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.EI

This is to certify that the

thesis entitled

A BICIIHEMICAL AND MOLECULAR ANALYSIS
OF RAF CNCGEENE TRANSFEII'IE) RAT LIVER
EPITHEILIAL CELLS

presented by

SHUCHEINIU

has been accepted towards fulfillment
of the requirements for

_M.,3.__degree in MEDICAL PMRAM

 

flag/Ale xi

Major professor

 

 

Date August 18, 1992

 

0-7539 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

A BIOCHEMICAL AND MOLECULAR ANALYSIS
OF RAF ONCOGENE TRANSFECTED RAT
LIVER EPITHELIAL CELLS

Shu Chen Lu

A THESIS

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

MASTER OF SCIENCE

Clinical Laboratory Science
Medical Technology Program
College of Natural Science
1992

ABSTRACT
A BIOCHEMICAL AND MOLECULAR ANALYSIS OF RAF ONCOGENE
TRANSFECTED RAT LIVER EPITHELIAL CELLS
by
Shu Chen Lu

Viral raf oncogene subcloneed into a mammalian expression vector,
EHneo(plasmid), was transfected into the non-tumon'genic and gap junctional intercellular
communication (GHQ-competent cell line, WB-F344, to study the effects of the
expression of the oncogene on GJIC and tumorigenicity of the tranfected cells. A total of
six G418-resistant clones has been isolated for analysis. One of the transfected clones
exhibited spindle-shaped morphology, while the others showed the same morphology as
the wild type parental cells. The morphologically transformed clone was the only one
which showed expressed v-raf mRN A and raf kinase signals as examined by northern blot
and western blot analyses, respectively. Among all the v-raf transfectants examined, only
those clones which had v-raf mRN A expression were capable of anchorage-independent
growth in sofi agar.

GJIC was examined in v-raf-, pSV-neo (plasmid control) and the parental WB-
F 344 cells using the scrape loading-dye transfer technique. It was found that the
transformed clone had reduced intercellular communication compared to the parental and
plasmid control cells. Northern analysis showed that the level of connexin 43 mRNA was
unchanged in the transformed cells compared to WB-F344 cells. Furthermore, the v-raf
transformed cells treated with TPA (100 ug/ml) overnight showed reduced GJIC
compared to the same cells without TPA treatment.

These results indicate that the expression of viral raf oncogene might induce
neoplastic transformation, although the frequency of such transformations is low. The
results also reveal that raf protein kinase might downregulate GJIC and affect the stability

of PKC translocation via directly or indirectly pathway.

To my parents with love

iii

ACKNOWLEDGMENTS

I wish to express my gratitude to my major professor, Dr. Burra V. Madhukar for
his support, guidance and encouragement. My thanks are also due to Dr. Chia-Cheng
Chang, committee member, for his valuable advice in both research and life.

I also wish to thank the valuable input provided by the other members of my
committee, Drs. Douglas Estry and John Gerlach. I wish to thank Drs. Emmanuel Dupont
and Yuh-Shan Jou for guiding me in various techniques in molecular biology. I want to
thank Dr. Diane Matesic for her technical help and friendship. My sincere thanks are also
due to Dr. Ulf R.Rapp of the National Cancer Institution, for providing the v-raf plasmid
used in this study. In addition, I wish to thank Tyng-Tyng Yang and Chien-Yuan Kao for.
their true fiiendship and encouragement. I also wish to acknowledge Ching-Fen Lin and
Ming Shia Yeh for their help in processing the thesis.

Finally, I wish to extend my deepest appreciation to my parents, my sisters and my
brother for their encouragement and support.

This research was supported by US Air Force-Office of Scientific Research grant
no. 89-0325.

iv

TABLE OF CONTENTS

INTRODUCTION ....................................................................................................... 1

LITERATURE REVIEW ............................................................................................ 3
Oncogenes ....................................................................................................... 3
Classification of Oncogenes .............................................................................. 3
Activation of Oncogenes .................................................................................. 5
Oncogene and Gap Junction Mediated Intercellular Communication ................. 6
Raf Oncogene .................................................................................................. 7

MATERIALS AND METHODS ................................................................................. 14
Preparation ofcompetent E. coli l4
Transformation of competent cells with raf plasmid cDNA ............................... 15
Small-scale preparations of plasmid DNAs by alkali lysis method ...................... 15
Large-scale preparations of plasmid DNA by alkali lysis method ....................... l6

Purification of plasmid DNA by precipitation with polyethylene

glycol(PEG) ..................................................................................................... 16
Cell culture ............ . .......................................................................................... 17
Transfection of WB cells with viral raf oncogene .............................................. 17
Scrape-Loading/Dye Transfer Technique to study GJIC ................................... 18
Isolation of total RNA from cells ...................................................................... 19
Northern Blot Analysis ..................................................................................... 19
Western Blot Analysis ...................................................................................... 22
Procedure for Anchorage Independent Growth (AIG) ...................................... 24
RESULTS ................................................................................................................... 25
A. Selection of v-raf transfectant ..................................................................... 25
B. In vitro Morphology ................................................................................... 25
C. Expression of v-raf mRNA ......................................................................... 25

D. Intercellular Communication ....................................................................... 26

E. Expression of raf p74 protein kinase in raf transfectant ................................ 26
F. Anchorage Independent Growth (AIG) ....................................................... 26
DISCUSSION ............................................................................................................. 47
REFERENCES ............................................................................................................ 51

INTRODUCTION

Oncogenes are being extensively studied for the mechanism of carcinogenesis. Raf
oncogenes encode proteins with protein-serine/threonine kinase activity. There is ample
evidence that raf protein plays an important role in transmitting signals from cell surface
receptors to the nucleus. However, both the exact mechanisms by which raf is functioning
and the down stream targets of raf proteins remain to be elucidated.

One form of intercellular communication is mediated by membrane channels
known as gap junctions. The junctional communication has been implicated in the
regulation of tissue homeostasis, cell growth and differentiation (Vitkauskas et al. 1985).
Many tumor cells showed either significant reduction or functional absence of I gap
junctions. The complete loss of GJIC of the tumor cells has also been correlated with
their metastatic potential (Leiberman et al. 1991). Furthermore, many non-genotoxic
chemicals such as the phorbol ester, TPA were shown to inhibit GJIC and were termed,
tumor promoters. The phobol ester tumor promoter, TPA is also a potent inhibitor of
GJIC in many cell types as well as an activator of protein kinase C (PKC) , a
serine/threonine kinase. The activated PKC presumably phosphorylates gap junction
protein, resulting in blocking the intercellular communication (Oh et al. 1988). It was
found that treatment cells with TPA, the activity of raf protein kinase increased as well as
protein kinase C (Morrison et al. 1988). The similarity between PKC and raf protein is
thus suspected. In the studies presented in this thesis study, the v-raf transfected cells
were examined to determined whether stable expression of v-raf kinase can downregulate
GJIC .

Mutational activation of the c-raf-l gene has been observed in many hepatocellular
carcinomas and neoplastic nodules (Beer et al. 1988; Ishikawa et al. 1985). Thargiersson

and associates at the National Cancer Institution demonstrated that a rat liver epithelial

(RLE) cell infected with v-raf (361 l-MSV) virus alone were only weakly tumorigenic and
were not capable of growing in soft agar (Worland et al. 1990).

The WB-F344, rat liver epithelial cell line was isolated from the liver of an adult
male Fisher-344 rat by Dr. Grisham of the Department of Pathology, University of North
Carolina, Chapel Hill (Tsao et al. 1984). There are two major reasons that the WB-F344
cells were chosen for this study. First, these cells are known to perform high degree of
GJIC. Second, the cell line is immortal but non-tumorigenic. The introduction of other
oncogenes,i.e. activated H-ras, and Neu, have been found to transform these cells into
tumorigenic cells (De Feijter et al. 1990).

In this study, the oncogenically active v-raf was transfected into the non-tumorigenic liver
epithelial cell line, WB-F344 ,to determine if the expression of the v-raf protein might

down regulate GJIC and induce neoplastic transformation of these cells.

