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dissertation entitled

Role of Bel—2 in Carcinogenesis

presented by
Nestor David Deocampo

has been accepted towards fulfillment
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Ph.D. degree in Genetics

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ROLE OF BCL—2 IN CARCINOGENESIS

By

Nestor David Deocampo

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Genetics Program, College of Natural Science

200 1

ABSTRACT
The Role of Bel-2 in Carcinogenesis
By
Nestor David Deocampo

Carcinogenesis has been described as a multi-step process
involving three ordered temporal phases, initiation, promotion, and
progression. The promotion phase has been speculated to include the
selective accumulation of an initiated cell via mitogenesis, and the escape
from apoptosis. Many oncogenes and tumor promoting chemicals have
been shown to contribute to mitogenesis and blockage of apoptosis. In
addition. these same oncogenes and tumor promoters have been shown
to block intercellular signaling through gap junctions. The objective of
this study was to test the hypothesis that cooperation of proliferation.
apoptosis, and gap junctional intercellular communication (GJIC) can
lead to the neoplastic transformation of normal cells. To accomplish this.
oncogenes that influenced proliferation and apoptosis were transfected
independently and together into normal epithelial cells and subsequently
assayed for neoplastic potential and GJIC.

In order to examine the involvement of apoptosis in carcinogenesis
and subsequent affects on GJIC WB-F344 cells, a normal rat liver
epithelial cell line, were transfected with the bcl-2 gene. Additionally. to
test the synergistic roles of blocking apoptosis and inducing mitogenesis,

a previously established WB.v-myc cell line was also

transfected with the bcl-2 gene. All cell lines were assessed for cell
proliferation, contact inhibition saturation density, anchorage
independent growth (used as a surrogate marker for neoplastic
transformation), invasion, and GJIC.

The results obtained from this investigation demonstrated that bcl-
2 and myc, when co-expressed, were able to cooperatively induce the
neoplastic transformation of normal WB-F344 cells. Those cell lines
expressing myc and bcl-2 demonstrated a loss in contact inhibition, a
reduction in apoptosis and a reduction in proliferation rates. These cell
lines also showed varying abilities to grow in soft agar that did not
correlate to bcl-2 protein expression. In order to test if there was a
correlation between neoplastic ability and GJIC. scrape load dye transfer
and fluorescent recovery after photobleaching were measured. The
results showed a relationship between the reduction in GJ 1C and
neoplastic ability, in that. those cell lines that demonstrated a reduction
in GJIC showed an increase in neoplastic ability.

Additionally, it has been previously hypothesized that cancer Is a
disease of blocked differentiation (Potter, 1978). To test this cell lines
were induced to differentiate using sodium butyrate. The results
demonstrated that differentiation was reduced when myc or bcl-2 were
expressed independently. Those cell lines that expressed both myc and
bcl-2 were unable to differentiate, which suggests that blockage of

differentiation is a progressive event in carcinogenesis.

This work is dedicated to my family, especially my wife, Diana, for their
love and support

iv

ACKNOWLEDGMENTS

It is my pleasure to acknowledge the many people whom assisted
me over the course of my graduate work. Firstly, my undying gratitude
goes to Dr. James E. Trosko, my mentor, whose guidance and patience
allowed me to develop into the scientist I am today. His role model as a
dedicated scientist not only served as an example of how to be a good
scientist, but most importantly, how to be a good educator. Secondly, I
would like to acknowledge my committee comprised of Dr. Chia-Cheng
Chang, Dr. Pamela Fraker, Dr. Jay Goodman, and Dr. Norbert Kaminski.
Their suggestions and critiques were critical in helping me focus and
complete my research.

I would also like to acknowledge those who have contributed to my
technical training towards the completion of this work. Dr. Melinda
Wilson, whose instruction in basic DNA techniques, transfections, and
help in planning made this work possible. I would like to express
appreciation to Beth Lockwood and Heather de-Feijter-Rupp whose
knowledge of cell culture and cell-cell communication assays was
essential toward the completion of my work. I would also like to give
acknowledgement to Dr. Brad Upham, whose encouragement reinforced
my technical abilities.

Lastly. I would like to express my deepest gratitude to my friend

and wife, Diana.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS x
INTRODUCTION 1
Initiation/Promotioanrogression Model of Carcinogenesis 4
The “Stem Cell” Theory of Carcinogenesis 8
Gap Junctional intercellular communication 12
The Role of Oncogenes 16
The Role of Apoptosis in Carcinogenesis 22
Bel-2 Prom-oncogene and Carcinogenesis 27
Experimental Rationale 38
Experimental Approach 43
WORKINQ H YPOTHESI§ 43
PE IF] MS 44
MATERIALS AND METHODS 45
Cell Culture 45
Transfection of bcl-2 45
Protein Extraction 48
Western Blot Analysis 49
Measurement of Saturation Density 50
Measurement of Cellular Proliferation 50
Apoptosis 51
Anchorage Independent Growth 51
In Vitro Invasion Assay 52
Cell-Cell Communication Assays 53
Glutathione Determination 55
Tyrosine Aminotransferase Assay 56
RESULTS 57
I. Synergistic effects of Bel-2 and V-myc in the expression of neoplastic

phenotype of Rat Liver Epithelial cells 57
Isolation and selection of bcl-2 and myc/bcl-Z transfectants 57
Characterization of Transfectants 60
Western Blot Analysis of Bcl-2 68
Anchorage Independent Growth 73
In Vitro Invasion Assay 76
Gap Junctional Intercellular Communication (GJIC) 77
Connexin 43 Expression 90
Apoptosis 90
Glutathione Expression 95

II. Differentiation Potential of Bel-2 and V-myc transformed Rat Liver epithelial
cells 100
Morphology 102
Tyrosine Aminotransferase (TA T) Assay 102
Connexin 32 Expression . 108

 

Discussion

 

APPENDIX A
Publication
References

 

 

 

111
128
128
I64

 

Table 1.
Table 2.
Table 3.
Table 4.

Table 5.

LIST OF TABLES

Maximum Saturation Density ............................................. 63
Doubling times of transfected cell lines. ............................ 67
Gap junction intercellular communication. ....................... 85
Apoptosis frequency ............................................................ 96
Tyrosine arninotransferase activity .................................. 107

viii

LIST OF FIGURES

Figure 1. The initiation/ promotion / progression model of carcinogenesis.

........................................................................................................ 6
Figure 2. Graphical representation of gap junction formation between two
neighboring cells. ........................................................................... 13
Figure 3. Summary of the Bel-2 family of apoptosis modulators. ......... 28
Figure 4. Diagrammatic illustration of the evolutionarily conserved
caspase cascade which leads to apoptotic cell death ....................... 32
Figure 5. Schematic diagrams of the various vectors used in this
research. ....................................................................................... 46
Figure 6. Morphological features of the WB-F344 cell line, before and
after transfection with bc1-2 and myc/bc12 ..................................... 61
Figure 7. Incorporation of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to assess proliferation. ............ 65
Figure 8. Westem blot analysis of human bc1-2 protien expression ....... 69
Figure 9. Western blot analysis of v-myc protein expression in the
WB.myc/bcl-2 cell lines. ................................................................ 71
Figure 10. Anchorage independent growth ........................................... 74
Figure l l. Graphical representation of invasion assay as measured by
the absorbance at 620 nm. ............................................................ 78
Figure 12. A representation of the SL/DT assay. .................................. 80
Figure 13. Scrape load dye transfer. .................................................... 83
Figure 14. Fluorescent recovery after photobleaching and scrape load
dye transfer. .................................................................................. 86
Figure 15. Anchorage independent growth correlates to cell-cell
communication. ............................................................................. 88
Figure 16. Western blot analysis of connexin 43 expression .................. 91
Figure 17 . Representation of data analyzed from FACS ......................... 93
Figure 18. Glutathione Expression. ..................................................... 98
Figure 19. Morphological features of the WB-F344, WB.bcl-202, and
WB.bcl-203 cell lines. .................................................................. 103
Figure 20. Morphological features of the myc/bcl-2 (WB.MB3 and
’ WB.MB19) and myc/ras transfected (WB.MR42) cell lines. ........... 105
Figure 21. Western blot analysis of connexin 32 expression ................ 109
Figure 22. Graphical representation of proposed model for
carcinogenesis. ............................................................................ 125

LIST OF ABBREVIATIONS

ANT ....................................................... adenine nucleotide translocator
B-HLH-LZ...basic motif, a helix-loop-helix motif, and leucine zipper motif
BH ................................................................................. bcl-2 homology
Ca2 +,Mg2+ -PBS ................................... PBS containing Ca2+ and Mg2+
CAD ....................... carbamoyl-phosphate synthase/aspartate

carbamoyl transferase/dihyroorotase guanosine triphosphatase (GTPase)
CREB ................................. cyclic-AMP response element binding protein
EGF .................................................................. epidermal growth factor
eIF—2a and eIF-4E .......................................... eukaryotic initiation factor
EMS ...................................................................... E-box Myc Sequence
Erkl and Erk2 ............................... extracellular signal regulated kinases
FACS .................................................... fluorescent activated cell sorting
FBS ........................................................................... fetal bovine serum
FRAP ....................................... fluorescent recovery after photobleaching
G418 ...................................................................................... geneticin
GJIC ..................................... gap junctional intercellular communication
GSH ........................................................................ reduced glutathione
kDa ....................................................................................... kilodalton
MAPK ................................................... mitogen activated protein kinase
MEKl and MEK2 ..................................................... MAPK/ERR kinases
MEM ............................................................. minimal essential medium
MTI‘ ............ 3-(4,5-dimethylthiazol-2-yl)-2.5-dipheny1tetrazolium bromide
myc ............................................................................ melogrtomatosis
ODC .................................................................. ornithine decarboxylase
PBS ............................................................... phosphate buffered saline
PDGF ........................................................ platelet-derived growth factor
PMSF ....................................................... phenylrnethylsulfonyl fluoride
PT ....................................................................... permiability transition
SDS ................................................................... sodium dodecyl sulfate

SL/DT .......................................................... scrape loading dye transfer

TAT ............................................................... tyrosine arninotransferase
TFE3 .................................................................. transcription factor E3
TPA ................................................. 1 2-tetradecanoylphorbol- 1 3-acetate
USF ............................................................ upstream stimulatory factor
VDAC .................................................. voltage-dependent anion channel

INTRODUCTION

Carcinogenesis has been described as a multi-step, multi-
mechanism process (Pitot et al., 1981; Dragan et al., 1993). This process
consists of three distinct temporal phases: initiation, promotion and
progression. The initiation / promotion/ progression model of
carcinogenesis is an in vivo model that cannot be fully demonstrated in
vitro due to the artificial nature of cell culture. However, in vitro models
may be used to demonstrate how biochemical mechanisms could
contribute to the initiation/ promotion/ progression model of
carcinogenesis and subsequent neoplastic transformation. Initiation is
characterized by an irreversible event, genetic or epigenetic, in a single
cell that has the potential to proliferate, Le. a stem or progenitor cell.
Promotion is a reversible, interruptible process that brings about the
clonal expansion of the initiated cell, through a chronic administration of
a stimulus that is mitogenic, e. g. chemical tumor promoters. Promotion
consists of those events that disrupt cellular homeostasis.

Homeostasis is described as a balance between positive and
negative regulatory signals that exist to allow for the proper development
and maintenance of stem/ progenitor cells and their differentiated
daughter cells in the tissues of multicellular organisms. Disruption of

homeostasis would then involve restrictions in the response of cells to

those positive and negative regulatory signals. This disruption would

involve the loss of the ability of cells to differentiate, the loss of the ability
of cells to respond to cell death signals, and the loss of the cell’s ability to
control growth or be contact inhibited. These disturbances imply some
form of communication between cells and within the cell itself. Internal.
intracellular, communication is accomplished through diffusible
elements restricted to the intracellular space. The cell to cell
communication between cells involves either or both extracellular
communication or intercellular communication. Extracellular
communication can be described as regulation between distal points
through hormones, growth factors, neurotransmitters, cell surface
proteins, and extracellular matrices. Intercellular communication
involves the direct transfer of signals between cells. One form of
intercellular signaling is through specialized protein structures in the
membranes of contiguous cells called gap junctions. This gap junctional
intercellular communication (GJIC) represents an important means by
which cells can interact directly with each other to equilibrate and
exchange ions, nutrients, and other regulatory molecules 1000 daltons
or less in size (Loewenstein, 1981). Many tumor cells have shown the
absence of gap junction coupling (Chang et al., 1987 ; Fentiman et al.,
1979; Corsaro and Migeon, 1977; Loewenstein and Kanno, 1966; Tosko
and Ruch, 1998) and a reduced response to apoptotic stimulus. These
observations led to the speculation that in solid tissues gap junctions are

required for the proper transfer of apoptotic signals between cells (Trosko

and 01

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and Goodman, 1994). Uncoupling of gap junctions between cells may be
a necessary event in promotion, however it is not the only step involved.
Additionally, the promotion phase of carcinogenesis involves the loss of
cells ability to respond to cell death signals and the loss of the cells
ability to control growth. GJIC appears to be necessary event in both of
these processes but it is not the sole event. In order for promotion to
occur a rnitogenic signal with concomitant loss of apoptosis needs to
occur. Thus if an initiated cell is uncoupled from its neighbor without
any specific signaling the cell will remain quiescent. However, if a
mitogerric signal is present, these cells would grow in an unconstrained
manner. Conversely, if an apoptotic stimulus was present in neighboring
cells, then these cells would persist as their neighbors died. Thus. it
would follow that, if blockage of apoptosis and induction of proliferation
were sufficient for neoplasia, the induction of these states might be
expected to down regulate GJIC. when expressed in a normal gap
junction competent cell.

The research in this study was undertaken to investigate if bc1-2.
an anti-apoptosis gene, plays a role in neoplastic transformation process
and could block gap junctional intercellular communication when co-
expressed with a proliferative oncogene like myc. To accomplish this, the
human bcl-2 oncogene was introduced into a normal rat liver epithelial
cell line, WB-F344, and in an already established v—myc expressing WB-

F344 cell line. These cells were tested for their ability to proliferate. to

contact inhibit, to express neoplastic characteristics (as measured by
anchorage independent growth in soft agarose, and invasion) and to
modulate gap junctional intercellular communication. The expression of
the bcl-2 protein was measured using western blot analysis with an

antibody specific for human bcl-2.

Initiationfl’romotion/Progression Model of Carcinogenesis

Multistage carcinogenesis was first demonstrated by repeated
chemical treatments of mouse skin patches (Berenblum, 1941). It was
found that some chemicals were able to cause tumors with repeated
treatments. These were called complete carcinogens. Other chemicals
could only induce tumors following treatment with an initiator, these
chemicals were termed tumor promoters. It is now known that initiators
and complete carcinogens are able to induce DNA lesions. In addition to
causing DNA lesions initiators and complete carcinogens also provide
growth (mitogenic) signals that allow for the expansion and accumulation
of initiated cells. Therefore, it is believed that tumor promoters are only
able to produce the mitogenic signals associated with complete

carcinogens.

Figure 16. Western blot analysis of connexin 43 expression. Bands at 43
kDa represent unphosphorylated (Po) and phosphorylated(P1 and P2)
connexin 43 protein.

91

Pitot and co-workers (1981) refined this concept and characterized
cancer as being three distinct stages: 1) initiation; 2) promotion; and 3)
progression (Figure l). The first step of this process is the alteration of a
“normal” cell into an “initiated” cell, which can be specifically selected
and clonally amplified given the proper conditions (Nowell 1976. Fialkow
1979). This phase is characterized by a fixed error occurring on a genetic
level. Changes at a genetic level would include mutations to the genetic
code, altering protein function or changes in gene expression that alters
cellular expression in such a way that the cells are able to have some
selective advantage over neighboring cells.

The promotion phase is most notably characterized by the clonal
expansion of the initiated cell or cells; however, this involves a couple of
cooperative mechanisms. The first is the clonal amplification of the
initiated cells by mitogenesis and the second is the blockage of cell
death, which, under normal circumstances, would eliminate the initiated
cells. The important distinction of promotion is its reversibility, in which
removal of the mitogenic stimulus could arrest the development of focal
lesions, and reapplication of the mitogenic stimulus causes the
resumption of growth of the focal lesion. The mitogenic stimuli could be
non-cytotoxic chemicals like Phenobarbital, removal of which induces
regression of tumors in the multi-stage rat liver carcinogenesis model, or
even hormones like testosterone, removal of which induces regression of

prostate tumors in rats and humans.

Figure l. The initiation/ promotion/ progression model of carcinogenesis.

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Additionally the mitogenic stimuli could be cytotoxic agents that
induce concomitant hyperplasia through necrotic cell death and
induction of mitogens associated with an inflammatory response (Aldaz
et al., 1985; Casale et al., 1997).

The blockage of cell death, specifically apoptosis, which is
programmed cell suicide and does not elicit an inflammatory response, is
most notably associated with the B-gell Lymphoma gene, Bel-2
(Tsujimoto et al., 1985). This gene allows for the accumulation of cells
that would normally progress to death thus leading to the lymphoma.
However, chemical tumor promoters, like l2-tetradecanoylphorbol- 13-
acetate (TPA) not only produce mitogenic signals, but also, they are able
to block apoptosis (Magnelli et al., 1995; Rodriguez and Lopez, 1989). In
this manner promotion results from mitogenesis and blockage of
apoptosis leading to the accumulation of initiated cells which would have
normally been excluded.

Progression is characterized by the significant acquisition of
alterations in an initiated cell such that it becomes promoter
independent, invasive into the surround tissues and, lastly, metastatic

into distal tissues.

The “Stem Cell" Theory of Carcinogenesis

The initiation / promotion / progression model of carcinogenesis does
not speculate as to the origin of the cell that is initially targeted for
carcinogenesis. “The stem cell theory” posits that the target cells are the
presumptive stem cells. These cells are characterized by their ability to
undergo asymmetrical cell division, during which, one daughter cell
retains parental “stem-cell” like characteristics and the other daughter
cell is committed to a differentiation pathway.

In order to understand this concept, it is necessary to describe the
hierarchical nature of multi—cellular organisms (Brody, 1973). This is the
idea that the whole is greater that the sum of its parts. The concept
involves atoms forming molecules, which form proteins, which are
organized into specialized organelles, which make up cells, which are
then organized to form tissues. These tissues then form the organs.
whose functions integrate to form the systems, whose features carmot be
found in any of the individual components.

