein, is phosphorylated, an event
coincident with it's association with p21“5. Inhibition of
this phosphorylation prevents protection from apoptosis by
Bcl-2. In other words, phosphorylated Bcl—2 provides
protection from apoptosis in Jurkat cells (Chen and Faller,
1995). The molecular weight of this protein corresponds to p28
identified in HCT 116 cells.

p345“2 fOrms a complex with cyclin B, that though
indetectable at interphase, rises abruptly at.Cb/M transition
to induce mitosis. After the synthesis of cyclin B, the
suppression of the complex has been attributed to the
phosphorylation of p34Cdcz. When cyclin B accumulates to a
critical threshhold level, it sets off a cascade of events
that leads, after a lag period, to a switch in the balance
between kinases and phosphatases such that the inhibitory
phosphorylations on p34““2 are suddenly removed. The 36 kDa
and the 37 kDa protein identified as having increased tyrosine
phosphorylation in response to DMH administration in rats fall
within the molecular weight range of p34wd. The probability
of it being the same protein seems limited, however, in view
of the fact that the tyrosine phosphorylation of p34md would
be expected to go down in response to DMH, a stimulator of
proliferation, whereas the p36 and p37 seem to have a higher

level of tyrosine phosphorylation.

69

The human Cdk Activating protein Kinase 1(CAK l) is
reported to be a Cdc-2 related cyclin-dependent protein kinase
with a molecular weight of 42 kDa. If this protein is
regulated in a manner similar to other cdc2 related proteins,
it would be expected that it associates with other regulating
partners, that is, cyclins and is is further regulated by site
specific phosphorylation or dephosphorylation. Microsequence
analysis after limited proteolysis has confirmed that CAK does
bind p35, a protein that demonstrates a great deal of
similarity to cyclin H. The two 42 kDa proteins identified in
HCT 116 cells and the 42 kDa protein identified in rats could
be the same protein as p42 CAK.

In addition, the p42 MAP kinase is known to be tyrosine
phosphorylated and activated in the T cell lymphoma cell line
171CD4+ when the antibody binds to the CD4 surface antigen
(Ettehadieh et al., 1992). The p42/p44 MAPK kinaes have been
identified by several groups (Gomez and Cohen, 1991;
Rossomando et al., 1992; Ahn et al., 1991). The MAPKK is a 45-
46 kDa dual specificity' protein, that autophosphorylates
tyrosine and threonine residues on itself in response to
activation by v—Raf (Dent et al., 1992).

p57 is a cytoplasmic protein found in colon carcinoma
cells that has recently been identified as a new MAP kinase
(Lee et al., 1993b) or an extracellular signal regulated

kinase (Rossomando et al., 1989; Boulton et al., 1991;

7O

Kyriaskis and Avruch, 1990). Stimulation of undifferentiated
colon carcinoma cells with DAGs diolein or dimyristin or with
TPA leads to phosphorylation of tyrosine on p57 within 1-2
minutes (Lee 6%: al., 1993b; Marian et en”, 1989). Signal
transduction through p57 has also been reported to involve c-
src in colon carcinoma cells (Lee et al., 1993b) which can be
expressed at higher levels in colon cancer (Aaronson, 1991).
The two 56 kDa proteins identified in HT-29 cells and the 58
kDa protein in HCT 116 cells could possibly be the same as
p57.

The 60 kDa protein identified in HCT 116 cells as having
decreased levels of tyrosine phosphorylation after rising
briefly lies within the molecular weight range of pp60°f”'
Dephosphorylation of Tyr-527 is required for the activation of
Src (Cooper et al., 1986) and src has been reported to be
activated in human colon carcinoma (Bolen et al., 1987).