 

LITERATURE REVIEW

Oncogenes

Oncogenes are a critical set of genes that become altered in the course of
carcinogenesis (Hartmut et al. 1983). They were first described as retrovirus-encoded
genes that produced tumors in birds and rodents. These genes were later shown to be
dominant mutated forms of host genes (proto-oncogenes) that might have been picked up
by the retroviruses (Bishop, J.M., 1983). It is generally believed that proto-oncogenes are
normal cellular genes and have an essential physiological role. They have been shown to
be involved in the regulation of cellular proliferation and differentiation (Rijsewijk 1987,
Muller 1986, Bar-Sagi 1985 and Liotta 1991). However, it appears that the ability of
many oncogenes to induce neoplastic transformation arises fi'om inappropriate expression

or function of proto—oncogenes that normally regulate cell proliferation.

Classification of Oncogenes

Oncogenes can be broadly grouped on the basis of their presumed firnction and
predominant properties. The presently known oncogenes can be catergroized into five
classes: growth factors (eg. sis), protein-tyrosine kinases (eg. src), Guanine Nucleotide
binding proteins (eg. ras), nuclear oncogenes (eg. erbA), and protein-serine/threonine
kinases (eg. rat). As mentioned previously, cellular oncogenes play very important roles
in cellular homeostasis and growth control.

(a) Growth Factors

Cellular sis oncogene encodes a protein which is homologous to the B-chain of
platelet-derived growth factor (PDGF) (Robbins 1983, Waterfield 1983). When the sis
gene is activated, the B-chain of PDGF is constitutively synthesized. These cells are then

stimulated to proliferate, and become independent of one or more exogenous growth

factors.
(b) Protein-tyrosine kinase

Some proto-oncogenes (eg. src, v-erbB) encode proteins that are located near to
the inner surface of the cell membrane and have kinase activity(Downward 1984, Collett
1978). This membrane-associated protein kinases phosphorylate cellular proteins on
tyrosine residues, thereby act as essential components of a messenger system, transmitting
and applying signal fi'om the surface to the interior of the cell.

(c) GTP-binding proteins

Ras family genes are the representatives in this group. Normal ras protein
associates with the inner surface of the plasma membrane, where it interact with effector
molecules to control cell division. This interaction is controlled by the conformation of
the ras protein, which is either a GTP-bound active state or a GDP-bound inactive state.

In normal cells, active ras proteins are rapidly converted to inactive forms by an
intrinsic guanosine triphosphatase (GTPase) activity(Hall 1990). In tumor cells, the
mutant ras proteins remain in their active conformation for abnormally long periods and
have an important step in the transformation process. In neurofibromatosis, the GTPase
activating protein (GAP) is disabled and cannot hydrolize ras-bound GTP, resulting in
prolonged ras activation, uncontrolled cell growth and tumor formation (Xu et al. 1990).
(d) Nuclear Oncogene

There are many kinds of nuclear oncogenes, such as myc, fos, myb, and jun. The
proteins which are encoded by these oncogenes act as stimulator of gene expression; for
example, the oncogene erbA functions as a transcription factor.

(e) Protein-serine/threonine kinase
The raf oncogene is the major candidate in this group. The roles which raf

proteins play in cell growth control will be discussed below.

Activation of Oncogenes

It is now believed that cancer is a disease involving malfunctioning cellular genes.
In fact, several mechanisms of activation have been identified from an analysis of DNA
extracted from human tumors:
(a) Chromosomal translocation

It has been known that non-random chromosomal aberrations occur in human
cancers. A good illustration is provided by studies of Burkitt's lymphoma(BL), a B-cell
malignancy common in Africa. It was found that 100% of the lymphoma cells in BL
exhibit a reciprocal translocation involving chromosome 8‘ at band 8q24. The
translocation appears to affect the expression of c-myc by a number of mechanisms, the
overproduction of the myc gene product via a translational mechanism may be a common
feature for these cells (Siato et al. 1983). The translocation is now known to involve the
c-abl oncogene on chromosome 9 and the bcr gene on chromosome 22 resulting in a bcr-
abl protein which posses phosphotyrosine kinase activity (Heisterkamp 1983).
(b) Gene mutation

Mutation within the coding sequence of an oncogene is an important mechanism of
activation, resulting in the production of aberrant protein. Such mutations can be induced
by exogenous mutagens such as benzopyrene (carcinogen in tobacco) in lung cancer
(Takahashi et al. 1991), aflatoxin in hepatocellular carcinoma and ultraviolet light in skin
cancer (Brash et al. 1991), or by exogenous mechanisms (Loeb 1989). To date, all the
ras genes investigated have been activated by a single point mutation, altering the amino
acid either at position 12 or around position 61 in the 189-amino acid ras protein product.
In vitro mutagenesis has revealed that other codons in the human c-Ha-ras-l gene where
amino acid substitutions can also lead to a gene with transforming activity. Therefore, the
alterations which "activated" the ras genes must therefore be critical determinants of the

malignant phenotype in the tumor cells.

(c) Gene Amplification

The abnormalities result in the amplification of cellular genes: a single-copy proto-
oncogene in a normal cell may be amplified up to 100 times in a tumor cell. The increased
expression of the oncogene, resulting from its amplification, is believed to contribute to
the malignancy. Examples now exist are multiple copies of Ki-ras in primary lung
carcinomas and the amplification of the myc family in leukemic cells and tumors of neural
origin (Varmus, 1984). Interestingly,the degree of amplification correlates with the stage
of progression of the tumor: greater amplification correlates with advanced stage and a

poor clinical prognosis (Schwab et al. 1984, Slamon et al. 1987).

Oncogene and Gap Junction Mediated Intercellular Communication

One form of intercellular communication is mediated by the membrane channels
known as the gap junctions. These channels link two adjacent cells to allow the exchange
of ions, nutrients and regulatory molecules. Gap junctions are constructed from hexamers
of transmembrane proteins that called connexins. Three major forms of connexins,
connexin 43, connexin 32, and connexin 26, were well characterized at the biochemical
and molecular level.

The junctional communication has been implicated in the regulation of tissue
homeostasis, cell growth and difl‘erentiation, as well as synchronization of tissue fimction
and regeneration. Several modulators provided substantial evidence for an important role
gap junctions might play in carcinogenesis. Many tumor cells showed absence of gap
junctional coupling that the ability to communicate with normal cells via gap junctions.

The correlation between the neoplastic phenotype and the absence of gap
junctional intercellular communication became more significant following the discovery
that tumor promoters, e. g. phorbol esters, inhibit this important membrane mediated
function (Yotti et al. 1979, Trosko and Chang, 1988). TPA(12-O-tetradecanoylphorbol-

l3-acetate), a potent tumor promoter, is one of the major promoter shown to block
intercellular communication. TPA is a structural analogue of diacylglycerol ,which is an
endogenous activator of protein Kinase C generated by hydrolysis of phosphatidylinosotol
4,5-bisphosphate (PIP2) by Phospholipase C. It was believed that the activated protein
Kinase C can phosphorylate gap junction protein so that the communication is inhibited
(Oh et al.1988). This evidence indicated that tumor promoters might act by blocking gap
junctional coupling between normal cells and initiated cells thus facilitating clonal
amplification of initiated cells.

Recently, inhibition of gap junctional communication has been also shown in
oncogene transfected cells. Chang et al (1985) were the first to demonstrate that in NIH
3T3 cells transfected with v-src oncogene, GJIC was downregulated and this inhibition
was correlated with increased protein Kinase C activation. Another oncogene product,
p21 protein, generated by ras oncogene was also shown to abolish gap junctional
communication in rat liver epithelial cells. Furthermore, it was found that cell-cell
communication was blocked in v-raf/v-myc transfectant (Kalirni et al.1992).

Similar to protein kinase C, the raf protein is also a serine/threonine specific
kinase. It was found that treatment cells with TPA, the activity of raf protein kinase
increased as well as protein Kinase C(Morrison et al. 1992). Therefore, it was suspected
that raf protein kinase might be able to downregulate GJIC in a manner similar to PKC. In
the current investigations, v-raf transfectants were studied and the results will be discussed

later.

Raf Oncogene
A.Viral raf oncogene

Cellular raf oncogene was first identified in its oncogenic form, v-raf, the oncogene of the

acutely transforming virus 36ll-murine sarcoma virus (361 l-MSV) (Rapp et al. 1983).