Maintenance of the hierarchy of the multi-cellular organism
involves positive and negative regulation of molecular, biochemical,
cellular and physiological information (i.e., “homeostasis”) and requires
three processes of communication: 1) intracellular, those mechanisms
which contribute to the signaling within an individual cell; 2)
intercellular communication, those mechanisms which contribute to the
signaling between neighboring cells: and 3) extracellular, which

contribute to signaling between distal cells, tissues and organs. When

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these communication systems functions normally, homeostasis or
balance is achieved. In the case of higher multi-cellular organisms, like
human beings, the hierarchy starts with the original diploid cell, the
fertilized eg, that gives rise to the trillions of cells that make-up the
human being. This original cell is denoted as being totipotent, for it has
the capacity to form all the cells in the organism. This totipotent cell
gives rise to what many term as the “pluri-potent stem cell”. The “pluri-
potent stem cell” is restricted to only produce cells of a specific lineage
under normal conditions. This restriction may be confined to cell types
of specific tissue or organ. These pluri-potent stem cells give rise to
daughter cells, which are themselves committed to only produce a
specific cell type within a specific tissue or organ. This daughter cell, or
progenitor cell, can follow a specific differentiation pathway to form cells
of specialized function. Thus, in any given tissue or organ, there are
great numbers of functionally specialized cells but only a few progenitor
cells and even fewer “stem cells”.

Nakano and coworkers (1985). provided experimental evidence for
the putative stem cells as being the targets for neoplastic transformation
by demonstrating that a sub-population of less differentiated cells were
more susceptible to chemical induction of neoplastic transformation.
These same cells were also characterized as being contact inhibition
insensitive. In other words, these cells continued to grow even when

Other cells in the population were growth inhibited at the same cell

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density. Partially differentiated cells demonstrate contact inhibition
sensitivity in that they reach a specific cell density that predeterrnines
the size of the tissue. “Stem cells” are contact inhibition insensitive
because they need to be able to replenish those cells that are lost
through the normal process of programmed cell death, apoptosis. which
eliminates terminally differentiated cells that are either no longer
function, have provided their functional requirement, or are damaged
(Levine et al., 1965). Interestingly, similar to stem cells, most tumors are
contact inhibition insensitive (Abercrombie, 1979; Borek and Sachs,
1966).

Additional evidence for the putative role of stem cells in cancer
comes from experiments in the isolation of “stem cells” in culture. Chang
and coworkers were among the first to isolate. culture, and characterize
human epithelial stem-cells in vitro (Chang et al., 1987; Kao et al., 1995).
These human breast pluri-potent stem cells were not only contact
inhibition-insensitive, but they also lacked functional GJ 1C, resulting
from a lack of connexin gene expression. These GJIC deficient cells have
been shown to be more susceptible to neoplastic transformation (Sun et
al., 1999). Significantly, most cancer cells also demonstrate a lack of
functional GJIC (Yamasaki, 1990; Loewenstein, 1981). These results of
loss of contact inhibition and lack of functional GJIC demonstrate
striking similarities and suggest that cancer cells may owe their origin to

stem cells.

11

Gap Junctional intercellular communication

GJIC is established between cells by the formation of gap
junctions. Gap junctions are intercellular channels that allow for the
free passage of small molecular weight molecules (less than 1 kDa)(Beyer
et al., 1990). A gap junction is comprised of plaques of connected
connexons, one in each adjoining membrane (Figure 2). A connexon, the
structural unit of the gap junction is a cylinder with a hydrophobic
channel formed by the association of six individual protein subunits.
which are called connexins. Gap junctions are formed when connexons
of two closely-opposed cells align end-to-end forming intercellular
channels which are responsible for GJIC.

'IVvo descriptions of GJIC are 1) homologous communication with
cells of same origin; and 2) heterologous communication with cells of
different origin (Hotz-Wagenblatt and Shalloway, 1993). Very early data
demonstrated that several tumor cell lines displayed reduced GJIC. but
this was not proven to be a universal characteristic (Y amasaki, 1990;
Loewenstein, 1981). More importantly, it was later demonstrated that.
While some tumors showed homologous communication they did not
dISplay heterologous communication (Loewenstein, 1979; Yamasaki,

1990). For example. in vitro transformed BALB/c 3T3 cells showed no

alterations in GJIC (Enomoto and Yamasaki, 1984). However, these cells

12

Figure 2. Graphical representation of gap junction formation between two
neighboring cells. The gap junction is represented as a pairing of
connexons in opposing plasma membranes. These paired connexons
form the gap junctions and subsequent channel. The connexon is
comprised of six individual connexin proteins.

13

First Cell

 

 

 

 

 

 

 

 

 

 

 

Connexon

v

Connexin

Figure 2

14

were unable to establish heterologous GJIC with their surrounding
neighbors. These results were repeatable regardless of transformation
conditions, i.e. chemical carcinogens, UV irradiation, or oncogenes
(Yamasaki et al., 1987). Importantly, these results were repeated in vivo
using the multistage rat liver carcinogenesis model (Krutovskikh et al.,
1991). Preneoplastic foci in this model showed a significant lack of GJIC
with the surrounding parenchyrnal cells.

The most striking evidence for the role of GJIC in promotion may
come from research into chemical tumor promoters. Chemical tumor
promoters were the first tools used to demonstrate multi-phase
carcinogenesis (Berenblum, 1941). Many chemicals, like the phorbol
ester, TPA, can promote initiated cells. TPA-induced cell proliferation
and tumor promotion have been shown to be mediated through the
activation of PKC (protein kinase C) (Yuspa, 1994; Dlugosz, 1994). This
leads to the subsequent activation of MAPK cascades, one of which is the
ras-raf-MAPK pathway (described in detail later in this report), which
eventually causes the transcriptional induction of the growth related
transcription factors fos and jun (Seger and Krebs; 1995).

An additional, and equally important, mechanism by which these
Chemicals act is by reducing/blocking gap junction-mediated

flntercellular communication (Y otti et al., 1979; Murray and Fitzgerald,
1979). This discovery has since led to the use of GJIC inhibition as a

Predictive indicator for the detection of tumor promoting activity of

15

environmental agents (Swierenga and Yamasaki, 1992: Budunova and
Williams. 1994). More recently GJIC has been used as a predictor for
carcinogenic activity (Rivedal et al., 2000; Rosenkranz et al., 1999).
Interestingly, using a structure—activity relationship system as a
predictive model, the blockage of GJIC has a higher correlation to
carcinogenicity than did mutagenicity, meaning blockage of GJIC was a
better predictor of a chemicals ability to act as a carcinogen than its
ability to form mutations (Rosenkranz et al., 1999).

To further demonstrate the importance of GJIC in carcinogenesis.
it has been shown that, in addition to their other properties many
oncogenes also inhibit GJIC (Yamasaki, 1990; Trosko et al., 1990; de-
Feijter et al., 1990; de-Feijter et al., 1996). Consistent with these
findings, the evidence that tumor suppressor genes and anti-carcinogens
act in an opposing manner by upregulating GJIC (de-Feijter-Rupp et al.,

1998; Zhang et al., 1992; Mehta et al., 1989; Ruch et al., 1989).

The Role of Oncogenes

We have previously discussed how promotion involves not only
mitogenesis and GJIC. but have also speculated to the role of blockage of
apoptosis. Therefore, it is not surprising that these are demonstrated

processes affected by many of the known oncogenes. Oncogenes are
genes that induce cellular and morphological alterations leading to

nec>plastic transformation. Oncogenes were first discovered as the

16

element in transforming viruses responsible for causing tumors (Rous.
1910: Rous, 191 1). Viral oncogenesis hypothesis implied that all
vertebrate cells contain genes, called proto-oncogenes, analogous to the
genes found in the transforming viruses (Huebner and Todaro. 1969;
Todaro and Huebner, 1972; Marshall, 1986). The proto-oncogenes have
been evolutionarily conserved in all metazoan organisms (Shilo and
Weinberg, 1981). This conservation of prom-oncogenes underlines their
great importance in normal cellular function. Proto-oncogenes can
become activated by one or more of four mechanisms, namely; 1)
inappropriate expression during stages of cell proliferation or
differentiation, 2) gene amplification, 3) chromosome translocations, and
4) mutations. Radiation, chemical mutagens, error-prone DNA
replication or even viruses can induce mutations. Once activated
oncogenes are thought to contribute directly to mitogenesis.

One way to classify oncogenes is to group them based on the
cellular localization of their gene products. By this manner, two major
classes were established: 1) nuclear and 2) cytoplamsic / membrane
bound oncogenes (W einberg, 1985). Oncogenes could therefore produce
a mitogenic signal by upregulating growth related genes in two ways: 1)
directly initiating DNA transcription. and 2) initiating signal cascade
mechanisms which eventually lead to initiation of DNA transcription.

The myc oncogene is an example of mitogenesis produced by direct

initiation of DNA transcription. Myc is a nuclear protein that shares

17

._.__.__

u;

ff.
N

many structural characteristics with known transcription factors, mainly
a protein structure that contains a basic motif, a helix-loop-helix motif.
and leucine zipper motif (B-HLH-LZ). This B-HLH-LZ structure
demonstrated high homology with the transcription factors USF
(upstream stimulatory factor), and TFE3 (transcription factor E3) (Gregor
et al., 1990: Bechmarm et al., 1990). The ability of myc to function as a
transcription factor was confirmed using both in vitro yeast and
mammalian cell systems (Amati et al., 1992; Kretzner et a1. 1992). The
role of myc in cell proliferation was demonstrated in 3 ways: 1) myc
expression patterns in normal cells, 2) deregulated myc expression
effects on normal cells, and 3) genes specifically targeted by myc for
transcriptional activation.

The expression pattern of the normal proto—oncogene provides
valuable evidence as to the role that an activated gene has in
proliferation. Normal Myc expression patterns showed that there was a
rapid increase in myc mRNA following mitogenic treatment (Cochran et
al., 1983; Cochran et al., 1984; Kelly et al., 1983: Greenberg and Ziff.
1984; Greenberg et al., 1985; Lau and Nathans, 1987). Myc expression
alone was then demonstrated to be sufficient to induce G0 to G1 cell
cycle transition (Coppola and Cole, 1986; Eilers et al., 1991).
Conversely, microinj ection of antisense oligonucleotides to myc mRNA

blocked progression beyond G1 (Coffey et al., 1988; Heikkila et al., 1987;

18

Holt st

of my:

CALC

eial

EMS

cat:

Holt et al., 1988, Loke et al., 1988). These results suggested that the role
of myc in proliferation was to drive the transition through G 1 from G0.

The overexpression of myc produced cells displaying shortened G 1.
and increased growth rates (Karn et al., 1989; Palmieri et al., 1983;
Langdon et al., 1988). Myc overexpression was even shown to be capable
of partially transforming rat fibroblasts (Kohl and Ruley, 1987:
Mougneau et al., 1984). However. complete malignant transformation
required two oncogenes (Land et al., 1983: Lugo and Witte, 1989).

Myc, when activated, recognizes and binds the DNA sequence
CA(C/T)GTG. which is termed the E-box Myc Sequence (EMS) (Blackwell
et al., 1993; Blackwood and Eisenmann, 1991; Prendergast et al., 1991;
Solomon et al., 1993). The genes that are specifically targeted by myc:
EMS binding and subsequent transcription can be classified into two
categories: 1) cell growth, and 2) cell-cycle progression. Those genes that
have been associated with cell growth are ODC (ornithine decarboxylase).
which is responsible for polyarnine biosysthesis necessary for S phase of
the cell cycle, elF—2a and eIF-4E (eukaryotic initiation factor), which is in
turn required for transcription, and CAD (carbamoyl—phosphate
synthase/aspartate carbamoyl transferase / dihyroorotase) , which is
responsible for pyrimidine biosynthesis (Bello-Fernandez et al., 1993:
Wagner et al., 1993: Rosenwald et al., 1993; Jones et al., 1996;

Miltenberger et al., 1995).

19

r"? the

norm,-
mot
PDGF

Ml.

The genes directly controlled by myc that have been associated
with the cell cycle are CDC25A, which removes inhibitory phosphates
from CDK2, p271‘1P1, which is a CDK inhibitor and is negatively regulated
by myc, and the transcription factor c/EBP-a, which is responsible for
cell cycle arrest and is also negatively regulated by myc (Galaktionov et
al., 1996: Steiner et al., 1995; Freytag and Geddes, 1992). Myc acts to
remove inhibitory signals allowing the cell cycle to proceed.

Cytoplasmic and membrane associated oncogenes function in
mitogenesis through the induction of signal transduction cascades which
activate the transcription of growth related genes. The best known
example of this is the ras oncogene. Ras is a guanosine triphosphatase
(GTPase) which has a GDP-bound inactive form and a GTP-bound active
form. In a similar fashion to myc. ras function in proliferation was
demonstrated by examining normal and over-expression of ras protein in
normal cells. The normal function of ras was found to be associated with
cytokine receptor kinases like the EGF (epidermal growth factor) and
PDGF (platelet-derived growth factor) receptors (Molloy et al., 1989; Ellis
et al., 1990: Kaplan et al., 1990: Satoh et al., 1990).

Activation of receptor kinases, by ligand binding leads to the
activation of ras (for a detailed review see Campbell et al, 1998). Briefly,
ras functions by complexing with the Raf gene product, after which raf is
subsequently activated. Raf then phosphorylates MEKl and MEK2

(MAPK/ERK kinases). MEKs phosphorylate tandem threonine and

20

tyrosine residues of the MAPKs (mitogen activated protein kinase) Erkl
and Erk2 (extracellular signal regulated kinases). These MAPKs can then
directly induce the gene expression by phophorylating transcription
factors, like Elkl or CREB (cyclic—AMP response element binding
protein). that can then induce growth related transcription factor genes
like fos and jun (Marais et al., 1993: T‘reisman, 1996; Ginty et al., 1994).
The MAPKs can also activate other kinases like p90RSK a
serine/threonine kinase which can also induce fos and jun gene
expression (Blenis, 1993). Oncogenic ras mutations lock the ras protein
in its active GTP-bound form keeping the cascade always induced (Satoh
et al., 1992; Boguski and McCormick. 1993: McCormick, 1994).
Mutations in ras genes are some of the most frequent mutations
observed in human cancers (Barbacid. 1987 ; Hunter, 1997).

In addition to their proliferative function, oncogenes, like ras and
the putative oncogene, bcl-2, can reduce or even block apoptosis. The
bcl-2 gene’s classification as an oncogene is based on its function as an
apoptosis suppressor (Hockenbery et al., 1990). However. several
oncogenes like Myc and the tumor suppressor gene, p53, can increase
apoptosis and promote cell cycle progression. The opposing functions of
the Myc oncogene in its stimulation of both proliferation and apoptosis
may explain its inability to induce signigicant neoplastic transformation
(Hayashi et a1. 1998), in that, as the cellular population increases so to

does the apoptosis. A precarious disruption between the birth and death

21

of cells is formed. Conversly, those chemical tumor promoters. like TPA.
and oncogenes. like ras, that reduce /block apoptosis and GJIC. alone
are not sufficient for complete neoplastic transformation of normal cells.
However, when these chemical promoters or oncogenes act in
cooperation with deregulated myc expression, the cells become
neoplastically transformed (Land et al., 1983; Storer et al., 1988; Dotto et
al., 1985; Hsiao et al., 1984; Yancopoulos et al., 1985) presumably

through the additional blockage of apoptosis and GJIC.

The Role of Apoptosis in Carcinogenesis

Apoptosis, commonly referred to as programmed cell death, has
been speculated to be a normal pathway to eliminate initiated or pre-
malignant cells thus preventing carcinogenesis (Schulte-Hermann et al.,
1995). Apoptosis and necrosis were described by pathologists as two
morphologically distinct process by which cells die (Kerr et al., 1972).
Necrosis is a passive osmosis-driven process, which is characterized by
cellular swelling. The cellular swelling observed in necrosis is proceeded
by the loss of ion-pumping activity, and distention of the endoplasmic
reticulum. The nucleus swells and becomes electron transparent with
small-scattered masses of chromatin. During later stages, the
mitochondria swell and the plasma membrane ruptures (Haukins et al.,

1972).

22

In stark contrast to necrosis, apoptosis is an active energy
requiring process, which is characterized by cellular shrinkage. The early
stages of apoptosis occur in the nucleus where chromatin condenses into
one or multiple masses lining the nuclear membrane (Wyllie, 1981). As
apoptosis proceeds, the nucleus splits into separate round bodies, the
nuclear envelope becomes uneven, the cisternae become dilated, and the
nuclear pores migrate to areas adjacent to uncondensed chromatin
(Arends et al., 1990). Later stages of apoptosis are manifested by a
volume loss in the cytoplasm. The cell shrinks and begins blebbing
outward. forming blebs around the cell. This event is proposed to be the
primary cause of shrinkage (Kerr et al., 1987). As the cell further
shrinks, it splits into small round apoptotic bodies that are usually
phagocytosed by adjacent cells. Inward blebbing is also known to occur
(Clarke, 1991). In these instances the cell membrane is endocytosed to
form clear vesicles that are indicative of lysosomal activation.

This initial description led to the observations that apoptosis was
the primary event that led to regression of not only non-neoplastic
tissues (Wyllie et a1, 1973; Kerr and Searle, 1973) and preneoplastic foci
in hepatocytes (Bursch et al., 1984). but also for the hormone-dependent
regression of mammary (Gullino, 1980). pancreatic (Szende et al., 1989)
and prostate (Kerr and Searle, 1973) cancer. Interestingly, Kerr and
Searle (1972) also attributed the slow growth rate of basal cell

carcinomas, which contain numerous mitotic markers. to apoptosis.

23

A”;

the

Int

11*]

Of

Animal studies demonstrated that food restriction eliminated 20-30% of
the normal liver cells through apoptosis. More significant is the fact that
in these same animals, putative preneoplastic lesions preferentially
underwent apoptosis, thereby reducing their numbers by 85% and
showing the selective role that apoptosis plays in initiated cell exclusion
(Grasl—Kraupp, 1994). However. not until the isolation of the _B_-_(_:ell
Lymphoma gene 2, Bel-2 (Cleary et al., 1986), that directly affected this
pathway. was apoptosis accepted as having a significant role in cancer.
Bel-2 was discovered as a result of a translocation between
chromosomes 14 and 18 present in most follicular lymphomas
(Tsujimoto et al., 1984; Bakhshi et al., 1985). Vaux and colleagues
(1988) demonstrated that bcl-2 could not only block the cell death that
was associated with cytokine withdrawal, but its over-expression could
also block the cell death associated with the over-expression of the myc
oncogene. Bel-2's effect on cell death and prolonged cell survival was
confirmed with transgeneic mice, where bcl-2 over—expression expanded
the B-lymphoid compartment 4 to 5 fold (McDonnell et al., 1989).
However, unlike other genes that were reported to be involved in
carcinogenesis, bcl-2 did not affect the proliferation rates, it only delayed
or blocked cell death (Strasser et al., 1991; Sentman et al., 1991).
Additional findings reinforced the importance of apoptosis in
cancer progression by demonstrating that chemotherapeutic anti-cancer

agents like cis-platin, and etoposide. which were previously thought to

24

L

HUI

10C

‘Uu

a l

kill cells by causing irreversible metabolic damage, actually functioned as
inducers of apoptosis (Searle et al., 1975; Kaufrnann, 1989; Eastman,
1990). Consistent with these finding were the observations that many
tumors possessed the ability to escape apoptotic stimulus (Hoffman and
Liebermann, 1994). In fact, using bcl-2 as the paradigm for anti-
apoptotic genes, it was shown that its over-expression was sufficient to
confer a resistance to chemotherapeutic drugs (Miyashita and Reed.
1992). Therefore. bc1-2 over-expression in cancer has been associated
with poor prognosis as seen in neuroblastoma (Castle et al., 1993), colon
cancer (Hague et al., 1994), and especially prostate cancer (McDonnell et
a1. , 1992).

