1M

 
 

 

  
 

 
 
 

 

 
 
 

 

 

 
 

 
 
 
 

 

 

 

 

 

 
 

 
 
 

 

 
 

 
 

 

 

 

 
 

 

 
 
 
 
 
 
 
 
 

 

 
 
 
 
 
 
 
 
 

 

 

 
 
 
 
 
 
 
 
 

 

 

 
 

 

 
 

 

 
 
 
 

 
 

 
 

 
 

 
 
 

 

 

 

 
 

 

 
 
 

 

 
 

 

 

 

 

 

 
 
 

, V . V V .-V . , . , , .. o, V .
V V , .V .V , . , . . V . , V V . V V V
,. . , V . ,3 ,y ,, V..V . VV .. . : .V V
. , . ... V. ., . . .
u . V .. V. V . _. . V VV .
A , . V V . V V. VVV V . V , V V .
. V, V V .. , , . V V V, V , , V
I , . . . V - , .. . , .VVV, V V . V, . \.
c V , V V, . V ,. .V ... V, . V.. .. V . V V . V .
a ,. . , . V. . V. , ..V ,. . , . V V , V V . V . .. V..V
. V, . , V .. ....,.V.. .. .. V. V . . .V V, . V . V V V V . . n , V
. . . . V . V . V .. V V. ,V . .. V . VV .‘V . . V.. . V V . . V x
.. V V VV. .. V . . ,V,.. V.V V . .. . , V V V V. . V
VV V . a. ... V . . VV . V . . V .V V . . V .. . V . . .V V .V .V... ..
, V . . .. . . ., V .V. . ., . , . . V . , .. V, V . V ..V... V
v . V V V . , VV . ,V...VV V .V V V . V . .V .1
. V , ..V.. V .V V V ..V V . .V . V . V .V V V . n r. .V V
V .V . ., . . V V VV . V. V. VV V
. . V . .V . V . . .. , .. V VVnV . . . V V .V
. V. V. . V. . VV. . . . .V.. VV V VV . . V V . v . ,. .4 . .
, h . . V. .. ,V V. . V ., V , ... .V. ,.s . u ,.. . . . . ., .

 

Ti-ififitfi

MICHIGAN STATE

m u: it I! llltllllllllllil’llllll

_' 3 1293 00917 5559

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

thesis entitled
Characterization of Gap Junction Gene
Impression in Normal and 'I'unorigenic
Human Breast Epithelial Cells

presented by

M9 -'IYng Yang

has been accepted towards fulfillment
of the requirements for

 

 

MaSter degree in SCienCe

by

The Program of Clinical Laboratory Science
College of Natural Science

Co" 361m ((am
c < j 5»—

Major professor

 

Dec. 15, 1992
Date

 

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

1.333;...“447
Michigan State
University

 

1

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

DATE DUE DATE DUE DATE DUE
'niUL~—-+-l9967
\k/

4|
fl

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
omens-9.1

CHARACTERIZATION OF GAP JUNCTION
GENE EXPRESSION
IN NORMAL AND TUMORIGENIC
HUMAN BREAST EPITHELIAL CELLS

By

Tyne-Tyne Yang

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

CHARACTERIZATION OF GAP JUNCTION GENE EXPRESSION IN NORMAL
AND TUMORIGENIC HUMAN BREAST EPITHELIAL CELLS

by
Tyns-Tyng Yang

Recently in our laboratory, we have identified two types of normal human breast
epithelial cells, one is deficient in gap junctional intercellular communication (GJIC)
(Type-I) and the other is competent in GJIC (Type-II). Furthermore, Type-I cells were
found to express luminal epithelial cell markers (i.e. epithelial membrane antigen, EMA,
and cytokeratin 18) and to be induced to difi‘erentiate by cholera toxin into Type-H cells
which express myoepithelial cell-specific antigen (i.e. cytokeratin 14). The primary
objective of this thesis is to characterize the expression of different gap junction (GJ)
genes in these two types of cells and three representative breast cancer cell lines.

The results indicate that Type-H cells express cx43 and cx26 but not cx32 6]
genes by Northern blot analysis. In contrast, Type-I cells and GJIC-deficient breast
carcinoma cell lines, MCF-7 and T47D, did not express any of these GJ genes. The GJIC
competent carcinoma-sarcoma cell line, HSOS78T, expressed cx43 but not cx26 or cx32
genes. In Western blot analyses, all these three breast cancer cell lines appear to contain
low levels of cx43 protein. Immunofluorescence studies, however, showed that only
HSOS78T cells displayed punctate fluorescence along borders of cell-cell contact.

Experiments were also carried out to restore GJIC in MCF-7 cells by transfection
with an amplifiable plasmid carrying a mutated dihydrofolate reductase gene and the
cloned cDNA for rat cx26, cx32 or cx43. The results indicate that GJIC can be restored
by all of the three GJ genes. After extended growth, most of these transfectants, however,
lost the ability to perform GJIC. Northern blot analysis showed that these transfectants

express mRN A of transfected rat GI genes.

To:
My Parents and Grandmothers
For Their Love and Support

ACKNOWLEDGMENTS

I wish to express my gratitude to my major professor, Dr. Chia-Cheng Chang and
Dr. Burra V. Madhukar for their guidance, support and encouragement. They have
provided me with the intellectual and technical training needed to accomplish my thesis
research in just a little more than a year.

I am deeply appreciative of the valuable input provided by the other members of
my committee, Drs. John Gerlach and Douglas Estry. Also, I wish to express my
gratitude to the faculty of the clinical laboratory science program for their time and effort
in my training. My sincere thanks also due to Dr. James E. Trosko for his valuable advice.
I wish to thank Dr. Yuh-Shan Jou for providing me the plasmids carrying various gap
junction cDNA used in this study. I am deeply indebted to Drs. Emmanuel Dupont and
Diane Matesic for guidance and assistance in various molecular biology techniques. My
appreciation also goes to Drs. Chien-Yuan Kao and Koichiro Nomata for their fi‘iendship
and encouragement. In addition, I like to acknowledge Ming-Shia Yeh for his valuable
help in processing the thesis.

Finally, I wish to extend my deepest appreciation to my parents, grandmothers,

sisters and brother for their encouragement, support and love.

O
I"

TABLE OF CONTENTS

INTRODUCTION ....................................................................................................... 1
LITERATURE REVIEW ............................................................................................ 3
General Properties of Gap junctions ...................................................................... 3
Structure of Gap Junction ..................................................................................... 4
The Role of GJIC in Normal Cells ........................................................................ 5
I. Embryogenesis and Development ............................................................. 5
11. Maintenance and Growth of Cells and Organs ......................................... 5
III. Synchrony of Contraction ...................................................................... 6
IV. Coordination of Response ...................................................................... 7
Regulation of Intercellular Communication ........................................................... 8
I. Intracellular Modulation of Junctional Permeability ................................... 8
II. Endogenous Modulation of Junctional Permeability ................................. 10
III. Exogenous Modulation of Junctional Permeability ................................. 11
GJIC and Expression of Gap Junction Genes in Normal and Tumorigenic
Human Breast Epithelial Cells ............................................................................... 13
MATERIAL AND METHOD ..................................................................................... 15
Cell Culture Medium ............................................................................................ 15
Cells ..................................................................................................................... l6
Transfection and Selection of MCF-7 WITH CONNEXIN GENES ...................... 17
Measurement ofGJIC ........................................................... 18
Isolation of Total RNA from Cells ........................................................................ 18
Northern Blot Analysis ......................................................................................... 19
I. Electrophoresis of RNA in Gel Containing Formaldehyde ......................... 19

11. Northern Blot .......................................................................................... 20

III. Hybridization and Autoradiography ........................................................ 20
Immunofluorescence staining of Gap Junctions ..................................................... 21
Protein Isolation and Quantification of Protein ...................................................... 22
Western Blot Analysis .......................................................................................... 22

I. SDS-Polyacrylamide Gel Electrophoresis of Protein ................................ 22

II. Transfer of Protein from SDS-Polyacrylamide Gel to Solid Support ........ 24

III. Irnmunoblotting ..................................................................................... 24
Anchorage of Independent Growth (AIG) ............................................................. 25

RESULTS ................................................................................................................... 26
I. GJIC and Gap Junction Gene Expression in Normal Human Breast
Epithelial Cells ...................................................................................................... 26

a. Ability of Two Types of Normal Human Breast Epithelial Cells to

Perform GJIC .............................................................................................. 26

b. Expression of mRNA for Gap Junction Genes in Two Types of

Normal Human Breast Epithelial Cells ......................................................... 26

c. Immunofluorescence Staining of Gap Junction ......................................... 34

II. GJIC and Gap Junction Gene Expression in Three Breast Cancer Cell

Lines .................................................................................................................... 34
a. Ability to Perform GJIC ........................................................................... 34
c. Expression of Connexin Proteins .............................................................. 37
d. Immunofluorescence Staining o f Gap Junction ........................................ 37

III. Transfection of GJIC-Deficient MCF~7 Cells with Cloned cDNA for Rat

Connexin Genes .................................................................................................... 40
a. Selection of Transfectants ........................................................................ 40
b. Gap-Junction Mediated Dye Transfer ....................................................... 40

vi

c. Gap Junction Gene Expression ................................................................. 45

c. Anchorage Independent Growth (AIG) .................................................... 45
DISCUSSION ............................................................................................................. 50
REFERENCES ............................................................................................................ 54

vii

INTRODUCTION

Gap junction channels provide a hydrophilic pathway for exchange of ions,
metabolites and second messenger molecules between coupled cells. This kind of
junctional communication has been implicated in the regulation of tissue homeostasis, cell
grth and differentiation and other functions such as synchrony of contraction
(Vitkauskas et aI.,1985). On the other hand, loss of junctional intercellular
communication is suggested to play a role in carcinogenesis especially in promotion phases
by facilitating the clonal amplification of initiated cells by releasing them from the
suppressive control of surrounding normal cells (Yotti et al., 1979; Yamasaki, 1990).

Breast cancer is the leading malignancy of American women. It was estimated that
there were 150,000 new cases and 44,000 deaths from female breast carcinoma in 1990
(Silberberg et al., 1990). Almost all of these cancers are adenocarcinomas and, of these
tumors, the vast majority are ductal adenocarcinoma of luminal epithelial cell origin (Page
and Anderson, 1987). Normal human breast epithelial cells are capable of performing
gap-junctional intercellular communication (GJIC) (Fentiman, 1980; Eldridge et al.,l989)
and have been reported to express gap junction genes for Cx26 and Cx43 (Tomesetto et
al.,l99l; Lee et al., 1992). In contrast, most breast carcinoma cell lines were found to be
deficient in GJIC (F entiman et al., 1979) with the exception of one report (Eldridge et al.,
1989) which showed that non-immortal cells derived from breast tumors were proficient in
GJIC whereas in 211m chemically immortalized cell lines were deficient in GJIC.

Recently in our laboratory we were able to culture two types of human breast
epithelial cells derived from reduction mammoplasty: one is deficient in GJIC (Type-I), the
other is capable of performing GJIC (Type-H). Furthermore, Type-I cells were found to
express luminal epithelial cell specific antigens (i.e. epithelial membrane antigen, EMA,

and cytokeratin 18) and to be induced to differentiate into Type-II cells which express

cytokeratin 14, a myoepithelial cell marker. The primary objective of this thesis research
is to determine which gap junction genes are expressed in these two types of cells.

In addition, since MCF—7 breast carcinoma cells are deficient in GJIC (Fentiman
and Taylor-Papadimitriou, 1977) and since transfection of GJIC-deficient human
hepatoma cells with gap junction genes has been shown to retard tumor growth “m ,\_ri_v_q in
conjunction with restoration of GJIC (Eghbali et al., 1990), experiments were carried out
to determine if GJIC in MCF-7 can be restored by the transfection of three rat gap
junction genes (i.e. Cx26, Cx32 and Cx43) cloned in mammalian expressible and

amplifiable vectors in our laboratory. These results are also presented in this thesis.

LITERATURE REVIEW

general Properties of Gap junctions

Gap junctions are composed of a cluster of membrane proteins which form
intercellular channels facilitating the transfer of molecules between coupled cells. This
kind of cell-cell communication was first demonstrated in parallel structural and electro-
physiological studies of synapse between neurons underlying the escape mechanism of
crayfish (Furshpan & Potter, 1959). In addition to the passage of ions carrying electric
impulse, physiological studies have shown that gap junctions also provide a pathway for
the transfer of small molecules, such as nucleotides, nutrients, cAMP other regulatory
molecules, between adjacent cells. Moreover, the net transfer of molecules and ions
through junctional channels is a passive process, depending only on electrochemical
gradients (Sheridan, 1987). Experiments with fluorescent tracer molecules suggested that
the channel can accommodate molecules of up to 10-15 kDa (Alberts et al., 1989).

