Differential Up-Regulation of Gap Junction Connexin 26 Gene in Mammary and Uterine Tissues: The Role of Sp Transcription Factors
Zheng Jin Tu,
Rahn Kollander and
David T. Kiang
Breast Cancer Research Laboratory Department of Medicine
University of Minnesota Medical School Minneapolis, Minnesota
55455
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ABSTRACT
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The mRNA and protein expressions of connexin
26 (Cx26) in rat mammary gland and uterus can be
up-regulated during pregnancy as well as by the administration of human
CG (hCG). In the present study, we found that the time course and
magnitude of Cx26 induction by hCG was different in these two tissues.
The molecular mechanism underscoring this difference was therefore
investigated. We had previously demonstrated that both Sp1 and Sp3
transcription factors play a functional role in Cx26 expression. By the
electrophoretic mobility shift assay, nuclear extracts from both virgin
mammary gland and uterus were capable of binding to a labeled
oligonucleotide probe that contained the proximal GC box and formed
three protein-DNA complexes (C1, C2, and C3). In the mammary
gland, pregnancy enhanced the intensity of all three complexes, whereas
in the uterine tissue there was a decrease in the C2 and C3 complexes
and an emergence of a new major component, C4 complex. In the
supershift study, the C1 complex could be supershifted only by an
antibody against Sp1, whereas C2, C3, and C4 could all be supershifted
by an antibody against Sp3, suggesting a potential presence of Sp3
isoforms of various sizes. We therefore conclude that the basal Sp
profiles in virgin mammary gland and uterine tissue are similar.
However, in response to pregnancy, the changes in Sp profile are tissue
specific and may account for the temporal and quantitative differences
between these two tissues in Cx26 induction.
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INTRODUCTION
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Gap junctional intercellular communication serves a critical role
in facilitating physiological function as well as in maintaining tissue
homeostasis (1, 2, 3). Gap junctions are formed by the assembly of
connexin (Cx) proteins (Ref. 4 , for review). The type and degree of Cx
expression in each tissue is cell-, age-, and stage-dependent, and such
regulation is tightly controlled (5, 6, 7). One of the well known
physiological functions of Cx in the reproductive system is a reduction
of Cx43 in uterine myometrium during pregnancy that is presumably
responsible for maintaining uterine quiescence before parturition (8, 9). At term, however, Cx43 expression abruptly increases 5- to 9-fold
to synchronize a simultaneous contractile activity during labor (9, 10).
It has been shown that Cx43 in mammary myoepithelial cells is
up-regulated during parturition, resulting in coordinated contraction
and therefore milk ejection (11). Consistent with this finding is our
recent observation that the Cx43 expression was down-regulated during
pseudopregnancy when induced by the administration of human CG (hCG)
(12). One may therefore postulate that such Cx43 down-regulation in
breast tissue may serve a similar physiological function in preventing
a premature milk ejection before parturition. In mammary luminal
epithelial cells, however, Cx26 is the major Cx. It is markedly
up-regulated during pregnancy and lactation, presumably to facilitate
and coordinate all mammary epithelial cells to pursue their
intended physiological function, i.e. milk production
(13, 14). This up-regulation of Cx26 in luminal epithelial cells could
also be induced by daily administration of hCG (12).
During tumorigenesis, the transformed and neoplastic cells are
frequently associated with a down-regulation of various Cx expressions
and functions, so that the aberrant cells in their pursuit of
autonomous growth will receive less influence from surrounding normal
cells (15, 16, 17). A loss of intercellular communication and a
down-regulation of Cx26 expression occurs in most breast cancer cells.
Of interest, several recent reports support the contention that Cx26 is
a candidate suppressor gene (5, 18, 19, 20, 21); when introduced into MCF-7
breast cancer cells by transfection, the Cx26 gene confers
not only a reduction of tumor growth potential but also a restoration
of cellular differentiation (20).
