| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
1 Connexin 43 Gap Junctions Are Decreased in Human Adrenocortical Tumors1
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine (S.A.M., K.D.), Pittsburgh, Pennsylvania 15261; the Medical Research Service, Veterans Affairs Medical Center and Division of Endocrinology, Department of Medicine, University of Miami School of Medicine (L.M.F.), Miami, Florida 33125; and the Department of Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (S.R.B.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Sandra A. Murray, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 connexins 43 gap junction protein levels in normal
adrenal glands to those in benign and malignant adrenocortical human
tumors. In addition, gap junction protein levels were studied in a
human adrenal cancer cell line (H295). In both normal and neoplastic
adrenal tissues, only 1 connexin 43 could be detected,
whereas ß1 connexin 32 and ß2 connexin 26
were not found. In the normal adrenal gland, the zona fasciculata was
demonstrated to have the highest number of gap junctions per cell
(mean ± SEM, 13.78 ± 1.93). In contrast, in
benign adrenocortical adenomas, the number of gap junctions per cell
compared to that detected in normal adrenal glands was significantly
reduced (mean ± SEM, 4.6 ± 1.17;
P 
0.05), and the lowest number was found in
malignant adrenocortical tumors (1.42 ± 0.58;
P 0.05). Similarly, there were few or no
1 connexin 43 gap junctions in the H295 population.
There was a progressive decrease in gap junction plaques in
adrenocortical cancer cell populations compared to those in normal cell
populations. Therefore, analysis of gap junction protein may be helpful
for the differential diagnosis of benign and malignant adrenal tumors.
The induction of gap junctions in malignant cells may provide a novel
therapeutic strategy for adrenal cancer. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Loss of intercellular communication through gap junctions is thought to be involved in the metastatic events characteristic of carcinogenesis in many tissues (5, 6, 7, 8). These gap junctions are membrane specializations that provide low resistance pathways for direct intercellular exchange of molecules smaller than 1000 Da (9, 10, 11). The molecules transferred have been postulated to include signals that modulate growth, differentiation, and various functions in recipient cells (12, 13, 14). Alterations in the amount of regulatory molecules and/or alterations in cell-cell communication of regulatory molecules through gap junctions may be one mechanism of tumor development. However, although the absence of gap junctions has been linked to the loss of growth regulation in many cancers (5, 6, 7, 8), changes in adrenal gland gap junctions have not been reported in adrenal malignancies.
Incorporation of 1 connexin 43 gap
junction complementary DNAs into the genome of rapidly growing
cancerous cells has been shown to retard the cell proliferation rate
and return cells to a more normal phenotype (15, 16), an observation
that supports the role of gap junctions in the loss of regulated growth
and in neoplastic development. In contrast, reducing
1 connexin 43 gap junctions by transfecting
cells with antisense complementary DNA against
1 connexin 43 has been shown to increase
adrenal cortical cell proliferation (17).
Although the distribution of connexin in the human adrenal gland has
not been previously reported, the rodent adrenal gland demonstrated an
inverse relationship between the presence of gap junctions and
proliferation rates in the adrenal zones (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). For example, the
highest amounts of 1 connexin 43 gap junction
protein were found in the inner, slower growing, cortical zones (zona
fasciculata/zona reticularis) (18, 19, 20). In contrast, the cells of the
outer, more rapidly dividing, cortical layer (zona glomerulosa) (18)
had relatively few gap junctions (18, 19, 20). In addition, when the cells
that demonstrated the highest gap junction expression (zona
fasciculata/zona reticularis) were placed into primary culture, ACTH
(40 mU/mL) treatment resulted in an increase in gap junction protein
expression and a corresponding decrease in proliferation (21). It can
be suggested from these types of experiments that gap junction channels
are important in regulation of normal adrenal homeostasis and growth.
Loss of growth control in the adrenal cortex may correlate with
alterations in cell-cell communication and the development of both
benign and malignant tumors.
Insight into the potential for adrenal neoplastic cell growth would be
facilitated by increased knowledge of gap junction distribution and the
capacity for cell-cell communication. For example, recent gene therapy
studies demonstrate that tumor cell growth can be inhibited via gap
junction-mediated communication with cells containing adenoviral
tumor-restricted promoter-toxic gene constructs (22, 23, 24). The
possibility therefore exists that adrenal tumors in the future could be
treated via gap junction communication with bioengineered cells.