The first gap junctional protein cDNA was isolated from rat liver expression library
by using an antibody against isolated gap junctions in 1986 (Paul,1986). To date, the
complete cDNA corresponding to three major different gap junction proteins have been
cloned: the cDNA codes for a 32 kD protein (connexin 32) from rat liver (Paul, 1986), a
26 kD protein (connexin 26) from rat liver (Zhang & Nicholson, 1989), and a cDNA
codes for a 43 kD protein (connexin 43) from rat heart (Beyer et al.,l988). Furthermore,
several corresponding connexins have been found in human, chicken, and the African frog,
Xenopus (Bennett et al., 1991).

This gap junction-mediated intercellular communication (GJIC) is believed to play
an important role in normal tissue grth and development (Loewenstein, 1981). The
disruption of junctional communication at early stages was shown to result in specific

developmental defects in Xenopus (Warner et al., 1984). GJIC may also be required for

synchronized contraction and metabolic coordination of cells in a tissue (Vitkauskas and
Canellakis, 1985). In addition, GJIC has been proposed to be involved in the promotion
phase of carcinogenesis. The evidence came fi'om studies which showed 12-O-
tetradecanoyl phorbol-13-acetate (TPA) and other tumor promoters inhibited GJIC (Yotti
et al., 1979; Murray and Fitzgerald, 1979; Trosko and Chang, 1988). By blockage of
GJIC, TPA and other tumor promoters might interdict the flow of growth-inhibitory
signals from normal cells to an initiated neighbor, thus giving the latter an opportunity to
proliferate (Yotti at al., 1979).

§tructure of Gap Junction

The basic structural unit of the gap junction channel is the connexon, comprised of
six protein subunits (connexins) which are organized as a regular hexagonal lattice. Two
connexons of coupled cells form a junctional channel providing a hydrophilic pathway to
connect the cytoplasms of these two neighboring cells. The gap junction hemichannel can
open or close by a lateral circular movement of connexin proteins. The firnctional pore
size of the G] is 1.5 nm in diameter and the interacting plasma membranes of this junction
are separated by a "gap" of 2 to 4 nm (Alberts et al., 1989).

From the information of gap junction protein sequence and studies using
monoclonal antibodies, the connexin topology has been proposed. While amino termini
and carboxy termini are located inside the cytoplasm, the protein traverses through the
membrane four times to form an M shape with two extracellular loops and one
cytoplasmic loop (Unwin and Zamphigi, 1980; Dermietzel et al., 1990). The cytoplasmic
regions are more variable among different connexins within a species and may confer
firnctional and regulational differences such as the presence or absence of phosphorylation
sites for CAMP—dependent kinase, tyrosine kinase or protein kinase C (Hertzberg and
Johnson, 1988). However, the sequences of comparable isoforms are extremely well

conserved across species. The striking feature of the two extracellular loops is the

presence of three cysteines in each. These particular amino acids may form disulfide
bonds with cysteines in adjacent hemichannels, contributing to the formation of the

intercellular junction (Bennett, 1991).

The Role of GJIC in Normal Cells

1. Embryogenesis and Development

The role of gap junction-mediated intercellular communication in embryogenesis
was first experimentally studied in embryos of lower vertebrates. Gap junctions were
first observed at the 8-cell stage of newt (Ito and Hori, 1966) and Xenopus (Slack and
Palmer, 1969) when electrical coupling was detected. Warner, et al. (1984) microinjected
a polyclonal antibody against purified rat liver-derived gap junction protein into one
identified cell of the 8-cell stage Xenopus embryo. This treatment selectively disrupts
both dye transfer and electrical coupling between the progeny cells. Moreover, it led to
specific developmental defects of the injected embryo, as indicated by the absence of eye
and deformed shape of the head ( Warner et al., 1984).

In mammals, GJIC has been monitored in pre- and postimplantation mouse
embryos. The development of regional pattern was observed, which led to the formation
of "communication compartments". Cells in a given compartment are well coupled with
one another, whereas dye coupling is severely restricted between various compartments
(Lo, 1980). This type of compartmentation was suggested to represent early tissue
determination (Welsch, F., 1990).

11. Maintenance and Growth of Cells and Organs

Normal cells of most adult tissues exchange metabolites through gap junctions.
Therefore, they share a very similar internal environment with other cells of that organ
(Vitkauskas and Canellakis, 1985). GJIC was first proposed by Loewenstein (1979) to

have an important role in grth control. An example of total metabolic dependence

upon neighboring cells is the lens of the eye. The eye lens do not contact with the blood
system directly but they are nourished totally by junctional communication with the
neighboring epithelial cells which are in contact with the blood supply (Rae, 1982).
According to the metabolic cooperation experiments, Gilula et al. (1978) reported that the
uridine which is used for RNA synthesis by the oocyte was derived from its neighboring
cumulus cells.

By using communication-competent mutant cells (i.e. auxotrophs for a metabolite)
in in \_Ii_t_r_g studies, it was demonstrated that auxotrophic mutants can survive in a medium
lacking their essential metabolite if they are placed in contact with communication-
competent normal cells (Subak-Sharp et al., 1969; Hertzberg and Gilula, 1981). Similarly,
communication-competent cells that are sensitive to a drug do not die in the medium

containing the dnrg, if they are cocultured with communication-competent drug-resistant

cells (Ledbetter and Lubin, 1979).

III. Synchrony of Contraction

It is important and essential for some organs to fiinction in synchrony. In the
heart, individual myocytes are connected to each other by extensive arrays of gap
junctions (De Mello, 1982). Recently, an enzymatic isolation of viable heart cells has been
developed and it allowed electrophysiological study of cell pairs (Kameyame, 1983). By
taking the advantage of that method, all published studies have confirmed that electrical
transmission in the heart muscles is by way of low resistance couplings (Metzger and
Weingart, 1985; White et al., 1985; Noma and Tsuboi, 1987). Therefore, GJIC provides
an efi'ective means for electrical synchronization of the cardiac fibers.

Synchrony of contraction is also characteristic of the uterus during parturition.
Gap junctions in these tissues from nondelivering animals are small and rare. However,
GJs in the myometrium increase in size and numbers one day before parturition and are at

their highest peak during delivery (Garfield, 1984). In delivering tissues, each cell has

about 1000 615 with an average size of about 0.2 pm in diameter (Garfield et al., 1980).
Afterward, the junctions decline sharply 24 hr following delivery and disappear until
nonpregnancy levels are again attained (Garfield, 1984). It was suggested that increased
electrical coupling in the myometrium at term facilitates improved current flow, permitting
synchronization and coordination of electrical and contractile activities in the individual

muscle fibers and normal delivery of the fetus(es) (Garfield et al., 1989).

IV. Coordination of Response

In multicellular organs, cells can communicate with each other by various
mechanisms, including the formation of low-resistance junctions. Intercellular
communication via gap junctions allows some tissues to respond in a coordinated manner
to hormones or growth factors. One example is the in M study of dye coupling among
cells in prolactin stimulated rat pancreatic islets. B cells of the isle of Langerhans, which
produce insulin, are interconnected by gap junctions with a limited number of their
surrounding cells of difi‘erent fimction. While prolactin elevated in serum, under the
condition of hyperinsulinemia, the B cells increase their number of gap junctions by
approximately 2-fold and the extent of intercellular communication with their neighboring
cells by as much as 10 to 20-fold (Michaels, 1982).

In another study Lawrence et al. (1978) cocultured rat ovarian granulosa cells and
mouse myocardial cells. Following exposure of the cocultures to epinephrine, a hormone
specific for myocardial cells, not only did the heart beat become faster, but the ovarian
granulosa cells also responded by releasing luteinizing hormone. On the other hand, upon
stimulating the coculture with a hormone specific for ovarain granulosa cells, follicle-
stimulating hormone, the myocardial cell also showed a response. These studies suggest
that cross-stimulation might result from intercellular communication of a common

mediator. It means that when a hormone stimulates a cell within a target tissue, a

coordinated response by other cells within the population might be achieved by the gap

junctional transfer of low-molecular weight mediators (Lawrence et al. 197 8).

Regulation of Intercellular Communication

The rate of gap junctional communication is proportional to junctional area,
namely the number of connexon pairs (Sheridan, 1973). However, gap junctions are

dynamic structures, which can be modulated by many endogenous and exogenous factors.

I. Intracellular Modulation of Junctional Permeability
a. Calcium

Intracellular injection of calcium solutions into cells of the salivary gland of the
insect Chironomous decreased both electrical and dye coupling. This condition can be
reversed by lowering calcium concentration (Rose et al., 1977). By using the calcium-
sensitive compound, aequorin, as a probe, Rose and Loewenstein showed that calcium
levels need to become elevated only in the region of the junction of the Chironomous cells
to decrease the transfer (Rose and Loewenstein, 1976).

Calcium is considered an important cell "decoupler" under pathological conditions
(De Mello, 1987). For instance, when heart fibers are damaged, a high resistance barrier
is immediately established near the damaged area protecting the fibers from further
damage. It is assumed that the movement of Ca++ through the damaged cells increases
the resistance of the gap junctions in neighboring cells (De mello,1972). This is described
as a "healing-over" phenomenon which appears to be a homeostatic mechanism by which
undamaged cells are protected from the harmful effect (Vitkauskas and Canellakis, 1985).

The calcium down-regulated GJIC may be mediated by protein kinase C, a

calcium-dependent protein kinase (Murray and Gainer, 1989).

b. pH

Decreases in intracellular pH have been associated with reversible uncoupling in a
variety of cells. When embryonic cells of Xenopus were exposed to 100% C02, the
intracellular pH was reduced fi'om 7.7 to 6.4, the membrane potential was decreased and
the electrical coupling was abolished (Turin et al., 1977). In cardiac Purkinje fibers the
intracellular injection of H+ also causes cell decoupling (De Mello, 1980). Most of the
time, lowering the pH causes a corresponding increase in the Cai'+ concentration which is
known to decrease electrical coupling (Rose and Rick, 197 8).
c. Voltage

In experiments involving the rectifying synapse of crayfish, depolarization on the
presynaptic side was found to increase junctional conductance (Furshpan et al, 1959). In
amphibian blastomeres, transjunctional voltage applied from either side decrease the
junctional conductance (Harris et al., 1981).
d. Cyclic adenosine 3 ',5 '-monoplrosplrate

The junctional permeability of various cultured mammalian cell types depends on
the concentration of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) (Wiener
and Loewenstein, 1983). The permeability rises when the cells are exposed to exogenous
cAMP or when they are stimulated to synthesize cAMP by choleragen or hormones. The
permeability decreases when cAMP is lowered by the addition of serum or by the increase
in cell density. For example, Cl-lD cells, a mouse cancer cell type, are unable to perform
GJIC in ordinary confluent culture but they became electrically coupled and capable of
transferring fluorescent tracer molecules (carboxyfluorescein, 376 daltons) upon
administration of cAMP, dibutyryl cAMP (db-cAMP), db-cAMP and caffeine or cholera
toxin (Azamia et al., 1981). It was hypothesized that junctional permeability is dependent
on protein kinase A which is a cyclic AMP-activated enzyme (Wiener and Loewenstein,

1983).

10

II. Endogenous Modulation of J unctional Permeability
a. Hormones

Gap junctions are hormonally responsive in several types of tissue. In the female
reproductive system, the granulosa cells of the ovarian follicle have gap junctions that
increase with estrogen and decrease with ovulatory doses of human gonadotropin
(Burghardt and Anderson, 1979; Larsen et al., 1981). Another example is the increase of
GJIC in pancreatic B-cells when stimulated to secrete by insulin (Meda et al., 1979). It

was suggested that these hormonal factors may exert their action through cAMP.

b. Growth Factors

Gap junctions are suspected to play a role in contact inhibition. Growth factors,
by definition, are capable of inducing mitogenesis and overcome contact inhibition.
Furthermore, many oncogenes are known to code for growth factor (e. g. sis) or growth
factor receptor (e. g. erb B). As will be describe later, several oncogenes have been shown
to block GJIC. Therefore, it is reasonable to suspect that GJIC might be blocked by
certain growth factors. Indeed, EGF and TGF-B have been reported to block GJIC
(Madhukar et al., 1989).

c. Oncogenes

A large body of evidence has been accumulated showing the important role of
oncogenes in the process of malignant progression. In addition, many tumor cells are not
able to perform GJIC. It is reasonable, therefore, to suspect the involvement of oncogene
expression in modulation of gap junctions. To date, several oncogenes have been shown
to modulate GJIC in various cell types. Rous sarcoma virus or v-src was first
demonstrated to inhibit GJIC in different cell types (Atkinson et al., 1981, 1986; Azamia
and Loewenstein, 1984; Chang et al., 1985) Azamia et al. (1988) reported that over

expression of the cellular src gene in NIH/3T3 cells causes reduction of cell-to-cell

ll

transmission of molecules in the 400- to 700-dalton range. By comparing the
phosphorylation of connexin 43 in uninfected and Rous sarcoma virous (RSV)
transformed mammalian fibroblasts, it was observed that connexin 43 protein in RSV
transformed fibroblasts contained both phosphotyrosine and phosphoserine but in
untransfected cells connexin 43 protein was phosphorylated only on serine residue. Thus,
the presence of phosphotyrosine in connexin 43 was correlated with the loss of GJIC
observed in RSV transformed fibroblasts (Crow et al., 1990). The ras oncogene was also
found to down-regulate GJIC (El-Fouly et al., 1989; Vanhamme et al., 1989; De Feijter et
al., 1990; De Feijter et al., 1992).