 

The primary structure of v-raf was identified by Mark et al. in 1984. V-raf is expressed as
a myristylated gag-raf fiJsion protein consisting of the amino-terminal 384 amino acids of
Gag and the carboxyl-terminal 323 residues of mouse c-raf. (Rapp, 1983; Schultz, et al.
1985). Transfection of NIHBT3 cells with cloned 3611-MSV proviral DNA lead to highly
efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-
MSV virus. It was also demonstrated that 3611-MSV was capable of transforming the rat
liver epithelial cells (Garfield et al. 1988). However, the other experiment showed that
cells infected with v-raf alone were only weakly tumorigenic and were morphologically
more similar to control cells than those transformed with a combination of v-raf and v-
myc, which were aggressively tumorigenic and showed increased refractivity and
spindlings as well as decreased attachment (Hampton et al. 1990).

B.Localization and expression of raf gene family

The raf family of prom-oncogenes consists of three active members: A-raf-l, B-raf and
C-raf-l. They are dispersed over different chromosomes and are expressed in different
tissues. The mammalian C-and A-raf genes have 16 coding exons, which span 40 and
20kb, respectively. B-raf gene is larger and extends over 46kb (Bonner, T. et al. 1984).
Raf-l has been mapped to chromosome 3p25 in humans, a region frequently altered in
small cell lung carcinoma, familial renal cell carcinoma, and ovarian caner(Kozak et al.
1984). In addition, c-raf-l gene is ubiquitously expressed. Among the transcripts that
have been detected in all cell lines and all normal and tumor tissues examined, the highest
levels of expression observed was in striated muscle, cerebellum, and fetal brain (Storm, et
al. 1990). A-raf was characterized by Huleihel in 1986 and localized to the x
chromosome between p21 and qll (Huebner et al. 1986). The A-raf gene is expressed at
high levels in the urogenital system, including epididymis, ovary, kidney, and urinary
bladder (Storm et al. 1990). The most recently identified member, B-raf, was discovered
as a transforming gene in N1H3T3 cell transfection assays of human Ewing sarcoma DNA

(Ikawa at al. 1988). B-raf expression is confined to fewer tissues than either c-raf-l or A-

raf, with high message levels being observed in cerebrum and fetal brain. (Storm, 1990).
C. Raf protein

The raf oncogene family shows a high degree of evolutionary conservation. The c-raf-l
and A-raf genes encode cytoplasmic serine/threonine protein kinases of 68 and 74 kDa,
respectively, which are 60% identical overall. The B-raf gene product has not yet been
completely characterized. However, its predicted protein product is 76% identical to c-
raf-l and 74% identical to A-raf. The raf protein contains three structural domains
(Heidecker et al. 1986), each of which is highly conserved in all raf genes.

The deduced amino acid homologies of these three regions are shown in Table 1.
(Stephen et al. 1990). The first conserved region (CR1), located in the amino-terminal
portion of the molecule, is a cysteine rich domain and contains the consensus sequences C-
X2-C-X9-C-X2-C (Heidecker et al. 1989). This structure is similar to the ligand-binding
domain of protein kinase C, CR1 comprises the presumed ligand binding site although a
ligand has not yet been identified for Raf. A second highly conserved region, CR2, also
located in the amino-terminal portion is rich in serine and threonine residues. The third
conserved region, CR3, is located in the carboxyl-tenninal portion of raf protein and is the
serine/threonine kinase domain. The amino-terminal of the protein is thought to regulate

the catalytic activity of the carboxy-temrinal kinase domain.

D. Raf oncogene in Signal Transduction

There is considerable evidences that the raf family serine/threonine protein kinases have
the hallmarks of signal transmitters involved in communicating between mitogen receptors
at the cell surface and the nucleus. First, treatment of cells with several grth factors
(eg. PDGF, EGF) increased both the level of phosphorylation and the intrinsic protein
kinase activity of the raf protein (Morrison et al. 1989). Second, in PDGF-treated cells,
Phospholipase C-y associates with ligand-activated PDGF receptors in a complex that
includes raf-l, PI-3 kinase, GTPase activating protein (GAP), and several src family

-w- -

10

members (Kaplan, 1990). Third, treatment with phorbol esters to stimulate protein kinase
C also leads to increased phosphorylation of raf protein and its kinase activity (Morrison,
1988). Furthermore, cells containing activated raf have an altered transcription pattern,
and raf oncogenes behave as transcriptional activators for Ap-l/PEA 3-dependent
promoters in transient transfection assays (W asylyk et al. 1989). Finally, raf-transformed
cells can proliferate in the absence of ras function (Smith et al). That is, the raf protein

kinase can fiinction in signal transduction independent of ras.

E. Activation of Raf protein kinase
There is ample evidence that raf protein plays an important role in transmitting signals
from cell surface receptors to the nucleus, however, both the exact mechanisms by which
raf is activated and downstream targets of the activated molecule remain to be elucidated.
There are four possible models for the activation of raf serine/threonine kinase (proposed
by Ping Li, et al, 1991). First, the kinase may be activated by a small second messenger
molecule. The amino-terminal domain of Raf-1 is similar to the ligand-binding domain of
PKC and thus may be a potential binding site for a lipid second messenger. In the second
model, the raf kinase is activated by an upstream kinase, a so-called "Kinase kinase." The
third model, the most direct route of activation, is via direct phosphorylation and
consequent activation by a tyrosine kinase. Several experiments suggest this direct
activation may occur. For example, tyrosine phosphorylation of Raf-l is seen in vivo afler
PDGF treatment of fibroblasts, and a portion of the Raf-1 in the cell is found in a
complexes with the activated receptor (Morrison et al. 1989). The forth model is that
tyrosine phosphorylation brings about a transient change in Raf-l conformation so that the
raf kinase is activated.

Structural analysis of naturally occurring oncogenic raf (Parker et al. 1986), and
those that arise during the course of N1H3T3 cell DNA transfection assays (Stanton et

al.1987: Ishikawa et al. 1987) indicate that the amino-terminal domain of Raf-l is

11

important in the regulation of Raf-1 is important in the regulation of Raf-l activity
(Morrison, 1990). Therefore, there are also some presumed mechanisms of Raf-1
activation by oncogenic and mitogenic events regarding regulatory domain (Morrison,
1990). Mutational analysis has revealed three oncogenic events that can generate a
transforming Raf-1 protein: (1) removal of CR1 and CR2 by amino-terminal truncation of
the protein; (2) disruption of CR2 by linker insertion; and (3) fusion of the intact protein
to unrelated sequences. V-raf which expresses gag-raf fusion protein was thought to be
activated by this mechanism. Mitogenic events can also activate Raf-1. As noted above,
this occurs through phosphorylation of serine or tyrosine residues or through ligand

binding to CR1.

F. Rafprotein kinase and Protein kinase C

Protein kinase C was first identified in 1977 as a protein kinase (Inoue at al. 1977; Takai
et al. 1977). It is also a serine/threonine kinase which plays roles in signal transduction
pathways related to cell proliferation. The activation of protein kinase C is connected with
phospholipase C. In other words, it was identified as an effector enzyme of the inositol
phospholipid second messenger pathway and activated by diacylglycerol. The
involvement of protein kinase C in tumor promotion was proved by the demonstration that
protein kinase C is the major intracellular receptor for phorbol esters and other promoters.

The serine/threonine kinase encoded by protein kinase C has a catalytic domain and a
regulatory domain. The catalytic domain is localized to the carboxy-terminal half of the
protein, which the regulatory domain comprises the amino-terminal half of the molecule,
which includes the phorbol ester binding site. This structure is highly analogous to the raf
kinases, which are also similar to protein kinase C in size. Therefore, there is a strong
analogy between activation of protein kinase C catalytic activity by binding ligand to its
amino-terminal regulatory domain and activation of raf transforming potential by deletion

of its amino-terminal domain as discussed above.

