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Department of Radiation Oncology, Temple University School of Medicine, Philadelphia, PA 19140
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INTRODUCTION |
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 TOP INTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Callahan and Campbell 1989
). Overall, these human tissue and animal
studies suggest that mammary tumors arise in a multistep process and
may involve the activation and inactivation of multiple oncogenes and
tumor suppressor genes.
Tumor suppressor genes are considered likely to be important in human
breast cancer (Marshall 1991
, Sager 1992
). In contrast to oncogenes, tumor suppressor genes inhibit
cell proliferation, and their loss is associated with tumorigenesis. A
screen for tumor suppressor genes from human mammary epithelial cells
resulted in the cloning of a gene encoding a gap junction protein
(Lee at al. 1991
). It has long been speculated that gap
junctions mediate cell-cell interactions important in growth
suppression and regulation (Loewenstein 1979
). For
example, coculturing of transformed cells with normal cells can
suppress cell proliferation and other characteristics associated with
the transformed phenotype (Mehta et al. 1986
,
Stoker et al. 1966
). In one study, growth suppression
was correlated with the efficiency of gap-junctional coupling
between the transformed and nontransformed cells (Mehta et al. 1986
). Other studies also showed that the transfection of
connexin gene expression vectors into tumor cells can restore normal
cell growth in vitro (Hirischi et al. 1996
, Mehta et al. 1991
, Zhu et al. 1991
) and reduce
tumorigenicity in vivo (Eghbali et al. 1991
, Rose et al. 1993
).
Gap junctions are composed of proteins known as the connexins
(Bennett et al. 1991
, Beyer et al. 1990
,
Kumar and Gilula 1992
). One of the most widely expressed
connexin genes, connexin (Cx) 43, is also found in the mammary
epithelium (Lee et al. 1991
and 1992
). In addition, the
mammary epithelium also contains Cx26 (Lee et al. 1991
and 1992
), a connexin gene often coexpressed with Cx43 or Cx32.
Moreover, gap junction gene expression in human breast cancer tissue
and cells is consistently down-regulated (Lee et al. 1992
, Wilgenbus et al. 1992
). A role for
gap-junctional perturbation in mammary tumorigenesis is suggested
by the finding that all mammary tumors are deficient in gap junctions
(Wilgenbus et al. 1992
), and cells derived from mammary
tumors do not exhibit gap junction–mediated cell-cell
communication (Lee et al. 1992
). These findings further
indicate the possible importance of gap junctions in the regulation of
mammary epithelial cell proliferation.
Chemoprevention is a new area of breast cancer research that shows
great promise. In particular, tamoxifen, progestins and retinoids have
been shown to be effective in lowering the risk of breast cancer
(Dickens and Colletta 1993
). Another class of compounds
that may be useful for breast cancer chemoprevention is the trypsin and
chymotrypsin protease inhibitors derived from various vegetables. The
efficacy of protease inhibitors in cancer prevention is well documented
(Kennedy 1993a
). For example, protease
inhibitors have been shown to suppress carcinogen-induced malignant
transformation of tissue culture cells (Kennedy 1993
).
There is also a large body of data from animal studies that
demonstrates the anticarcinogenic activity of protease inhibitors
(Kennedy 1993
).
In this study, we examined a soybean-derived protease inhibitor,
the Bowman-Birk inhibitor (BBI), as a possible breast cancer
chemopreventive agent. Bowman-Birk inhibitor suppresses
tumorigenesis in several experimental carcinogenesis model systems
(Kennedy 1993a
). In addition and of particular
significance to these studies is the observation that a soybean diet
rich in protease inhibitors (e.g., BBI) lowered the breast tumor
incidence in irradiated rats (Troll et al. 1980
). Our
interest in BBI is further motivated by several considerations of a
more practical nature. First, BBI, a product derived from a natural
food source (soy beans), is likely to be nontoxic to humans. Second,
the animal studies of Kennedy and co-workers (Kennedy 1993b
, Oreffo et al. 1991
) have shown
the efficacy and safety of BBI as a cancer chemopreventive agent.
Third, these studies also demonstrated that BBI can maintain its cancer
chemopreventive potency even when administered as a dietary supplement
(Kennedy et al. 1993
). Hence, it is likely that BBI will
be eminently suitable as a cancer chemopreventive agent for use in
humans.