Even though numerous evidence shows that oncogene products decrease
intercellular communication, not all studies published seem to obey this nrle. The mutated
T24 ras protooncogene transfected into NIH/3T3 cells does not alter the extent of
metabolic cooperation of these cells from that of the parental NIH/3T3 cells (Vitkauskas
and Canellakis, 1983). Myc and fos oncogenes, when introduced into NIH/3T3 cells, did
not decrease the extent of metabolic cooperation between these cells (Vitkauskas et al.,
1983). These results suggest that at least some proto-oncogenes may afi‘ect intercellular
communication different fiom those of the corresponding viral oncogenes (Vitkauskas and
Canellakis,1985), or that there might be a threshold level of the oncogene products needed
to down regulate the GJIC (De F eijter et al., 1992).

III. Exogenous Modulation of Junctional Permeability

a. Inhibition of GJIC by Tumor Promoting agents

Blockage of GJIC was believed to play an important role in carcinogenesis since
defective or selective GJIC was observed in many transformed or tumor cells
(Loewenstein, 1979; Kanno, 1985; Yamasaki et al., 1987). In 1979, Yotti et al. reported
that the mouse skin tumor promoter, TPA can eliminate the metabolic cooperation

between 6-thiogunaine-resistant and wild type Chinese hamster V79 cells (Yotti et al.,

12

1979). Murray et al. (1979) working independently, also showed that tumor promoters,
TPA and phorboI-12, l3-didecanoate (PDD), inhibited the transfer of radioactively labeled
chemicals fi'om HEL/37 mouse epidermal cells to 3T3 cells during coculture. In addition,
in yilg studies showed that the liver tumor promoters, phenolbarbital(PB) and p,p'-
dichlorodiphenyltrichloroethane (DDT), reduced gap junctions of rat hepatocytes fi'om
fieeze-fiacture analysis (Sugie, et al., 1987). Anther group, using a GJ protein cDNA and
northern blot analysis, demonstrated that the level of GJ protein mRN A in liver was
markedly reduced after a few weeks of phenobarbital exposure (Mesnil, et al., 1988).

Actually, there are some confusing and contradictory observations in the literature.
In culture, TPA did inhibit GJIC of mouse epidermal cells (Pasti et al., 1988), however, it
did not inhibit GJIC of mouse epidermal cells when mice were painted with TPA (Karn
and Pitts, 1988). However, Kalimi and Sirsat (1984) it reduce gap junction in TPA-
treated mouse epidermis. In addition, transforming growth factor 13 did enhance
transformation of BALB/c3T3 cells but failed to inhibit GJIC (Hamel et al., 1988). On the
other hand, it has been shown to inhibit GJIC i_n yitr_o (Madhukar et al., 1989). Tumor
promoters, such as okadaic acid or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also
failed to block GJIC (Boreiko et al., 1986, Abemethy et al., 1985).

This apparently contradictory evidence suggest that different tumor promoters may
operate via different mechanisms. Alternatively, it is more likely that details of difl‘erent
protocols are the basis for the differences. There is also the possibility that some
promoters might affect only initiated cells and not the normal cells. In the case of the
negative efi'ect of TCDD, it needs to be shown that the cells tested had TCDD receptors.
Some have suggested that tumor promotion may involve multiple mechanism and GJIC
inhibition may not be the sole mechanism of tumor promotion evoked by various
chemicals (Yamasaki, 1990). For example, it was postulated that the tumor-promoting
property of okadaic acid is the result of significant cytotoxicity to the mouse epidermis,

with the resulting regenerative hyperplasia acting as the true promoter (Mordan, 1991).

13

However, this explanation does not rule out the role of gap junctions, since the dead cells
(or removal of cells by surgery) would result in the lack of communication (Trosko et al.,

1983)

b. Enhancement of GJIC by Anti-Tumor Promoters

Some studies show that certain anti-tumor promoting agents, such as cyclic AMP,
retinoic acid, and glucocorticoids can antagonize the inhibitory effect of TPA on gap-
junctional communication (Yamasaki and Enomoto, 1985). In addition, anti-tumor
promoters can induce junctional communication between normal cells and chemically or
virally transformed cells, resulting in the growth inhibition of the transformed cell types
(Mehta et al, 1986). For example, retinoids are presently undergoing widespread clinical
trials as cancer chemopreventive agents (Bertram et al., 1987). In the range of 108-1010
M, the retinoids are up-regulators of GJIC in both 10T1/2 cells and their carcinogen-
initiated counterparts. This up-regulation was correlated strongly with inhibition of
transformation (Hossain et al., 1989). When db-cAMP, fluocinolone acetonide, retinoic
acid, or dexamethasone was added during the induction of BALB/c3T3 transformation by
3-methylcholanthrene, there was a 65-95% decrease in the number of transformed foci.
These chemicals have been shown to reestablish intercellular communication between

transformed and normal cells (Yamasaki et al., 1988).

GJIC and Expression of Gap Junction Genes in Normal and Tumorigenic
Human Breast Epithelial Cells

Normal primary breast epithelial cells have been shown to communicate with
homologous cells (Fentiman and Taylor-Papadimitrious, 1977; Fentiman, 1980; Eldridge
et al., 1989). In contrast, most human breast cancer epithelial cell lines were found to be
deficient in GJIC (Fentiman et al., 1979), including the MCF-7 (Fentiman and Taylor-

Papadirnitrous, 1979) used in this study. The few breast cancer cell lines competent in

14

cell-cell communication (i.e. MDA231 and HSOS78T) were difl‘erent from normal breast
epithelial cells in showing non-selective communication (ability to communicate with calf
lens cells) (Fentiman et al., 1979). Eldridge et al. (1989) compared GJICs in normal,
immortal and non-immortal tumor-derived human breast epithelial cells. They reported
that both normal and early passage tumor-derived cells were competent in GJIC in
contrast to 'm fitr_o immortalized cell lines which were deficient in GJIC.

- Thus far, many difl‘erent gap junction genes have been identified: Cx26 (Zhang and
Nicholson, 1989); Cx31 (Hob et al., 1991); Cx32 (Paul, 1986); Cx43 (Beyer et al., 1987)
and Cx46 (Beyer et al., 1988). Four of these genes have been mapped on human
chromosomes; ch. 13 (Cx26), ch. x (Cx32), ch. 6 (Cx43) and ch. 13 (Cx46) (Wilecke et
al., 1990; Hsieh et al., 1991). Using Northern blot analysis, mRNA species of Cx26 and
Cx43 were found to be expressed in cultures of normal breast epithelial cells but not in
tumor-derived breast epithelial cell lines including MCF-7 (Tomassetto et al., 1991; Lee et
al., 1992). In contrast to normal breast epithelial cells, these breast carcinoma cell lines do
not perform GJIC (Lee et al., 1992). Immunofluorescence studies using antibodies against
different gap junction proteins have shown that normal breast epithelial cells express Cx43
but not Cx26 and Cx32 (Wilgenbus et al., 1992). None of these Cx genes were expressed
in invasive ductal or lobular carcinoma (Wilgenbus et al., 1992). Based on the differential
expression of the gap junction gene in normal and tumorigenic breast epithelial cells, Lee
et al. (1991) proposed that Cx26 may be a tumor-suppressor gene of breast cancer.
Similarly, transfection of GJIC-deficient mouse or human cells with Cx43 and Cx32
respectively have been shown to inhibit cell growth i_n m (Mehta et al., 1991) and to

retard tumor growth i_n mp (Eghbali et al., 1990) in conjunction with restoration of GJIC.

MATERIAL AND METHOD

Cell Culture Medium

In this study two difl‘erent tissue culture media, a modified Eagle's minimal
essential medium (MEM) and a modified MCDB 153, were used. The modified Eagle's
MEM consisted of Earle's balanced salt solution, 50% increase of all vitamins and essential
amino acids except glutamine, 100% increase of all non-essential amino acids and
supplemented with 1 mM sodium pyruvate (referred to as D medium) and 5% fetal bovine
serum (FBS) (Chang et al., 1981) was used to culture MCF-7, T47D and HSOS78T cells.
These tumor cell lines were available in our laboratory and were from America Type
Culture Cell (ATCC) originally.

The MCDB 153 was modified by increasing the concentrations of calcium (0.15
mM) and six amino acids (Pittelkow and Scott, 1986) and adding recombinant human
epidermal growth factor (EGF) (1 ng/ml), insulin (10 ug/ml), hydrocortisone (1 ug/ml),
human transferin (10 ug/ml), ethanolamine (0.1 mM), and phosphoethanolamine (0.1
mM). A 1:1 mixture (v/v) of the modified MCDB 153 and the modified Eagle's MEM
(without FBS) (referred to as MSU-1 medium) (developed by CC. Chang) was used to
develop the primary human breast epithelial cells fi'om reduction mammoplasty and for
culture of Type-II cells. Type-I cells can be cultured in the MSU-1 medium supplemented
with 5% FBS.

To select for methotrexate-resistant transfectants of MCF-7 cells, the modified
Eagle's MEM was supplemented with 10% dialyzed fetal bovine serum. The Amicon DC2
system with Diaflo hollow fiber cartridge H1P10 (10,000 MW cut-off) was used to
prepare dialyzed FBS. Dialysate was circulated at a high rate around the fibers to remove

salts and other small solute by diffusion. When the total volume of the serum was

15

l6

reduced by 50%, PBS was added to restore the initial volume. After 5 cycles, about 97%

of small molecules can be removed.

Cells

 

Human breast tissues were derived from reduction mammoplasty. They were
minced to small fragments with scalpels and dissociated to single cells or cell aggregates
with collagenase (1 mg/ml) in medium 199 (0.1 g tissue per ml) overnight at 37°C. Then,
the cell suspensions were pelleted at 600 g for 10 min and incubated in MSU-1 medium
supplemented with 5% FBS. To reduce fibroblasts in culture, the floating cell aggregates
were transferred to new flasks alter two hours. The next day, the medium was changed to
serum-free MSU-1 medium. The cell culture developed in a week, with medium change
once every 2 days, the cells could be dissociated with trypsin (0.0l%)-EDTA (0.02%) in
phosphate buffered saline (PBS) (calcium, magnesium-lice) for subculture or storage in
liquid nitrogen.

The differential rates of attachment on plastic surface by different types of cells
were used to separate epithelial cells from fibroblasts. Cells taken from the freezer were
plated in a 25 cm2 flask containing 10 ml MSU-1 medium with 5% FBS and allowed to
attach for 2 hours. The unattached cells in the supernatant were then transferred to a new
flask which contained mostly epithelial cells. The original flask containing enriched
fibroblasts was discarded. The next day, cells in the supernatant which contain mostly
Type-I cells were transferred to a 100 mm culture plate. Ten ml MSU-1 medium (serum-
free) was added into the flask which contain mostly type II cells.

Three human breast carcinoma cell lines were also used in this study, i.e. MCF-
7,T47D and HSOS78T. All these cell lines were cultured in the modified Eagle's MEM
containing 5% FBS. All cultures were incubated in 37°C incubators supplied with

humidified air and 5% C02-

17

Trangfection and Selection of MCF—7 WITH CONNEXIN GENES

Plasmids (pSV(RI)-DHFR containing rat connexin 43, 32 or 26 cDNA insert) (Jou
et al., 1993) were introduced into MCF-7 cells by using the cationic liposome-mediated
transfection method. Lipofectin reagent (BRL cat no. 8292SA) is a 1:1 (v/v) liposome
formulation of the cationic lipid N-[l-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium
chloride (DOTMA), and dioleyl phosphatidyl-ethanolamine (DOPE) in filter sterilized
water. Positive charged lipid, DOTMA, can form liposomes and interact spontaneously
with DNA to form a liposome/polynucleotide complex with complete entrapment of the
DNA. The fiision of the complex with cell membrane can deliver functional nucleic acid
molecules to the tissue cultured cells.