Ihi mouse NIH-3T3 cells transformed vniji an activated
allele of c-src in which the regulatory tyrosine is replaced
by phenylalanine, tyrosine phosphorylation of a 68 kDa protein
was found to be greatly enhanced (Fumagalli et al., 1994).
Purification and microsequencing of p68 showed that it was
related to the p62 GAP associated protein that has been
described before (Ellis et al., 1990). It appears that during
mitosis src binds via it's SH2 and SH3 domains to tyrosine

phosphorylated p68 (Park and Cartwright, 1995). The two 67 kDa

71
proteins identified in rat colon to be tyrosine phosphorylated

have the possibility to be the same as p68.

SUMMARY AND CONCLUSIONS

Hyperproliferation, an early bio-marker for colon
cancer, is presently detected in histological samples by
immunohistochemistry or radio-labeling. This study was
conducted to look for tyrosine phosphorylated proteins that
could be used as markers for hyperproliferation.
Dimethylhydrazine administration was used to induce
hyperproliferation in the colon mucosa of rats.

Synchronized HT—29 and HCT 116 cell cultures were used
to detect changes in tyrosine phosphorylation in cells at
different stages of the cell cycle. In HT-29 cells, two
proteins of molecular weights 50 and 56 KD were found to be
tyrosine phosphorylated to a greater extent at the 0.5 hr
time point compared to the 0 hr and 8 hr time points.
Another 56 KD protein was less tyrosine phosphorylated at
0.5 hr time point compared to the 0 and 8 hr time points and
a 77 KD protein was tyrosine phosphorylated to a detectable
level only at 0.5 and 8 hr time points.

In HCT 116 cells, five proteins with approximate

molecular weights 43, 51, 58, 60 and 92 KD were found to be

72

73
more tyrosine phosphorylated at 0.5 hr time point compared
to the 0 and 8 hr time points. The 41 and the 59 KD proteins
were phosphorylated to a greater level at 0.5 as well as 8
hr time points compared to 0 hr time point.

Two proteins were identified that behaved in identical
fashion between the two cell lines. These proteins were of
molecular weights of approximately 50 and 57 KD.

It was concluded from this study that 2-DE followed by
western blotting followed by probing the membranes with
anti-phospho-tyrosine was an effective technique to detect
changed tyrosine phosphorylation of total proteins of
cultures colon cancers cells, HT-29 and HCT 116's.

For the next study, rats were given a single injection
of DMH and were sacrificed on the third and seventh day
after the injection. The control (no DMH) rats were
sacrificed on third day after the injection. The DMH
injection resulted in increased tyrosine phosphorylation
compared to control on day three, but not day seven in four
proteins of approximate molecular weights 36, 37, 42 and 44
KD. A 54 KD protein was more tyrosine phosphorylated
compared to the control on day three and seven after
injection. A 67 KD protein was found to be less tyrosine
phosphorylated on day seven compared to day three and
control. Another 67 KD protein was found to tyrosine

phosphorylated at much lower level on day three and seven

74

after the injection compared to control.

IREKKnflHENEHTTICRHB FCHIIFUHKHRE IUESEEJUIH

1. Present work has suggested several proteins whose
tyrosine phosphorylation could potentially be used as
markers for hyperproliferation in colon mucosal tissue.
Possibly some of these proteins are related to the cell
cycle regulatory proteins. In recent years many of these
proteins have been identified and monoclonal antibodies
against them are commercially available. In future research
these antibodies could be used to identify the proteins
indicated by present study.

2. Since this work has exhibited that 2-DE can be used to
detect changes in protein tyrosine phosphorylation, this
ability should be used to test for the effect of certain
alleged tyrosine kinase inhibitors, such as genistein in
diet, on tyrosine phosphorylation in tissues. Genistein, an
isoflavanoid found in some foods, e.g., soybeans, has been
reported to inhibit cell proliferation, but the mechanism by
which it does so is not completely understood. One of the
possible mechanisms suggested for it's action is by
inhibition of tyrosine phosphorylation. This can be studied

75

76

using the technique used in this study.

BIBLIOGRAPHY

BIBLIOGRAPHY

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