Although the mechanisms by which protease inhibitors suppress
carcinogenesis are unknown, they are thought to act as antipromotional
agents in many of the in vitro and in vivo two-stage carcinogenesis
experimental models (Kennedy 1984
, Troll et al. 1970
). The suppression of malignant transformation by BBI and
other anticarcinogenic protease inhibitors has been suggested to be
mediated via their effects on the expression of c-myc and
other genes or oncogenes involved in the initiation, promotion or
progression of the malignant phenotype (St. Clair and St. Clair 1991
).
In this paper, we report our finding that the expression of a gap
junction gene Cx43 is induced in human mammary epithelial cells after
BBI treatment. This observation is particularly intriguing because gap
junctions are proposed to play a role in growth suppression and tumor
promotion. Moreover, as mentioned above, human breast cancer cells have
been shown to be deficient in gap junctions (Lee et al. 1992
, Wilgenbus et al. 1992
).
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MATERIALS AND METHODS |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells were grown in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and antibiotics (penicillin, 100 U/mL; streptomycin, 100 mg/L). Each cell line was cultured under two different conditions. The control condition was growth in their respective media. The treated condition was growth in their media with the addition of BBI (100 mg/L). Cells were grown to 80% confluence and then starved of serum for 24 h. They were subsequently serum stimulated for 4 h in medium containing twice (20%) the initial serum concentration. The cells were scraped, pelleted and flash-frozen at the end of the 4 h for subsequent RNA isolation for Northern analysis.
Northern analysis.
We isolated total cellular RNA by the RNAzol B Method (Cinna/Biotecx, Houston, TX). Total RNA (10 µg) were denatured and resolved by agarose gel electrophoresis and then blotted onto Duralose-UV nitrocellulose membranes (Stratagene, La Jolla, CA). The blots were hybridized to human Cx43 riboprobes. In addition, each Northern blot was rehybridized to a riboprobe for the ribosomal protein gene rpl32 (obtained from S. Liebhaber, Washington University, St. Louis) to control for possible variations in RNA loading.
Functional analysis in cell lines.
Gap-junctional communication was examined by using microelectrode impalements to iontophoretically inject fluorescent dye tracer (such as carboxyfluorescein or Lucifer yellow) into individual cells and then monitoring the movement of the fluorescent dye between cells. The efficiency of gap-junctional communication was quantitated by determining the rate at which cells that were 1st-, 2nd- and 3rd-order removed from the impalement site were filled with the fluorescent dye tracer.
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RESULTS |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In preliminary experiments, we observed by Northern blot hybridization
that treatment of a rodent fibroblast cell line (C3H/10T1/2) with the
protease inhibitor BBI resulted in a four- to fivefold increase in the
expression of transcripts from the gap junction gene Cx43 (Fig. 1A
,
B
). A similar induction of Cx43 transcripts was seen when these cells
were treated with a synthetic protease inhibitor, Antipain (Fig. 1A
, B
). In contrast, we noted that there was only
a low level of Cx43 transcripts in the radiation-transformed
C3H/10T1/2 cell line (Fig. 1B
), a characteristic of
transformed cell lines in general. We also determined that the
BBI-mediated increase in Cx43 transcripts was correlated with an
increase in the level of gap-junctional coupling in BBI-treated
C3H/10T1/2 cells (data not shown). This was demonstrated with the
examination of dye coupling using intracellular microelectrode
impalements to inject the fluorescent dye tracer carboxyfluorescein.
These microelectrode impalement studies showed that the BBI-treated
cells exhibited an increased level of gap-junctional communication,
as indicated by the very extensive spread of the fluorescent dye
tracer. In comparison, the nontreated cells exhibited only a low level
of dye transfer.
|
). In contrast, MCF-7 and BT-20 are
tumorigenic cells derived from human breast cancer tissues
(Soule et al. 1973
). Northern blot analysis revealed
that BBI treatment of MCF-10 led to an induction of Cx43 transcript
expression, whereas no Cx43 transcripts were detected in MCF-7 or
BT-20, either with or without BBI treatment (Fig. 2
) These findings are particularly intriguing because in previous
studies, BBI was shown to exert its anticarcinogenic effects only at
the initiation and promotional stages of tumor progression, with no
suppressive effects elicited on fully malignant cells or tumors
(Kennedy 1984
, Troll et al. 1970
).
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|
DISCUSSION |
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|
TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), suggesting that gap junctions may be categorized
as tumor suppressor genes. Overall, these preliminary results suggest
that the MCF-10 mammary epithelial cell line is an excellent model
system for examining the effects of BBI on gap junction gene
expression. We hypothesize that gap junctions may play a role in early
events in the progression of mammary tumorigenesis and that these
events may be modulated by protease inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from the American Institute for Cancer Research.
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REFERENCES |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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