12

Since there is structural similarity between raf protein kinase and protein kinase C and
since both of them play important roles in signal transduction pathway, it is considered
that these two kinases are functionally interrelated. One evidence is that treatment of cells
with phorbol esters to stimulate protein kinase C also leads to increased phosphorylation
of raf protein and its kinase activity. There are two hypotheses regarding the correlation
between protein kinase C and raf protein kinase. First, the path of activation is parallel.
They may be activated separately by kinase including recruitment of second messengers
derived from tyrosine-stimulated lipid turnover and direct phosphorylation by tyrosine and
serine/threonine-specific protein kinases. Second, raf protein kinases are downstream to
protein kinase C. The PDGF receptor is associated with phospholipase C, which appears
to be a substrate for phosphorylation by the receptor tyrosine kinase. The activity of PLC
may be stimulated by phosphorylation, leading to increased hydrolysis of
phosphatidylinositol 4, 5-bisphosphate (PIPZ) and production of the second messengers
diacylglycerol (DAG) and inositol 1, 4, S-triphosphate (IP3). The combination of
diacylglycerol and calcium will activate protein kinase C. The activated PKC then activate

raf protein which transduce signal to the nucleus.

G. Rafoncogenes in carcinogenesis

The 3611-MSV retrovirus which transduce the raf gene has been studied in
carcinogenesis. Newborn mice treated with the 3611-MSV virus developed severe
erythroleukemia after 4-12 weeks and only occasionally do myelogenous and
lymphoblastic neoplasms occur simultaneously with the erythroleukernias. (Klinken,
1991). The other experiments have been done in human tumors. A NIH 3T3 transfection
assay using DNA from a cell line established from a radioresistant human larynx carcinoma
led to isolation of a 5‘-truncated human Raf-1 gene. (Kasid, et al. 1987).

Since Raf-1 has been mapped to chromosome 3P25 it has been of particular interest to

study the role it plays in tumors that show consistent or high frequency loss of 3P, such as

13

small cell lung carcinoma (SCLC), renal cell carcinoma, ovarian carcinoma, and mixed
parotid gland tumors. Comparing 11 samples of small cell lung cancer to the
corresponding normal tissue, a consistent loss of chromosome 3P alleles was found.(Rapp,
et al. 1988). In addition, among 73 human lung cancer cell lines were examined, a highly
significant loss of one Raf-l allele was detected in all 42 SCLC cell lines. These results

demonstrated that one allele of Raf-l is consistently lost in small cell lung carcinoma.

(Rapp, et al. 1988).

MATERIALS AND METHODS

The viral raf plasmid cDNA (Figure 1) used in this research was kindly provided by Dr.
Rapp who is from Laboratory of Viral Carcinogenesis, National Cancer Institute,
Frederick, Maryland. The cellular and viral plasmid DNAs were then transfected into E.
coli strain, DH5a . The following step is to isolate large amount of those plasmid DNAs
from bacterial using alkaline lysis method. The DNAs were used to be transfected into
WB-F344, rat liver epithelial cells, using lipofectin. Because the constructs of raf
oncogenes contained neomycin resistant gene, neomycin was used to select those cells
which contain the integrated raf genes. The DNAs, RNAs and proteins were isolated from
the transfectants to study the level and the expression of raf gene and raf protein using
northern blot analysis and western blot analysis. Scrape Loading/Dye Transfer Technique
was applied to study the effect of viral oncogene in intercellular communication. The
method of anchorage independent growth(AIG) was used to assess the tumorigenicity of

v-raf transfectants. These methods will be described in greater detail later.

Preparation of competent E. coli
A colony of E. coli strain DHSa was picked up by a sterile wire from LB plate (1%

NaCl, 1% tryptophan, 0.5% yeast extract, 1.5% Agar) and grew in a 5 ml of LB medium
in 15 ml polystyrene tubes (Falcon 2099). After incubating the culture overnight at 37°C,
1 ml of the culture was transferred to a 25 ml LB medium in a sterile, disposable 50 ml
polypropylene tubes (Falcon 2098) and incubated at 37°C. The OD550 was checked
every 20-30 minutes until the value of OD5 50 was equal to 3.0. The culture was cooled
on ice for 10 minutes and then centrifuged at 3000 rpm for 10 minutes at 4°C. the cells
were resuspended in 25 ml of ice-cold 100 mM CaC12 and centrifuged again. The cells

were resuspended in 2.5 ml of ice-cold 100 mM CaClz . These cells were then used for

14

15

transformation and stored at ~70°C containing 15% glycerol.

Transformation of competent cells with raf plasmid cDNA

One hundred microliter of competent cells from above were transferred into a 17x100mm
polypropylene tube. Twenty nanograms of raf plasmid DNA isolated as described below
was added to the tube. The tube was then placed on ice for 30 minutes with occasional
mixing. After heat shock at 42°C for 2 nrinutes, the cells were kept at room temperature
for 10 minutes. The transformed cells were then transferred to 1 ml of LB medium and
incubated at 37°C for 1 hour. One hundred microliter of the culture was streaked on a

LB plate containing 50 ug/ml of Ampicillin and incubated at 37°C overnight.

Small-scale preparations of plasmid DNAs by alkali lysis method

A single bacterial colony from above step was transferred into 2 ml of LB medium
containing 50 ug/ml of Ampicillin in a 15 ml tube and incubated ovemight at 37°C with
vigorous shaking. A 1.5 ml of the culture was poured into a 1.5m] eppendorf tube and
centrifirged at 12,000 rpm for 30 seconds at 4°C. The medium was removed by
aspiration, leaving the bacterial pellet as dry as possible. The bacterial pellet was
resuspended in 100 ul of ice-cold solution I (50 mM glucose, 25 mM Tris.Cl (pH8.0), 10
mM EDTA (pH8.0)) by vigorous votexing. Then 200 ul of freshly prepared solution 11
(0.2N NaOI-I, 1% SDS) was added followed by 150 ul of solution III (3M NaOAC,
pH5.2) and stored on ice for 30 minutes. The tube was centrifuged at 12,000 rpm for 10
minutes at 4°C and the supernatant was transfered to a flesh tube. Two volumes of ice-
cold ethanol was added to precipitate the double-stranded DNA. After 15 minutes at -
70°C, the tubes centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was
removed by gentle aspiration and the pellet was washed by 70% ethanol. The air dried
nucleic acids were redissolved in 20 ul of TE (10 mM Tris, lmM EDTA pH8.0).

l6

Large-scale preparations of plasmid DNA by alkali lysis method
500 ml of LB containing 50 ug/ml ampicillin in a 2-liter flask with 1 ml of the late-log-

phase culture was incubated at 37°C overnight (12-16 hours) with vigorous shaking (250
cycles/minute on a rotary shaker). The bacterial cells from the 500 ml culture were
harvested by centri‘fiigation at 10,000 rpm for 10 minutes at 4°C. The bacterial pellet was
resuspended in 10 ml of solution I followed by adding 20 ml of freshly prepared solution 11
described above. Fifteen milliliters of ice-cold solution III were then added and mixed by
inverting several times. After keeping on ice for 30 minutes, the bacterial lysate was
centrifuged at 10,000 rpm for 10 minutes in a Sorvall RC2B-high speed centrifilge using a
SS-34 rotor. Two volumes of ethanol were added into the supernatant and stored in a
freezer(-20°C) for 1 hour. The nucleic acids were recovered by centrifirgation at 10,000
rpm for 10 minutes at 4°C. The pellet was rinsed by 70% ethanol and dissolved in 1.5 ml
of TE(pH8.0). [The solution I, 11, HI are the same as the small-scale preparation].

Purification of lasmid DNA b reci itation with ol eth lene I col PEG

One volume of 5 M LiCl was added to the DNA isolated above and stored on ice for 20
minutes. After centrifugation for 15 minutes at room temperature, 1 volume of
isopropanol or 2 volumes of ethanol was added to the supernatant and nucleic acids were
repelleted by centrifugation at 10,000 rpm. The pellet was rinsed with 75% ethanol and
resuspended in TE (pH 8.0). These DNAs were digested with 20 ug/ml of Rnase for 1
hour. One volume of 13% PEG 8,000 and 1.6M NaCl were added to remove protein and
RNA. The pellet was resuspended in 200 ul TE(pH8.0) after kept on ice and span 15
minutes at room temperature. The DNAs were extracted once with 1 volume of phenol,
once with one volume of phenol/chloroform and once with one volume of chloroform to

remove the RNAs and other proteins. The supernatant was precipitated by adding 1 vol.