However, these treatment methods would be possible only in tumors that
express gap junctions and are communication competent. A knowledge of
gap junction distribution in different types of tumors is therefore
needed. It is proposed that study of the type and distribution of gap
junctions in normal and neoplastic adrenal tissue may have potential
for the development of novel treatments as well as diagnostic markers
for prognosis. In this study we characterize for the first time
1 connexin 43 gap junction protein
distribution in normal human adrenal glands and measure and compare gap
junction protein amounts in adrenal specimens removed from patients
with adrenal tumors.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The adrenals were removed from the donors and processed for protein analysis by immunohistochemistry (18, 19).
Patients
Of the nine tumor specimens examined, three were adenoma with autonomous cortisol secretion, and six were carcinomas (malignant tumors) associated with the production of various steroids. Three normal adrenal specimens (obtained from patients undergoing nephrectomy) were also examined. None of the patients was receiving exogenous steroids. The cortical origin of the tumors was confirmed by immunohistochemical staining against cytokeratin, vimentin, synaptophysin, and D11 protein (25). Malignancy was diagnosed or excluded according to the criteria of Hough et al. (26), Weiss et al. (27), and Slooten et al. (28). The criteria for adrenal malignancy included increased mitotic rate, nuclear grading, atypical mitosis, clear cells comprising 25% or less of the tumors, diffuse patternless sheets of cells, necrosis, invasion of venous structures, invasion of sinusoidal structures, invasion of the capsule of tumor, and the presence of metastasis. The six carcinomas did not express major histocompatibility complex class II, a marker that we have shown previously to be absent in adrenal cancer (25).
Cell line
The NCI-H295 human adenocarcinoma cells were obtained as a gift from Dr. William Rainey (University of Texas Southwestern Medical Center, Dallas, TX). Cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) that contained hydrocortisone (3.6 µg/L), insulin (5 mg/mL), transferrin (10 mg/mL), estradiol (2.7 µg/L), selenium (1 ng/mL), and 2% FCS (all obtained from Sigma, St. Louis, MO). H295 cells were grown at 37 C in a 5% C02 humidified atmosphere with routinely changed medium. Cells were grown in plastic tissue culture flasks (25-cm2 flask, Falcon Plastics, Oxnard, CA). In preparation for immunocytochemical or dye communication analysis, the cells were seeded onto coverslips and given 24 h to attach to the substrate.
Antibody description
Affinity-purified polyclonal rabbit antibodies (IgG) were gifts
from Drs. Norton B. Gilula and Nalin Kumar. Preparation and
characterization of antibodies directed against synthetic peptides
corresponding to cytoplasmic domains between transmembranes 2 and 3 or
the carboxyl end of the connexin (29, 30, 31, 32, 33) of the following three
different gap junction proteins were performed:
1 connexin 43 peptide extending from residues
370–381 and corresponding to the carboxyl-terminus of the
1 (connexin 43) (31), ß1 peptide
(connexin 32) extending from residues 262–280 and corresponding to the
carboxyl-terminus of ß1 connexin 32 gap
junction protein (32), and ß2 (connexin 26)
protein extending from residues 112–115 and corresponding to the
carboxyl-terminus of ß2 (connexin 26) (33). The
preparation and characterization of these antibodies have been
previously described, and immunocytochemical staining with these
antibodies has been demonstrated in skin (ß1
connexin 32 and ß2 connexin 26) (33, 34) or in
adrenal cell populations (1 connexin 43)
(18, 19, 20, 21).
Immunohistochemistry of gap junction protein
To demonstrate gap junctions in cell cultures, adrenal cells were grown on coverslips until they were 85% confluent. The culture medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS) and fixed for 20 min in 3% formaldehyde. The cells were then washed three times in PBS and permeabilized in acetone for 7 min at -20 C in preparation for incubation with the gap junction antibody. Adrenal glands were prepared by rapidly freezing them in OCT embedding medium compound (Miles, Inc., Elkhart, IN) and cutting frozen sections on a cryostat (Minotome, International Equipment Co., Boston, MA). Sections were collected on gelatinized slides and incubated in PBS at room temperature for 5 min. The sections were incubated in a blocking solution containing 3% BSA and 3% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in PBS (10 mmol/L sodium phosphate, pH 7.5, and 0.9% NaCl) for 1 h at room temperature to reduce nonspecific binding. All immunocytochemical staining was performed with a standard immunocytochemical protocol (18). At the end of the 1-h incubation, the cell and gland preparations were rinsed and incubated on a drop of Cy3 affinity-purified goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:300 in PBS. In some specimens, the sections and cells were then costained with Hoechst dye solution (0.5 mg/mL) to demonstrate DNA within the nucleus. In addition to observations on adjacent sections, after the immunocytochemical analyses the coverslips were removed, and the specimens were counterstained with hematoxylin and eosin to aid in visualization of the tissue. All photographs were taken with a Nikon microscope (Nikon, Melville, NY) using Kodak T Max 400 black and white film (Eastman Kodak Co., Rochester, NY). Some data were collected with computer-assisted imaging.