MCF-7 cells were seeded in a loo-mm tissue culture dish in 10 ml of D medium
with 5% fetal bovine serum. The cells were incubated until they were 40-50% confluent.
For each loo-mm dish, 20 ug plasmid and 40 ug lipofectin reagent were diluted to 1.5 ml
serum-free D medium separately and then were combined in a polystryene tube and mixed
gently. The mixture was allowed to stand for 15 min at room temperature before it was
added to the cultured cells which were washed twice with 5 ml serum-free medium prior
to the addition. Cells were incubated for 16 hr before returning to D medium with 5%
FBS (3 ml per plate). Twelve hours later, the cells were washed with serum-free medium
again and exposed to selective medium ( D medium supplemented with 10% dialyzed FBS
and 400 ng/ml methotrexate-MTX).

The plasmids used contained mutant dihydrofolate reductase (DHFR) gene.
DHFR catalyzes the NADPH-dependent reduction of folate to tetrahydrofolate which is
an essential cofactor in the synthesis of glycine, purines and thymidine. The antineoplastic
agent --MT X, a folate analogue, is a competitive inhibitor of DHFR, therefore, a
deficiency of nucleotide pools and cell death may be induced by MTX. In transfectants

that express the mutated DHFR gene, abundant mutant DHFR with reduced affinity for

l8

mtx will be sufficient to neutralize MTX inhibition and to confer MTX-resistance to these

cefls.

Measurement of GJIC

The ability of cells to perform gap junctional intercellular communication was
determined by the scrape loading and dye transfer method (El-Fouly et al., 1987). The
dye used is Lucifer yellow (MW457.2, Sigma, cat. no. L0259) which is an intensely
fluorescent 4-aminophthalimide and it can be detected with epifluorescence microscope at
low, non-cytotoxic concentrations. Lucifer Yellow cannot diffuse through intact plasma
membrane but its low MW permits it's transmission from one cell to another through gap
junctions.

Confluent cultured cells grown in 35 mm culture plate for 2 days were washed
several times with PBS. With 2 ml Lucifer Yellow solution (0.5 mg/ml) in the plate
several scrape lines on the monolayer were made using a surgical blade. Alter 3 minutes
incubation at room temperature the dye solution was decanted and the cell monolayers
were rinsed several times with PBS. The cells in PBS or medium were examined under a

Nikon epifluorescence microscope to determine the extent of dye distribution.

Isolation of Total RNA from Cells

Total RNA was extracted by using the modified single-step method of RNA
isolation which was initially described by Chomczynski and Sacchi (1987). Under acidic
condition, total RNA can be extracted in a single step, free of DNA, by using guanidinium
thiocyanate, phenol and chloroform.

Confluent cell cultures in a 75 cm2 flask were washed twice with PBS and then
exposed to 1.5 ml of Solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH
7.0, 0.5% sarcosyl, 0.1 M B-mercaptoethanol) After making sure the cells were lysed, the

flask was rocked for 5 min. The cell lysate was transferred to two 2.2 ml eppendorf tubes

l9

equally and 75 ul of 2 M sodium acetate, pH 4.0, was added to each tube. The solution
was vortexed and then 0.75 ml water-saturated phenol and 200 pl chloroform (
chloroform: isoamyoalcohol=49: l) were added. The mixture was vortexed after addition
of each solution and cooled on ice for 10 min. The sample was centrifiiged at 10,000xg
for 15 min at room temperature. The aqueous phase was transferred to a fresh tube,
mixed with one volume of isopropanol and then placed at -20°C for at least 1 hr to
precipitate RNA. The insolubilized RNA was pelleted at 10,000xg for 30 min. The pellet
was resuspended in 200 ul of 4 M LiCl and gently vortexed for 10 min. Sedimentation at
10,000xg for 10 min was again performed and the resulting RNA pellet was dissolved in
200 ul of 0.5% SDS. 200 ul chloroform was then added and mixed to the aqueous phase
by vortexing more than 1 min. After centrifugation at 10,000xg for 15 min, the upper
aqueous phase was collected and precipitated by 2.5 volume of absolute ethanol and 1/10
volume of 3 M sodium acetate, pH 5.2. The pellet was washed with 75% ethanol,
sedirnented, air dried and dissolved in 30 ul of 0.5% SDS. The concentration and purity
of RNAs was measured by reading at 260 nm and 280 nm.

Northern Blot Analysis

I. Electrophoresis of RNA in Gel Containing Formaldehyde

One gram of agarose was added to 100 ml of DEPC-treated ddHZO and boiled in a
microwave oven until about 30 ml of H20 had evaporated. After cooling to 60°C, the gel
solution was supplemented with 10 ml of 10 x MOPS (3-[N-Morpholino]propane-sulfonic
acid) and 18 ml of formaldehyde to make a 1 x MOPS, 3% formaldehyde gel. Afier
solidification, the gel was submerged in running buffer (1 x MOPS, 3% formaldehyde in
DEPC-treated ddeO). Twenty micrograms of each RNA sample in 20 ul of sample
buffer (50% formamide, 1 x MOPS, 6% formaldehyde, 1 ul of tracking dye and 1 rig/ml

20

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 90 volts for 4 hours.

II. Northern Blot

When the migration was complete, the gel was rinsed in excess DEPC-treated
ddHZO for 45 min to remove the formaldehyde. A picture of the gel along side with a
transparent mler was photographed above an ultraviolet illumination box. The gel was
then soaked in 20 x SSC for 45 min with agitation. The RNAs were transferred from the
gel to a nylon membrane (HybondTM-N, 0.45 micron; Amersham) by capillary transfer
overnight. The membrane was rinsed with 2 x SSC and baked at 80°C in a vacuum oven
for two hours. Furthermore, the side of the membrane carrying the RNA was exposed to

a low dose of UV (4.5 min) on a UV transilluminator to crosslink RNA to the membrane.

III. Hybridization and Autoradiography

The whole length rat cDNAs for Cx43, Cx32 and Cx26, which were provided by
Dr. Emmanuel Dupont, were used for making probes. Probes were made by using a
random primed DNA labeling kit (Molecular Biology, Bochringer Mammheim)

Fifty nanograms of cDNA in 18 ul ddeO were denatured by heating at 95°C for
10 min and subsequently chilled on ice. The following components were added to the
denatured cDNA: 6 ul of dATP/ dGTP/ dTTP (1:121), 4 ul of reaction buffer, 10 ul of [or-
32P]dCTP (50 uCi) and 2 ul of Klenow enzyme. The mixture was incubated at 37°C for
1 hour and the reaction was terminated by adding 10 ul of 0.2 M EDTA, pH 8.0. The
reaction mixture was purified using a Bio-spin column (Bio-Rad, cat. no. 732-6006). The
purified labeled-probe was denatured by heating at 95°C for 10 min and then chilled on ice
just before being used.

During the time of making a probe, the membrane was pre-hybridized in a sealed
bag containing 10 ml of hybridization buffer (5 x SSC, 5% Dextran Sulfate, 0.5% SDS, 5x
Denhardt's and 250 ug/ml Honing sperm DNA) at 65°C for 1 hr. For hybridization, the

21

labeled probe was added to the bag and continuously incubated at the same temperature
overnight. Next day, the membrane was taken out from the bag , rinsed with 2 x SSC ,
and washed twice with 0.5 x SSC at 65°C for 30 min. Autoradiographies were done at -

70°C by using Kodak X-OMAT and regular screen.

lmmunofluorescence staining of Gap Junctions
Cells were seeded on glass cover slips and cultured until they approached

confluence. The cells were rinsed with 2 ml of Ca“ and Mg” containing PBS for 3
times. After the last wash, the washing solution was removed as much as possible. The
cells were fixed in 2 ml of 4% paraforrnaldehyde in 100 mM PIPES, pH 6.8, 1 mM MgC12
and 3% sucrose for 15 min. Fixation solution was freshly made by boiling
paraforrnaldehyde in a solution of 100 mM {PIPES and 1 mM MgC12 until dissolved, and
then adding adequate sucrose. The solution was allowed to cool at room temperature.
After fixation, the cultured cells on cover slips were rinsed thoroughly with 2 ml of 3%
sucrose in PBS 2 times. Cells were perrneabilized with 2 ml of -20°C methanol for 30 sec
and washed subsequently with PBS twice. After these steps, the cells were incubated in 2
ml of 2 mg/ml sodium borohydride for 10 min, and followed by washing twice with PBS.
Non-specific binding sites were blocked by incubating these cells in 10% normal goat
serum (NGS) in PBS plus 0.05% Tween-20 at room temperature. One hour later, the
solution was thoroughly removed by suction. The cells were incubated with 300 ul of
primary antibody (1° Ab) (purified by Dr. Dupont) (1:50 dilution in 5% NGS, 0.3%
gelatin and 0.05% Tween-20 in PBS) at room temperature for 2 hr. Diluted antibodies
were centrifuged at least 15 min in a microcentrifuge before using. For the preirnmune
control, the diluted 1° antibody was incubated with corresponding peptide antigen (1: 100
dilution) for 30 min before being used to incubate with cells. Afterward, the cover slip
was rinsed twice, 5 min each, with 2 ml of 0.3% gelatin and 0.05% Tween-20 in PBS

(washing buffer). The second incubation was carried out for 1 hr at room temperature in

22

the presence of Rhodamin-labeled conjugate afinipure Ducking anti-rabbit 1g G
(ImmunoResearch laboratory, Inc. cat. no. 711-025-132) (1:200 dilution in the same Ab
dilution buffer used for 1° Ab). After being rinsed with 2 ml washing buffer for 4 times,
the cover slip was mounted onto a slide using Polysciences’ Aqua/polymount. The cells

were examined under an epifluorescence microscope using the G filter.

Protein Isolation and Quantification of Protein

Confluent cells culture in 25 cm2 flask were rinsed twice with 5 ml of PBS then
lysed in 0.5 ml of lysis buffer (20% SDS, 0.1 M Tris, pH 7.5 and 20 mM EDTA). The cell
lysate was then transferred to a 2.2-ml eppendorf tube and sonicated for 30 sec.
Subsequently, 100 pl of cell lysate was taken out for measuring the concentration of
protein and 4 pl of B-mercaptoethanol was added to the remaining lysate.

A modified Bio-Rad DC protein assay was used for determining the protein
concentration of the samples. First, samples were diluted 1:20, meanwhile, six protein
standard solutions containing fi'om 25 ug/ml to 800 ug/ml were prepared in the same
buffer as the sample. 25 ul of standard or sample was pipeted into a 1.5-ml eppendorf
tube, and mixed with 125 pl of reagent A' and 1 ml of reagent B (Bio-Rad cat no.500-
0116). The tubes were vortexed alter adding each reagent. Alter 15 min, the absorbance
was read at 750 nm. The average absorbances of triplicate determination was used to

calculate protein concentration.

Western Blot Analysis

I. SDS-Polyacrylamide Gel Electrophoresis of Protein
The Laemmli discontinuous buffer system ( Laemmli, 1970) was used with 4.5%

stacking gels and 12.5% separating gels. The apparatus was obtained from Bio-Rad Inc.

23

Fifteen ml (for two gels) of 12.5% separating gel(4.2 ml acrylamide/Tris (30:0.8),
2.5 ml 1.5 M Tris, pH 8.8, 2.9 ml ddHZO and 0.1 ml 10% SDS) was prepared and
degassed for 15 min before being used. Then, 30 u] of ammonium persulfate (AP) and
270 pl of 1% N,N,N,N-Tetramethylethylenediamine (TEMED) were added to the gel
solution to initiate polymerization. As soon as mixing well, the acrylamide solution was
poured into the gap of glass plates. Isopropanol was spread over the gel to prevent
oxygen fi'om diflirsing into the gels and inhibiting polymerization. After polymerization
was complete (about 40 min), the overlay was poured off and the top of the gel was
washed several times with deionized water to remove any unpolymerized acrylamide.
While the separating gel was polymerizing, the stacking gel (1.5 ml Acrylamide/Tris
(3020.8), 2.5 ml 0.5M Tris, pH 8.3, 5 ml of ddeO and 0.1 ml 10% SDS) was prepared
and degassed for around 10 min. The gel mixture was swirled rapidly when 150 pl of
10% AP and 750 pl of 1% TEMED were added. Without delay, the stacking gel solution
was poured directly onto the surface of the polymerized resolving gel. Immediately, a
clean Teflon comb was inserted into the stacking gel solution. More stacking gel solution
was added to fill the space of the comb completely. The Teflon comb was removed
carefully alter polymerization was done (~ 20 min). The gels were then mounted in the
electrophoresis apparatus. Moreover, Tris-glycine electrophoresis buffer was added to the
top and bottom reservoirs. It was important to remove all bobbles which became trapped
at the bottom of the gel between the glass plates. Finally, 20 pg of each of the samples
which were prepared in 1 x SDS gel-loading buffer was loaded into the bottom of the
wells. An equal volume of 1 x SDS gel-loading buffer was loaded into any wells that were
unused. The gel was electrophoresed at 50 v until the dye front had moved into the
resolving gel, the voltage was increased to 200 v. When the bromophenol blue reached

the bottom of the resolving gel, the power was turned off.