To help visualize the role of apoptosis in cancer one can view it as
a mathematical equation where A equals the accumulation of cells, P
equals proliferation rate and D equals cell death,

A = P - D
Homeostasis could then be defined as.

P = D, and A = 0 (no net change in accumulation of cells)
It is also possible to demonstrate the effects of oncogenes on the
accumulation of cells using this equation. Myc is known enhance
proliferation, but is only very mildly turmorigenic, however it also
sensitizes cells to limitations in nutrients. In this scenario as cells over-
expressing Myc accumulate, they also begin to die as their energt

sources become depleted. Therefore P is only slightly larger than D

25

P > D, A = P - D
and there is only a small accumulation of cells. Ras, which enhances

proliferation and inhibits apoptosis, is significantly more tumorigenic

than Myc. In the Ras scenario, P is significantly larger than D.

P>>>D,A=P-D

Here the accumulation of cells is similar to the proliferation rate. Using
this equation, it is also possible to visualize how bcl-2, by blocking
apoptosis could increase the accumulation of cells by blocking cell death
where.

P>>D, A = P - D
Here, however, unlike Ras, there is little or no effect on proliferation and
the accumulation of cells is dependent on extracellular effectors of
proliferation. Therefore, if an initiated cell, over-expressing a Bel-2 like
gene. failed to undergo apoptosis, it would escape the normal cell
exclusion and be in a position for clonal expansion. If myc
overexpression was coupled to Bel-2 overexpression. then the rate of
proliferation would be disparagingly higher than cell death and effectively

accumulation of cells would equal proliferation.

P>>>>n, A = P - o, effectively A = P

26

Bel-2 Proto-oncogene and Carcinogenesis

The importance of apoptosis is not just limited to cancer,
homologues of the bcl-2 gene have been identified in metazoans and even
viruses, demonstrating the importance and conservation of apoptosis in
multicellular organisms (Thompson, 1995). The bcl-2 proto-oncogene
codes for a 239 amino acid protein that contains a ligand domain, a
receptor domain, a pore formation domain. and a membrane insertion
domain (membrane anchor) (Vaux et al., 1988). The bc1-2 protein can
form homodimers and, in the cell, is localized to the mitochondrial inner
and outer membranes, as well as the nuclear envelope and endoplasmic
reticulum. The bc1-2 proto-oncogene is a member of a larger bcl-2
related family of genes (Figure 3). The bcl-2 family members are
comprised of both pro-survival inhibitors and apoptogenic promoters of
cell death. The two predominant members are bcl-2 representative of
pro-survival, and bax representative of apoptogenic cell death (Gross et
al., 1999). Bel-2 and its relatives inhibit apoptosis, while bax and its
relatives promote cell death.

Members of the inhibitor and promoter subfamilies typically share
three of the four characteristically conserved bcl-2 homology (BH)
sequence motifs, BHl, BH2 and BH3 (Muchmore et al., 1996; Sattler et
al., 1997). Additionally, because of the similarities in sequence,

members of both the bcl-2 and bax subfamilies can form similar protein

27

Figure 3. Summary of the Bel-2 family of apoptosis modulators.
Topology of the bcl-2 protein is depicted, showing regions of significant
function and location of the bcl-2 homolgy (BH) domains. Additional,
bcl-2 family members are shown illustrating regions of conservation of
BH domains.

28

Inhibitors of Apoptosis (Bel-2 Relatives)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ligand Pore Formation Membrane
Domain Domain Anchor
I—_|
Em [:ij IBHl I [swim
LHydrophobic Groove (Receptor Domain) I
E: L J 7 3*“ I I II
CHI 1 I [-2]
I H I 3H1 I I I
w j I 3H1 I I I ]
CE I 3*“ I I I
“3' mg“) II: L I I 3*“ I I I -:I
(AdenoviruS) E_E J I BHI I I I
(Ebstein Barr) ] am I fl I
(Kaposi’s sarcoma) I I Bill I I I
(Kaposi’s sarcoma) L I BHI I I I
(RSV) En J Hm I I i-]
(””9”“) I It I I 3*“ I I E-:I

 

 

Promoters of Apoptosis (Bax Relatives)

 

I

E; J 713le I

 

I:

flBHII

I

 

F

”I IBHII

 

ii!

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BH3 only members

I

]

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L flBHII

 

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Hill

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[

I

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mm—rgfimmw

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Figure 3

29

I

Bel-2

Bcl-XL
Bcl-W
Mel-1
Bil-l
Diva
Cod-9
19 kDa ElB
BHRF-l
Bcl-Z-KS
ORF-16
NR-13
Borg-1

(Drosophila)

 

Nip3
Nix/BNIP3
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conformations. This similarity in protein conformation between the
opposing subfamilies readily leads to heterodimerization, presumably
allowing for initiation of apoptosis through the direct sequestration of
bcl-2 family members’ by the bax family members (Suzuki et al., 2000).
This sequestration is achieved through the interaction of the ampipathic
BH3 a helix of the apoptosis promoters with the hydrophobic groove.
formed by the a helices in the BH3, 8H], and BH2 regions, of the
apoptosis inhibitors (Sattler et al., 1997). However, there is a third
subfamily, bearing little resemblance to each other or to the bcl-2 and
Bax subfamilies, which contains only a BH3 domain and are promoters
of apoptosis, suggesting that only the BH3 domain is necessary for the
sequestration of pro-survival inhibitors (Kelekar and Thompson, 1998).

The proto-oncogenic importance of bcl-2 was demonstrated by
genetic manipulation of the nematode Caenorhabditis elegans. Deletion
of the ced-9 gene, a bc1-2 homologue, led to early embryolethality due to
excessive developmental cell death (Hengartner et al., 1992). The effect
of this deletion was rescued by the insertion of a human bcl-2 transgene
(Vaux et al., 1992). This suggested a conservation of function between
the mammalian bcl-2 and the metazoan ced-9 genes. Further studies
have delineated that the bcl-2 proto-oncogene is involved in the
regulation of a highly conserved cell death pathway, the caspase cascade,
found in multicellular organisms ranging from metazoa to insects to

mammals (reviewed in Budihardjo et al., 1999). Bel-2 and its

30

homologues function as integral members of the caspase cascade. which
serves as the culmination of apoptotic stimulating pathways (Figure 4).
The caspases are a family of proteases that are responsible for the actual
execution of apoptosis. Activation of the caspase cascade occurs with an
apoptotic stimulus, ranging from radiation to DNA damage to cytotoxic
chemicals, which activates apoptogenic proteins, inactivates pro-survival
proteins. leading to the release of the cofactors. dATP and cytochrome C
(Liu et al., 1996). These cofactors stimulate a caspase activator (initiator
caspase) complex, referred to as the apoptosome, which leads to the
activation of an executioner caspase and subsequent apoptosis (Hu et
al., 1998; Pan et al., 1998).

How bcl-2 functions biochemically in apoptosis and the caspase
cascade is still unclear. Putatively, bcl-2 can function in 3 ways; 1)
direct interaction with non—bc1-2 members of the caspase cascade. 2)
stabilization or perturbation of new or existing mitochondrial membrane
channels, 3) other functions yet to be determined. Evidence from C.
elegans supports a direct sequestration model of bcl-2 action, in which.
ced-4, the caspase activator co-localizes with ced-9, and only when a
death stimulus occurs is ced-4 displaced (Conradt and Horvitz, 1998).
Since bcl-2 can rescue ced-9, it has been proposed that a similar survival
mechanism ezdsts in mammals (Hengartner and Horvitz, 1994).

The role of bcl-2 in the mitochondrial membrane is more empirical.

Bel-2 over-expression has been shown to block all disturbances

31

Figure 4. Diagrammatic illustration of the evolutionarily conserved
caspase cascade which leads to apoptotic cell death. Typical proteins
mediating five steps are depicted in boxes. Mammalian genes are listed
first followed by the C. elegrans homologous gene then the Drosophila
homologous gene. A nematode homologue of Caspase 3 has yet to be
identified.

32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cytotoxic/Apoptotic Triggers
DNA Damage Radiation Chemotherapy
Glucocorticoids Hypoxia Growth Factor Deprivation
Loss of adhesion Ceramide Spindle Disruption
PAS/1‘ NF/Reaper Heat Metabolic inhibition
Developmental Cues Trail Microtuble Disruption
A tosis
£30m“ Bax/EGL-l/Debcl
l Mitochondiral
A tosis interactors
hfifiim BCl-Z/ CCd-9/dBorg-1 ““0"“ in the
release of
cytochrome C and
dATP
Caspase Cytoplasmic
Activators Apaf' 1/ ced'4/D ar k effectors initiated
by cytochrome C.
Have been found
to form a pre-
initiation complex
Initiator with Diva termed
Caspases Casp-9/Ced-3/Dronc the Apoptosome
Executioner 0
Cl 8 bstrates:
l/ im'fivage u PARP
a-fodrin Lamins
. boactin proIL-IB
UlSnRNP
ApoptOSIS Topoisomerase-l
(Cell Death)
Figure 4

33

associated with the mitochondria that are attributed to apoptosis.
such as changes in membrane permeability, pH, and overall integrity
(Green and Reed, 1998). Additionally, the three dimensional
crystalographic structure of the bcl-xi, gene, a splice variant of bcl-2, is
similar to pore forming bacterial toxins (Muchmore et al., 1996). These
findings suggested that bcl-2 might function to either: 1) form new pores
in the mitochrondrial membrane, which has yet to be shown in cells or
under physiological conditions, or 2) effect (either stabilize or perturb)
existing channels, like the permeability transition pore (171‘) comprised of
a voltage-dependent anion channel (VDAC) in the outer membrane and
the adenine nucleotide translocator (ANT) in the innner memebrane
(Crompton, 1999). The interaction of the bcl-2 family members and
mitochondrial pores has been shown using VDAC channels in liposomes
(Shirnizu et al., 1999). Bax was able to stabilize an open conformation
that allowed for the free passage of cytochrome C, while bcl-xL perturbed
the VDAC channel keeping it in a closed conformation. Additionally, bax
has also been shown to interact with the ANT channel (Marzo et al.,
1998).

Bel-23 role in apoptosis may involve molecular interactions yet to
be fully understood. As yet. no functions have been associated with the
localization of bcl-2 to the endoplasmic reticulum (ER) or the nuclear
envelope. It has been suggested that bcl—2 localization to the nuclear

envelope and ER may involve intracellular Ca2+ stores. The major

34

storage site for Ca2+ resides between the nuclear membrane and its
continuity with the ER lumen. There has been growing evidence for the
role of bcl-2 in the modulation of these ER Ca2+ stores. He and
colleagues ( 1997) were able to demonstrate that the depletion of the ER
calcium is a possible trigger of apoptosis, and that bcl-2 overexpression
inhibits ER calcium loss. In this role bc1-2 appears to be able to assist in
the maintenance of the ER calcium pool, thereby blocking apoptosis at a
step prior to caspase activation and independent of mitochondria.
Recently, bcl-2 has been shown to actively decrease the free Ca2+ in the
cytoplasm by upregulating the ER calcium pump (Kuo et al. , 1998) and
capacitative Ca2+ entry (Williams et a1. 2000). These two systems
represent the major processes by which calcium is pumped into the ER.

Less studied is the role of bcl-2 localization to the nuclear
envelope. Early electron micrographs showed that bcl-2 formed highly
organized patches resembling nuclear pore complexes (Kraj ewski et al.,
1993) suggesting an active role for bcl-2. Recent evidence supports this
finding in that bcl-2 was able to inhibit wild-type p53 nuclear import
following DNA damage (Beham et al., 1997). This suggests that bcl-2
may act as some form of a “gatekeeper” selectively controlling protein
traffic into the nucleus.

Lastly, bcl-2 localization to the mitochondria, nucleus and ER,
suggests that bcl-2 may act as an oxidative stress sensor monitoring free

radicals, for these compartments are postulated as the major areas of

35

free radical production (Thompson. 1995). Oxidative stress has been
defined as “a disturbance in the prooxidant-antioxidant balance in favour
of the former, leading to potential damage” (Sies, 199 l). The major agent
for the prooxidant state is the free radical. A free radical is a chemical
species that possesses one or more unpaired electrons. These free
radicals tend to be highly reactive and can readily lead to DNA damage.
Many carcinogens function to induce DNA damage through free radical
generation, however this has the side effect of inducing apoptosis
(Clemens, 1991; Witz, 1991; Boref, 1991). Initial experiments showed
that bcl-2, in addition to blocking apoptosis, was able to protect cells
against hydroxyl radicals formed from gamma-radiation (Sentman et al..
1991; Strasser et al., 1991). Additionally, bc1-2 has been shown to
attenuate lipid peroxidation which can lead to DNA damage (Hockenbery
et al., 1993; Kane et al., 1993: 'Iyurina et al., 1997). Exactly how bcl-2
functions in free radical protection is not known. One mechanism by
which oxidative stress and free radicals function is to cause fluxes in
intracellular calcium levels, which as earlier discussed, can be blocked
by bcl-2. However, and more importantly, bcl-2 has been demonstrated
to upregulate the genes that act as free radical scavengers such as
intracellular thiols (Bojes et al., 1997), like thioredoxin, and reduced
glutathone (GSH) (Kane et al., 1993; Mirkovic et al., 1997; Voehringer et
al., 1998). These proteins function in the cell as the major intracellular

antioxidents.

36

Interestingly, oxidative stress has also been speculated to play a
role in the promotion phase of carcinogenesis (Perera et al., 1987;
Cerutti, 1989). Many known liver tumor promoters have been shown to
induce oxidative stress (Cattley and Roberts, 2000; Roberts et al.. 2000;
Perera et al., 1987) at levels that do not apparently affect apoptosis.
Additionally, down regulation of GJIC has also been speculated to be
associated with oxidative stress signals, suggesting a possible
mechanism by which tumor promoters affect gap junctions (U pham et
al., 1997).

In conclusion, promotion involves mitogenesis, blockage of
apoptosis, and blockage of GJIC. Classical tumor promoters can
function to induce mitogenesis, block apoptosis, and down regulate
GJIC. speculatively through oxidative stress. Oncogenes can function in

similar manners as tumor promoters to obtain the same results.

37

Experimental Rationale

If one accepts the stem cell theory of carcinogenesis (Trosko and
Chang, 1989), then the blockage of terminal differentiation (Potter, 1978)
may be involved in the “initiation” phase of carcinogenesis, while clonal
expansion (mitogenesis), inhibition of apoptosis, and down regulation of
GJIC would contribute to the “promotion” phase (Shulte-Herman et al.,
1995; Trosko and Goodman, 1994). Therefore, over expression of various
oncogenes , cooperating with each other, might be involved in all phases
of the initiation / promotion / progression model to carcinogenesis.

The cooperation of oncogenes was one of the first important
molecular concepts of carcinogenesis. One of the first cooperative
interactions to be reported was between the myc and ras oncogenes
(Land et al., 1983; Lee et al.. 1985). The myc oncogene, a transcription
factor, has been associated with the cellular functions of proliferation,
differentiation and apoptosis (Gandarillas, 1997; Evan et al., 1994;
Koskinen and Alitalo, 1993; Evan et al., 1992; Askew et al., 1991).
Disruption of any of these basic cellular functions could contribute to the
multi-step, multi-mechanism process of carcinogenesis as was
demonstrated by the association of myc over-expression with many types
of tumors. (Cole, 1986; Kelly and Siebenlist, 1986) Similarly the ras
oncogene, being a member of the G- protein family, has been shown to be

involved in one of the many signal transduction pathways affecting

38

mitogenesis, diflerentiation and apoptosis (Campbell et al., 1998). Ras,
as with myc. has been shown to be activated in many types of tumors
(Suarez, 1989; Nishirnura and Sekiya, 1987).

In addition, the demonstration that phorbol esters, such as TPA,
seemed to act in a manner similar to the ras oncogene, in that they could
cooperate with the myc oncogene to induce a neoplastic phenotype
(Connan et al., 1985). Thus, 'IT’A, by activating protein kinase C, triggers
signal transduction pathways and can act as a modulator of mitogenesis.
differentiation and block apoptosis, in affect acting as a surrogate “ras”
gene (Magnelli et al., 1995; Nishizuka, 1992; Rodriguez and Lopez, 1989).

Our laboratory and others have shown that, while ras and TPA
efiect signal transduction, they also effect gap junction-mediated
intercellular communication (GJIC) (de-Feijter et al., 1992; de-Feijter et
al., 1990; Murray and Fitzgerald, 1979; Yotti et al., 1979). Gap junctions
are channels that directly link the interiors of neighboring cells allowing
for the free diffusion of small molecular weight molecules (Spray and
Bennet, 1985). Most tumors demonstrate a reduction in GJIC activity.
either between themselves (homologous GJIC) or with other cell types
(heterologous GJIC) (Loewenstein, 1981). Presumably, down regulation
of GJIC activity would lead to the removal of growth inhibitory signaling.
thereby providing a selective advantage to the initiated or neoplastic

cells. TPA was the first chemical agent shown to reduce GJIC activity

39

(Yotti et al., 1979; Murray and Fitzgerald, 1979). Rat liver epithelial cells
stably transfected with Ha-ras demonstrate a dose-dependant reduction
of GJIC with increasing levels of ras T24 protein (de-Feijter et al., 1992;
de-Feijter et al., 1990; El-Fouly et al., 1989). Recently our laboratory
reported that Ha-ras and v-myc could cooperate to down regulate GJIC
and this down regulation correlated to the cells malignancy (Hayashi et
al., 1998).