As important as it appears to be, our knowledge of transcriptional
regulation on Cx in general and Cx26 in particular is surprisingly
limited. Little is known of Cx26 regulation owing to a lack of
information on its promoter region. We have recently mapped and
characterized the basal promoter of the human Cx26 gene (22)
as well as the rat Cx26 gene (GenBank accession number
AF015311). The promoter regions in both species are extremely GC rich
and highly conserved. The GC consensus boxes at the proximal promoter
region and Sp transcription factors have been shown to play an
important role in regulating the Cx26 expression (Z. J. Tu, Z. Gong,
and D. T. Kiang, manuscripts in preparation).
Because Cx26 is a major Cx in mammary epithelial and uterine
endometrial cells, we investigated the molecular mechanisms
underscoring the up-regulation of the Cx26 gene during
pregnancy, lactation, and hCG administration, particularly the
transcriptional regulation at the promoter region of the rat
Cx26 gene.
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RESULTS
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Cx26 Up-Regulation in Pregnancy and Lactation
A marked up-regulation of Cx26 mRNA and protein was seen during
pregnancy in both mammary epithelial and uterine endometrial cells.
These changes are demonstrated by Northern blot analysis (Fig. 1A
), in situ hybridization
(Fig. 1B
), and by fluorescent immunocytochemistry using a monoclonal
antibody against Cx26 (Fig. 1C
). During pregnancy, there was a 15-fold
induction of steady-state Cx26 mRNA in uterine tissues, far greater
than the 8-fold increase in the mammary glands. When rats reached the
lactation and postlactation stages, the Cx26 expression in mammary
gland remained elevated, whereas it was attenuated to below its basal
level in uterine tissue (Fig. 1A
). These data clearly demonstrated a
differential tissue-specific response of the Cx26 gene at
various physiological stages. Using the in situ
hybridization technique, the enhanced Cx26 mRNA expression could be
localized in the luminal epithelial cells of the lactating mammary
gland (Fig. 1B
). The induction of Cx26 protein in the mammary
epithelial cells during pseudopregnancy were previously reported by our
group (12). An enormous induction of Cx26 protein could also be
demonstrated in the endometrial cells of the pregnant uterus by the
fluorescent immunocytochemical staining (Fig. 1C
).

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Figure 1. Up-Regulation of Rat Cx26 Expression in the Mammary
Gland and Uterus
A, Northern blot analysis on the Cx26 mRNA expression in rat mammary
gland and uterus at virgin (V), pregnant (P), lactating (L), and
postlactating (PL) stages. The 28S and 18S rRNAs were used as loading
controls. B, Cx26 mRNA expression in virgin (vm) and lactating (lm) rat
mammary glands using the in situ hybridization technique
with digoxigenin labeling. There was no labeling in the control when
sense probe was used (magnification, 400x). C, Fluorescent
immunocytochemical staining of Cx26 protein expression in virgin (v)
and pregnant (p) rat uterus. Mouse IgG was used as control primary
antibody (c) (magnification, 640x).
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Differential hCG Effects on Cx26 Regulation in Different
Tissues
The kinetics of Cx26 induction by the administration of hCG were
further examined. During the hCG treatment, rats were killed at
different time intervals. By Northern analysis, a 5-fold induction of
Cx26 in the uterus was seen after 3 days of hCG administration (Fig. 2
), whereas the induction lagged behind
in mammary gland and took 5 days of hCG to reach a similar intensity
(Fig. 2B
). In the uterine tissue, Cx26 induction reached its maximum
level (a 18-fold increment) by day 8 and then declined to near the
basal level by day 21, a time point equivalent to natural parturition.
In the mammary gland, however, the effect on Cx26 induction reached and
maintained its maximal level (a near 11-fold increment) from day 8
through day 21 of hCG administration. Therefore, regardless of whether
it is natural pregnancy (Fig. 1
) or hCG-induced pseudopregnancy (Fig. 2
), the results consistently show that the time course and the
magnitude for Cx26 induction between these two tissues are
different.