24

II. Transfer of Protein from SDS-Polyacrylamide Gel to Solid Support

Four pieces of 3 MM paper and two pieces of polyvinylidene difluoride (PVDF)
membrane were cut to the exact size of the SDS-polyacrylamide gel. One corner of the
filters was marked with a soft-lead pencil.

These 3 MM papers were soaked in a shallow tray containing transfer bufi‘er. The
PVDF membranes were pre-wetted in methanol then soaked in transfer bufl‘er. The
transfer apparatus was set up in the following order: anode, porous pad, 3 MM paper,
membrane, gel, 3 MM paper, porous pad and cathode. A glass pipette was used as a
roller to squeeze out any air bubbles between layers. Twenty volts overnight were
applied to transfer the protein.

Following transfer, the gels were stained with 0.1% Coomassie Brilliant Blue in
methanoleZO (1 : l)/glacial acetic acid (9:1). Meanwhile, the membranes were rinsed
with ddHZO then stained with Ponceau S until the bands of protein were visible. The
membranes were washed in several changes of deionized water. The standards were cut

out fi'om the membranes before irnmunoblotting.

III. Immunoblotting

Non-specific binding sites were blocked by soaking the PVDF membranes in 5%
milk solution (40 mM Tris, pH7.5, 5% nonfat milk and 0.02% SDS) for at least one hour
at room temperature with gentle agitation. The purified rabbit anti-rat connexin (Cx 43,
Cx 32 or Cx 26) antibody, provided by Dr. Dupont, was diluted 1:25 with 5% milk
solution. The control peptide was prepared in 1:100 dilution with Ab solution and
preincubated for 30 min at room temperature. The membranes were incubated in a
solution containing 1°Ab or 1°Ab and control peptide at room temperature for one hour.
Afterward, these membranes were washed in 10 ml of 4% milk solution (40 mM Tris, 4%
nonfat milk and 0.1% Tween 20) four times for 5 min each. The membranes were then

incubated with 5 ml of a 1/500 dilution of biotinlayted anti-rabbit IgG (species-specific

25

F (ab)2 fiagment) (Amersham, cat. no. RPN 1064) in 4% milk solution for 1 hr and
subsequently washed 4 times with the same solution. The membranes were incubated with
a 1:5000 dilution of alkaline phosphatase-conjugated streptavidin (Amersham, cat. no.
RPN 1234) in the solution mentioned above for one hour and sequentially washed four
times with the solution of 20 mM Tris, 0.5 M NaCl and 0.1% Tween 20. Finally, these
membranes were developed with the 5-bromo-4-chloro-3 -inclonyl phosphate (BCIP)/
nitro blue tetrazolium (NBT) color development solution which was prepared by
dissolving a 10 mg tablet of NET in 30 ml of alkaline phosphatase buffer (0.1 M Tris, pH
9.5, 0.1 M NaCl and 5 mM MgC12) and adding 100 pl of 5% BCIP.

Anchorage of Independent Growth (AIG)

Untransforrned cells derived from solid tissues usually require a substrate to attach
and to proliferate. In contrast, most tumor cells are capable of anchorage independent
growth (AIG). The following procedure was used to test the ability of AIG.

A sufficient volume of 3.3% agarose (Sigma A-6013 Type-1 Low EEO) in PBS
was prepared first and was kept at 45°C in a waterbath. Meanwhile, enough medium (D
medium with 10% FBS or D medium with 10% dialyzed FBS and 400 ng/ml MTX) was
warmed to 39°C in a waterbath. For the 0.5% hard agar layer, 0.45 ml of 3.3% agarose
and 2.55 ml warm medium were mixed thoroughly by using a warm pipet and the mixture
was immediately plated into a 60-mm dish. The hard agar layer was left at room
temperature for 30 min to solidify. The 0.33% of soft agar was prepared by mixing 2.2 ml
of medium, 0.3 ml of 3.3% agarose and 2x104 cells in 0.5 ml of medium. This was then
overlayed on the hard agar. Five milliliter of medium was added the second day and
renewed every 4 days. Four weeks later, the culture was stained for 24 hours at 37°C
with a solution containing 1 mg/ml of the tetrazolium salt, which is 2-(P-iodophenyl)-3-
(nitrophenyl)-5-phenyltetrazolium chloride (Sigma I-83 77) in 0.9% of sodium chloride.

The stained colonies in agar were dried (on a warming plate) and counted.

RESULTS

1. GJIC and Gap Junction Gene Expression in Normal Human Breast

Epithelial Cells

a. Ability of Two Types of Normal Human Breast Epithelial Cells to Perform GJIC

Two primary human breast epithelial cell cultures (HMEll and HME14) derived
from reduction mammoplasty from two different women were used in this study. The
initial cultures were developed in MSU-1 medium. Both contain two morphologically
distinguishable epithelial cell types. The first cell type is less reflective and less distinctive
in cell boundaries (Fig. 1A and Fig. 2A for HMEll and HME14 respectively). This cell
type (Type-I) is also characterized by its inability to perform GJIC based on studying using
the scrape loading/dye transfer technique (Fig. 3A and Fig. 4A for HMEll and HME14
respectively). In contrast, the second cell type is cobblestone-shaped and is more
reflective with distinctive cell boundaries (Fig. 1B and Fig. 2B for I-IMEll and HMEl4
respectively). More specifically, the second cell type (Type-II) is competent in GJIC (Fig.
3B and Fig. 4B for HMEll and HMEI4 respectively).

b. Expression of mRNA for Gap Junction Genes in Two Types of Normal Human
Breast Epithelial Cells

The expression of gap junction genes was studied by Northern blot analysis using
cloned cDNA for rat Cx26, Cx32 and Cx43 as probes. The results indicated that Type-I
cells do not express gap junction genes for Cx43 (Fig. 6A, lane 3 and 5 for HMEll and
HME14 respectively) and Cx26 (Fig. 7, lane 3 and 5 for HMEll and HME14
respectively). In contrast, Type-II cells were found to express genes for Cx43 (Fig. 6A,
lane 4 and 6 for HMEll and HME14 respectively) and Cx26 (Fig. 7, lane 4 and 6

respectively). The breast stromal fibroblasts derived from the initial HME14 culture were

26

27

A

' ..\,\I.~I.\(p

l\ til

n!
/,
l\

A...” ./ ‘rL/VV/\

bV
\ 0
EV...

V rVQrP

\ S. ,.r VV
\\..?m9e.o. .

:(

a”‘

' 34%,”;

Q

r?»

rm, “1L

6

 

The morphology of two types of normal human breast epithelial cells. The

Figure 1.

photographs shown here are cells derived from HME 11: (A) Type-I cells (B) Type-II

cells.

28

 

    
   

‘-
.i’

I

\

t
a

3.1;.“

w

gaff“ «W ‘6' al.-n as
l .. it «- 19% w

24 a ‘5. ‘0 ’ I" A? .‘ {P T
(I

 

1': 5g;

‘ I‘sa

'ffl'.“ ’
Nee; - ,

War

\‘ l'

l "'47:

i , a» .
‘

Figure 2. The morphology of two types of normal human breast epithelial cells from the
primary culture, HME 14: (A) Type-I cells (B) Type-II cells.

29

‘i. \- “‘"'V,7/

‘-
/

 

 
 
 
   
  

3‘. 5. ' "3“ 1‘ r -. r ‘VI‘
'(-.._Y('!‘, , In: -o‘1vA’¢l‘V';'t§-fi
1.314)] ‘ ‘ J:.\“%'§ 7"
\ s‘ “ ”49‘ r‘...‘3"‘. .- . 5
Iii/:1?) " 5%., ‘3‘. "\.fi 6.
....,. ho“. A \

‘ d4! .’ q ‘

g .

   

    

 

Figure 3. Gap junction mediated dye transfer was revealed by the scrape loading /dye
transfer technique. (A) Type-l cells of HME 11 under a phase-contrast (right) or an
epifluorescence (left) microscope. (B) Type-II cells of HME llunder a phase-contrast

(right) or an epifluorescence (left) microscope.

30

    

.. \\ 'th‘ -‘\ (_
.‘ . - ‘ N i'
«‘uszanCWVMv
' ..l ‘ ‘I‘\ . ,\
I‘lfl\‘\\\\\‘\‘\\§\‘. -.d “‘lk

I \ \
. ’H'.\\‘.\\\
'1‘, “‘V“‘

0 § .
\i v v‘.‘ ..
’13:?!" ‘l

\ ,

I

    

‘0 ‘V’ r \f."
"Q: I\‘\/

Figure 4. The ability of cells to perform GJIC was shown by the scrape loading /dye
transfer technique. Type-I cells (A) and Type-II cells (B) of HME14 under a

phase-contrast (right) or an epifluorescence (left) microscope.

31

 

Figure 5. Gap junction mediated dye transfer of normal breast stromal fibroblasts
derived fi’om same culture as HME14. Same area of cells was observed under a
phase-contrast (bottom) or an epifluorescence (top) microscope.

32

Kb1234567891011

3.5- I . - . -Cx43

§_V
i_-
a
5.
i_e
i_-
E4
5.:
:1

III
I

ll

 

Figure 6. (A) Northern blot analysis of the expression of Cx43 gap junction gene in
various cells. 1. Rat liver WB cells (positive control), 2. Blank, 3. I-IMEll type-I cells,
4. HMEll type-ll cells, 5. HME14 type-I cells, 6. HME14 type-II cells, 7. HME14
stromal fibroblasts, 8. Blank, 9. HSOS78T cells, 10. MCF-7 cells, 11. T47D cells.
The amount of RNA loaded in each cell type or cell line was shown (B).

33

Kb 1234567891011

2.7- v i
_ V -Cx26
2" D E -Cx26

Figure 7. Northern blot analysis of Cx26 mRNA expression in various cell types and
cell lines: 1. Rat liver WB cells, 2.Blank, 3. MEll type-l cells, 4. I-IMEll type-II cells,
5. HME14 type-I cells, 6. HME14 type-II cells, 7. HME14 stromal fibroblasts,

8. Blank, 9. HSOS78T, 10. MCF-7, ll. T47D.

34

competent in GJIC (Fig. 5) and were found to express Cx43 gene (Fig. 6A, lane 7) but not
Cx26 gene (Fig. 7, lane 7). All these breast epithelial cells (HMEll and HME14) and
stromal fibroblasts did not express the Cx32 gene (data not shown). As a control, the rat
liver WB cell line expressed both Cx43 (Fig. 6A, lanel) and Cx26 (Fig. 7, lane l)genes but
not the Cx32 gene was used. The differential expression of gap junction mRN A in

difl‘erent cell types was studied under identical conditions using equal amounts of RNA
(Fig. 6B).

c. Immunofluoreccence Staining of Gap Junction

The presence of gap junctions on contiguous cell membranes was studied by
irnmunofluorescence staining using specific antibodies against Cx26 or Cx43. No specific
labeling for either connexin was observed for Type-I cells of HMEll (data not shown).
The Type-H cells of HMEI4, however, displayed punctated fluorescence along borders of
cell-cell contact after staining with either Cx43 (Fig. 8A) or Cx26 (Fig. 8B). The

preirnmune controls did not show any observable fluorescence staining (data not shown).

ll. GJIC and Gap Junction Gene Expression in Three Breast Cancer Cell

Lines

a. Ability to Perform GJIC

Two breast carcinoma cell lines, MCF-7 and T47D, and a breast carcinoma-
sarcoma cell line, HSOS78T, were used in this study. The two breast carcinoma cell lines
were deficient in GJIC as studied by the scrape loading /dye transfer technique (Fig. 9B
and 9C for MCF-7 and T47D respectively). In contrast, the breast carcinoma-sarcoma

cell line HSOS78T was competent in GJIC (Fig. 9A).

35

 

Figure 8. Immunocytochemical analysis of the presence of gap junction protein in
cultured cells. (A) HME l4 type-II cells stained with antibodies against Cx43. (B) HME
l4 type-II cells stained with anti-Cx26 antibodies.

 

 
 
 

e‘
" e
‘ 9' P _ f): NV“:

   
 

‘ l \ O — 1

‘ 4 ‘VA‘ “\u'-' ‘ r ‘.V\
‘i‘ .3513; l?!” 0 s' T“. ( \‘w "
‘ \ ‘.II‘-‘~t$r4"" ‘ ' V' ‘w‘

v. , \ .zvglg‘. ,' t . .. . 1'1,
‘ ‘\ _ ‘b’v .' t'oo
ifzeele

. 5

      
 

 

n

   

It

     

;: -
F e‘ ;
{5? .jp y

  
 

2' v3
1%? 4:.
W’O":

we. ,’

"is
l
s

O r
a! -
'1‘tu gII
7.43: ch“
‘3; '

‘0
{\

  

I".
t.
\!.-
-o
1'1

I

(20.)?