Ras and myc oncogenes have also been implicated in apoptosis
(Hoffman and Liebermann, 1998; Sakai et al., 1994; Arends et al., 1993).
Where myc seems to confer a susceptibility to apoptosis (Donzelli, 1999;
Hoffman and Liebermann, 1998; Evan et al., 1992), ras appears to
reduce the cell’s response to apoptosis (Sakai et al., 1994; Arends et al.,
1993). Therefore, in cells in which both myc and ras are activated, signal
transduction cross-talk interacts to block differentiation, triggering
cellular proliferation, resistance to apoptosis, and subsequent down
regulation of GJIC. In effect, these unregulated disruptions of interacting
signals bring about the appearance of the tumor.

The bcl-2 protooncogene has been one of the major genes
implicated in the apoptotic process (reviewed by Korsmeyer, 1999). Early
experiments in transgenic animals overexpressing bc1-2 under
immunoglobulin promoter control showed increased frequency of

follicular hyperplasia and B-cell survival thus suggesting the role of bcl-2

4O

 

as an anti-apoptotic gene (McDonnell et al., 1989). Since this work,
many have demonstrated that bcl-2 is not only involved in tumors of
lymphoid origin but also in many tumors of epithelial origin (Dellas et al..
1997; Wang et al., 1998, Jiang et al., 1995; Gee et al., 1994; Hague et
al., 1994; Leek et al., 1994; Lu et al., 1993;. McDonnell et al. 1992).
Additionally, bcl-2 has also been demonstrated to synergistically interact
with TPA to transform cells (Amstad et al., 1997). Thus, by blocking
apoptosis, bcl-2 seems to be acting as a surrogate “ras” gene. However,
bcl-2 itself is unable to induce proliferation or neoplastic transformation
(Del-Bufalo et al., 1997; Lu et al., 1995; Nataray et al., 1994).

Many investigators have studied the co-expression of myc and bcl-
2 in a variety of cell types and transgenic models (Jager et al., 1997,
Fanidi et al., 1992; Strasser et al., 1990; Vaux et al., 1988). These
studies were able to show that bcl-2 and myc can cooperate and increase
or enhance the tumor incidence and formation. However, the cooperative
assays studying myc and bcl-2 have focused on cells of lymphoid origin,
fibroblasts, and targeted promoter controlled vectors, but not in other
epithelial systems. The experiments described in this study were
designed to test whether the bcl-2 oncogene in cooperation with v-myc
could function, neoplastically, to transform the WB-F344 cell line, a
normal rat liver epithelial cell line. Our laboratory has previously

reported the establishment of a WB-F344 cell line stably expressing the

41

VII

inc

W0 ‘

v-myc oncogene (Hayashi et al., 1998). This cell line demonstrates an
increase in proliferation and cell saturation density but it did not form
colonies in soft agar or tumors in nude mice. This cell line was used as
the target for transformation by bcl-2. In this manner, this report
investigated the effect that bcl-2 alone and in cooperation with v-myc

would have on neoplastic transformation and GJIC.

42

Experimental Approach

WORKING HYPOT HES] S

Carcinogenesis has been described as a multi-step process
involving three ordered temporal phases, initiation, promotion, and
progression. Initiation is characterized by an irreversible genetic event in
a single cell that has the potential to proliferate, i.e. the stem cell, which
renders that cell sensitive to clonal expansion. The promotion phase has
been speculated to include the selective accumulation of the initiate cell
via mitogenesis (proliferation), and the escape from apoptosis, which
would normally eliminate the initiated cell. Many oncogenes and tumor
promoting chemicals have been shown to contribute to mitogenesis and
blockage of apoptosis. In addition, these same oncogenes and tumor
promoters have been shown to block intercellular signaling through gap
junctions. Gap junctional intercellular communication has been
postulated as the means by which cells communicate and transfer
signals that would suppress growth and induce apoptosis. The objective
of this study was to test the hypothesis that cooperation of proliferation,
apoptosis, and GJIC can lead to the neoplastic transformation of normal
cells. To accomplish this, oncogenes that influenced proliferation and
apoptosis were transfected independently and together into normal
epithelial cells and subsequently assayed for neoplastic potential and

GJIC in the following manner:

43

SPECIFIC AIMS

1. Construction of WB cell lines that express bcl—2 and bcl-2 plus myc.

A. Transfection of the bcl-2 plasmid along with appropriate vector
controls, into rat liver epithelial cells, WB-F344, and WB.myc
cell lines.

B. Isolation of multiple clones (This will eliminate the
interpretation complications associated with “insertional
mutagenesis”.).

2. Characterization of clonal cell lines: WB.bcl-2; WB.myc/bcl-2,
A. Determine bcl-2 expression by western blot analysis.

B. Test for phenotypes of neoplastic transformation, using soft
agar assay for the determination of anchorage independent
growth and an in vitro invasion assay to determine cellular
motility.

C. Test GJIC by the scrape loading dye transfer technique. and
flourescent recovery after photobleaching (FRAP), in those
clones expressing bcl-2.

D. In those clones demonstrating altered GJIC, determine the level
of CX43 protein.

3. Quantitate apoptosis frequency using fluorescence activated cell sorting
(FACS).

4. Compare the differentiation potentical of bcl-2 and v-myc transformed
clones with the parental WB-F344 cells.

44

MATERIALS AND METHODS

Cell Culture

WB-F344 rat liver epithelial cells, WB.myc and all subsequent bcl-
2 transfectants were cultured in a modified Eagle’s minimal essential
medium (MEM) (Chang et al., 1981), Formula 78-5470EF, Life
Technologies Inc. (Gibco/BRL, Gaithersburg, MD), supplemented with
7% fetal bovine serum (Life Technologies Inc. (Gibco/BRL, Gaithersburg,
MD) and appropriate antibiotic drug for selection. All cells were

incubated at 37°C in a humidified atmosphere containing 5% C02.

Transfection of bcl-2

The human Bcl-2 cDNA cloned into the pSFFV vector (kindly
provided by Drs. Gabrial Nunez and Valerie Castle, University of
Michigan, Ann Arbor, MI), was transfected into WB-F344 cell line and co-
transfected with pTK-Hyg (Clontech Laboratories Inc. , Palo Alto, CA) into
the WB.myc (see Figure 5 for vector maps) cell using lipofectin (Roche
Molecular Biochemicals, Indianapolis, IN). Specifically, 40 pg of
Lipofectin was added to 1.5 milliliters of D-Medium (serum free) and 20
pg of DNA was added to a separate 1.5 ml of D-medum. The two samples
were gently mixed and incubated at room temperature for 1 5 minutes.
This mixture was then added to sub-confluent cultures grown in 100 mm

dishes that had been rinsed twice with serum free D-medium. The plates

45

Figure 5. Schematic diagrams of the various vectors used in this
research. pSVv-myc was purchased from the American Type Culture
Collection. Briefly, pSVv-myc was created by ligating an EcoRI-Kpnl
Restriction fragment from the provial DNA into the the EcoRI and Kpnl
sites of pSV2gpt (Land et al., 1983). This step effectively deletes the gpt
selection and inserts the viral two long terminal repeats (LTRs). Then an
entire provial fragment of the avian MC29 was ligated into the EcoRI site.
The remaining plasmid contains the gag-myc viral genes flanked by two
LTRs, and a SV40 early promoter in the opposite polarity to the direction
of transcription of the viral DNA. The plasmid pTK-Hyg (Genbank
accession number U40398), was purchased from Clonetech Laboratories
(Palo Alto, CA) and contains a hygromycin resistance gene flanked by a
herpes simplex virus TK promoter (for transcriptional initiation) and a
HSV TK polyadenylylation signal. The pSFFVbcl-2 vector was kindly
provided by Dr. Valerie Castle and Dr. Gabrial Nunez at the University of
Michigan. Briefly, pSFFVbcl-2 was created by ligating a fragment from
pFP502 containing the Friend spleen focus-forming virus 5‘ LTR into
pBluescript (Stratagene, La Jolla, CA.). Then a fragment from pSV2cat
containing the SV40 early splice region and late polyadenylylation signals
was ligated into SFFV/Bluescript. For mammalian selection a fragment
for pSV2neo containing the neomycin resistance gene was ligated into
SFFV/SV40/Bluescript, creating pSFFVneo (Fuhlbridgge et al., 1988).
Bcl-2 was then cloned into the EcoRI site of pSFFVneo. All plasmids
used contained ampicillin resistance gene for selection in bacteria.

46

     

pSFFVbcl-2
8.1 kb

‘2'
e
5;
g b
6 9‘36
a3 ~
$6“ ”99?”
or”

47

were then incubated overnight at 37 °C in a humidified chamber. The
following day D-medium containing 7% fetal bovine serum (FBS) was
added directly to the plates and incubated an additional 24 hours.
Transformed cells were then split and subsequently plated with
appropriate drug selection. Individual clones were then isolated using
cloning rings, and subsequently recloned. Due to enhanced apoptotic
properties of the WB.myc cells, co-transfection with pTK-Hyg was
performed as described above with the following modifications: first, the
culture was allowed to reach confluency; second, 20 pg of DNA in a ratio
of 1:20 pTK-Hyg:pSFFV.bcl-2 was used to assure that selection using
hyromycin B would yield bcl-2 protein positive cells. Preliminary
screening of any clones was carried out using a modified dot blot assay.
Putative clones were cultured in 35 mm dishes and extracted as outlined
in the following section. Extracts were diluted with an equal volume of
sterile distilled water, dot blotted onto Immobilon-P PVDF membrane
(Millipore Corporation, Bedford, MA) and proceeded to detection as
described later in the Western Blot Analysis section. Human bc1-2

protein positive clones were identified and subsequently recloned.

Protein Extraction

Total protein was extracted from confluent cell cultures grown in

25 cm2 flasks using 20% sodium dodecyl sulfate (SDS) containing 2mM

48

phenylrnethylsulfonyl fluoride (PMSF), 1 pM aprotinin, 1 uM leupeptin
(Roche Molecular Biochemicals, Indianapolis, IN 46250), 1 uM antipain.
5 mM sodium fluoride (Fluka, Milwukee, WI), 0. 1 mM sodium
orthovanadate (Aldrich, Milwukee, WI). Protein extracts were
subsequently sonicated three times at five-second intervals, aliquoted
and stored at -20°C. The protein concentration was determined by
diluting the extracts 1:5 and assayed using the Bio-Rad DC protein assay

(Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis

Total proteins extracts were loaded in equal amounts (10- 15 mg)
into each well of a 10% SDS—polyacrylamide, in accordance with Laemmli
(1970). The gels were electrophoresed at 200 volts for approximately 45
minutes, removed and equilibrated in transfer buffer and transferred to
lmmobilon-P PVDF membrane (Millipore Corporation, Bedford, MA).
Human Bel-2 was detected with hamster anti—human bc1-2 monoclonal
antibody (Pharmingen, San Diego, CA), v-myc was detected with anti-v-
myc polyclonal antibody (Caltag Laboratories, Burlingame, CA), using the
supersignal ultra substrate (Pierce Chemical Co. Rockford, IL) and

appropriate peroxidase labeled secondary antibody.

49

Measurement of Saturation Density

Multiple 60-min dishes were seeded with 1x105 cells in D-medium
containing 7% FBS. Plates were tiypsinized and cell counts were made
3, 4, 5, and 6 days after initial plating. The saturation density
represents the maximum number of cells per cm2 for each cell type and
is presented as the average density of quadruplicate plates from three

independent experiments.

Measurement of Cellular Proliferation

Cellular proliferation (growth) was assayed by staining cell cultures
with 3-(4,5-dimethylthiazol-2—yl)-2,5-diphenyltetrazolium bromide (M11)
as previously described by Alley et al. (1988). A MTI‘ stock solution was
made to a final concentration of 5 mg/ ml in phosphate buffered saline
(PBS). Cell cultures are plated in 96 well microtiter plates at a density of
5x103 cells per well. Four hours prior to assaying, 50 pl of l :5 dilution of
the MIT stock is added to each well and incubated at 37°C. Each well
was then aspirated and 150 pl of dirnethyl sulfoxide (DMSO) was added
to each well to extract the MIT from the cells. Plates were then shaken
for 1 minute to thoroughly solubilize the MIT stain. Plates were then
measured for absorbance of the wells on a Titertek plate reader at 540
nm. The data reported represents quadruplicate wells from triplicate

experiments.

50

Apoptosis

Cultured cells are trypsinized and centrifuges for 5 minutes at
1000g. Cells are then washed twice in PBS by alliquoting 500 pl of PBS
and pipetting repeatedly to mix, then centrifugation for 5 minutes at
1000g. The final wash was decanted and the cells were fixed in 70%
ethanol overnight at 4°C. Samples were then transferred to -20°C for
long term storage. Samples were stained in propidium iodide and run on

a FACS according to Fraker and co-workers (1995).

Anchorage Independent Growth

To assess anchorage independent growth, cells were grown in soft
agarose as previously described (Kao et al., 1995). Briefly, an initial hard
agarose layer (0.5% agarose 6013, Sigma Chemical Co.), made in D-
medium containing 7% FBS, was plated in a 60 mm dish. Individual cell
lines were then trypsinized, counted, and diluted to a final concentration
of 1x104 total cells in D-Medium containing 0.33% agarose and 7% FBS
and was subsequently plated on top of the 0.5% agarose layer.
Additional medium was added 3 days later and changed every 3 days for
28 days. Colonies were stained using 1 mg/ml 2-(p—iodophenyll-3-

(nitrophenyl)-5-phenyltetrazolium chloride (Sigma) in 0.9% NaCl, and

51

counted and is reported as the percentage of a positive control from

triplicate plates assayed in quadruplicate experiments.

In Vitro Invasion Assay

The ability cells to invade through Matrigel (Collabrotive
Biochemicals), a reconstituted basement membrane, was examined using
a Boyden chamber in vitro invasion assay as previously described (Bello
et a1. 1997). The assay was conducted as follows: on the day prior to
running the invasion assay, the cell lines were plated in T-75 flasks in 10
ml of D-medium with 7% FBS. Matrigel was diluted 1:20 with cold (4°C),
sterile distilled water on ice for a final concentration of 500 pg/ml. Each
Nuclepore filter was coated with 25 pg Matrigel in 50 pl and left to dry
overnight at room temperature under sterile conditions. Cells were
rinsed with PBS 24 hours after plating and incubated with 3 ml of 1 mM
EDTA for 8- 10 min, dislodged by tapping and recovered by
centrifugation. Pellets were re-suspended to obtain 2x106 cells per
millfliter. The bottom chamber contained 220 pl of NIH / 3T3 cell
conditioned medium, which served as a chemo-attractant. For preparing
conditioned medium, subconfluent N IH/ 3T3 cultures in 100 mm plates
were fed with 7 ml of serum-free DMEM containing 50 ug/ ml ascorbic
acid. Conditioned medium was collected 24 h later, centrifuged to

remove cell debris and stored at -20°C. A cell suspension containing

52

4x105 cells in 200 pl medium was added to the top chamber and allowed
to remain undisturbed for 5 min before overlaying with 650 pl of
medium. Cells were allowed to invade for 24 h at 37°C at which time the
filters were prepared for absorbance reading. The migrated cells were
fixed, stained with HEMA-3 and allowed to hydrate in distilled water.
Nuclear stain was extracted by placing individual filters into individual
wells of a 24-well plate containing 300 pl of 0. 1 N HCl for 15 min. 200 pl
from each well was then placed in a 96 well plate and the absorbance at
620 nm was measured using a Titertek microplate reader. In addition to
evaluating the number of migrated cells on the filter, cells that had
migrated to the bottom chamber were also counted. Medium in the
bottom chambers was mixed by pipetting five times and 1 00 pl were
taken from each of the three chambers, pooled into cuvettes containing
10 ml Isoton, mixed by inversion and counted on a Coulter ZM cell
counter. Three replicate invasion chambers were prepared per

treatment. All experiments were done in triplicate.

Cell-Cell Communication Assays

Two methods developed in our laboratory were used to assess gap
junction-mediated intercellular communication: (a) scrape-loading dye
transfer (SL/DT) (El-Fouly et al., 1987); and (b) fluorescence recovery

after photobleaching (FRAP) (Wade et al., 1986). The scrape-loading dye

53

transfer assay utilizes confluent cultures grown in 35-mm dishes. The
cultures are then rinsed three times with PBS containing Ca2+ and Mg2+
(Ca2+,Mg2+ -PBS). 1.5 milliliters of PBS containing 0.05% Lucifer yellow
CH (Molecular Probes Inc., Eugene, OR) were added, and several scrapes
(cuts) were made on the monolayer using a surgical scalpel. The
cultures were incubated for 3 minutes at room temperature in the dye
solution, and then rinsed three times with Ca2+, Mg2+-PBS (to remove
any background fluorescence). The cultures were then fixed with 1
milliliter of 4% formalin and visualized using an Ultima laser cytometer
(Meridian Instruments, Lansing, MI).

The scrape-loading dye transfer assay results were confirmed using
the FRAP assay. Briefly, cultures grown in 35-mm dishes were rinsed 3
times with Ca2+, Mg2+-PBS and then incubated with C324», Mg2+-PBS
containing 5,6-carboxyfluorescein diacetate (7mg/ml, Molecular Probes
Inc., Eugene, OR) at 37°C in a humidified incubator for 15 minutes. The
cells were then rinsed several times with Ca2+, Mg2+-PBS and analyzed
using the Ultima laser cytometer. Cells were randomly selected under a
microscope (four cells were selected per field plus one unbleached
control, five fields per scan), and photobleached with an argon laser
beam. The transfer of fluorescence was then monitored in 4-minute
intervals for a total of 12 minutes. The intensity of recovered

fluorescence of the individually bleached cells is then quantitated and

54

rates of dye transfer can be measured as the percentage of an
unbleached control cell (one per field).

Quadruplicate dishes were run per treatment group and the mean
percent cells dye couples was calculated. Statistical analysis using the
Kruskal-Wallis test followed by Dunnett’s post hoc test was used to
compare the control to treatment groups and all subsequent pairwise

comparisons.

Glutathione Determination

Cells were lysed with two milliliters of 0.05 normal perchloric asid
and the lysate was filtered through a 0.22 micron filter. The lysate was
fractionated using an 150 mm X 4.6 mm adsorbophere C18 5 p. MF-Plus
HPLC column (Alltech Associates, Deerfield, Ill.) and a mobile phase that
consisted of 50 mM sodium phosphate, 0.05 mM l-octanesulfonic acid,
and 2% acetonitrile at a pH of 2.70 and a flow rate of 1.0 ml/ minute.
The HPLC system used was a 580 solvent delivery module from ESA
(Chehnsford, MA). The glutathione was detected with a Coulochem
Model 5200 electrochemical detector (ESA, Chelmsford, MA) with applied
potentials of +400, +900, and +950 mV set for the screening, analytical,

and guard cell electrodes, respectively.