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Figure 2. The Kinetics of Cx26 Induction by hCG, a Comparison
between Mammary Gland and Uterus
A, Northern blot analysis of rat Cx26 expression in the mammary gland
and uterus. The rats were given hCG ip at a dose of 100 U daily up to
21 days. The 28S and 18S rRNAs were used as loading control. B,
Relative Cx26 mRNA expressions during hCG treatment in comparison with
the pretreatment levels (day 0) in the rat uteri (closed
circles) and mammary glands (open circles).
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Binding of Transcription Factors to the Cx26 Promoter
Because the promoter region of Cx26 is GC rich and our previous
experiments revealed that the proximal GC box is essential for the
basal function of human and rat Cx26, we decided to use a gel mobility
shift assay to analyze and compare the nuclear protein-binding profiles
of this promoter region. The physical structure of the rat Cx26
promoter region is shown in Fig. 3
.
There are four major transcription start sites at four consecutive
nucleotides. A TATA-like box is located at -39 and a GC box at -136,
relative to the first transcription start site; both are highly
conserved when compared with mouse and human Cx26.

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Figure 3. Genomic Structure of the Rat Cx26 Promoter Region
in Comparison with Those of Mouse and Human
The GC and TATA-like boxes are identified with
rectangles. Gaps (dotted lines) are
introduced to maximize the alignment. The dashed lines
represent aligned identical bases. The arrows denote the
major transcription start sites: four for rat Cx26 (rCx26), two for
mouse Cx26 (mCx26), and a single site for human Cx26 (hCx26).
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In the gel mobility shift assay, the labeled probe used was a rat Cx26
promoter oligonucleotide (-144 to -123) encompassing the critical
proximal GC box (Table 1
). Proteins in
nuclear extracts of the virgin mammary gland and uterine tissue bound
to this labeled probe and formed three complexes designated C1, C2, and
C3. Their intensities were C2 > C3
C1 (Fig. 4
). In the mammary gland, all of these
three complexes proportionally increased in abundance during pregnancy
and lactation and then returned to their basal level at the
postlactation stage. The uterine tissue had a quite different response
to pregnancy; the increase in the C1 complex was similar to that of
mammary gland during pregnancy; however, there was a marked decrease in
the C2 and C3 bands, and a new C4 complex emerged to become the
dominant component. It appears that the C2/C3 components were markedly
switched to a C4 complex during pregnancy, a unique response seen in
uterine tissues but not in mammary glands.

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Figure 4. EMSAs on Nuclear Proteins Extracted from the
Mammary Gland and Uterus in Virgin (V), Pregnant (P), Lactating (L),
and Postlactating (PL) Rats
A double-stranded oligonucleotide encompassing the promoter GC
box was used as the probe (sequence shown in Table 1 ). Four protein-DNA
complexes were observed and designated as C1, C2, C3, and C4.
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Differential Expression of Sp among Tissues during Pregnancy
Since the labeled probe contained a potential binding site for Sp
transcription factors, we performed a competitive assay using wild-type
or mutated self-oligonucleotides, as well as wild-type or mutated Sp1
consensus oligonucleotides as competitors (Table 1
). The nuclear
extracts from pregnant mammary gland again formed three complexes with
the DNA probe (Fig. 5
). These bindings
could be effectively competed by wild-type self-oligonucleotide or Sp1
consensus oligonucleotide, but not by the two mutated ones. Similar
results were also seen in pregnant uterus. Here, the additional C4
complex was also efficiently suppressed by wild-type self- and Sp1
oligonucleotides. Therefore, the major transcription factors that bind
to the Cx26 promoter region belong to the Sp family.