   

Gap junction mediated dye transfer of breast tumor cell lines. (A) HSOS78T
cells. (B) MCF-7 cells. (C) T47D cells. Same area of cells was observed under a phase-

Figure 9.

contrast (right) or an epifluorescence (left) microscope.

37

b. Expression of mRNA for Gap Junction Genes

Under the same experimental conditions as those used to study normal human
breast epithelial cells, the MCF-7 and T47D did not express mRNA for Cx43 (Fig. 6, lane
10 and 11 respectively), Cx26 (Fig. 7, lane 10 and 11 respectively) and Cx32 (data not
shown). The GJIC-competent HSOS78T cells were found to express Cx43 mRNA (Fig.
6, lane 9) but not Cx26 mRNA (Fig. 7, lane 9) and Cx32 mRNA (data not shown).

c. Expression of Connexin Proteins

Total cellular proteins of the three breast cancer cell lines were used to detect the
presence of connexin protein by Western blot analysis. The results showed that the three
cell lines do not express Cx26 and Cx32 proteins (data not shown). A very weak
expression of Cx43 protein, however, was Observed for all the three cell lines (Fig. 10,
lane 2, 3, 4 for MCF-7, T47D and HSOS78T respectively). The HSOS78T cells are
different from MCF-7 and T47D cells by the presence of an additional Cx protein of lower,

molecular weight.

d. Immunofluorescence Staining o f Gap Junction

Since the three breast cancer cell lines were found to express low levels of Cx43
protein, irnmunofluorescence labeling was done to determine if Cx43 proteins are
detectable on the membranes of these 3 cell lines. The results indicate that Cx43 protein
was present in HSOS78T cells as shown by the presence of punctated fluorescence along
the borders of cell-cell contact (Fig. 11A). No Cx43 proteins were detected for MCF-7
and T47D by the immunofluorescence staining (Fig. 11B and 11C respectively). The
presence of Cx43 appears to correspond to the gap junction function of these three cell

lines.

38

 

Figure 10. Western blot analysis of the expression of Cx43 protein in breast tumor cell
lines. The membrane was incubated with antibodies against Cx43 protein.

1. Molecular weight marker. 2. Molecular weight marker. 3. MCF-7. 4. T47D.

5. HSOS78T. 6. HME14 Type-II cells. 7. Ml3SV-2. 8. Rat heart.

39

 

Figure l 1. Immunostaining of breast cancer cell lines using antibodies against cx43. A.
HSOS78T. B. MCF-7. C. T47D.

40

Ill. Transfection of GJIC-Deficient MCF—7 Cells with Cloned cDNA for Rat

Connexin Genes

a. Selection of Transfectants

The transfectants were selected in D medium containing 10% dialyzed fetal calf
serum and 0.4 ug/ml methotrexate since the plasnrid carries not only the connexin gene but
also the DHFR gene. When surviving colonies reach 5 mm in diameter, colonies were
randomly selected to test their ability to perform GJIC by the scrape loading /dye transfer
technique. Among 15 rat Cx43 gene transfectants tested, 7 were found to acquire the
ability to perform GJIC. Five out of 21 Cx32 cDNA transfectants tested were competent
in GJIC. For Cx26 cDNA transfectants, 3 out of 14 have restored their ability for GJIC.
For plasmid controls similarly selected by methotrexate, 6 colonies were examined and
none of them were capable of GJIC. All the positive clones showing GJIC and plasnrid

controls were isolated and propagated for firrther studies.

b. Gap-Junction Mediated Dye Transfer

The ability of rat gap junction cDNA transfectants to perform GJIC was restored
in confluent cultures of early passage cells as shown by studies using the scrape loading
/dye transfer technique. All colonies that were tested positive in preliminary screening still
retained their ability for gap junction mediated dye transfer. Three representative Cx43
transfectants are shown in Fig. 12 (A. MCF-7-43-10; B. MCF-7-43-16; C. MCF-7-43-3).
Similarly, three representative Cx32 transfectants are shown in Fig. 13 (A. MCF-7-32-2;
B. MCF-7-32-11; C. MCF-7-32-20) and three Cx26 transfectants are shown in Fig. 14 (A.
MCF-7-26-4; B. MCF-7-26-6; C. MCF-7-26-8). A representative plasmid control
deficient in GJIC is shown in Fig. 15. These results clearly show that GJIC can be
restored by transfection with either of the three cloned cDNA for rat Cx26, Cx32 or

Cx43 .

 

Figure 12. Gap junction-mediated dye transfer in three Cx43 transfectants:
(A) MCF—7-43-10. (B) MCF-7-43-l6. (C) MCF-7-43-3. Same area of cells was
observed under a phase-contrast (right) or an epifluorescence (left) microscope.

i

Pei; .

at
u t’

s.

'. . . l
‘ I
.I ’ "r
V , g
\ ‘fl
\ . 1‘
a V
. .
.' ‘ ‘ .

3;:

 

Figure 13. Gap junction-mediated dye transfer of three Cx 32 transfectants:
(A) MCF-7-32-2. (B) MCF-7-32-1l (C) MCF-7—32-20. Same area of cells was

observed under a phase-contrast (right) or an epifluorescence (left) microscope.

43

 

Figure 14. Gap junction-mediated dye transfer of three Cx26 transfectants:

(A) MCF-7-26-4. (B) MCF-7-26-6. (C) MCF-7-26-8. Same area was observed under a

phase-contrast (right) or an epifluorescence (left) microscope.

44

 

as. warm '
#3:;zy'm r .
353‘???"
e- ".'.' '

 
  
  
 
   

. ,' us.
;.é‘.3.e~ .,

.35, :o {.53 '.
'2’ \‘tke‘v‘ ' E

‘1) A”A‘.I2..‘.-‘H. Q

Figure 15. Gap junction-mediated dye transfer of a MCF-7 clone transfecting with
plasmid pSV(RI)-DHFR (MCF-7-DI-IF R). Same area of cells was observed under an
epifluorescence (top) or a phase-contrast (bottom) microscope.

45

The ability of these transfectants to perform GJIC, however, was found to decline
after extended culture for all clones except one (MCF-7-43- 10) which retained the ability
after extended growth. Comparing the level of GJIC by the three different Cx gene
transfectants at early passage, the Cx43 cDNA transfectants appear to have the highest
level of GJIC.

c. Gap Junction Gene Expression

I Five clones of Cx43 cDNA transfectants, three clones of Cx32 transfectants and
three clones of Cx26 transfectants were analyzed by Northern blot for expression of
transfected connexin genes. Total RNAs extracted from these cells were hybridized with
radiolabeled rat Cx43, Cx32, Cx26 cDNA probes. The results show that all clones of
transfectants proficient in GJIC expressed the respective mRN As of the transfected
connexin gene (Fig. 16, Cx43 cDNA transfectants; Fig. 17, Cx32 cDNA transfectants; fig.
18, Cx26 cDNA transfectants). The parental MCF-7 and plasmid control did not express
any of these genes (Fig. 16-18, lane 1 for MCF-7; lane 2 for MCF-7-DHFR plasmid
control). Two transcripts of different sizes were observed in these transfectants. The
amount of the two transcripts was about equal for all Cx26 and Cx32 cDNA transfectants.
For Cx43 cDNA tranSfectants, the low molecular weight transcript is negligible except for
one clone (MCF-7-43-10). The presence of these two transcripts might have resulted
from the differential processing of the mRNA transcribed from the connexin gene plasmid

which carried an intron (about 0.61 Kb) at the 3' end of the connexin gene.

c. Anchorage Independent Growth (AIG)

To test if expression of gap junction genes might affect tumor cell phenotype, three
clones of two different gap junction gene transfectants (i.e. Cx43 cDNA: MCF-7-43-3,
MCF-7-43-10, MCF-7-43-16 and Cx26 cDNA: MCF—7-26-4, MCF-7-26-6, MCF—7-26-8)
were examined for their ability of AIG. The plasmid control, MCF-7-DHFR, was included

in the experiment. After 4 weeks of growth, the colonies formed in the agar were counted

46

under a microscope. The plasmid control had 39.5% colony-fomring efficiency. For the
Cx43 and Cx26 cDNA transfectants the average colony-forming efficiency was 28.5% and
38% respectively. Clearly, the expression of these two gap junction genes did not
significantly affect the AIG of these cells.

47

A.
Kb 1 2 3 4 5 6 7
'IV
2.6- ..‘..-Cx43.
1.9— . -Cx43
B.

3.234567

ll‘ '1‘ e
O. ' it

. ... ‘ .. -Actin

Figure 16. Northern blot analysis of Cx43 transfectants. (A) The membrane was
hybridized with the rat Cx43 cDNA probe. (B) The same membrane was hybridized with
actin DNA probe. 1. MCF-7. 2. MCF-7-DHFR. 3. MCF-7-43-2. 4. MCF—7-43-3. 5.
MCF-7-43-4. 6. MCF—7-43-10. 7. MCF-7-43-l6.

48

A
Kb 1 2 3 4 5

l
2-3' i ‘ -Cx32
2°" a , -Cx32

12345

- Actin

 

Figure 17. Northern blot analysis of Cx32 transfectants. (A) The membrane was
hybridized with the rat Cx32 cDNA probe. (B) The same membranes was hybridized with
actin DNA probe. 1. MCF-7. 2. MCF-7-DHFR. 3. MCF-7-32-2. 4. MCF-7-32-l l. 5.
MCF-7-32-20.

Mi 2 4

{ Kb 1 ~‘ & On 050

l

l

J _

l
2.5- - Cx26
19" - Cx26

B.‘

- Actin

 

Figure 18. Northern blot analysis of Cx26 transfectants. (A) The membrane was
hybridized with the rat Cx26 cDNA probe. (B) The same membrane was hybridized with
actin DNA probe. 1. MCF—7. 2. MCF-7-DI-IFR. 3. MCF-7-26-4. 4. MCF-7-26-6. 5.
MCF—7-26-8.

DISCUSSION

The objectives of this thesis research were: 1) to determine what gap junction
genes are expressed in normal human breast epithelial cells and 2) to determine if GJIC
can be restored in MCF-7 cells by transfection with cloned cDNA for difi‘erent rat gap
junction genes. The major results obtained from this study may be summarized as follows:
1) The two primary human breast epithelial cell cultures derived fi'om reduction
mammoplasty from two women (i.e. HMEll and HME14), like other normal breast
epithelial cell cultures developed and studied by this laboratory, contain two
morphologically distinctive cell types: one is deficient in GJIC (Type-I) and the other is
competent in GJIC (Type-II); 2) The Type-I cells from both cultures did not express gap
junction genes for Cx26, Cx32 or Cx43 as revealed by Northern analysis; 3) The Type-II
cells from both cultures expressed the transcript for Cx26 and Cx43 but not Cx3 2; 4) The
human breast carcinoma cell lines, MCF-7 and T47D, are deficient in GJIC and do not
express detectable amount of mRNA for Cx26, Cx32 and Cx43, whereas, the breast
carcinoma-sarcoma cell line HSOS78T is competent in GJIC and expressed mRNA for
Cx43 but not Cx26 and Cx32 genes similar to normal human breast stromal fibroblasts; 5)
The GJIC can be restored in MCF—7 cells by transfection with cloned cDNA for rat Cx26,
Cx32 and Cx43; 6) Most MCF-7 transfectants lost the acquired GJIC gradually after
extended culture and 7) These MCF-7 transfectants expressing gap junction genes do not
seem to significantly alter their growth rate i_n m and ability of AIG.

Previous studies have shown that normal human breast epithelial cells are
competent in GJIC (F entiman and Taylor-Papadirrritriou, 1977; Fetirnan, 1980;Eldridge et
al., 1989) and expressed Cx26 and Cx43 genes (Lee et al., 1992). These phenotypes are
similar to those exhibited by Type-II cells of our normal breast epithelial cell culture.
There is good reason to believe that those cultures (e. g. Eldridge et al., 1989; Lee et al.,
1992) are Type-II cells since the media (i.e. MCDB 170, DFCI-l) used in their studies are

50

51

known to promote the growth of Type-II cells (personnel observation). The Type-I cells
were first identified and reported by our laboratory (Chang et al., 1990) and apparently
have not been studied by other laboratory. F urtherrnore, the Type-I cells can be induced
by cholera toxin (present in the DFCI-l medium) to difi‘erentiate into Type-H cells (Chang
et al., 1991) and can be converted to Type-II cells by culturing in the MCDB 170 medium
(Clonetics MEGM medium) (personnel observation).