55

Tyrosine Aminotransferase Assay

Cells were cultured for 6 days in D-MEM medium containing 7%
FBS and 3.75 mM sodium butyrate. All cultures were supplemented
with lle7 M dexamethasone for the last 24 hours as previously
described (Coleman et al., 1994). Cells were harvested by trypsinization
and counted prior to lysis in a buffer containing 140 mM KCL, 125 mM
KPO4 (pH 7.6), 1 mM EDTA, and 1% NP-40. Insoluble material was
removed by centrifugation, and protein concentrations were determined
by the Biorad DC protein assay (Bio-Rad Laboratories, Hercules, CA).
Tyrosine arninotransferase activities were determined using the method
of Granner and Tomkins (1970), as modified from Diarnondstone (1966).
Enzymatic activities were calculated using the molar extinction
coefficient for p-hydroxyphenylpyruvate formed per minute per 1x105

cells.

56

RESULTS

1. Synergistic effects of Bcl-2 and V-myc in the expression of
neoplastic phenotype of Rat Liver Epithelial cells

Isolation and selection of bcl-2 and myc/bcl-Z transfectants

The initial strategr to investigate the interaction between v—myc
and bcl-2 in neoplastic transformation and GJIC was to generate clonal
cell lines over-expressing human-bc1-2 alone and co-expressing human
bcl-2 and v-myc by transfecting previously established normal WB-F344
and WB.v-myc, cell lines respectively. This methodologl required the
cloning of the human bcl-2 gene into a different vector, namely pSV2rrnrr.
This approach was necessary because the established WB.v-myc cell line
already possessed geneticin (G418) resistance. This same mammalian
selection marker was also contained within the pSFFV.bcl-2 vector
(provided by Dr Valerie Caslte and Gabriel Nunez). Hence, if this vector
had been transfected into WB.v-myc, it would not be possible to
distinguish between the untransfected and transfected colonies.
Accordingly, the bcl-2 insert was excised from the pSFFV vector using
the restriction enzyme EcoRI and ligated into the pSV2dnrr vector. Upon
examination of the amplified plasmid, the insert had incorporated in the
anti-sense position. Subsequent recloning was attempted without
success. Therefore, a different approach was used to study the

interaction between v-myc and bcl-2 oncogenes by co-transfecting WB.v-

57

myc with pSFFV.bc1-2 simultaneously with an additional plasmid that
would contain a suitable selection marker. Since this method required
no additional cloning transfections were performed on the normal WB-
F344 cells in conjuction with the transfections being performed on WB.v-
myc. WB-F344 cells were transfected with either pSFFV vector control or
pSFFV.bcl-2 vector by lipofectin treatment and subsequently followed by
selection in (G418) for 14 days. Single colonies were isolated, and tested
for bcl-2 expression using Western dot blot screening and an anti-bcl-2
antibody. All bcl-2 positive clones were subsequently re-cloned and
stored in liquid nitrogen for preservation.

Several initial attempts to co-transfect WB.v-myc were made using
the pSFFV.bcl-2 vector and the pKT203 vector. which contained a
hygromycin resistance gene as a mammalian selection marker. These
attempts failed to yield any hygromycin resistant cells. Due to the
success in obtaining WB.bcl-2 clonal cell lines, any errors that may have
been associated with pSFFV.bcl-2 were ignored and a different vector
with hygromycin resistance was sought. PTK-Hyg, purchased from
Clontech Laboratories (Palo Alto, CA), was used for its specific
optimization in co-transfection studies. Again, several attempts failed to
transfect WB.v-myc cells. In order to determine the possible cause(s) for
these failures, close scrutiny to the culture conditions was observed
during the co-transfections with pT‘K-Hyg. WB.v-myc cells were

monitored for the initial 24 hours after serum-free lipofectin treatment

58

and microscopic evaluation was performed every hour noting any
alterations in the culture appearance. Under these conditions WB.v-myc
cells appeared to be undergoing a great deal of cell death attributed to
apoptosis, based on cellular morphological appearance. Additionally, the
cells were not able to recover when medium containing serum was added
to the culture.

Thus, a different strategr was employed to co-transfect the cells.
namely the cells were allowed to reach saturation density to increase the
actual number of cells and the transfection time was reduced to twelve
hours to limit the induction of apoptosis caused by the serum-free
conditions. This transfection protocol proved to be successful. A finally
transfection was performed where WB.v-myc cells were co-transfected
with pSFFV.bcl-2 and pTK-Hyg at a ratio of 20:1 to assure the selection
of bcl-2 positive cells. Transfection of WB.v-myc was followed by
selection in G418 (400 pg/ml) plus hygromycin B (50 pg/ ml) for 14 days.
All bcl-2 protein positive clones identified, using Western dot blot
screening, were subsequently re-cloned, expanded and frozen at low
passage number in liquid nitrogen. Vector control, four WB.bc1-2 and six
WB.myc/bcl-2 clones were selected for further studies. Cultures
between passages fifteen and twenty-five were consistantly used in

experiementation and not used past one month in culture.

59

Characterization of Transfectants

The morphological appearances of the selected clones were viewed
using a Nikon Diaphot phase contrast microscope (Figure 6). The WB-
F344, WB.v-myc, and WB.bc1-2 cells demonstrated growth in uniform
monolayers of polygonal cells. The Wb.myc /bc1-2 clonal cell lines
demonstrated different morphologies: uniform polygonal (WB.MB3),
spindle-shaped (WB.MB19), and multinucleated (WB.MB39).

Cultures were then allowed to reach maximal cell density, assaying
for the cells ability to be contact inhibited. Neoplastic cells lose their
ability to be contact inhibited and are subsequently able to grow in an
unrestrained manner. All cell lines chosen for this study demonstrated
contact inhibition but varied greatly in their maximal saturation
densities (Table 1). WB-F344, WB.neo (vector control), and WB.bcl-2 cell
lines all demonstrated similar contact inhibition saturation densities of
approximately one hundred thousand cells per square centimeter. WB.v-
myc attained a saturation density approximately 1 .4 fold greater than
that seen in the normal, vector control and bcl-2 expressing cell lines.
The myc/bcl-2 expressing clones demonstrated saturation densities in a
range from approximately 3 to 5 fold greater than WB-F344 and 2 to 3.5

fold greater than WB.v-myc.

6O

Figure 6. Morphological features of the WB-F344 cell line, before and
after transfection with bcl-2 and myc/bcl2. a. WB-F344 b.WB.v-myc c.
WB.bcl-202 (bcl-2) d. WB.mb03 (myc/bcl-2) e. WB.mbl9 (myc/bcl-2) f.
WB.mb39 (myc/bcl-2). All panels are of equal magnification: X 200

61

 

62

Table 1- We nsiy

      

   

Cell line Cell Density (x 105/cm2)

 

 

 

 

WB-F344 0.955 :1: 0.053
WB.v-myc 1.379 1 0.053
WB.bcl-20 1 1.008 i 0.159
Bel-2 WB.bcl-202 0.950 :i: 0.055
alone WB.bcl-203 1.061 1: 0.2 12
WB.bcl-204 1.008 t 0.103
WB.MB3 2.759 t 0.159
v-Myc WB.MBG 3.130 LI: 0.135
and WB.MB 19 4.722 :1: 0.106
Bel-2 WB.MBZ8 2.492 t 0.043
WB.MB39 4.456 :I: 0.096
WB.MB50 4.234 1: 0.280

 

63

Proliferation studies were conducted next using 3-(4,5-
dimethylthiazol-Z-yl)-2,5-diphenyltetrazolium bromide (MIT) (Mosmann.
1983; Wiedermann et al., 1990). This is a non-radioactive colorimetric
assay based on the reduction of the MTI‘ tetrazolium salt to a colored
formazan. This reduction can only occur in metabolically active viable
cells. Thus, by plotting a time course of cell growth from an initial
seeding it is possible to calculate the proliferation rate (doubling time) for
the absorbance directly correlates with cell number. Figure 7 is a
representation of the proliferation analysis using M'I'I‘. It shows the
growth curves for WB-F344, WB.bcl-201, WB.v-myc, WB.MB3, and
WB.MB19. From these curves, doubling times were calculated, using
two separate areas (four time points) during log phase growth to
determine the growth rate and are shown in Table 2. WB.v-myc
demonstrated the fastest growth rate of fourteen-hour doubling time.
while the next was the normal WB-F344 cells had a doubling time of
twenty-one hours. Interestingly the WB.bcl-2 and WB.myc/bcl-2 clones
showed the slowest doubling rates of approximately 24 hours and 33

hours, respectively.

64

Figure 7. Incorporation of 3—(4,5-dimethylthiazol-2-yl)-2.5-
diphenyltetrazolium bromide (M'IT) to assess proliferation. Graph
represents aborbance at 540 nm versus hours in culture. 1x104 cells
were initially plated in each well of a 96 well plate.

65

Absorbance 540 nm (1 10 '3)

 

2500

aoooi

1800 ~

1000 -

500 -

 

-9 ..WB-F344
—I - WB.bcl-201
_.—WB.v-myc

I Ob C WB.mb03

—0— WB.mb 19

 

H ours

Figure 7

66

 

 

100

Table 2. Doubling times of transfected cell lines. Rates of
doubling times as calculated from proliferation curves of MIT
inco oration.

 

 

 

 

Cell line Hours
WB-F344 2 1:0. 1
WB.v-myc l4:l:0.2
WB.bcl-201 241:0.2
Bcl-2 WB.bc1-202 23:0. 1
alone WB.bc1-203 25:0.3
WB.bcl-204 24i0.5
WB.MB3 341:0.6
v—Myc WB.MB6 3310.2
and WB.MB 19 32:03
Bel-2 WB.MB28 3010.1
WB.MB39 3 11:04
WB.MBSO 3 1:0.3

 

67

Western Blot Analysis of Bcl-2

The expression of human bcl-2 was assessed using Western blot
analysis with an anti-bcl-Z monoclonal antibody (Pharmingen, San
Diego, CA) specific for human bc1-2 protein. Figure 8a shows high
expression of a 26 kilodalton (kDa) band, corresponding to transfected
human bcl-2 protein. in the WB.bcl-2 cell lines. while the control cell
lines showed no expression. Figure 8b demonstrates the expression of
the human bc1-2 protein in the WB.v-myc/bcl-2 cell lines but not in
WB.v-myc and the tumorigenic WB.MR42 cell line which over-expresses
v-myc/Ha-ras (previously described by Hayashi et al., 1998). All
WB.myc/bcl-2 cell lines demonstrated similar levels of bcl-2 expression
except the WB.MB28 cell line, which showed very low levels of bcl-2
expression.

The presence of v-myc in the WB.myc/bcl-2 cell lines was assessed
with Western blot analysis with an anti-v-myc polyclonal antibody to
verify that transfection of bcl-2 had no discemable affect on v-myc
expression. Figure 9 shows the presence of a l 10 kDa band that
corresponds to the gap-pol-myc region of the v-myc oncogene. Its
expression is consistent between the controls and transfected clones,
suggesting that over-expression of bcl-2 has not effected the expression

of v-myc from the parental WB.v-myc cell line.

68

Figure 8. Western blot analysis of human bcl-2 protien expression.
WB.bcl-2 cell lines; b) WB.myc/bcl-Z cell lines. 26 kDa band represents
human bcl-2 protein.

69

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Figure 8

70

Figure 9. Western blot analysis of v-myc protein expression in the
WB.myc/bcl-2 cell lines. 110 kDa band corresponds to gag-myc protein
expressed by full length v-myc transcript.

71

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72

Anchorage Independent Growth

Anchorage independent growth, commonly referred to as growth in
soft agarose. has often been used as a marker for neoplastic
transformation and to identify tumor cells (Stoker. 1968; Stoker et al..
1968; Hamburger and Salmon, 1977; Marshall et al.. 1977). Anchorage
independent growth appears to be a necessary, but not sufficient
condition for tumorgenicity of cells. To assess if the bcl-2 gene was
conferring neoplastic characteristics to the normal rat liver epithelial
cells. we tested their ability to grow in soft agarose. Using a previously
described v-myc/Ha-ras transfected tumorigenic WB-F344 cell line
(designated MR-42) as a positive control (Hayashi et al., 1998). 1x103
cells were plated on 60 mm dishes in triplicate. Following 28 days in
culture plates were stained with 2-(p-iodophenyl)~3-(nitrophenyl)-5-
phenyltetrazolium chloride, and colonies were counted. The MR—42 cell
line was used as a plating (technique) positive control, which was
previously demonstrated to form colonies in soft agarose with one
hundred percent efficiency. Therefore all results were normalized to this
positive control and colony forming efficiencies where determined as a
percentage of the positive control. Figure 10 demonstrates that the
control cell lines. as well as the bc1-2 only clonal cell lines, were unable

to form colonies in soft agar.

73

Figure 10. Anchorage independent growth. Data represent colony
formation in soft agarose as a percentage of a positive control WB.MR42.
a known tumorigenic cell line, derived from the WB-F344 cells.

74

11096
10096
9096
8096
7096
0096
0096
4096
3096
2096
1096

 

096

Figure 10

75

 

 

However, the v-myc / bc1-2 clones were able to form colonies in soft agar
and they also demonstrated clonal differences in their colony forming
efliciency ranging from 3 to 90 percent. When the v-myc/bcl-2 clonal
cell lines colony forming efficiencies were compared to their respective

bcl-2 expression level no correlation was found.

In Vitro Invasion Assay

The in vitro invasion assay (Albini et al., 1987) is based on the
early observations of tumors both in vivo and in vitro which suggested
that invasion through basement membranes represented a critical step
toward progression (Liotta. 1984; Terranova et al., 1986; Mignatti et al.,
1986). Basement membranes are thin extracellular structures
surrounding epithelial tissue, as well as the lining of most blood and
lymph vessels (Martin and Timple. 1987; Timple and Dziadek. 1986).

Basement membranes are comprised of larninin. collagen type IV,

fibronectin, and heparin sulfate proteoglycan (Laurie et al.. 1982). The in

vitro invasion assay uses a basement membrane reconstituted onto a

filter in a Boyden chamber in which a chemoattractant, 3T3 conditioned

medium, is placed below the filter and a cell suspension is placed above
the filter. TWO measurements are determined in this assay; 1) the

number of cells that have invaded to the chemoattractant side of the

filter; and 2) the number of cells that have invaded and detached. These

quantifies are averaged together and determine the invasive ability.

76

.44-

 

Figure 1 la demonstrates that WB.MBIQ had the highest amount
of invasion to the other side of the membrane. while WB.MR42 had the
highest amount of cells that were able to detach away from the filter
(Figure 1 lb). However, when the data are taken together, the trend that
was observed with anchorage independent growth assay was repeated in
the invasion assay (Figure 11c). WB.MR42, the tumorigenic cell line had
the highest average rate of invasion, followed by the WB.MB 19 cell line.
WB.MBOS, WB.v-myc, WB.bcl-201, and WB-F344 all showed nominal

invasion.

Gap Junctional Intercellular Communication (GJIC)

In order to begin to explain the differences in growth in soft
agarose and invasion demonstrated by the myc/bcl—2 cell lines. the
individual cell line’s ability to communicate through gap junctions was
assessed by the scrape loading dye transfer technique (Figure 12). The
WB-F344 cell line is used as the control for communication with the dye
traveling about 68 cell layers. When WB-F344 cells are compared to the
WB.mb 19 clone containing v-myc and bcl—2. there is an approximate
50% decrease in the cells ability to transfer dye (3-4 cell layers). This
difference in dye transfer can be measured as an average fluorescent
area for a fixed magnification, where the control WB-F344 fluorescence

almost fills the total area and WB.MB 19 only fills half of the area.

77

Figure l 1. Graphical representation of invasion assay as measured by
the absorbance at 620 nm. Top graph represents those cells that
invaded and stayed attached to the chemoattractant side of the filter.
The middle graph is a representation of those cells that detached and
were free floating in the chemoattractant medium. Bottom graph
represents the average of the top and middle graphs.

78

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WB.v-myo
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via-r344

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0 0. l 0.2 0.3 0.9 0.8 0.0 0.7 0.0 0.9

Aboorbanco (640 nm)

Figure 11

79

 

Figure 12. A representation of the SL/DT assay. Note the normal WB-
F344 cell line transfers the dye approximately 6-8 cells, while the
myc/bcl-2 clone MB. 19 is only able to transfer the dye 2-3 cells on either
side of the scrape line.

80

 

Figure 12

81

To reduce subjective measurements, average fluorescent area for
any given SL/DT line at a fixed magnification was assessed using the
Ultima laser cytometer (Figure 13). The data show that WB.F344. vector
control. WB.v-myc, and bcl-2 transfect cell lines all demonstrated normal
cell-cell communication. The WB.myc/bcl-2 cell lines demonstrated
either normal or various reductions in cell-cell communication. These
results were confirmed using the fluorescent recovery after
photobleaching assay (FRAP)('I‘able3). This assay allows for the
monitoring of individual cells in the population assessing an activity for
the whole population. Figure 14 shows the OJ 1C activity (both SLDT and
FRAP) of the various cell lines. The data represented in Figure 14
demonstrate similar findings between both the SLDT and FRAP assays.
however none of the WB.bcl-2 cell lines showed any reduction in cell-cell
communication. Only WB.MR42, WB.MB 19. WB.MB39, and WB.MBSO
showed reductions in cell-cell communication.

When AIG and GJIC, as measured by the two communication
assays, are compared. they appear to be inversely correlated. Those
WB.myc/bcl-2 cell lines, that showed no significant changes in GJIC.
demonstrated only minimal ability to form colonies in soft agarose.
However, those cell lines, that demonstrated marked reduction in cell-
cell communication (WB.MB 19. WB.MB39, and WB.MBSO) demonstrated

an enhanced or increased ability to form colonies in soft agar (Figure 15).

Figure 13. Scrape load dye transfer. Data represent average fluorescent
area normalized to 100% of the control cell line WB-F344.

83

 

12096

 

10on - _,,~ ; 7 7 *7 7777777

 

Figure 13

84

 

Table 3. Gap junction intercellular communication. Cell-cell
communication of transfected cell lines as measured by scrape
load dye transfer and fluorescent recover after photobleaching

assay S

 

 

 

 

 

Cell line SL/DT % FRAP
WB-F344 0.934: 0.035 100: 3 (404)
WB.v-myc 0.925: 0.046 102: 5 (208)
WB.bcl-ZOl 0.947: 0.068 98: 8 (192)
Bel-2 WB.bcl-202 0.964: 0.01 l 98: 7 ( 160)
alone WB.bcl-203 0.886: 0.163 96: 12 (172)
WB.bcl-204 0.992: 0.022 107: 5 ( 184)
WB.myc/ras (MR42) 0.093: 0.018 2: 8 (92)
WB.mb3 0.845: 0.082 93: 6 (320)
v-Myc WB.mb6 0.803: 0.093 96: 3 (290)
and WB.mb 19 0.524: 0.037 54: 5 (308)
Bel-2 WB.mb28 0.897: 0.136 93: 2 (286)
WB.mb39 0.598: 0.038 70: 6 (380)
WB.mbSO 0.782: 0.027 76: 3 (328)

 

( ) Denotes the number of cells assayed

85

Figure 14. Fluorescent recovery after photobleaching and scrape load
dye transfer. Graph compares the two assays for gap junction-mediated
intercellular communication.