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Figure 5. Competitive Suppression of Nuclear Protein Binding
to the Labeled Probe
The nuclear extracts were either from pregnant mammary glands or
pregnant uteri. The competitors were either nonlabeled GC, or consensus
Sp1 oligonucleotides, or their mutated counterparts (mGC and mSp1). The
sequences of the labeled probe and its competitors are listed in Table 1 .
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To further delineate the type of Sp transcription factors involved in
the binding, as well as the dynamic changes of Sp during pregnancy, we
performed supershift assays using antibodies against Sp1, Sp2, and Sp3
(Fig. 6
). The antibody against Sp2 did
not supershift any of these complexes. However, the C1 complex was
clearly supershifted by Sp1 but not by Sp3 antibody in both pregnant
mammary gland and uterus. On the contrary, C2, C3, and C4 could be
supershifted by an antibody against Sp3, suggesting that these three
complexes could contain Sp3 isoforms of various sizes, with C4 having
the smallest Sp3. In comparing the gel mobility shift pattern between
virgin and pregnant uterus, there was a nearly total switch in dominant
Sp3 complexes, from C2 and C3 in virgin uterus to C4 during
pregnancy.

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Figure 6. Supershift Analysis of Protein-DNA Complexes Using
Polyclonal Antibodies (Ab) against Sp1, Sp2, and Sp3 Transcription
Factors
The nuclear extracts were from rat mammary glands or uteri. The
protein-DNA binding complexes, C1 to C4, were designated as in Fig. 4 .
The two supershift bands are identified as S1 and S2.
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DISCUSSION
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Gap junctional intercellular communication plays a pivotal role in
developmental functions. There are dynamic changes in the types as well
as in quantities of connexins in the reproductive system. Our primary
interest was to examine the molecular mechanism underscoring the Cx26
up-regulation in mammary gland and uterus. We have found that Cx26 was
up-regulated in both tissues, but there are differences in time course
as well as in quantity between these two tissues in response to
pregnancy and lactation.
Transcriptional regulation has been shown to serve as the primary
control of connexin gene expression (23, 24, 25, 26). By reporter gene
analysis, the basal promoter regions of mouse Cx43 and rat
Cx32 have been mapped and characterized. An activator and a
repressor have been identified in the 5'-flanking region of the mouse
Cx43 gene (25), and a liver-specific binding complex has
been demonstrated to be an essential component of rat Cx32 basal
promoter (24). Although GC-like sequences have been described in these
connexin gene promoters, they are not Sp1 binding sites (24, 25).
In our study, Sp1 and Sp3 are clearly involved in the induction of Cx26
during pregnancy and lactation when the mammary gland is fully
differentiated. It is worth noting that similar phenomenon also
occurred in p21Cip1/WAF1, another tumor suppressor gene
intimately related to terminal differentiation and growth arrest. Sp1
and Sp3 activate the p21 promoter, but only Sp3 overexpression enhances
promoter inducibility during keratinocyte differentiation (27). In our
study, Sp3 is also the dominant transcription factor that bound to the
promoter of Cx26. It forms C2, C3, and C4 protein-DNA complexes at the
site of GC box and activates the transcription of Cx26 during
pregnancy.
Sp3 is a bifunctional transcription regulator. It is capable of acting
either as a repressor or an activator in a promoter-dependent manner
(Ref. 27 and references therein). When Sp3 binds to a single site of
the promoter, the repressor activity is lost. Therefore, the positive
or negative regulation is determined by the number of binding sites
present in the promoter region. Activation or repression of Sp3 has
been studied in promoters with various numbers of functional GC boxes,
e.g. the H4 histone gene with a single functional GC box,
the thymidine kinase promoter with multiple GC boxes but only one
functional, and the dihydrofolate reductase promoter that contains
multiple functional GC boxes. Only the dihydrofolate reductase promoter
displays Sp3 repression of Sp1 activation (28), whereas in the former
two promoters with only one functional GC box, the primary function of
Sp3 acts as an activator. Although the human Cx26 promoter has two GC
boxes, only one of them is capable of Sp binding. Therefore, with both
human and rat Cx26 promoters having only one functional GC box, one
would predict that Sp3 in this case would serve as an activator.