. Type-I cells were deficient in GJIC and did not express gap junction genes for
Cx26, Cx32 and Cx43. These phenotypes are also shared by the breast carcinoma cell
lines, MCF-7 and T47D and other cell lines studied by Lee et al. (1992). The in vivo
study also indicates that the Cx43 gene was not expressed in invasive ductal carcinoma
(Wilgenbus et al., 1992). This correlation might indicate that most human breast
carcinomas are derived fi'om Type-I cells rather than fi'om Type-II cells. Similarly, the
breast sarcoma cell line, HSOS78T, and normal human breast stromal fibroblasts were
competent in GJIC and expressed Cx43 but not Cx32 and Cx26 genes. This might
indicate that the HSOS78T cell line is of fibroblast origin instead of epithelial cell origin.
With firrther substantiation, the above observation might be used as a marker to
distinguish breast sarcoma from breast carcinoma.

Based on differential expression of the Cx26 gene in normal and tumorigenic
breast epithelial cells, Lee et al. (1991) proposed that the Cx26 gene may be a tumor-
suppressor gene of breast cancer. The major characteristics of a tumor suppressor gene is
its expression in normal cells and the absence of its function in tumor cells. By this
criterion and in light of the fact that Cx26 and Cx43 gap unction genes are not expressed
in Type-I normal human breast epithelial cells which express luminal epithelial cell specific
antigens (i.e. EMA and cytokeratin 18) (Chang et al., in preparation), the hypothesis is
clearly in doubt. We have observed that the differentiation of Type-I to Type-II cells
involves a wholesale transition of gene expression, the most notable among them are

morphological change, growth requirements, keratin gene and gap junction gene

52

expression. Therefore the gap junction gene expression might be correlated with cell
growth and differentiation at the transcriptional level. The blockage of this differentiation
in Type-I cells might have implications in carcinogenesis. Furthermore, since blockage of
GJIC might be a mechanism for tumor promotion, one reason that breast carcinoma is of
ductal luminal origin might be the deficiency in GJIC of Type-I cells which are suspected
as the putative breast epithelial stem cells.

The ability to perform GJIC in communication-deficient MCF-7 cells can be
restored by transfection with cloned cDNA for rat Cx26, Cx32 or Cx43 gap junction
genes. Since MCF-7 cells do not express any of these genes, the results indicated that
functional cell-cell communication can be carried out by any one of the 3 gap junction
genes. Difl‘erent from the rat liver WB cells GJIC-deficient mutants transfected with the
same Cx43 gene and plasmid (Jou et al., 1993), the restoration of GJIC in MCF-7 cells did
not require amplification of the transfected gap junction gene. The discrepancy might be
due to the difference in mechanisms of GJIC deficiency between the two types of cells.
Unlike the MCF-7 cells, the WB cell mutants are defective in post-translational
phosphorylation of Cx43. Most MCF-7 transfectants, however, were unable to sustain the
initial level of GJIC after extended growth. The loss of GJIC in MCF-7 transfectants
could be at transcriptional or translational level. Further studies involving DNA
methylation and Western blot analysis should be able to discern the possible underlying
mechanisms. In addition, since the transfected gap junction genes are carried by an
amplifiable vector, it is possible to study whether the loss of GJIC in MCF-7 transfectants
might be prevented by amplification of the transfected gap junction genes.

The MCF-7 gap junction gene transfectants do not seem to change their ability of
AIG. Since most of these transfectants were unable to sustain the restored GJIC, the
effect of GJIC on these phenotypes may not be accurately studied. Although tumorigenic
cells are usually AIG competent, the AIG competent cells are not necessarily tumorigenic.

The MCF-43-10 clone was found to maintain GJIC after extended growth. This cell line

53

should be usefirl to study whether restoration of GJIC might afi'ect the tumorigenicity of

MCF-7 cells in immune-deficient mice.

REFERENCES

Abemethy, D.J., W.F. Greenlee, J.C. Huband and OJ. Boreiko. 1985. .2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3I-I/10T1/2 cells.
Carcinogenesis 6:651-653.

Alberts, B., D. Bray, J. Lewis, M. Rafl‘, K. Roberts, and JD. Watson. 1989. Molecular
Biology of the cell, p 798-802. Garland Publishing, Inc. New York & London.

Atkinson, M.M., A.S. Memko, R.G. Johnson, J.R. Sheppand, and JD. Shenidan. 1981.
Rapid and reversible redunction of junctional permeability in cells infected with a
temperature-sensitive mutant of avian sarcoma virus. J. Cell Biol. 91:573-578.

Atkinson, M.M., S.K. Anderson, J.D. Sheridan. 1986. Modification of gap junctions in
cells transfected by a temperature-sensitive mutant of rous sarcoma virus. J. membr. Biol.
9:53-64.

Azarinia, R., G. Dahl, and W.R. Loewenstein. 1981. Cell junction and cyclic AMP: III.
promotion of junctional membrane permeability and junctional membrane particles in a
junction-deficient cell type. J. Membrane Biol. 63: 133-146.

Azamia, R., and W.R. Loewenstein. 1984. Intercellular communication and the control of
growth: X. alternation of junctional permeability by the src gene. A study with
temperature-sensitive mutant rous sarcoma virus. J. Membr. Biol. 82: 191-205.

Azamia, R., S. Roddy, T.E. Kmiecik, D. Shalloway and W.R. Loewenstein. 1988. The
cellular src gene product regulates junctional cell-to-cell communication. Science 239:
398-400.

Bennett, M.V.L., L.C. Barrio, T.A. Bargiello, D.C. Spray, E. Hertzberg, and JG. Saez.
1991. Gap junctions: new tools, new answers, new qutestions. Neuron 6:305-3 20.

Bertram J.S., N.L. Kolonel, and F .L. Jr Meyskens. 1987. Rationale and strategies for
chemoprevention of cancer in humans. Cancer Res. 47:3012-3031.

Beyer, E.C., D.C. Paul, and DA. Goodemough. 1987. Connexin 43: a protein from rat
heart homologous to a gap junction protein from liver. J. Cell Biol. 105:2621-2629.

Beyer, E.C., D.A. Goodenough, D.C. Paul. 1988. The connexins, a family of related gap
junction proteins. In Gap Junctions (ed., E.C. Hertzberg and R.G. Johson), p 167-175.
Alan Liss. New York.

Boreiko, C.J., D.J. Abemethy, J.H. Sanchez and EH. Dorman. 1986. Effect of mouse
skin tumor promoters upon [3H]uridine exchange and focus formation in cultures of
C3H/10T1/2 mouse fibroblasts. Carcinogenesis 7: 1095-1099.

54

55

Burghardt, RC. and E. Anderson. 1979. Hormonal modulation of ovarian interstitial cells
with particular reference to gap junctions. J. Cell Biol. 81: 104.

Chang, C.C., J.A. Boezi, S.T. Warren, C.L.K. Sabourin, P.K. Liu, L. Glatzer and IE.
Trosko. 1981. Isolation and characterization of a UV-sensitive hypermutable aphidicolin-
resistant Chinese hamster cell line. Somat. Cell Genetics 7:23 5-253.

Chang, C.C., J .E. Trosko, H.J. Kung, D. Bombick and F. Matsumura. 1985. Potential role
of the src gene product in inhibition of gap junctional communcation in NIH/3 T3 cells.
Proc. Natl. Acad. Sci. USA 82:5360-5364.

Chang, C.C., S. Nakatsaka, G. Kalinri, J.E. Trosko and CW. Welsch. 1990.
Characterization of two types of normal human breast epithelial cells that are either
deficient or proficient in gap-junctional intercellular communication. J. Cellular Biochem.
Supplement 14B p.331.

Chang, C.C., C.Y. Kao, S. Nakatsuka, J.E. Trosko and CW. Welsch. 1991.

Identification of a subpopulation of normal human breast epithelial cells with stem cell
characteristics. Proc. Am. Assoc. Cancer Res. 32:40

Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chlorofonn extraction. Analytical Biochem. 162: 156-
159.

Crow, D.S., E.C. Beyer, D.L. Paul, S.S. Kobe and AF. Lau. 1990. Phosphorylation of
connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed
mammalian fibroblasts. Mol. Cell. Biol. 10:1754-1763.

De Feijter, A.W., J.S. Ray, C.M. Weghorst, J.E. Klaunig, J.I. Goodman, C.C. Chang, R.J.
Ruch, and J .E. Trosko. 1990. Infection of rat liver epithelial cells with v-Ha-ras:
Correlation between oncogene expression, gap junctional communication, and
tumonigenicity. Mol. Carcinogenesis 3:54-67.

De Feijter, A.W., J.E. Trosko, D.B. Krizrnan, RM. Lebovitz, and M.W. Lieberman. 1992.
Correlation of increased levels of Ha-ras T24 protein with extent of loss of gap junction
function in rat liver epithelial cells. Mol. Carinogenesis 52205-212.

De Mello, WC. 1972. The healing-over process in cardiac and other muscle fibers. In
electrical phenomena in the heart (ed. W.C. De Mello), p 323. Academic Press Inc, New
York.

De Mello, WC. 1980. Influence of intracellular injection of H+ on the electrical coupling
in cardiac Purkinjier fibers. Cell Biol. Int. Rep. 4:51-57.

De Mello, WC. 1982. Cell-to-cell communication in heart and other tissue. Prog.
Biophys. Mol. Biol. 39: 147-182.

56

De Mello, WC. 1987 . Modulation of junctional permeability. In cell-to-cell
communication (ed., W.C. De Mello), p 43. Plenum Press, Inc. New York, New York.

Dermietzel, R., T.K. Hwang, and BS. Spray. 1990. The gap junction family: structure,
function, and chemistry. Anal. Embryo]. 182:517-528.

Doolittle, R.F., M.W. Hunkarpiller and KC. Robbins. 1983. Simian sarcoma virus
oncogene v-sis, is derived fi'om the gene(s) encoding a platelet derived grth factor.
Science 221:275-277.

Downward, J., Y. Yasden and E. Mays. 1984. Close similarity of epidermal growth
factor receptor and v-erb B oncogene protein sequences. Nature 307 :52 1-527.

Eghbali, B., J .A. Kessler, and DC. Spray. 1990. Expression of gap junction channels in
communication incompetent cells after stable transfection with cDNA encoding connexin
32. Proc. Natl. Acd. Sci. USA 87:1328-1331.

El-Fouly, M.H., J.E. Trosko and CC. Chang. 1987. Scrape-loading and dye transfer, a
rapid and simple technique to study gap junctional intercellular communication. Exp. Cell
Res. 168:422-430. 1

El-Fouly, M.H., J.E. Trosko, C.C. Chang, S.T. Warren. 1989. Potential role of the human
Ha-ras oncogene in the inhibition of gap junctional intercellular communication. Mol.
Carcinogenesis 2: 13 1-135.

Eldridge, S.R., T.W. Martens, C.A. Sattler, and MN. Gould. 1989. Association of
decreased intercellular communication with the immortal but not the tumorigenic
phenotype in human mammary epithelial cells. Cancer Res. 49:4326—433 1.

Fentiman, 1.5., and J. Taylor-Papadinritriou. 1977. Cultured human breast cancer cells lose
selectivity in direct intercellular communication. Nature (London) 269:156-158.

Fentiman, 1.8., J. Hurst, R.L. Ceriani, and J. Taylor-Papadimitriou. 1979. Junctional

intercellular communication pattern of cultured human breast cancer cells. Cancer Res.
39:4739-4743.

Fentiman, LS. 1980. Cell communication in breast cancer. Ann. R. Coll. Surg. Eng,
62:280-286.

Furshpan, E.J., and DD. Potter. 1959. Transmission at the giant moter synapses of the
crayfish. J. Physiol. (London) 145:289-325.

Garfield, R.E., D. Merrett, and AK. Grover. 1980. Gap junction formation and regulation
in myometrium. Am. J. Physiol. 239:c217.

57

Garfield, RE. 1984. Control of myometrial function in preterm versus term labor. Clin.
Obstet. Gynecol. 27:572-591.

Garfield, R.E., W.C. Cole, and MG. Blennerhassett. 1989. Gap junctions in uterine
smooth muscle. In cellinteractions and gap junctions volume 11. (ed. N. Specelakis and
WC. Cole), p 247. CRC Press, Inc. Boca Raton, Florida.

Gilula, N.B., M.L. Epstein, and W.H. Beers. 1978. Cell-to-cell communication and
ovulation, a study of the cumulus-oocyte complex. J. Cell Biol. 78:58-75.

Hamel, E., F. Katoh, G. Mueller, W. Birchmeier and H. Yamasaki. 1988. Transformation
growth factor B as a potent promoter in two-stage BABL/c3 T3 cell transformation.
Cancer Res. 48:2832-2836.