86

12096

 

.SLDT
. DFRAP
10096 ,,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 14

87

Figure 15. Anchorage independent growth correlates to cell-cell
communication. This graph represents the comparison of average cell-
cell communication with AIG.

88

 

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89

Connexin 43 Expression

Due to the reduction of cell-cell communication the levels of
connexin 43. the predominant connexin expressed in WB-F344 cells. was
assessed using Western blot analysis. Figure 16 demonstrated that
those cell lines that had normal cell-cell communication had normal
connexin 43 expression, which is characterized by multiple bands at 43
kDa. These bands represent unphosphorylated and phosphorylated
protein. WB.mb 19 and WB.mb39, which had a 60% reduction reduced
cell-cell communication, demonstrated very low expression of connexin

43 protein consistent with a reduction GJIC.

Apoptosis

To determine if bcl-2 was functioning as a pro-survival factor in
the bcl-2 containing cell lines apoptosis was assayed using fluorescent
activated cell sorting (FACS). Apoptosis was examined using the Becton
Dickinson FACS Vantage. Cultures, which were grown in twenty five
square centimeter flasks, were rinsed twice with phosphate buffered
saline and serum-free medium was added to flasks. Forty-eight hours
later cells were trypsinized. fixed in seventy-percent ethanol, and stored
at minus 20°C. Twenty-four hours prior to analysis on FACS, cells were
stained with propidium iodide. Data were analyzed using two software
protocols (Figure 17). The first software was the PC-LYSYS TM version 1.0

and the second was WinMIDI version 2.8.

90

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92

Figure 17. Representation of data analyzed from FACS. Top two graphs
represent data analyzed using PC-LYSYS software. Bottom four graphs

represent data analyzed using WinMIDI software. Regions denoted by R
represent G2 / G1 /Apoptotic peaks reading from right to left.

93

Number

0011

WB-F344 0 Hour Control WB.bcl-203 0 Hour Control

 

 

Cell Number

 

 

 

 

 

DNA Con ten ‘ DNA Content

WB.v-myc 0 Hour Control

 

 

 

 

 

 

E
B
WB.v-myc 24 Hour Serum Deprivation
l—l
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94

Both software packages allow for the identical analysis of the data. with
the exception that WinMIDI was less complicated. The data
demonstrate, that when expressed alone bc1-2 was able to function and
block apoptosis associated with serum deprivation and caused G1 cell
cycle arrest (Table 4). However, when co-expressed with myc. cell cycle
arrest is not seen, even though there is an apparent block in apoptosis.
In fact. it appears that there is a slight increase in cell cycle progression
demonstrated by an increase the WB.myc/bc1-2 cell lines G2 population.
This suggests that cell cycle progression induced by myc can be

enhanced by bcl-2 possibly through the blockage of apoptosis.

Glutathione Expression

Glutathione (GSH) is a tripeptide, whose primary function is to
form conjugates with reactive electrophiles rendering them inactive and
represents the major soluble intracellular antioxidant. Glutathione
peroxidase uses GSH to convert organic peroxides, like hydrogen
peroxide, into water and alcohols. The role of GSH in cell death was first
investigated by studying the action of menadione, a powerful oxidant, in
the induction of necrosis (Thor et al.. 1982). This work demonstrated
that cell death required the depletion of intracellular GSH. Since this
initial work. many researchers have shown that GSH depletion also
occurs during apoptosis (Fernandez et al., 1995; Slater et al., 1995).

GSH has been proposed to function as a scavenger of free oxidants in the

95

Table 4. Apoptosis frequency.

 

 

 

 

Cell line Apoptosis Gl /G2
(Percent) (Percent)
Serum + - + -

WB-F344 1.3 3.9 517/206 529/150
WB.bcl-201 0.4 0.5 863/86 806/96
Bel-2 WB.bcl-202 0.3 0.5 827/95 798/102
alone WB.bcl-203 0.5 0.5 83.5/7.9 81.2/8.4
WB.bcl-204 0.3 0.5 84.2/8.1 80.1/9.1
WB.v-myc 1.6 8.2 489/247 498/146
WB.MBB 0.5 0.5 309/31.4 391/234
V_Myc WB.MB6 0.4 0.6 286/293 403/281
and WB.MBlQ 0.3 0.5 317/304 408/265
Bel-2 WB.MB28 0.6 0.7 321/316 415/227
WB.MB39 0.5 0.5 299/307 389/249
WB.MB50 0.4 0.6 31.4/31.8 41.2/254

 

96

cytoplasm. Macho and colleagues (1997) extended these findings by
demonstrating that GSH depletion was an early event in apoptosis,
taking place before much of the protein signaling. Additionally. Bel-2
and its relatives have been shown to elevate GSH levels and protect cells
against oxidant-induced apoptosis (Kane et al., 1993; Bojes et al., 1997:
Mirkovic et al., 1997; Voehringer et al.. 1998).

To determine if elevation of GSH was responsible for the reduction
in apoptosis seen in the bcl-2 containing cell lines. GSH concentration
was assessed using a HPLC technique and an electrochemical detector.
Standards of GSH were injected into the injection port of an ESA solvent
delivery module (ESA, Inc., Chelmsford, MA). GSH was detected using
and BSA Choulochem II dual electrode electrochemical detector.
Retention times were calculated from the time of injection to peak
appearance on a chart recorder. Concentration was calculated by
determine the area under the curve and represented as percent of the
normal control WB-F344. The results demonstrate the GSH was
significantly reduced in the WB.bcl-2 cell lines (Figure 18). This result
was contradictory to all previous reports that showed an increase in GSH
concentration in cells over-expressing bcl-2. Due to the inexplicable
nature of these results, further studies were abandoned in favor of

examining the bc1-2 containing cell lines differentiation potentials.

97

Figure 18. Glutathione Expression. Graph represents glutathione
concentration normalized to protein content and expressed as a
percentation of the control cell line WB-F344.

98

 

 

 

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Figure 18

99

II. Differentiation Potential of Bel-2 and V-myc transformed Rat
Liver epithelial cells

The adult liver, in mammalian species. contains many different cell
types. The main cell types are hepatocytes. bile duct epithelium, stellate
cells, Kupffer cells, vascular endothelium. fibroblasts and leukocytes
(Desmet, 1994). The predominant cell type is the hepatocyte which
comprises approximately ninety percent of the weight of the liver.
Hepatocytes are large cuboidal epithelial cells that function to exchange
metabolites with the blood and secrete bile into the bile duct. The
hepatocytes are responsible for the metabolism of amino acids, lipids.
carbohydrates, and the detordfication of xenobiotics as well as the
synthesis of serum proteins. A significant. large fraction of the mature
hepatocytes are binucleated, with some nuclei being tetraploid (Digemes
and Blound, 1979; Medvedev, 1988). While heptocytes are capable of
proliferation. they are self-limited in culture. Therefore. several in vitro
models for hepatic progenitor cell growth and differentiation have been
developed from the mouse (HBO-3: Rogler, 1997; Ott et al., 1999). the pig
(PICM-19: Talbot et al., 1993). the human (AKN -1: Nussler et al., 1999),
and the rat (WB-F344: Grisham, 1980). The most important of these
systems is the WB-F344 cell line which was clonogenically derived from
non-parenchymal cells. These cells can be cultured indefinitely and can
be induced to form hepatocytes in culture. More importantly, this cell

line is the only system that has been demonstrated to differentiate into

100

normal hepatocytes when transplanted back into rats (Colman et al..
1997; Colman et al., 1993; Grisham et al., 1993).

Initial morphological observations of the WB-F344, cells
transfected with myc/bcl-2. used in this study demonstrated very
difl'erent morphologies. The WB.mb39 cell line possessed cells that were
very large, multinucleated and cuboidal, highly reminiscent of the
description of hepatocytes. This cell line prompted the investigation into
determining if bcl-2 affected the differentiation potential of WB-F344
cells. Differentiation was induced using 3.75 mM sodium butyrate (SB).
Previous work demonstrating SB ability to induce differentiation in WB-
F344. as well as other hepatic progenitor cells made it the ideal
candidate for this study. The mechanism by which SB functions to
induce differentiation is unclear. However, SB has been demonstrated to
block histone deacetylases (HDAC) activity. HDAC activity results in
histone hypoacetylation. chromatin condensation and transciptional
repression (reviewed in Ng and Bird, 2000). Therefore by blocking HDAC
activity, SB stimulates transcriptional activation and cells are released to
differentiate. Using the cell lines described in the previous section.
alterations in differentiation potential were investigated. The criteria
used in the determination of differentiation were changes in morphology.
activation of the hepatocyte specific tyrosine amino transferase enzyme.
and expression of the heptocyte specific gap junction protein connexin

32.

101

Morphology

Cells were treated with 3.75 mM SB for six days. On the fifth day
water soluble dexamethasone was added to a final concentration of 1x10'
7 M. Figure 19 represents the morpholog of untreated and treated WB-
F344. WB.bcl-202, and WB.bcl-203 that was seen during the treatment.
These cell lines all demonstrated similar induced morphological
alterations of increased size, and multinucleation. WB.myc/bcl-2 and
the tumorigenic WB.myc/ras cell lines all show an inability to sustain
morphological alterations induced by sodium butyrate (Figure 20). These
cells lines demonstrated an early response to the treatment but cells

rapidly died during the prolonged exposure.

Tyrosine Aminotransferase (TAT) Assay

The morphological results suggested that the WB-F344 and
WB.mcl-2 cell lines were behaving in a manner consistent with
hepatocyte differentiation. To confirm that these cells were indeed
differentiating into hepatocytes, TAT activity, which is specific to viable
hepatocytes, was measured. SB was able to induce TAT activity in WB-
F344. WB.v-myc. and WB.bcl-2 cell lines (Table 5). However the Bcl-2
cell lines showed a slightly reduced response to treatment when

compared to the normal WB-F344 cell line.

102

Figure 19. Morphological features of the WB-F344. WB.bcl—202, and
WB.bcl-203 cell lines. Images are before and after treatment with 3.75
mM sodium butyrate for 6 days and a pulse of dexamethasone (1x10'7 M
final concentation) for the last 24 hours. (Magnification: X200)

103

Treated (SB)

Untreated

 

Figure 19

104

Figure 20. Morphological features of the myc/bcl-2 (WB.MB3 and
WB.MBIQ) and myc/ras transfected (WB.MR42) cell lines. Images are
before and after treatment with 3.75 mM sodium butyrate for 6 days and
a pulse of dexamethasone (1x10'7 M final concentation) for the last 24

hours. (Magnification: X200)

105

           

Untreated Treated (SB)
,1' W3:
m . "‘“L’K'I 9o
2 . . _ ,
E. .

WB.mb19

 

WB. MR42
"l ‘

 

106

Table 5. Tyrosine arninotransferase activity. TAT activity of
transfected cell lines before and after treatment for 6 days with
3.75 mM sodium butyrate and 11:107 M dexamethasone for the

final twenty-four hours.

 

 

 

 

 

Cell line Basal SB Induced“
Activity Activity
WB—F344 27911.43 203101155
WB.v-myc 1.69il.65 73.03i2.65
WB.bcl-201 1.23:1.19 139.3 1:1.99
Bel-2 WB.bc1-2o2 1.321127 l42.24il.63
alone WB.bcl-203 1.53:1.49 140.84i2.96
WB.bcl-204 0.93:0.79 132.8 1:1 .02
WB.myc/ras (MR42) Oi 0.00 O i 0
WB.MBS Oi 0.00 O i O
v-Myc WB.MBG 01: 0.00 0 :l: O
and WB.MBIQ Oi0.00 0 i0
Bel-2 WB.MB28 Oi 0.00 O i 0
WB.MB39 0i 0.00 0 i 0
WB.MBSO Oi 0.00 O i 0

 

107

The myc/bcl-2 and myc/ras cell lines demonstrated no induction of TAT
assay. in fact they demonstrated absolutely no basal TAT activity, which

was detected in the normal, v-myc and bcl-2 containing cell lines.

Connexin 32 Expression

In addition to testing for activation of a hepatocyte specific enzyme
activity, expression of connexin 32 protein was used as a hepatocyte
specific marker. As previously mentioned, connexin 32 is the
predominant connexin protein expressed by hepatocytes. WB-F344 cells
do not express connexin 32. Figure 2 1 shows that connexin 32 was
induced in the normal WB-344. WB.bcl-2 and WB.v-myc cell lines.
Induction of connexin 32 was not observed in the WB.myc/bcl-Z cell

lines WB.mb3 and WB.mb19.

108

Figure 21. Western blot analysis of connexin 32 expression. Connexin
32 was assayed following induction by 3.75 mM sodium butyrate for 6
days and a pulse of dexamethasone (lxlO'7 M final concentation) for the

last 24 hours.

109

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110

Discussion

In the initiation / promotion / progression model of multi-stage
carcinogenesis, initiation is an irreversible genetic event that sensitizes a
cell to subsequent promotion (Figure 1). Promotion is then characterized
as the events that lead to the clonal expansion of the initiated cells.
Progression is described as the accumulation of alterations within an
initiated cell that leads to promotion-independent growth. The stem cell
theory of carcinogenesis proposes that the target of initiation is the stem
cell, where initiation involves the blockage of the stem cells ability to
differentiate. Under normal conditions, these “initiated” cells would be
contact inhibited by being coupled to the surrounding normal cells
through gap junctions. Promotion would then cause the down-regulation
of GJIC (leading to the loss of contact inhibition) and the induction of
gene expression related to cell proliferation. Additionally, promotion
would also involve the escape from apoptosis, which would normally
eliminate initiated cells. Therefore. by down-regulating GJIC, stimulating
proliferation, and blocking apoptosis, an initiated cell could become
neoplastically transformed. The objective of this study was to determine
if the oncogene, bcl-2, could act cooperatively with the myc oncogene in

the neoplastic transformation of normal epithelial cells. Furthermore.

111

 

the importance of GJIC. proliferation. and apoptosis in promotion was
examined.

To test the hypothesis that the inhibition of apoptosis, in
conjunction with a proliferation signal and subsequent down regulation
of GJIC. lead to neoplastic transformation, the bc1-2 and myc oncogenes
were expressed alone and together in the WB-F344 cells, a normal rat
liver epithelial cell line. The molecular biologi associated with the
beginning of this project was extremely labor intensive. Eventually,
several WB.bcl—2 clones were established and subcloned. However, many
problems occurred when transfecting the WB.v—myc cells. It was
determined that WB.v-myc cells were especially sensitive to the
transfection conditions. Eventually WB.myc/bcl-2 cell lines were also
established.

Clones were initially screened by their human bcl-2 protein
expression. using a western dot blot protocol. Subsequent
characterization of subclones for bcl-2 protein expression showed a
single band at 26 kDa. indicating the presence of bcl-2. This result is
very interesting due to the fact that Bel-2 has been traditionally shown to
form complexes with itself and other members of the bcl-2 family.
therefore. under most conditions multiple bands can be visualized. In
this pro] ect. all electrophoretic conditions consistently produced similar
results, a single band at 26 kDa. It is possible that the extraction

methods and electrophoretic conditions precluded the observation of

112

 

protein interactions. In this system, if bcl-2 was acting as a monomer. it
would call into question the mechanism by which bcl-2 is thought to
function. Bel-2’s proposed functions are based on the proposition that it
is the formation of complexes with other proteins and itself that allows
for the sequestering of other proteins or perturbations in mitochondrial
channels. The results presented in this study suggest that in this cell
system. bcl-2 might be acting through alternative mechanisms than
those that others have reported. Similarly, no one has actually reported
findings. either in vivo or under physiological conditions. that bcl-2 is
functioning as a multimer. What these mechanism are cannot be
delineated without further investigation; however they will most likely be
related to the subcellular localization of bcl-2.

Contact inhibition functions to ensure the harmonious
development of the whole tissue, affecting the way tissues organize.
control their size. their morphology and synchronize their function
(Jones, 1980; Lieberman and Glaser, 1981). Tumor cells have been
Characterized by their lack of contact inhibition. In cell culture, the loss
of contact inhibition is indicated by the ability of cells to reach a higher
maximum or saturation density. Transfected cells were allowed to reach
their maximum density under the assumption that the more neoplastic
Characteristics a cell line possesses, the more cells would occupy the
same space at its maximum density as compared to normal cells. It is

1IIIPOI'tant to note that the cells were continuously provided with fresh

113

 

 

medium during the course of this experiment because it has been shown
that density-depend effects can be affected by decreases in available
growth factors (Alberts el al, 1994). The myc/bcl-2 transfected cells were
contact inhibited at a higher density than the normal WB-F344, WB.v-
myc and WB.bcl-2 cell lines. Independently, neither myc nor bcl-2 was
sufficient to eflect contact inhibition. However, when these two
oncogenes cooperate, they are able, significantly, to effect contact
inhibition, indicating that their co-expression is contributing to the
neoplastic transformation of WB-F344 cells. Thus, an initiated cell.
lacking contact inhibition, and under the stimuli being provided by bcl-2
and myc, could escape the constraints being signaled by the surrounding
cells of its tissue of origin.

Interestingly. when growth rates were examined, clones containing
bcl-2 showed a reduction in proliferation. Although at the time these
results were novel, they were confirmed shortly thereafter by several
publications which demonstrated growth arrest in cells over expressing
bcl-2 (Uhlmann et al, 1996: Vairo et al, 1996; O’Rielly et al, 1996).
Similarly, the results from this project and others demonstrated that bcl-
2 expression caused an accumulation of cells at G1 (Borner. 1996; Mazel
et al, 1996). Bcl-2 and myc/bcl-2 cell lines equally demonstrated a
reduction in proliferation. which suggest that. unlike the ras oncogene.
bcl-2 does not provide myc with a cooperating mitogenic signal.

Furthermore. these cell lines may prove to be very important for the

114

 

investigation into the non-apoptotic functions of bcl-2, such as the
interactive role with mitogens in proliferation and cell cycle.