Indeed, during pregnancy, there is not only an increase in Sp3 quantity
but also a qualitative shift in Sp3 complexes in association with a
marked induction of Cx26.
In the present study, there is a switch of Sp3 complex from C2/C3 to C4
in the rat uterus during pregnancy. It is possible that through
protein-protein interactions Sp3 may form different complexes with
various cofactors. An alternative explanation is the presence of Sp3
isoforms. It has been shown that these isoforms are not generated by
proteolysis, but rather via the mechanism of different internal
translational initiation within Sp3 mRNA (29). Most recently,
independent activation and repressor domains of Sp3 have been
characterized (30). The repressor activity has been mapped to the
5'-end of the DNA-binding domain. At this moment, we can only speculate
that the Sp3 in the C4 complex may lose the 5'-repressor domain and
result in a robust activation of the Cx26 gene. Among these
three Sp3 complexes, the C4 may exert the strongest activating
function, and its abundant presence in uterus, but not in mammary
gland, during pregnancy may account for the temporal and quantitative
differences between these two tissues in Cx26 induction.
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MATERIALS AND METHODS
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Animals
Female Sprague Dawley rats were purchased from Harlan Farm
(Indianapolis, IN). The pregnant rats were 1518 days postconception.
The lactating rats were approximately 7 days postpartum with litters.
The postlactation rats were those whose litters had been removed 7 days
previously. For the hCG experiment, 90-day-old female rats
received hCG (Profasi from Serono Laboratory, Norwell, MA) 100 U/day ip
for 21 days. The dosage chosen (100 U/day) was based on our previous
experience (12) as well as dosages described in the literature
(31).
The rats were housed at a constant temperature (22 C) and in an imposed
diurnal cycle with the light period from 0600 to 1800 h. Food
(rodent pellets, Harlan Tek-Lad, Madison, WI) and water were available
ad libitum. Mammary and uterine tissues were dissected out
and frozen immediately in liquid nitrogen. They were stored in liquid
nitrogen until used for Northern blot analysis, in situ
hybridization, and immunocytochemistry staining.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from rat mammary glands and uteri by the
method of Chomczynski and Sacchi (32) using phenol and guanidine
isothiocyanate. RNA (20 µg/lane) was loaded on a 1.5% agarose gel,
separated by electrophoresis, and transferred to a positively charged
nylon membrane (Ambion, Austin, TX). For hybridization,
32P-labeled antisense RNA probes were generated using rat
Cx26 cDNA (provided by David Paul, Harvard University). The
hybridization was performed at 65 C overnight, and washing was done
according to the manufacturers instructions (Ambion). After washing,
autoradiography was performed with intensifying screens at -70 C. The
amounts of 28S and 18S RNA were determined as loading controls.
In Situ Hybridization Analysis for Cx26 mRNA
Expression
Six-micron cryosections of the frozen tissues were fixed in
3.7% formaldehyde in PBS, pH 7.2, for 1 h. The slides were washed
with diethyl pyrocarbonate-treated PBS and treated with 1
µg/µl proteinase K for 8 min at 37 C. After dehydration and
delipidation with a series of ethanol and chloroform washes, the slides
were rehydrated with an ethanol and 2xsaline sodium citrate
series and acetylated with 0.25% acetic anhydride, 0.1 M
triethanolamine, pH 8.0, for 10 min. Hybridization was carried out
overnight with digoxigenin (Boehringer Mannheim, Indianapolis,
IN)-labeled antisense transcripts from a Cx26 cDNA insert (nucleotides
+501 to +860, relative to the ATG site) that was subcloned in pGEM4Z
(Promega, Madison, WI). The labeling of the cRNA probe was done
according to the manufacturers instructions (Promega). Hybridization
was followed by ribonuclease (RNase) treatment (20 µg/ml, 30 min, 37
C) and subsequent washing (2xsaline sodium citrate, 50%
formamide, 50 C, 30 min). After blocking in Tris-buffer-saline
(TBS), pH 7.5, containing 2% normal sheep serum and
0.05% Triton X-100, the slides were incubated with
antidigoxigenin-AP-antibody (Boehringer Mannheim), 1:500 in TBS, pH
7.5, 1% normal sheep serum. After being washed in TBS, pH 7.5 and pH
9.5, the slides were incubated in color-developing solution (nitroblue
tetrazolium and x-phosphate in TBS, pH 9.5) in dark jars for at least
3 h. The color reaction were stopped by placing the slides in
Tris-EDTA, pH 8.0, and the slides were then counterstained with
0.01% neutral red and mounted in aquamount.