17

of isopropanol and 0.1 volume of 3M sodium acetate (pH5.2). The pellet obtained by
centrifiigation was rinsed with 75% ethanol and air dried . The nucleic acids were
dissolved in 500 ul of TE (pH8.0). An aliquot of this solution was diluted 10-fold with
distilled water and the absorbance was recorded using a Gilson UV-Spectrophotometer.
The ratio of OD260 : OD230 usually ranged from 1.8-2.0 indicating DNA free of protein

contamination.

M

The ‘WB-F 344, rat liver epithelial cells and other raf-transfected cells were
routinely grown in a modified Eagles' MEM, which contains Earle's balanced salt solution
with a 50% increase of vitamins and essential anrino acids, a 100% increase of non-
essential anrino acids and l Mm sodium pyruvate, 5.5 Mm glucose, 14.3 Mm NaCl,
11.9mM Nal-ICO3 , pH7.3] This medium was supplemented with 10% fetal bovine serum
(GIBCO, BRL) and 50 ug/ml gentamicin (Quality Biological Inc. MD). For transfected
cell line, 400 ug/ml of neomycin was added as selective agent. The cells were maintained
at 37°C in a humidified atmosphere of 5% C02 and 95% air and subculture whenever

they were confluent.

Transfection of WB cells with viral raf oncogene

DNA transfection is commonly used to introduce DNA into eukaryotic cells for the study
of gene expression. A variety of DNA transfection procedures have been developed,
while in this research, the cationic liposome-mediated transfection method was used.
Lipofectin reagent (BRL, cat. No. 82928A) is a 1:1 (w/w) liposome formulation of the
cationic lipid N-(l-(2,3-dioleyloxy) propyl)-N,N,N-trimethylammonium chloride
(DOTMA) and dioleyl phosphatidyl-ethanolamine (DOPE) in membrane-filtered water.

18

This reagent is taken up phagocytically by cells and interacts spontaneously with DNA to
form a lipid-DNA complex. The fusion of the complex with tissue culture cells results in
expression of the DNA.

In this method, WB cells were seeded in three 60-mm tissue culture dishes in 5 ml of D
medium (described above) supplemented with 10% fetal bovine serum. These cells were
incubated at 37°C in 5%-C02 environment until they are 40-50% confluent. For each 60-
mm dish, 10ug of raf plasmid DNA and SOug of lipofectin reagent was diluted to 1.5 ml of
serum free medium separately. 1.5 ml each of the DNA and the lipofectin reagent
dilutions were combined in a polystyrene tube and mixed gently . Then this complex was
added to the dishes after washing the cells twice with 3 ml serum-flee medium. The cells
were incubated for 16 hrs at 37°C in C02 environment. 3 ml of growth medium
supplemented with 10% fetal bovine serum were added to the cells. After 12 hrs growth,
those cells were split into three of 100 mm cell culture dishes and started selection the
following day.

Since the viral raf plasmid DNA contain neomycin resistant gene, G418 (GIBCO , NY )
was used to select the clone which contain viral raf oncogene, 300 ug/ml of G418 was

used for selection.

Scrape-LoadingLDye Transfer Technigue to study GJIC

Cells were grown to confluence in 35 mm plates. The plates were washed with PBS
several times. 2 ml of Luciffer yellow solution (0.5 mg/ml in PBS; Sigma, cat. No.
L0259) was added to the plates and three scrape lines were made on the cell monolayer
with a surgical blade. After 3 minutes to allow dye uptake and transfer at room
temperature, the cells were washed several times with PBS and fixed in 10% phosphate-
buffered formalin. Photomicrographs of the fluorescence images of the dye loaded cells

were taken to determined the extent of dye transfer of the clones.

19

Isolation of total RNA from cells

Total RNA was isolated by the single-step method using Acid Guanidinium Thiocyanate-
Phenol-Chloroform Extraction (Chomczynski, et al. 1987), as described below:

Confluent cultures of cells in 25 cm2 flasks were rinsed with PBS and lysed in 0.75 ml of
solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH7.0; 0.5% sarcosyl,
0.1 M 2—mercaptoethanol) after removing the PBS. The lysate was subsequently
transferred to a 2.2-ml eppendorf tube. Sequentially, 75 ul of 2 M sodium acetate, pH4.0,
0.75 ml of phenol, and 200 ul of chlorofonn-isoamyl alcohol mixture(49:l) were added,
with thorough mixing by votex after the addition of each reagent. Samples were
centrifuged at 14,000 rpm for 15 minutes at room temperature. The aqueous phase was
transferred to a fresh tube, mixed with 1 ml of isopropanol, and placed at -20°C for 1
hour to precipitate RNA. The tubes were centrifuged at 10,000 rpm for 30 minutes at
room temperature, 200 ul of 4 M LiCl was added to the pellet with slowly shaking over a
20-minute period. Sedimentation at 14,000 rpm for 10 minutes was again performed and
the resulting RNA pellet was dissolved in 200 ul of 0.5% SDS. The RN As were extracted
once with 200 ul of chloroform. The aquous phase was precipitated using 20 ul of 3M
sodium acetate, pH5.2 and 400 ul of ethanol. After centrifugation, the resulting RNA was
washed with 75% ethanol and subsequently dissolved in 20-50ul of 0.5% SDS. The

concentration of the RN As were measured by reading OD260.

Northern Blot Analysis
I. Electrophoresis of RNA through gels containing formaldehyde

One gram of agarose was added to 100 ml of DEPC-treated ddHZO and boiled in
microwave until the volume was reduced to about 70 ml. Ten milliliters of 10X MOPS
(3-[N-Morpholino] propane-sulfonic acid) and 18 ml of formaldehyde were then added to

20

the flask to prepare a 1X MOPS, 3%-formaldehyde gel. The gel solution was poured into
a gel tray to make about 5 mm-thick gel. After polymerization in about 1 hour, the
running buffer (1X MOPS, 3% formaldehyde in DEPCddHZO) was poured into an
electrophoresis box to cover the gel with the buffer. Twenty micrograms of each sample
RNA in 20 ul of sample buffer (50% forrnarnide, 1X MOPS, 6% formaldehyde, 1 ul of
tracking dye, 1 ug/ml of Ethidium Bromide) was heated at 68°C for 10 min. and then
chilled on ice . The samples were loaded into wells and electrophoresed at 80 volts for 4
hours, or 10 volts overnight with buffer recirculation

II. Northern Blot

After electrophoresis, the gel was soaked in DEPC-treated ddeO for 45 minutes twice.
A picture of the gel was photographed by using ultraviolet illumination. The gel was
soaked in 20X SSC for 45 minutes with agitation and set up for capillary transfer of RNA
to nylon membrane. The RNAs were transfered from the gel to a nylon membrane
(HybondTM-N, 0.45 micron; Amersham corporation) which was pre-wet in ddHZO and
soaked in 20X SSC. After transfer overnight, the membrane was washed in 5X SSC
briefly and dried in the air for 10 minutes. The membrane then was baked at 80°C in a
vacuum owen(Fisher Scientific) for two hours and exposed to UV light(254 nm) to cross
link the RNA to membrane and prepared for hybridization with 32P—labelled c-DNA

probes.

1H. Hybridization and Autoradiography
(a) Preparation of a- 32P labelled c-DNA probes.

The raf-l gene is 290 bp fragment (Oncor, cat. P2115) and the connexin 43
cDNA was isolated from Blue-G2A plasmid. Probes were made using a random primed
DNA Labelling kit (Molecular Biology, Bochringer mammheim).