Image analysis and quantification
To better characterize gap junction distribution in the adrenal
specimen, computer-assisted microspectrofluorometric image analysis of
gap junction number per cell was performed. Zona reticularis cells
close to the medulla were selected for measurement, thus avoiding the
possibility of reading cells at the interface between the zona
reticularis and the zona fasciculata. Zona fasciculata cells near the
zona glomerulosa were selected for analysis. We did not analyze the
number or size of gap junctions in the zona glomerulosa, because very
little gap junction protein expression was detected in this zone. As
most adrenal carcinomas occur in the zona fasciculata, the average
readings taken from the zona fasciculata area were quantitated for
comparison of gap junction expression in the adrenal tumor tissue and
cell lines. Two sections of each tissue specimen were selected for data
analysis, and each data point represented 20 or more representative
areas within the tissue specimen or cell population. The number of
cells in an area was determined by counting the number of Hoechst
dye-stained nuclei. Gap junction number and distribution in adrenal
cell populations were characterized with a Nikon Microphot
FXA fluorescence phase microscopes interfaced to an Optimas Image
Analysis program (Media Cybernetics, Silver Spring, MD) run on a
Gateway (Gateway 2000 Inc., North Sioux City, SD) computer or by
counts made by the investigator. Statistical analysis between means was
calculated using Student’s t test. The data are expressed
as the mean ± SEM. P
0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate a possible correlation between distribution of gap
junction protein and adrenal tumorigenesis, it was necessary to
characterize gap junctions in the normal human adrenal.
1 connexin 43 gap junction protein was the
only member of the connexin family of gap junction proteins studied
(1 connexin 43, ß1
connexin 32, and ß2 connexin 26) found in the
adrenal tissues or adrenal cell line. The morphology of the gap
junctions observed with immunohistochemical techniques was that of
single fluorescent puncta. No staining characteristic of
1 connexin 43 gap junctions was observed with
preimmune incubated sections or sections stained for connexin 26 or 32
(data not shown).
The presence of 1 connexin 43 gap junction
antigen was differentially abundant in the normal adrenal cortex (Fig. 1
). Only a relatively small number of
1 connexin 43 gap junction protein plaques was
detected at sites of cell contact between the cells of the zona
glomerulosa (Figs. 1 and 2). The observed gap
junctions in the zona glomerulosa were mainly found on cords of
connective tissue infiltrating the zona glomerulosa from the
surrounding adrenal capsule (Fig. 1). In contrast,
1 connexin 43 gap junctions at areas of
cell-cell contact between cells in the zona fasciculata and zona
reticularis regions were more abundant than those gap junctions found
in the zona glomerulosa (Figs. 1 and 2). The average number of gap
junctions, as assessed with computer-assisted analysis or by manual
counting of gap junction plaques in the zona fasciculata, for example,
ranged from 2.1–26.8/cell, with a mean of 13.8 ± 1.9
SEM (Fig. 3). The average gap
junction plaque size (±SD was 2. 3 ± 0.93
µm2. 1 connexin 43 gap
junctions were not observed in the medulla.
|
|
|
Adenoma. Three different glucocorticoid-producing
adenomas were analyzed. 1 connexin 43 staining
was punctate and much more variable in the adrenal adenoma than in
either the zona fasciculata or zona reticularis of normal adrenal
cells. Although some cells in steroid-secreting tumor populations were
extensively connected with gap junctions, the majority of cells had
only a few gap junctions at their cell surface. The range was 0.2–9.1
gap junctions, with a mean (±SEM) of 4.6 ± 1.17 gap
junctions/cell nuclei. These specimens, in addition to typical punctate
staining for 1 connexin 43 gap junction
protein at the sites of cell contact, had large structures within the
cytoplasm that stained for 1 connexin 43 gap
junction protein. Gap junction plaque sizes in the adenoma populations
(mean ± SD, 2.3 ± 0.9
µm2) were not different from gap junction
plaque sizes measured in the normal adrenal gland. The number of nuclei
per counting area appeared to be similar in all of the adenomas
measured.