While bcl-2 was able to act as a pro-survival factor in both bc1-2
and myc/bcl-2 cells. the difference in apoptosis rates after serum
withdrawal between the transfectants and the normal WB-F344 cells was
small, exhibiting only a four-fold increase. WB.myc cells showed a
higher sensitivity to serum withdrawal induced apoptosis with an eight-
fold increase. These changes are admittedly small. These results are
consistent with the hypothesis that the WB-F344 cells are stem cells.
and their survival would be essential for they would be needed to
repopulate the tissue after significant trauma. Therefore, they might be
less susceptible to apoptosis. In addition, apoptosis of tissues under
food restriction is a slow process lasting months, where the serum
withdrawal in these experiments was only forty-eight hours.

The cell lines were also assayed for their ability to grow in an
anchorage independent manner, as assayed in tissue cell culture by their
propensity to form colonies in soft agarose. Anchorage-independent
growth measures a cell’s ability to grow in an environment lacking
attachment signals. Normal cells require attachment signals to establish
polarization and are therefore not able to grow in soft agarose. Tumor
cells lack the need for polarization and are able to form colonies in soft
agar, thus making AIG an assay for putative neoplastic transformation.

Of the cell lines tested only those cells that contained myc/bcl-2 were

115

able to form colonies in soft agarose. Although. this suggests that all the
cell lines containing myc/bcl-2 were neoplastically transformed, the
degree of transformation varied greatly as expressed by large differences
in their colony forming efficiencies. When the invasive ability was
examined using a Boyden chamber invasion assay, they again displayed
similar variability. In this context, invasion is being used a measure of
the cells ability to remodel its environment and is considered a
transitional neoplastic point between promotion and progression because
once a cell escapes the confines of the basement membrane it is then free
to metastasize. If over-expression of bcl-2 was the main factor in the
neoplastic transformation of the WB.v-myc cell line, a dose correlation
between bcl-2 protein expression and the cell lines colony forming
efficiencies or invasive ability would expected to be seen. No such
correlation between bcl-2 protein expression and colony formation or
invasion was seen. suggesting that a another mechanism was occurring
that was responsible for the differences observed.

Gap junction-mediated intercellular communication (GJIC) has
been shown to play an important role in tumor promotion and
progression. Presumably, as cells lose GJIC. they are effectively removed
from intercellular signals that can regulate proliferation, differentiation.
and apoptosis. When it was shown that tumor promoters, such as
phorbol esters (Y otti et al, 1979, Murray and Fitzgerald, 1979). could

reversibly inhibit GJIC. it was hypothesized that an “initiated” cell would

116

be growth suppressed by being coupled by gap junctions to surrounding
normal cells. The implication is that, while the initiated cell is
genotypically altered. it could still be contact inhibited by regulatory
signals from the surrounding normal cells. By triggering signal
transduction in the “initiated” and surrounding normal cells, growth
factors, hormones and tumor promoting chemicals. would cause the
down-regulation of GJIC and induce gene expression (Trosko et al, 2000).
While normal cells would proliferate, differentiate and apoptose, the
“initiated” cell would only proliferate, thereby increasing the number of
initiated cells, i.e. clonal expansion. As long as the external exogenous
chemical (tumor promoter) is applied, the clonal expansion of the
initiated cell will occur.

The results in this study indicate that there might be a strong
association between GJIC and promotion. Those cell lines that retained
normal GJIC were restricted in their ability to form colonies in soft agar.
However, as the cell lines progressively lost the ability to communicate
through gap junctions, they progressively increased in their ability to
form colonies in soft agarose. The results indicate that that GJIC may
be contributing to the cells ability grow in soft agarose. The utilization of
myc and bcl-2 was not meant to imply that all cells being initiated or
neoplastically transformed represent the

initiation/ promotion / progression model of carcinogenesis, but rather to

117

show the importance of proliferation, apoptosis and GJIC during that
process.

An early observation of the myc/bcl-2 cell lines was the striking
dissimilar morphologies being expressed. This led to the investigation of
the role that differentiation might play in multi-stage carcinogenesis.
Potter (1978) proposed that cancer was a disease in which differentiation
was blocked; “oncogeny as partially blocked ontogeny.” Therefore.
initiation may involve the blockage of a progenitor or stem cells ability to
differentiate and promotion would allow that cell to clonally expand
because it could not differentiate.

To test this. hypothesis the myc/bcl-2 cell lines were chemically
induced to differentiate. A loss in the ability of the bcl-2 containing
clones to undergo differentiation was observed. However this blockage
was not consistent between the cell lines. In fact, it appeared that those
cell lines that were described as being more neoplastic were less able to
respond to chemical differentiation. This represents a refinement of the
“oncogeny as partially blocked ontogeny” theory which suggests that
blockage of differentiation is an early event before promotion. The
results here could be interpreted as representing a progressive loss of the
ability to differentiate as neoplasia increases. This does not exclude that
blocked differentiation can be an initiation event. only that the inability

to difierentiate progresses with promotion.

118

Earlier in this report, promotion was described as an accumulation
of cells using the equation A = P + D, where P was proliferation rate and
D was cell death. Differentiation represents another function that
cannot be additively summed with P and D, therefore this simple
equation cannot be used. In addition to differentiation, another state
exists in which the cells are unable to replicate, but remain viable. This
is known as senescence. Cellular senescence represents another
mechanism that limits clonal growth. Normal cells (excluding stem cells)
have a limited proliferative life span. after which they arrest and lose the
ability to divide (Hayflick. 1965). This phenomenon, termed replicative
senescence has been proposed to contribute to tissue aging (Smith and
Pereira-Smith. 1996). It is very important to delineate between
differentiation, apoptosis and senescence. Differentiation is a functional
state of cells providing specific metabolic activities to its
microenvironment. Differentiating cells can begin to terminally
differentiate, which results first in an irreversible loss in the ability to
proliferate (senesce) and ultimately leads to apoptosis. The distinction
between apoptosis and senescence is that senescent cells are still viable
and remain metabolically active. They are only unable to initiate DNA
replication. Interestingly, the myc and ras oncogenes. that can effect
proliferation, apoptosis and differentiation, also block the cell‘s ability to

senesce (Dean et al., 1986; Delgado et al., 1986) .

119

Therefore, a cell can be exhibiting one of five functions that are
quiescence (homeostasis), proliferation, apoptosis, differentiation, and
senescence. In order to represent the findings of this report in a broader
scope, it is necessary to conceptualization these functions as unique
states that a cell might reside, in which, each state is defined by a set of
signaling pathways. The idea of defined signaling involved with a specific
function is not new. It is well known that the signaling pathways are
very different between proliferation and apoptosis even though they both
involve some of the same proteins. Because some of the same proteins
are involved, these states can interact. That is to say, a cell can enter
other states, depending in what signaling is present. For example.
replicating cells can differentiate, apoptose, senesce or quiesce. Similarly
differentiated cells can proliferate, quiesce. apoptose, or senesce.
However, all evidence indicates that apoptosis and senescence are
irreversible. Figure 22a, depicts a directed graph demonstrating the
intricate interactions of these states, in which each state is represented
as a vertices. This kind of graph shows the complexity of the system.
When using a directed graph such as this it becomes difficult to visualize
how initiation and promotion could occur.

To simplify the complexity of these interactions a
divergent/ convergent model is proposed (Figure 22 b-f). In this model
homeostasis (quiescence) is represented as a balance of the signaling

pathways involved in the functions of proliferation, apoptosis,

120

difierentiation, and senescence. In this model, a normal cell in
homeostasis could equally respond to any signaling pathways. In tissue
culture, it is difficult to demonstrate quiescence. In vitro culturing
conditions preclude the cells from entering this state. As cells reach a
density in culture that would induce contact inhibition and quiescence.
prolonged maintenance at this density begins to cause genetic
instabilities, thereby altering the signaling pathways. However, if a
stimulus is present then a divergent path from the cells is chosen and a
new state is assumed, i.e. quiescence transitioning into proliferation.

In tissue culture, this is represented as the normal passing of cells
at sub-confluent condition where a majority of the culture is quiescent.
When these cells are re-plated, they begin to replicate. An initiated cell
in this theory would be restricted in what pathways and subsequent
states that it could reside. This is demonstrated by WB.v-myc and
WB.bcl-2 cells, which are precluded from senescing and apoptosing.
respectively. Promotion would then be described as the blockage of
divergent signals leading to fewer and fewer pathways which are
available and the signals converge into a single state, for neoplastic cells
that is usually proliferation. In this study, bcl-2 and myc cooperate to
block differentiation and apoptosis and to reduce contact inhibition,
such that the cells are able to proliferate in an unrestrained manner.
That is to say, the initiated cell, when stimulated to proliferate, is

inhibited to apoptose, to differentiate, and is not able to senesce. As the

121

tumor grows, signaling changes and some cells can proceed to other
states, as is demonstrated by the heterogeneity of tumors in vivo. To
summarize, promotion in this model is the set of signals that allows for a
convergence of signals in the initiated cell, leading to the accumulation of
cells. When WB-F344, WB.v-myc, and WB.bcl-2 cell lines were treated
with sodium butyrate, the cumulative signaling led to differentiation.
However, in the WB.myc/bcl—2 cell lines, difi'erentiation and senescence
were blocked, and proliferation could not be initiated, so the cells could
only die, even though they were over-expressing bcl-2.

In this model, gap junctions would play an essential and critical
role, for they would serve as a means to balance the signaling between
cells. A reduction or block in GJIC would lead to an imbalance in
signaling, thus leading to restrictions in what divergent pathway a cell
can reside. The fact that gap junctions are so highly evolutionarily
conserved suggests their importance. Bel-2 is also conserved, however.
its conservation is based on the proposal that apoptosis is a critical event
to be controlled. While this is true, bcl—2 undoubtedly has other
functions.

This report and published data have shown that bcl-2 can effect
multiple pathways like cell growth and differentiation. As more research
is conducted into bcl-2, more functions will be found. However, the
demonstration that bcl-2 is involved in both difi'erentiation and

proliferation suggests that its conservation might be due to its functions

122

as an adaptive factor as opposed to a pro-survival factor. Bel-2 appears
to function in pathways that would allow for adaptation. This is partly
represented by bcl-2’s ability to facilitate the repair of DNA and inhibits
p53 entry into the nucleus. Thus damaged DNA could have altered gene
expression. By blocking apoptosis, slowing proliferation, bcl-2 allows for
the transcriptional expression to occur. For a less organized species, this
would be of profound benefit, allowing for adaptations to occur for
maximization to its environment. To human beings this proves to be
profoundly detrimental.

These cell lines might represent a novel model to investigate the
bcl-2 functions that are distinctly associated with monomeric bcl-2
protein expression and test the functionality of bcl-2. This work lays the
foundation for future studies needed to be conducted with regard to how
bcl-2 is functioning on a biochemical level using these cell lines.
Beginning with the determination of subcellular localization, this could
be accomplished by Western blotting of isolated organelle compartments
and by irnmunohistochemistry with anti-bcl-2 antibody to determine the
localization and relative amount of bcl-2. . Additionally, future analysis
of bcl-2 function and apoptosis in these cells should be considered
utilizing inducers that function to produce apoptosis via different
mechanisms, such as ionizing radiation or staurosporine. This would
aid in the characterization of bcl-2 action, by determining exactly which

apoptosis pathways were efiected in this system. In these experiments,

123

the WB.v-myc/bc1-2 cells were only measured against themselves and
found to be unable to have fully functional GJIC. Future studies will
have to be conducted to test if co-culture of these cells with normal rat
liver epithelial cells will behave difierently and if these cells, when placed
back into the rat liver, will either be suppressed or whether they can
grow into tumors.

In conclusion, this study attempted to demonstrate the roles of
proliferation, contact inhibition and apoptosis in promotion. Albeit, the
initiation / promotion/ progression model of carcinogenesis is an in vivo
model that cannot be fully investigated in tissue cell culture. This report
demonstrated that myc and bcl-2 could cooperate in the neoplastic
transformation of WB-F344 cells. These myc/bcl-2 cells demonstrated
characteristics consistent with a neoplastic phenotype being
characterized by anchorage independent growth, blockage of apoptosis.
and the loss of contact inhibition. All myc/bcl-2 cell lines grew in soft
agar. This growth was independent of bcl-2 protein expression level and
appeared to be related to the cell line’s ability to communicate through
gap junctions. This report also presented data investigating the role of
differentiation in multi-step carcinogenesis and suggests that the
progressive loss of differentiation is a characteristic of promotion. Lastly,
this report speculated its findings into a model of carcinogenesis and
suggests that bcl-2 evolutionary conservation might be related to an

adaptive response rather than just apoptosis.

124

Figure 22. Graphical representation of proposed model for
carcinogenesis. a) Direct graph demonstrating interaction of the five
states of existence for a cell (H=homeostasis, A=apoptosis.
D=difierentiaiton, S=senescence, P=Proliferation). b-f) Signal
divergence/ convergence model of promotion. b) Homeostasis is
represented by a balance of all signaling. c) Promotion is represented as
a cumulative blockage of signaling associated with D (differentiation), S
(senescence), and A (apoptosis). d) Differentiation is represented by a
cumulative blockage of signaling associated with P, S, and A. e)
Apoptosis is represented by a cumulative blockage of signaling
associated with D, S, and P (proliferation). f) Senescence is represented
by a cumulative blockage of signaling associated with P, D, and A.

125

 

 

 

 

 

 

 

 

 

 

a 9 b P
0
'5 ‘
(9‘ 0
® D A
(all pathvgzyesoigstfilanced)
c I d D

 

Promotion
(Signal accumulation
for proliferation)

A

Differentiation

A P

 

 

 

 

Apoptosis
(all other pathways blocked)

 

 

 

 

BAA

Senescence

 

Figure 22

126

 

APPENDICES

127

APPENDIX A

PUBLICATION

Deocampo, Nestor D., Wilson, Melinda R., and Trosko, James E. (2000).
Cooperation of bcl—2 and myc in the neoplastic transformation of normal
rat liver epithelial cells is related to the down-regulation of gap junction-
mediated intercellular communication. Carcinogenesis 2 1 , 1501- 1506.

128

TITLE

Cooperation of Bcl-2 and Myc in the Neoplastic
Transformation of Normal Rat Liver Epithelial Cells
is Related to the Down Regulation of Gap Junction-
Mediated Intercellular Communication.

Nestor D. DeoCampo, Melinda R. Wilson, and James E. Troskol

National Food Safety and Toxicology Center, Department of Pediatrics and Human

Development, and Genetics Program East Lansing, MI 48824

lCorrespondences to: Department of Pediatrics and Human
Development, Michigan State University, 246 Food Safety Building, East
Lansing, MI 48824

Phone: (517) 432-4130
Fax: (517) 432-6340
Email: trosko@pilot.msu.edu

Running Title: Bel-2 and Myc in Carcinogenesis

129

ABSTRACT

The objectives of this study were to isolate several rat liver epithelial cell
clones, containing the human bcl-2 and myc/bcl-2 genes, in order to
study their potential cooperative effect on neoplastic transformation and
gap junction-mediated intercellular communication and to test the
hypothesis that the loss of gap junction-mediated intercellular
communication leads to tumorigenesis. Using anchorage independent
growth as a surrogate marker for neoplastic transformation, we
transfected both the normal rat liver epithelial cells, WB-F344, and a
WB-F344 cell line overexpressing v-myc with human bcl-2 cDNA. Those
cell lines that only expressed v-myc or human bcl-2 were unable to form
colonies in soft agar. However, those cell lines that overexpressed both v—
myc and human bc1-2 showed varying ability to form colonies in soft
agar, which did not correlate with their human bcl-2 expression level. In
order to test if there was a correlation between the cell lines’ growth in
soft agar and their ability to communicate through gap junctions, we
performed scrape load dye transfer and fluorescent recovery after
photobleaching assays. Our results show that v-myc and human bcl-2
can cooperate in the transformation of normal cells, but the degree to
which the cells are transformed is dependant on the cells ability to

communicate through gap junctions.

130

Abbreviations: AIG, anchorage independent growth: FBS, fetal bovine
serum: FRAP, fluorescent recovery after photobleaching: GJIC, gap
junction-mediated intercellular communication; PBS, phosphate buffered
saline; PMSF, phenylrnethylsulfonyl fluoride; SL/DT, scrape load dye
transfer; SDS, sodium dodecyl sulfate

131

INTRODUCTION

If one accepts the stem cell theory of carcinogenesis (1), then the
blockage of terminal differentiation (2) might be involved in the
“initiation” phase of carcinogenesis, while clonal expansion (mitogenesis),
plus inhibition of apoptosis, might contribute to the “promotion” phase
(3). Within this conceptual framework, over expression of various
oncogenes , acting together with other genes, might be involved in either
the initiation or promotion phases.

The myc oncogene, a transcription factor, has been associated with
the cellular functions of proliferation, differentiation and apoptosis (4-8).
Disruption of any of these basic cellular functions could contribute to the
multi-step, multi-mechanism process of carcinogenesis. Over expression
of the myc oncogene has been associated with many tumors (9,10).

The ras oncogene, being a member of the G- protein family. has
been shown to be involved in one of the many signal transduction
pathways affecting mitogenesis, differentiation and apoptosis. Ras, as
with myc, has been shown to be activated in many types of tumors
(1 l , l2).

Cooperation of oncogenes is one of the first important molecular
concepts of carcinogenesis. One of the first interactions to be reported
was the cooperation between the myc and ras oncogenes (13). In

addition, the demonstration that phorbol esters, such as 12-

132

tetradecanoylphorbol- 13-acetate,TPA. seemed to act in a manner similar
to the ras oncogene, in that it could cooperate with the myc oncogene to
induce a neoplastic phenotype (14). TPA, by activating protein kinase C,
triggers signal transduction pathways and can act as a modulator of
mitogenesis, differentiation and apoptosis (15- 17).

Ras and myc oncogenes have also been implicated in apoptosis
(18-20). Where myc seems to confer a susceptibility to apoptosis
(6, 19,21), ras appears to reduce cellular responses to apoptosis ( 18,20).
Therefore, in cells in which both myc and ras are activated, signal
transduction crosstalk interacts to block terminal differentiation,
triggering cells to proliferate and become resistant to apoptosis. In effect.
these unregulated disruptions of interacting signals bring about the
appearance of the tumor.

The bc1-2 protooncogene has been one of the major genes
implicated in the apoptotic process (22). Early experiments in transgenic
animals overexpressing bcl-2 under immunoglobuhn promoter control
showed increased frequency of follicular hyperplasia and B-cell survival
thus suggesting the role of bcl-2 as an anti-apoptotic gene (23). Since
this work many have demonstrated that bcl-2 is not only involved in
tumors of lymphoid origin but also in many tumors of epithelial origin
(24-3 1). Bcl-2 has also been demonstrated to synergistically interact
with TPA to transform cells (32). Thus, by blocking apoptosis, bcl-2

seems to be acting as a surrogate “ras” gene. However, bcl-2 itself is

133

unable to induce proliferation or neoplastic transformation that is often
associated with the overexpression of other oncogenes, like myc or ras
(33-35).