Fifty nanograms (18ul) of the DNA was denatured by heating for 10 minutes at 95°C and

subsequently chilled on ice. The following compoents were added to the tube to set up

21

the reaction: 6 ul of dATP,dGTP,dTTP mixture (1:1:1 mixture of each dNTP), 4 ul of
reaction mixture (random hexanucleotides in 10X reaction buffer), 10 ul of [or-32P]dCTP,
50uCi, 3000 Ci/mmde and 2 ul of klenow enzyme. The reaction mixture was incubated
for 1 hour at 37°C and the reaction was terminated by adding 10 ul of 0.2 M
EDTA,pH8.0. The product of this reaction was purified by Bio-spin columns (Bio-Rad
,Cat. No. 732-6006).Before adding the mixture to the column, the column was warmed
to room temperature and was inverted several times to resuspend the settled gel. The top
cap of the column as well as the bottom stopper to allow the excess buffer to drain by
gravity. The column was then placed in a collection tube and centrifuged for 2 minutes in
a horizontal rotor at 1,100Xg. The sample was applied very carefully to the center of the
column, allowing the liquid to drain into the gel bed between successive drops of the
sample. The column containing the applied sample was placed in a collection tube and
centrifuged for 4 minutes at 1,100Xg. The purified labelled-probe in the collection tube
was denatured by heating for 10 minutes at 95°C and chilled on ice. This probe was then
used for hybridization.

(b) Hybridization with labelled c-DNA and autoradiography

The membrane containing RNA(Northem blot) was pre-hybridized in a sealing bag
containing 10 ml of prehybridization buffer (5X SSC, 5X Denhardt's, 0.5% SDS, 250
ug/ml Herring sperm DNA, 5% Dextran sulfate) at 65°C for 1 hour. For hybridization,
the 32P-labeled c-DNA probe was added to the prehybridization buffer and incubation
was continued overnight. The membrane was then washed twice with 2X SSC, 0.1%
SDS for 10 minutes at room temperature and twice with 0.5X SSC, 0.1% SDS for 30
minutes at 65°C. Autoradiographies were done at ~70°C using Kodak X—OMAT and
Kodak X-OMAT regular screen.

After exposure, the film was processed in an automatic X-ray fihn processor (Department

of Radiology, Michigan State University).

22

Western Blot Analysis

I. Isolation Protein from cells

Confluent cultures in 25 cm:2 flask washed with PBS several times and 0.5 ml of lysis
buffer (20% SDS, 0.1 M Tris, pH7.5, 20 mM EDTA, 5% 2-mercaptoethanol and 1 mM
PMSF) was added to lyse the cells. The cell lysate was transferred into 2.2-ml ependrof
tube and sonicated for 30 seconds. Then the protein samples were stored at -20°C.

11. SDS-Polyacrylamide Gel Electrophoresis of Proteins

SDS-PAGE was done according to Laemmli using a discontinuous buffer system (1970).
The apparatirs was bought from Bio-Rad Inc. The glass plates were assembled according
to the manufacturer's instructions. 10 ml (for two gels) of 10% separating gel (4 ml of
ddHZO; 2.5 ml of 1.5 M Tris, pH8.8; 100 ul of 10% SDS; 3.33 ml of Bis:Acrylamide
(0.8:30); 50 ul of 10% Ammonium Persulfate; 2.5 ul of N,N,N,N-
Tetramethylethylenediarnine (TEMED) was poured into the gap between the glass plates
and overlay the acrylamide solution with isopropanol to prevent oxygen from diffusing
into the gel and inhibiting polymerization.

After polymerization was completed (about 30-45 minutes), the isopropanol was poured
off and a clean comb was inserted into the space between two glass plates. 5 ml (for two
gels) of 4% stacking gel (3 ml of ddHZO, 1.3 ml of 0.5 M Tris, pH 6.8, 50 ul of 10%
SDS, 650 ul of Bis-Acrylamide (0.8:30), 25 ul of 10% Ammonium Persulfate, 4 ul of
TEMED) was filled into the spaces of the comb completely.

While the stacking gel was polymerizing, 20 ul of samples were prepared in 1X SDS gel-
loading buffer. After polymerization of the stacking gel, the comb was removed carefirlly
and the gel was mounted in the electrophoresis apparatus. Tris-glycine electrophoresis
bufi‘er (25 mM Tris, 250 mM glycine, 0.1% SDS) was added to the top and bottom
reservoirs and the samples were loaded into the wells using microliter-pipet tips.

Electrophoresis was done at a constant voltage of 100/cm. The gel was run until the

 

23

bromophenol blue reached the bottom of the resolving gel (about 2 hours). The gels
werecarefully removed from the glass plate and used for western blotting.

III. Transfer of Proteins from SDS-Polyacrylamide Gels to Polyvinylidene Difluoride
(PVDF) Membranes.

When the SDS-polyacrylamide gel was approaching the end of its run, one piece of
Irnmobilon PVDF membrane and four pieces of whatman 3MM paper were cut to the
exact size of the SDS-polyacrylamide gel.

The following precautions were taken to ensure the transfer of proteins from the gels to
the PVDF membrane. First, the 3MM paper was put in transfer buffer to allow it to wet
and the PVDF membranes need to be put in methanol first followed by transfer buffer.
Second, a glass pipet was used as a roller to squeeze out any air bubbles between gel and
membrane. Third, the 3MM papers and membrane were the same size as gel, otherwise,
there was a good chance that the overhanging deges of the paper and the filter will touch,
causing a short circuit that will prevent the transfer of protein fi'om the gel.

A voltage of 20 Volt/cm was applied to the gel to transfer overnight. The gel was stained
with 0.1% Coomassie Brilliant blue in methanoleZO/Aceteic Acid (1:1 V/V) while the
membrane was stained with Ponceau S.

IV. Immunoblotting

The non-specific binding sites were blocked by soaking PVDF membranes for 2 hours at
room temperature in phosphate buffered saline (PBS)/3% nonfat dry milk. The
membranes were incubated for overnight at 4°C with anti-mouse raf-l kinase which was
diluted to 3 ug/ml in 5 ml of buffer containing 4% nonfat dry milk, 40 nm Tris and 0.1%
Tween 20. The membranes were then washed four times with the same bufi‘er. The
membrane was incubated with 1/500 dilution of biotinlayted anti-mouse Ig (Species-
specific F(ab') fragment) for 1 hour and followed by washing three times with the same
buffer. The following step was to incubate the membrane with 1:2500 dilution (in 40 mM
tris, 0.1% tween 20, 4% milk and 0.5 M NaCl) of alkalinephosphatase-conjugated

24

streptavidin for one hour. Sequentially, the membranes were washed four times (each 5
minutes) with 20 mM tris, O. 1% tween 20 and 0.5 M NaCl. The last step was to develop
the color reaction at room temperature with 5-bromo-4-Chloro-3-inclolyl phosphate
(BC[P)/nitro blue tetrazolium (NBT) color development solution: One tablet of NET was
dissolved in 30ml of alkaline phosphatase buffer (0.1 M tris, 0.1 M NaCl and 5 mM
MgC12 , pH9.5) and 100ul of BCIP was added to the buffer just before color

development.

Procedure for Anchorage Independent Growth (AIG)

3.3% of Agarose was prepared in PBS and boiled using microwave oven, then was kept
in 45°C waterbath. 2.55 ml of medium (D medium supplemented with 10 % fetal bovine
serum) was warmed up at 39°C and mixed with 0.45 ml of 3.3% agarose solution with a
warm pipet to make 0.5% hard agar layer (0.5%) in 60 mm plate. This plate was put in
incubator to let it solidify about one hour. Then, the soft agar layer (0.33%) with cells
was prepared as follows: 2.2 ml of medium 0.3 ml of 3.3% agarose and 0.5 ml of cells in
medium (1X104 cells/ml, Table 2) were mixed well and were inoculated on top of hard
agar layer (the medium was warmed up in 39°C water bath). After these plates were
incubated at 37°C for three days, 3 ml of liquid medium was added and renewed every
three days. The cells were grown for four weeks and stained with 1 mg/ml of the
tetrazolium salt, 2-(P-iodophenyl)-3-(nitrophenyl)-5-phenyltetrazolium chloride (Sigma 1-
8377) in 0.9% sodium chloride for 24 hrs at 37°C.

 

RESULTS

A. Selection of v-raf transfectant

The construction of v-raf plasmid DNA contains neomycin resistant gene.
Neomycin is a bacterial antibiotic which interfere with prokaryotic ribosomes, while
mammalian cells are not affected by neomycin. The analogue, G418, now available
through Gibco as Geneticin will affect eukaryotic ribosomes. The neoR gene code for a
phosphotransferase which inactivates the G418. Therefore cells which carry the plasmid
and express this resistance marker (v-raf integrated) can survive in selective media. 300
ug/ml of Geneticin (Gibco) was used to select v-raf transfectant. The total number of

clones resulted from the selection was about 20/60mm plate .