Herris, A.L., D.C. Spray and M.V.L. Bennett. 1981. Kinetic properties of a voltage-
dependent junctional conductance. J. Gen. Physiol. 77:95-117.

Hertzberg, EL. and NB. Gilula. 1981. Gap junction communication. Ann. Rev. Physiol.
43:479-491.

Hertzberg, EL, and R.G. Johnson. 1988. Gap junctions. Alan R. Liss, Inc., New York.

Hoh, J.H., S.A. John, and JP. Revel. 1991. Molecular cloning and characterization of a
new member of the gap junction gene family, connexin 31. J. Biol. Chem. 266: 6524-
6531.

Hossairr, M.Z., L.R. Wilkens, P.P. Mehta, W. Loewenstein, and IS. Bertram. 1989.
Enhancement of gap junctional communication by retinoids correlates with their ability to
inhibit neoplastic transformation. Carcinogenesis 10: 1743-1748.

Hsieh, C.L., NM. Kumar, N.B. Gilula, and U. Francke. 1991. Distribution of genes for
gap junction membrane channel proteins on human and mouse chromosomes Somatic
Cell. Mol. Genetics 17: 191-200.

Ito, S., and N. Hori. 1966. Electrical characteristics of Triturus eggs during cleavage. J.
Gen. Physiol. 49:1019.

Jou, Y.S., E. Dupont, S.C. Lu, B.V. Madhukar, S.Y. Oh, J.E. Trosko and CC. Chang.
1993. Restoration of gap junction intercellular communication in a communication
deficient rat liver cell mutant by transfection with connexin43 cDNA. Mol. Carcinogenesis
(in press).

Kalimi, Gand S.M. Sirsat. 1984. The relevance of gap junctions to stage I tumor
promotion in mouse epidermis. Carcinogenesis 5:1671-1677.

58

Kalinri, 6., CC Chang, P. Edwards, E. Dupont, B.V. Madhukar, E. Standbridge and IE.
Trosko. 1990. Re-establishment of gap junctional communication in a non-tumorigenic
Hela-norrnal human fibroblasts hybrid. Proc. Am. Assoc. Cancer res. 31:318.

Kam, E. and J .D. Pitts. 1988. Effects of the tumor promoter 12-O-tetradecanoylphorbol-
13-acetate on junctional communication in intact mouse skin: persistence of homologous
communication and increase of epidermal-dermal coupling. Carcinogenesis 9: 1389-1394.

Kameyarne, M. 1983. Electrical coupling between ventricular paired cells isolated from
guinea-pig heart. J. Physiol. (London) 3362345.

Kanno, Y. 1985. Modulation of cell communication and carcinogensis. J. Physiol.
35:693-707.

Larsen, W.J., H.N. Tung and C. Polking. 1981. Response of granulosa cell gap junctions
to human chorionic gonadotropin (hCG) at ovulation. Biol. Reproduct. 25: 1 l 19.

Lawrence, T.S., W.H. Beers and NB. Gilula. 1978. Transmission of hormonal stimulation
by cell-to-cell communication. Nature 272:501-506.

Ledbetter, M.L.S. and M. Lubin. 1979. Transfer of potassium, a new method of cell-cell
coupling. J. Cell Biol. 80:150-159.

Lee, S.W., C. Tomasetto and R. Sager. 1991. Positive selection of candidate tumor-
suppressor genes by subtractive hybridization. Proc. Nat. Accd. Sci. USA 88:2825-2829.

Lee, S.W., C. Tomasetto, D. Paul, K. Kejomarsi, and R. Sager. 1992. Transcriptional
downregulation of gap-junction proteins blocks junctional communication in human
mammary tumor cell lines. J. Cell Biol. 118:1213-1221.

Lo, CW. 1980. Gap junctions and development. In "development of mammals" (ed,
M.H. Johnson) Vol. 4, p 39-80. Elserier, New York.

Loewenstein, W.R. 1979. Junctional intercellular communication and the control of
growth. Biochim. Biophy. Acta 56021-65.

Loewenstein, W.R. 1981. Junctional intercellular communication: the cell-to-cell
membrane channel. Physiol. Reviews 61:829-913.

Meda, P., A. Perrelet and L. Orci. 1979. Increase of gap junctions between pancreatic B-
cells during stimulation of insulin secretion. J. Cell Biol. 82:441.

Madhukar, B.V., S.Y. Oh, C.C. Chang, M. Wade and IE. Trosko. 1989. Altered
regulation of intercellular communication by epidermal growth factor, transforming
growth factor-B, and peptide hormones in normal keratinocyte. Carcinogenesis 10:13.

59

Mehta, P.P., A. Hotz-Wagenblatt, B. Rose, D. Shalloway, and W.R. Loewenstein. 1991.
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to
normalization of growth. J. Membr. Biol. 124:207-225.

Mehta, P.P., J .S. Bertramand W.R. Loewenstein. 1986. Growth inhibition of transformed
cells correlates with their junctional communication with normal cells. Cell 44: 187-196.

Mesnil, M., D.J. Fitzgerald, and H. Yamasaki. 1988. Phenobarbital specifically reduces
gap junction protein mRNA level in rat liver. Mol. Carcinog. 1:79-81.

Metzger, P., and R. Weingart. 1985. Electric current flow in cell pairs isolated fiom adult
rat hearts. J. Physiol. (London) 336: 177.

Michaels, R.L. 1982. Increased dye coupling among cells in prolactin stimulated
pancreatic islets. J. Cell Biol. 95:94a

Mordan, L]. 1991. Carcinogenesis models. Science 251: 142-143.

Murray, A.W., and DJ. Fitzgerald. 1979. Tumor promoters inhibit metabolic cooperation
in cocultures of epidermal and 3T3 cells. Biochem. Biophys. Res. Commun. 91:395-401.

Murray, A.W., and H.S.C. Gainer. 1989. Regulation of gap junctional communication by
protein kinase. In cell interactions and gap junction (ed., N. Sperelakis and WC. Cole), p
98. CRC Press, Inc. Boca Raton, Florida.

Noma, A., and N. Tsuboi. 1987. Dependence of junctional conductance on proton,
calcium and magresium ions in cardiac paired cells of guinea-pig. J. Physiol. (London)
382: 193.

Page, D.L., and TL. Anderson. 1987. Diagnostic I-Iistopathology of the breast.
Edinburgh, Churchill Livingstone.

Pasti, G., E. Rivedal, S.H. Yuspa, C.L. Herald, G.R. Pettitand P.M. Blumberg. 1988.
Contrasting duration of inhibition of cell-cell communication in primary mouse epidermal
cells by phorbol 12,13-dibutyrate and by bryostatin 1. Cancer Res. 48:447-451.

Paul, BL. 1986. Molecular cloning of cDNA for rat liver gap junction protein. J. Cell
Biol. 1032123-134.

Pittelkow, MR, and RE. Scott. 1986. New techniques for the in vitro culture of human
skin keratinocytes and perspectives on their use for grafting of patients with extensive
burns. Mayo Clin. Proc. 61:771-777.

Rae, II. 1982. Curr. Top. Eye Res. 35:598-609.

60

Rose, B. and W.R. Loewenstein. 1976. Permeability of a cell junction and the local

cytoplasmic free ionized calcium concentration: a study with aequorin. J. Membrane Biol.
28:87

Rose, B., 1. Simpson and W.R. Loewenstein. 1977. Calcium ion produces graded changes
in permeability of membrane channels in cell junction. Nature 267:625.

Rose, B. and R. Rick. 1978. Intracellular pH, intracellular free Ca and junctional cell-cell
coupling. J. Membrane Biol. 44:377-415.

Sheridan, J .D. 1973. Functional evaluation of low resistance junctions: influence of cell
shape and size. Am. Zool. 13:1119-1128.

Sheridan, J .D. 1987. Cell communication and growth. In cell-to-cell communication (ed.,
W.C. De Mello), p 188. Plenum Press, Inc. New York, New York.

Silberberg, B, CO Boring, T.S. Suives. 1990. Cancer Statistics, 1990. CA 40:8-26.

Slack, C., and IF. Palmer. 1969. The permeability of intercellular junctions in the early
embryo of xenopus laevis, studies with a fluorescent tracer. Exp. Cell Res. 55:416.

Subak-Sharpe, J.H., R.R. Burk and JD. Pitts. 1969. Metabolic cooperation behveen
biochemically marked mammalian cells in tissue culture. Cell Sci. 4:353-367.

Sugie, S., H. Mori, and M. Takahashi. 1987. Effect of in vivo exposure to the liver tumor
promoters phenobarbital or DDT on the gap junctions of rat hepatocytes: a quantitative
fieeze-fracture analysis. Carcinogrenesis 8:45-51.

Tomasetto, C., S.W. Lee, D. Paul, and R. Sager. 1991. Gap junction communication in
normal and tumoral mammary epithelial cells. J. Cell Biol. 115,190a.

Trosko, J .E., and CC. Change. 1988. Non-genotoxic mechanism in carcinogenesis: role
of inhibited intercellular communication. In: Banbury report 31: carinogen risk assessment:
new directions in qualitative and quantitative aspects, p 139-170. Cold Spring Harbor
Laboratory, New York.

Turin, L., and AB. Warner. 1977 . Carbon dioxide reversibly abolishes ionic
communication between cells of early amphibian embryos. Nature 270:56-57.

Unwin, P.N.T., and G. Zamphigi. 1980. Structure of the junction between communicating
cells. Nature 283:545-549.

Vanhamme, L., S. Rolin and C. Szpirer. 1989. Inhibition of gap-junctional intercellular
communication between epithelial cells transformed by the activated H-ras-l oncogene.
Exp. Cell Res. 180:297-301.

Vitkauskas, G.V. and ES. Canellakis. 1983. J. Cell Biol. 97:80a

61

Vitkauskas, G.V., and ES. Canellakis. 1985. Intercellular communication and cancer
chemotherapy. Biochim. Biophy. Acta 833: 19-34.

Warner, A.E., S.C. Guthrie, and NB. Gilula. 1984. Antibodies to gap-junctional protein
selectively discrupt junctional communication in the early amphibian embryo. Nature
311:127-131.

Welsch, F. 1990. Teratogens and cell-to-cell communication. In "cell Intercommunication"
(ed., W.C. De Mello), p137. CRC Press Inc. Boca Raton, Florida.

White, R.L., D.C. Spray, A.C. Campos de Carvalho, B.A. Wittenberg, and M.V.L.

Bennett. 1985. Some electrical and pharmacological properties of gap junctions between
adult remtricular myocytes. Am. J. Physiol. 249zc447.

Wiener, EC, and W.R. Loewenstein. 1983. Correction of cell-to-cell communication
defect by introduction of a protein kinase into mutant cells. Nature 305:433-435. -

Wilgenbus, K.K., C.J. Kirkpatrick, R. Knuechel, K. Willecke, and O. Traub. 1992.
Expression of Cx26, Cx32 and Cx43 gap junction ptroteins in normal and neoplastic
human tissues. Int. J. Cancer 51 :522—529.

Willecke, K., S. Jungbluth, E. Dahl, H. Hennemanh, R. Heynkes, and KR Grzeschik.
1990. Six genes of the human connexin gene family coding for gap junctional proteins are
assigned to four different human chromosomes. Eur. J. Cell Biol. 53:275-280.

Yamasaki, H. and T. Enomoto. 1985. Role of intercellular communication in BALB/c3T3
cell transformation. In Carcinogenesis vol. 9, a comprehensive survey-mammalian cell
transformation : mechanisms of carcinogenesis and assays for carcinogens (ed. J.C. Barrett
and R.W. Tennant) p. 179-194. Raven press,New York.

Yamasaki, H., M. Hollstein, M. Mesnil, N. Martel, and AM. Aguelon. 1987. Selective
lack of intercellular communication between transformed and nontransformed cells as a

common property of chemical and oncogene transformation of BALB/C3T3 cells. Cancer
Res. 47:5658-5664.

Yamasaki H. and F. Katoh. 1988. Further Evidence for the Involvement of gap-junctional
intercellular communication in induction and maintenence of transformed foci in
BALB/C3T3 cells. Cancer Res. 48:3490-3495.

Yamasaki, H. 1990. Gap junctional intercellular communication and carcinogenesis.
Carcinogenesis 1 1 : 1051-1058.

Yotti, L.P., C.C. Change, and J .E. Trosko. 1979. Elimination of metabolic cooperation in
Chinese hamster cells by a tumor promoter. Science 206: 1089-1091.

62

Zhang, IT, and BI. Nicholson. 1989. Sequence and tissue distribution of a second
protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J. Cell Biol.
109:3391-3401.

"1111111111111“