Immunocytochemistry
Six-micron cryosections from frozen tissues were fixed in
absolute ethanol for 10 min. After PBS washing and blocking with 1.5%
horse serum (Vector Laboratory, Burlingame, CA), they were incubated
with a monoclonal antibody against Cx26 (Zymed, San Francisco, CA) at
1:500 dilution and 4 C overnight. Biotinylated horse antimouse IgG
antibody, diluted at 1:200 in 3% horse serum/PBS was then added and
incubated at room temperature for 2 h. After washing with PBS,
streptavidin-fluorescein (1:1000 in PBS)(Amersham, Arlington Heights,
IL) was added. The degree of fluorescence was examined under a Zeiss
fluorescent microscope (Carl Zeiss, Thornwood, NY).
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay (EMSA)
Nuclear extracts from rat mammary glands and uteri were prepared
according to the method described (33). The nuclear extract was
aliquoted and quick frozen in liquid nitrogen before being stored at
-70 C. The protein concentration was measured using a protein assay
kit (Bio-Rad Laboratories, Hercules, CA) according to the directions
provided by the manufacturer. The stored nuclear extracts were used
only once after thawing.
For the EMSA, the oligonucleotides (see Table 1
for sequences) were
labeled with [
-32P]dCTP by filling 3'-recessed termini
using Klenow DNA polymerase I. The labeled probes (50,000 cpm,
0.5
ng) were incubated with 2 µg of the nuclear extract proteins and 1.0
µg of poly[d(I-C)] in a total volume of 20 µl of buffer
containing 10% glycerol, 15 mM HEPES-NaOH, pH 7.8, 5
mM MgCl2, 5 mM dithiothreitol, and
50 mM KCl. The reaction mixtures were incubated at room
temperature for 30 min and then were resolved on a 4% nondenaturing
polyacrylamide gel in 0.25x TBE buffer (1xTBE, 89 mM
Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) at
room temperature for 2 h at 200 V, and the products were detected
by autoradiography.
For competition studies, the specific oligonucleotides were synthesized
and their sequences are listed in Table 1
. The consensus Sp1
oligonucleotide and the mutated form of Sp1 oligonucleotide were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These
excess unlabeled double-stranded DNA competitors were added at
the same time as the probes in the EMSA. For the antibody supershift
assay, specific rabbit polyclonal antibodies against Sp1, Sp2, or Sp3
(all from Santa Cruz Biotechnology, Inc.) were added and incubated with
the reaction mixture for an additional 15 min at room temperature.
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ACKNOWLEDGMENTS
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We thank Dr. David Paul for providing the rat Cx26 cDNA probe
and Dr. B. J. Kennedy for his invaluable comments.
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FOOTNOTES
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Address requests for reprints to: David T. Kiang, M.D., Ph.D., Box 286, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, Minnesota 55455.
Supported by NIH Grant R01-CA72044 and Minnesota Medical Foundation,
Inc.
Received for publication June 24, 1998.
Revision received September 2, 1998.
Accepted for publication September 4, 1998.
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