B. In vitro Morphology

Among the clones obtained fi'om selection, only one clone was found to be
morphologically transformed (WB-v-raf I). The morphplogy of the other clones were
similar to the untransfected parental rat liver epithelial cells (Figure 2). The transformed
cells showed spindle-shaped morphology and loss of contact inhibition of growth (Figure
3 and Figure 4) . Interestingly, the transformed cells reverted to normal morphology

similar to parental cells after a few passages.

C. Expression of v-raf mRNA

Northern blot analysis of raf transcripts indicated the presence of a 3.3-kilobase
mRNA for endogenous c-raf in each cell line. Whereas only two out of six v-raf-
transfected clones (WB-v-raf A2 and WB-v-raf I) were found to have an additional 8.1-
kilobase band which is v-raf mRNA (Figure 5). It was appeared that the clones which had
v-raf-expression, also had increased level of endogenous c-raf-expression. The WB-v-raf

I cells which lost transformed morphology at latter passage were found to have lose v-raf

25

26

mRNA (Figure 6).

D. Intercellular Communication
(a) Scrape Loading/Dye Transfer

Using the technique of scrape loading/dye transfer, the relative levels of gap
junctional intercellular communication of all v-raf transfectant clones were determined. It
showed that WB-v-raf A2 clone had the same level of communication as parental WB
cells and the plasmid control cells (G418r) (Figure 7). While the WB-v-raf I showed
reduced communication (Figure 7). WB-v-raf I treated with TPA (100 ng/ml) overnight
showed reduced GJIC compared to the same cells without TPA treatment. In the same
experiment, WB-v-raf A2, the parental WB cells and the 64181" plasmid control showed
extensive GJIC (Figure 8).
(b) Expression of connexin 43 mRNA

Northern blot analysis of connexin 43 transcripts indicated the presence of a 3.1
kilobase mRNA in each cell line. The v-raf transfected G418-resistant clone with
transformed morphology (WB-v-raf I) showed the same level of connexin 43 mRNA as
did the wild type WB cells (Figure 9). It suggested that the reduction of intercellular

communication might be due to posttranslational level.

E. Expression of raf p74 protein kinase in raf transfectant

Western blot analysis of v-raf transfectants using monoclonal anti-c-raf antibody
showed that only the protein isolated from early passage of the morphologically
transformed clone (WB-v-raf I) reacted with the antibody and showed raf p74 protein

specific band (Figure 10).

F. Anchorage Independent Growth (AIG)
After the cells plated in soft agar for three weeks, the WB-v-raf I (with

transformed morphology and expression of v-raf mRN A and protein) and WB-v-raf A2

(with normal morphology and expression of v-raf mRNA) were found to be able to grow

27

in soft agar, while the wild type WB, WB-neor cells, WB-v-raf A1 (a G418; clone without
the expression of v-raf mRNA) cells can not grow (Figure 11). It is noticeable that WB-v-
raf I clone has higher frequencies of AIG+ colonies than WB-v-raf A2. The latter,

however, was found to form larger colonies (Figure 12).

28

XIII

SA
LTR R50 my-

43.! All! A "I"

me—m—e—H

Figure 1. Construction of v-raf in EHneo plasmid DNA. Neo-vectors contain the TnS
neomycin-resistance gene, which is expressed as a subgenomic RNA afier splicing at an
RSV promoter inserted in flour of it. Special features of the constructs are as follows: SD
and SA are splice donor and acceptor sites,respectively;the asterisk indicates the presence

of the myristilation site. (From U.R.Rapp et al. Cold Spring Harbor Symposia on
Quantitative Biology L111: 173- 183,1988).

29

 

 

Figure 2. Single clone resulted from selection
A. This clone showed same morphology as wild type(100X)
B. This clone showed morphological transformation(100X)

3O

. fl. ,, . ,- f /
- I 41- {9" Q .
l P,“ . ~ . '
T v JIM - 11L“

Figure 3. Morphological features of WB-v-raf I clone.

 

31

 

 

 

Figure 4. Foci formed by WB-v-raf clone which showed loss of contact inhibition.

32

N
NCO?
*NE<<<-—u—I-'-o
<<hu—g_u-u-u-F>i
moussessswe
owllllllol
322>>>>>>m>
Kb

8.0— . v-raf

3.3— ..."- .

c-raf

 

—18 S
—285

 

Figure 5. Northern blot analysis of v-raf transcripts in the various cell lines. Twenty

micrograms of total RNA were separated on a 1% agarose/2.2 M formaldehyde gel,
blotted onto nylon, and hybridize with 32P-labeled v—raf probes.

 

33

:: : x m
E 2 < LL LI. IL
;;&822
Kb
8.0 — —v-raf
3'3 _ . . U . . —c-rof
—ies
—2es

 

Figure 6. Northern blot analysis of v-raf transcripts in WB-v-raf I cells. Lane 1. WB-v-
rafI transformed cells. Lane 2. WB-v-raf I revertant cells. Lane 3. WB-v-raf I cells
treated with lOOng/ml of TPA for 48 hrs. Lane 4. WB-v-rafl cells treated with lOOnyml
of EGF for 48 hrs. Lane 5. WB-v- rafl cells treated with lOOng/ml of TGF-a for 48 hrs.
Lane 6. WB-v-rafll cells treated with 1 ng/ml of TGF-B for 48 hrs.

 

34

 

 

 

Figure 7(a). Gap junctional mediated dye transfer detected by Scrape-Loading/Dye
ransfer. '

A. Phase-contrast micrograph of WB-F344 cells.

B. Fluorescent micrograph showed Lucifer Yellow transfer to contiguous cells.

 

35

1,. \ ‘ " ~4_
I I ' 1 K, .‘ ‘(b ,
J .a ‘ I " ‘,. ‘ ‘. -' '
(P- . ' .‘ v 3"“ e' \n‘ ‘
I . e:- e. ', . . g '
". , _ —-.. 1.‘ . . '2 .
. .1 . .I ,. , ..
_ - . . , 1‘-
x, , ’. \ ".‘f. 1‘
‘. , o ‘ "'—' I
. I .
~ . ~ . ~,..,.‘.J. .’
‘ ,'. '5; f.“
- ' - " s
e I. "
. s 4 1‘ O
1" ‘.— I’-
. at

 

Figure 7(b). Gap junctional mediated dye transfer in pSVneo transfected WB cells.

 

 

 

36

 

Figure 7(c). Gap junctional mediated dye transfer in WB-v-raf Al cells.

 

 

37

 

 

 

 

Figure 7(d). Gap junctional mediated dye transfer in WB-v-raf A2 cells.

 

38

u
\
C
t
. a
.,
' 0
‘ 4' v‘ o
I
I
I' ' ,
4 .
0‘ I‘ o
\ t
f s s
r
I, - .-
0 -
A- '
‘ ‘ .
I
I
e I a
C
. . ..
.- o
r

 

Figure 7(c). Gap junctional mediated dye transfer in WB-v-raf A3 cells.

 

 

39

 

Figure 7(1).

Gap junctional mediated dye transfer in WB-v-raf A4 cells.

4O

.- ‘ - - v r
t ,‘ l , .' . .‘
.. . “ ., 1‘ I' --.
V- - " ‘ . t o 1"“ , ‘-
’ ( “.‘ ‘ . ..
. A; I p
I ‘19 " ~ \ I r ‘
. 4 g I. \ ' a ' ‘
. ‘ g ‘1. \ I. T r
. O. 'V‘ - ‘, .. a
a \ ‘ ‘ ' ‘ -
a ‘. I . I .
. a I ‘. “ v ‘. h.” e;‘ g.
‘ "‘ I 0‘ - b . ’ t .t T ‘ ‘
,. I I'i" I ' _- ~
' . . .v '. ,.t |' .
u . I
i .0 l g f’ ‘ _ e ._ ‘
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Figure 7(g). Gap junctional mediated dye transfer in WB-v-raf I cells.