Many investigators have studied the co-expression of myc and bcl-
2 in a variety of cell types and transgenic models (36-39). These studies
were able to show that bcl-2 and myc can cooperate and increase or
enhance the tumor incidence and formation. However, these
experiments studying myc and bcl-2 cooperation have focused on cells of
lymphoid origin, fibroblasts, and a whey acidic protein promoter
controlled bcl-2 vector in the mouse lactating mammary gland, but not in
other epithelial systems. The experiments we describe in this study were
designed to test whether the bcl-2 oncogene in cooperation with v-myc
could function to neoplastically transform the WB-F344 cell line, a
normal rat liver epithelial cell line. We have previously reported the
establishment of a WB-F344 cell line stably expressing the v-myc
oncogene (40). This cell line demonstrates an increase in proliferation
and cell saturation density but it did not form colonies in soft agar or
tumors in nude mice. This cell line will be used as the target for
transformation by bcl-2.

Our laboratory and others have shown that while ras and TPA
effect signal transduction, they also affect gap junction-mediated
intercellular communication (GJIC) (41-44). Gap Junctions are channels

that directly link the interiors of neighboring cells allowing for the free

134

difi'usion of small molecular weight molecules (45). Most tumors
demonstrate a reduction in GJIC activity, either between themselves
(homologous GJIC) or with other cell types (heterologous GJIC) (3).
Presumably, down regulation of GJIC activity would lead to the removal
of growth inhibitory signaling, thereby providing a selective advantage.
TPA was the first agent shown to reduce GJIC activity (43,44). Rat liver
epithelial cells stably transfected with Ha-ras demonstrate a dose-
dependant reduction of GJIC with increasing levels of ras T24 protein
(41.42.46). Recently our laboratory reported that Ha-ras and v-myc
could cooperate to down regulate GJIC and this down regulation
correlated to the cells malignancy (40). In this same manner, we have
investigated the efi'ect that bcl-2 alone and in cooperation with v-myc will
have on GJIC and look for any correlation to neoplastic transformation.
In this report we demonstrate that bcl-2 can cooperate with v-myc
to induce neoplastic transformation of a normal rat liver epithelial cell
line. We also demonstrate that the extent of neoplastic transformation is

dependant on the cells ability to communicate through gap junctions.

135

MATERIALS AND METHODS

Cell Culture
WB-F344 rat liver epithelial cells, WB.myc and all subsequent bcl-2

transfectants were cultured in D-media (Formula 78-5470EF, Life
Technologies Inc.(Gibco/BRL), Gaithersburg, MD), supplemented
with 7% fetal bovine serum (Life Technologies Inc.(Gibco/BRL),
Gaithersburg, MD). All cells were incubated at 37°C in a humidified

atmosphere containing 5% C02.

Transfection of bcl-2
The human Bel-2 cDNA cloned into the pSFFV vector (kindly provided by

Gabrial Nunez, University of Michigan, Ann Arbor, MI) was transfected
into WB-F344 cell line and co-transfected with pTK—Hyg (Clontech
Laboratories Inc., Palo Alto, CA) into the WB.myc cell using lipofectin
(Roche Molecular Biochemicals, Indianapolis, IN). Briefly, 40 [lg of
Lipofectin was added to 1.5 milliliters of D-Medium (serum free) and 20

[:1 g of DNA was added to a separate 1.5 ml of D-medium. The two
samples were mixed and incubated at room temperature for 15 minutes.
This mixture was then added to sub-confluent cultures grown in 100 mm
dishes that had been rinsed twice with serum free D-media. The plates
were then incubated overnight at 37°C in a humidified chamber. The

following day D-medium containing 7% fetal bovine serum was added

136

directly to the plates and incubated an additional 24 hours.

Transformed cells were then split and subsequently plated with
appropriate drug selection. Individual clones were then isolated using
cloning rings, and subsequently recloned. Due the enhanced apoptotic
properties of the WB.myc cells, co-transfection with pTK-Hyg was
performed as described above with the following modifications: first, the
culture was allowed to reach confluency; second, 20 mg of DNA in a ratio
of 1:20 pTK-Hyg:pSFFV.bcl-2 was used to assure that selection using

hyromycin B would yield bcl-2 positive cells.

Protein extraction
Proteins were extracted from confluent cell culture grown in 25 cm2

flasks using 20% sodium dodecyl sulfate (SDS) containing 2mM
phenylrnethylsulfonyl fluoride (PMSF), 1 DM aprotinin, 1 {3M leupeptin
(Roche Molecular Biochemicals, Indianapolis, IN 46250), 1 GM antipain,
5 mM sodium fluoride (Fluka, Milwukee, WI), 0. 1 mM sodium
orthovanadate (Aldrich, Milwukee, WI), and subsequently sonicated three
times at five second intervals, aliquoted and stored at -20°C. The protein
concentration was determined by diluting the extracts I :5 and assayed
using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules,

CA) .

137

Western Blot analysis
Proteins were loaded in equal amounts (IO- 15 D g) into each well of a

10% SDS-polyacrylamide, in accordance with Laemmli (47). The gels
were electrophoresed at 200 mV for approximately 45 minutes, removed
and equilibrated in transfer buffer and transferred to Immobilon-P PVDF
membrane (Millipore Corporation, Bedford, MA). Human Bcl-2 was
detected with hamster anti-human bcl-2 monoclonal antibody
(Pharmingen, San Diego, CA), endogenous rat bcl—2 was detected with a
polyclonal anti-bcl-2 antibody (Santa Cruz Biotechnology Inc., Santa
Cruz, CA), v-myc was detected with anti-v-myc polyclonal antibody
(Caltag Laboratories, Burlingame, CA), using the supersignal ultra
substrate (Pierce Chemical Co. Rockford, IL) and appropriate peroxidase

labeled secondary antibody.

Anchorage Independent Growth
To access anchorage independent growth cells were grown in soft agarose

as previously described (48). Briefly, an initial hard agarose layer (0.5%
agarose 6013, Sirnga Chemical Co)made in D-medium containing 7%
FBS was plated in a 60 mm dish. Individual cell lines were then
trypsinized, counted, and diluted to a final concentration of 100 total
cells in D-Medium containing 0.33% agarose and 7% FBS and was
subsequently plated on top of the 0.5% agarose layer. Additional

medium was added 3 days later and changed every 3 days for 28 days.

138

Colonies were stained using 1 mg/ ml 2-(p-iodophenyl)-3-(nitrophenyl)-5-

phenyltetrazolium chloride (sigma) in 0.9% NaCl, and counted.

Cell-Cell Communication assays
Two methods developed in our laboratory were used to assess gap

junction-mediated intercellular communication: (a) scrape-loading dye
transfer (SL/UI') (49), (b) fluorescence recovery after photobleaching
(FRAP) (50). The scrape-loading dye transfer assay utilizes confluent
cultures grown in 35-mm dishes. The cultures are then rinsed three
times with Ca2+, Mg2+- phosphate-bufi'ered saline (PBS). 1 .5 milliliters
of PBS containing 0.05% Lucifer yellow CH (Molecular Probes Inc.,
Eugene, OR) was added, and several scrapes (cuts) were made on the
monolayer using a surgical scalpel. The cultures were incubated for 3
minutes at room temperature in the dye solution, and then rinsed three
times with Ca2+, Mg2+-PBS (to remove any background fluorescence).
The cultures were then fixed with 1 milliliter of 4% formalin and
visualized using an Ultima laser cytometer (Meridian Instruments,
Lansing, MI).

The scrape-loading dye transfer assay results were confirmed using
the FRAP assay. Briefly, cultures grown in 35-mm dishes were rinsed 3
times with Ca2+, Mg2+-PBS and then incubated with Ca2+, Mg2+-PBS
containing 5,6-carboxyfluorescein diacetate (7 mg/ ml, Molecular Probes

Inc., Eugene, OR) at 37°C in a humidified incubator for 15 minutes. The

139

cells were then rinsed several times with Ca2+, Mg2+-PBS and analyzed
using the Ultima laser cytometer. Cells were randomly selected under a
microscope (four cells were selected per field plus one unbleached
control, five fields per scan), and photobleached with an argon laser
beam. The transfer of fluorescence was then monitored in 4-rninute
intervals for a total of 12 minutes. The intensity of recovered
fluorescence of the individually bleached cells is then quantitated and
rates of dye transfer can be measured as the percentage of an

unbleached control cell (one per field).

140

 

 

RESULTS

Characterization of Isolated Clones
To examine the interaction between v-myc and bcl-2 oncogenes in

neoplastic transformation and GJIC in rat liver epithelial cells, we

generated clonal cell lines, utilizing established normal WB-F344 and A
WB.v-myc cell lines, over-expressing human-bcl-2 alone and co-

expressing human bcl—2 and v-myc. The morphological appearances

under phase-contrast microscopy of the different cell lines are shown in

figure I. The WB-F344, WB.v-myc, and WB.bcl-2 cells grew in uniform

monolayers of polygonal cells and exhibited contact inhibition of growth.

The Wb.myc/bcl-2 cell lines demonstrated different morphologies, such

as seen between the different clones [uniform polygonal, spindle-shaped,

and multi-nucleated].

Western Blot Analysis of Bcl-2
The expression of human bcl-2 was assessed using western blot analysis

with an anti-bcl-2 monoclonal antibody (Pharmingen, San Diego, CA)
specific for human bcl-2 protein. Figure 2a shows high expression of a

26 kDa band corresponding to transfected human bcl-2 protein, while

 

the control cell lines show no expression. We also verified the presence
of v-myc using western blot analysis with an anti-v-myc polyclonal

antibody. Figure 2b shows the presence of a 110 kDa band that

141

corresponds to the gap-pol-myc region of v-myc. Its expression is
consistent between the controls and transfected clones, suggesting that
over-expression of bcl-2 has not effected the expression of v-myc from the

parental WB.v-myc cell line.

Anchorage Independent Growth
Anchorage independent growth, more commonly referred to as growth in

soft agar, has often been used as a marker for neoplastic transformation
and to identify tumor cells (51-54). While this assay does not confirm
that cells are indeed neoplastically transformed, it does demonstrate that
those cells that are able to grow contain a characteristic shared with all
tumors. To assess if the bcl-2 gene was conferring neoplastic
characteristics to the normal rat liver epithelial cells, we tested their
ability to grow in soft agar. Using a previously described (40) v-myc/Ha-
ras tumorigenic WB-F344 cell line (designated MR-42) as a positive
control (100% colony forming efficiency), we plated replicate plates with
100 cells per plate in soft agar. Figure 3 demonstrates that the control
cell lines, as well as the bcl-2 only clones, were unable to form colonies
in soft agar. However, the v-myc / bc1-2 clones were able to form colonies
in soft agar and they also demonstrated clonal differences in their colony
forming efficiency ranging from 7 to 90 %. When we compared the v-
myc/bcl-2 clones colony forming efficiencies to their respective bcl-2

expression level, we found no correlation.

142

 

 

 

Gap Junction-Mediated Intercellular Communication (GJIC)
In order to explain the differences in growth in soft agar demonstrated by

the myc/bcl-2 cell lines, we assessed the individual cell lines ability to
communicate through gap junctions. Figure 4a demonstrates typical
results obtained from the scrape loading dye transfer technique. The
WB-F344 cell line is used as the control for communication with the dye
traveling about 6-8 cell layers. When WB-F344 cells are compared to the
WB.mb 19 clone containing v-myc and bcl-2, there is an approximate
50% decrease in the cells ability to transfer dye (3-4 cell layers). These
results were confirmed using the fluorescent recovery after
photobleaching assay (FRAP). Figure 4b shows the GJIC activity (both
SLDT and FRAP) of the various cell lines. This assay allows for the
monitoring of individual cells in the population assessing an activity for
the whole population. When compared to colony forming efiiciency, the
two communication assays appear to be inversely correlated. Those cell
lines that showed no significant changes in GJIC demonstrated a marked
decrease in colony forming ability in soft agar. However those cell lines
that demonstrated marked reduction in cell-cell communication

demonstrated an enhanced or increased ability to form colonies in soft

agar.

143

 

 

 

DISCUSSION

In this study our objective was to determine if myc and bcl-2
could act cooperatively in the induction of tumorigenicity by the
inhibition of gap junction-mediated intercellular communication. To test
this idea, myc/bcl-2 genes were co-expressed in WB-F344 cells, a normal
rat liver epithelial cell line. The parental WB-F344 cells, myc
transformed, and bcI-2 transformed cells grew in uniform monolayers of
polygonal cells but did not grow in soft agar. The myc/bcl-2 transformed
cells demonstrated various phenotypes and the cells were able to form
colonies in soft agar. When these cell lines were compared to a cell line
with 100% colony forming efficiency, they demonstrated varying abilities
to form colonies is soft agar. The ranges in colony forming ability did not
correlate to the cell lines respective bcl-2 expressions. We further
characterized these myc/bcI-2 cell lines by examining their gap junction-
mediated intercellular communication. We found that there was an
inverse correlation between the cell ability to communicate and their
ability to form colonies in soft agar. Those cells lines that demonstrated
a reduced GJIC showed an increased ability to form colonies in soft agar.

The v-myc/bcl-2 transformed cells did indeed grow in soft agar.
while the controls (WB-F344, WB.v-myc, WB.bcl-2) did not grow. The
degree to which the cell lines were able to form colonies varied greatly. If

over-expression of bcl-2 was the main factor in the neoplastic

144

I ———_‘-.—_
l

 

transformation of the WB.v-myc cell line. we would expect to see a dose
correlation between bcl-2 and the cell lines colony forming efficiencies.
We did not see any correlation between bcl-2 expression and colony
formation, which suggests another mechanism occurring that was
responsible for the differences we were observing.

Gap junction-mediated intercellular communication (GJIC) has
been shown, by our group and others, to play an important role in tumor
promotion and progression. Presumably, as cells lose GJIC, they are
efi'ectively removed from intercellular signals that can regulate
proliferation, differentiation, and apoptosis. In this manner an “initiated”
cell could escape growth regulation.

. The role of GJIC in the regulation of cell behavior (e.g., growth
control, differentiation, apoptosis, and adaptive responses of
tenninally-differentiated cells), while still not known in detail, seems to
involve the transfer of ions and small molecular weight regulatory
molecules through the gap junction channels to act as either a “sink” or
“source” (55). GJIC is known to synchronize electronic or metabolic
responses between cells (56). Coupling of normal homologous or
heterologous cells could alter the behavior of the cells. When it was
shown that tumor promoters, such as phorbol esters (44), could
reversibly inhibit GJIC, it was hypothesized that an “initiated” cell (a
stem-like cell which has been prevented from terminally differentiating or

which has been “immortalized”) (57) would be growth suppressed by

145

 

being coupled by gap junctions to surrounding normal cells. The
implication is that, while the initiated cell is genotypically altered by the
“initiator”, it still could be “partially blocked from terminally
differentiating” (2) and be “contact inhibited” by regulatory signal
equilibration from surrounding normal cells. Growth factors, hormones
and tumor promoting chemicals, by triggering signal transduction in the
“initiated” and surrounding normal cells, would (a) cause the
down-regulation of GJIC (leading to the inhibition of contact inhibition)
and (b) induced gene expression related to cell

proliferation / differentiation (57). While the normal cells would
proliferate and differentiate, the “initiated” cell would only proliferate or
not die by terminal differentiation or apoptosis, thereby increasing the
number of initiated cells. As long as the external exogenous chemical
(tumor promoter) is applied, the clonal expansion of the initiated cell can
occur. If, and when, other genetic changes within the initiated cell occur
that can stably down regulate GJIC, external dependence on tumor
promoters declines and the cell becomes independent of the suppressing
effect of surrounding normal cells.

When we examined the myc/bc1-2 cell lines GJIC activity, we
found a striking correlation between GJIC and colony formation. Those
cell lines that possessed functional GJIC, formed very few colonies in soft
agar. However, those cell lines that demonstrated a reduction in GJIC

activity, showed dramatic increases in their ability to form colonies.

146

 

 

This suggests an active role for GJIC in contributing to neoplastic
transformation. The use of myc and bcl-2 was not used to imply that all
cells being initiated or neoplastically transformed represent the
initiation / promotion / progression model of carcinogenesis, but rather to
show the potential importance of GJIC during that process. The myc
transformed cell still has functional GJIC and could be suppressed by
surrounding normal cells. In these experiments, the WB.v-myc/bcI-2
cells were only measured against themselves and found to be unable to
have fully functional GJIC. Future studies will have to be conducted to
test if co-culture of these cells with normal rat liver epithelial cells will
behave differently and if these cells, when placed back into the rat liver.
will either be suppressed or whether they can growth into tumors.
These new clones demonstrate that bcl-2 can cooperate with myc
to neoplastically transform rat liver epithelial cells. However, the degree
to which the cells are neoplastically transformed is dependent on their

ability to communicate through gap junctions.

147

 

 

Acknowledements

Reasearch was supported by a National Cancer Institute grant, CA21104,
given to James E. Trosko.

148

 

 

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154

 

Fig. 1. Phase Contrast photornicrographs of WB-F344, control and
transfected cells. (A) WB-F344: (B) vector control, WB.neo: (C) WB.v-myc;
(D-F) WB.myc/bcl-2 clones, WB.mb3, WB.mb19, WB.mb39 respectively.

155

 

 

 

 

 

 

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156

Fig. 2. Analysis of expression of human bcl-2 and v-myc proteins. (A)
Detection of 26 kDa protein corresponding to human bcl-2 from extracts
of normal WB-F344 and transfected cell lines. (B) Detection of a l 10 kDa
protein corresponding to v-myc from extracts of normal WB-F344 and
transfected cell lines.

157

 

 

 

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158

Fig. 3. Assessment of anchorage independent growth using growth in
soft agar. The data represent the efiiciency of colony formation
represented as the number of colonies formed per 102 cells plated.

159

 

 

 

 

 

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Fig. 4. Analysis of GJIC expression using the scrape loading dye
transfer (SL/DT) and fluorescent recovery after-photobleaching (FRAP)
techniques. (A) A representation of the SL/D’l‘ assay. Note the normal
WB-F344 cell line transfers the dye approximately 6-8 cells, while the
myc/bcl-2 clone, MB. 19, is only able to transfer the dye 2-3 cells on
either side of the scrape line. (B) Assessment of GJIC using the FRAP
technique. The data represent the percentage of fluorescent recovery
after a single cell is photobleached amongst a population of cells.

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163

 

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