 

41

   
 
 
  
 
 
    
    

 
 
  

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Figure 8. Gap junctional mediated dye transfer. Cells were treated with 100 ng/ml of
TPA for 24 hrs. A. WB parental cells B. pSVneo plasmid control cells C. WB-v-raf
A2 cells D. WB-v-raf I cells.

 

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Figure 9. Northern blot analysis of connexin 43 transcript in the various cell lines.

Twenty micrograms of total RNA were s arated on a 1% agarose/2.2 M formaldehyde

e
gel, blotted onto nylon, and hybridize with sz-labeled cx43 cDNA probes.

43

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Figure 10. Western blot analysis of proteins in cells transfected with v-raf and pSV2neo.
Total lysates of cells were separated by SDS-PAGE, transferred to PVDF membrane, and
probed with anti-raf kinase. Bands were visualized with alkaline phosphatase.

44

 

Figure 11. Anchorage independent growth (AIG) of the various cell lines. A-l: WB-v-
raf A1 clone A-2: WB-v-raf A2 clone A-3: WB-v-rafl clone B-l: WB parental cells B-2:

pSVneo plasmid control B-3: MCF7 positive control cells.

 

 

Figure 12(a). Anchorage-independent growth (AIG) of the various cell lines. Five
thousand of cells were grown in 0.33% soft agar layer. Colonies were stained with
lmg/ml of 2-(p- iodophenyl)-3-(nitrophenyl)-5-phenyltetrazolium chloride in 0.9% NaCl
for 24 hours at 37°C. A.MCF-7 (positive control,40X) A'.MCF-7(positive control,100X)
B.WB parental cells(4OX) B'.WB(100X) C.pSVneo(plasmid control,40X)
C'.pSVneo(100X)

46

 

 

Figure 12(b). AIG of the various cell lines (cont). D.WB-v-raf Al(40X) D'.WB-v-raf
Al(lOOX) B.WB-v-raf A2(40X) B'.WB-v-raf A2(100X) F .WB-v-raf I(40X) F'.WB-v-raf
I(100X). '

DISCUSSION

Several results obtained fi'om this research revealed that the integration and
expression of v-raf in WB-F 344 cells resulted in their neoplastic transformation and
tumorigenicity. First, the v-raf transfected clone, WB-v-raf I, had transformed
morphology which is spindle-shaped. The cells expressed both v-raf mRNA and raf
protein kinase in northern and Western blot analyses respectively. Second, these cells
showed the ability of anchorage-independent growth. Such an ability is normally, but not
always, indicate of tumorigenicity. Third, the WB-v-raf I cells were found to revert to
normal morphology after a few passages and simultaneously lost the v-raf mRNA. In
other words, the expression of v-raf mRNA and transformed morphology seems to be-
reversible and correlated. These results are evidences that v-raf is responsible for
neoplastic transformation of the WB-F344 cells. This finding is consistent with earlier
observation done by Dr. Worland although the experiment was done with different cell
line (Worland et al.1990). Since v-raf is the oncogenic form of cellular raf oncogene, this
conclusion is in agreement with the finding that the activation of c-raf proto-oncogene can
lead to transformation of rat liver epithelial cells (Heidecker et al. 1990).

It has been suggested that neoplastic transformation and tumorigenicity normally
require the cooperation of at least two oncogenes. While this is true for primary cells
which require an immortalization step prior to transformation, this proposal may hold true
for already immortalized cell lines, since the latter have already escaped differentiation
owing to the expression of certain oncogenes, such as c-myc. For example, Trosko et al
have reported that WB-cells or RLE cells transfected with v-Ha-ras were neoplastically
transformed and were tumorigenic. Familiarly, many laboratories have reported
transformation of NIH 3T3 cells upon transfection with a single oncogene. More recently

cells transfected with v-raf oncogene were also shown to be neoplastically transformed

47

 

 

 

48

(W orland et al 1990). In all of these observation and that of the present study it is
probable that all the transformed clones may also express a cooperating oncogene. In
many such instances v-myc or c-myc has been implicated as the cooperating oncogene.

In the experiment done by Worland (worland et al. 1990) showed that the level of
v-raf expression in those v-raf transduced clones did not directly correlate with the degree
of the transformation or tumorigenicity since two v—raf transduced cell lines exhibited
marked differences in tumorigenicity despite similar levels of v-raf expression. Therefore,
they concluded although v-raf expression is presumably the initiating event in a process
that results in transformation of these cells, other unknown events are required for their
progression to a highly tumorigenic phenotype. In the present study, similar results were
obtained with WB-v-raf transfected cells. Unlike the WB-v-raf I clone, the other clone,
WB-v-raf A2, which had the same level of v-raf mRN A expression exhibited normal
morphology as parental cells. Anchorage independent growth (AIG) assay indicated that
WB-v-raf A2 cells were capable of proliferating in soft agar with fewer numbers of
colonies than the WB-v-raf I did. In addition, unlike in the case of WB-v-raf I cells raf
protein kinase was not detectable in WB-raf A2 cells in Western blot analyses. These
finding indicate that the level of raf protein kinase played significant role in neoplastic
transformation. This assumption is supported by the observation that colonies from the
soft agar expressed transformed morphology when plated on plastic dishes. However, we
do not know if these cells also express the v-raf protein.

In earlier study done by Worland and his associate (Worland et al.1990), it was
found that RLE (rat liver epithelial) cells transformed with the v-raf/v-myc combination
were capable of anchorage-independent growth in soft agar, while those cells transformed
with v-raf alone were not (W orland et al. 1990). In contrast, the WB cells transformed
with v-raf alone (WB-v-rafl) can form colonies in soft agar in our study. It was found that
the most consistent association between in vitro phenotype and tumorigenesis was the

ability of the cells to form colonies in soft agar, so the transformed clone might be

49

tumorigenic. Carcinogenesis is a complex process by which a normal cell undergoes
multistep changes toward transformation. This conversion is of a multistep nature as
determined by studies of tumor development and progression both in human, as well as in
experimental animals (Pitot et al. 1981). ‘An operational staging assumes three major
phases of carcinogenesis namely initiation, promotion and progression (Boutwell 1974,
Carins 1975). Thus the immortalized cell line, WB-F344, transfected with v-raf is a good
model for tumor progression and it allows in vitro study of genotypic and phenotypic
changes accompanying tumor progression.

In this study, it has also been shown that v-raf transfected cells have significantly

reduced levels of GJIC as measured by a dye transfer technique. This reduction was more
pronouced in WB-v-raf I clone than in the others. Downregulation of GJIC has been
implicated as one of the control events contributing to the promotion and progression of
tumorigenesis. Many cell lines transformed by oncogenes such as src, ras,neu were found
to be unable to communicate either homologously and/or heterologously.
Nicolson et a1 (1988) have suggested that highly metastatic tumorigenic cells fail to
communicate even homologously while less metastatic cells may communicate
homologously but not heterologously (with normal cells). Although several of the WB-v-
raf clones isolated in the present study still expressed high levels of homologous
communication, it is possible that they may not be able to show heterologous
communication and therefore can still be tumorigenic. In the current study we also found
that the raf transformed cells express CX43 gene similar to the parental cell line.

Another significant observation in the present study was the response of the v-raf
transfected clones to the tumor promoting phorbol ester, TPA. Oh et al (1989) reported
that in the parental WB-F344 cells, TPA (10 ug/ml) causes only a transient inhibition of
GJIC. Communication competence of the TPA treated cells reverts to normal level after
6-8 hrs inspite of the continued presence of TPA. However, these cells were refi'actory to

an additional treatment with TPA and GJIC was unafi‘ected. These authors showed that

 

 

 

50

the calcium and phospholipid-dependent protein kinase C was involved in the loss of
communication among those cells upon TPA-treatment. Presumably gap junction proteins
are subtracts for phosphorylation by PKC. It has also been suggested that cells continually
exposed to TPA became desensitized to this chemical due to the dissociation (proteolytic
cleavage) of PKC from the plasma membrane. In contrast to the transient loss of GJIC in
TPA-treated parental WB-cells, in the v-raf transfectant TPA's effect on GJIC was
sustained for at least up to 24 hrs. This observation suggests that v-raf transfection may
have resulted in the sustained activation of PKC. The mechanism for this change needs to

be investigated.

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