Carcinoma. The majority of cells in the carcinomas appeared
closely stacked together, with only a small amount of cytoplasmic
material surrounding the nuclei. The presence of more nuclei per
measured area could indicate that the rate of cell division in these
areas was greater than that in the normal tissue or the other tumor
types studied. Gap junction distribution within the tumor populations,
as in the adenoma, was highly variable, with some areas having more gap
junctions than others. However, even in areas with high numbers of gap
junctions, there were fewer plaques than in normal tissue. In adrenal
carcinomas the number of gap junction per cell ranged from 0.12–7.8
gap junctions/cell with a mean (±SEM) of 1.42 ± 0.58
(Fig. 3
). The carcinomas had a dramatic reduction in gap junction
number per cell compared to the zona fasciculata from the normal
adrenal glands. The average size (±SD) of gap junction
plaques (2.3 ± 0.8 µm2) was not different
from the sizes measured in the normal specimen or in the adenoma
tissue.
Description of human adrenal tumors cells in culture
The H295 cell line was established by Gazdar and colleagues
(35) from an invasive primary adrenocortical carcinoma of a patient who
showed symptoms of mineralocorticoid, glucocorticoid, and androgen
excess. The ability of the H295 cell to produce each of these three
zone-specific steroid groups suggests that these cells may act as a
pluripotent adrenocortical cell (36). The H295 carcinoma cell
populations did not have 1 connexin 43 gap
junctions (Fig. 2). The lack of 1 connexin 43
gap junctions is similar to the findings of few gap junctions in the
carcinoma specimen taken directly from the patients studied here.
ß1 and ß2 connexin
protein plaques were not found in H295 populations, and there was no
dye communication in this cell line (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 connexin
43 gap junction protein in normal and neoplastic human adrenal glands
and in a human adrenal tumor cell line. Neither connexin 26 nor
connexin 32 was present in the human adrenal gland. It was observed
that 1 connexin 43 was abundant in the inner
adrenal gland zones (zonae fasciculata and reticularis) and sparse in
the outer zone. These findings are similar to the distribution reported
in rodents (20) and suggest that a functional relationship exists
between adrenal gland zonation and gap junction distribution.
A major route for cell to cell communication resides in gap junctions
(8). In addition to providing a channel for communication of regulatory
molecules, gap junctions may serve to hold cells together, and the loss
of gap junctions may facilitate both migration and metastatic behavior.
Glomerulosa cell migration into the inner cortex may be facilitated by
the lack of gap junctions between the cells in the zona glomerulosa,
and the presence of gap junctions would perhaps limit such migration.
In addition, the inner two areas of the adrenal cortex proliferate more
slowly than the outer adrenal cortical layer (18), and the variations
in 1 connexin 43 expression within the adrenal
cortex may be related to the rate of adrenal cell proliferation. The
functional relevance of gap junction protein expression is additionally
supported by data demonstrating that the inner two zones vary both in
gap junction distribution and hormone responsiveness. The inner
glucocorticoid- and androgen-producing zona fasciculata/zona
reticularis, respectively, show characteristically high levels of gap
junction protein expression, proliferate slowly, and are highly
responsive to ACTH. In contrast, the mineralocorticoid-producing outer
zone has few gap junctions, grows rapidly, and shows little
responsiveness to ACTH (18, 19, 20). In support of a relationship between
ACTH response and the expression of gap junctions, primary adrenal
cells maintained in tissue culture increase the number of gap junctions
after ACTH stimulation (37). Mouse adrenal tumor cells (Y-1) in culture
also increase the number of gap junctions and decrease proliferation in
response to ACTH stimulation (38). In vivo measurements of
changes in gap junctions and adrenal zone proliferation rates as well
as measurements of hormone responses after increased ACTH levels are
needed to definitively establish these findings.
Because the outer zone of the adrenal cortex has fewer gap junctions and is thought to proliferate more quickly than the inner zones (18, 19, 20), the possibility of a relationship between proliferation and gap junctions might be anticipated in neoplasms. There is extensive evidence that tumor cells exhibit uncontrolled growth as a result of diminished ability to communicate with and respond to metabolic signals from surrounding cells (9, 12, 13, 16). Loss of growth regulation in some tumor cells involves two defects. First, cells may lose their ability to produce, transport, or effectively receive regulatory molecules (12, 13). Secondly, the loss of adhesion at the gap junction may allow cells to separate from one another and to metastasize.
A1though gap junctions were found in the adrenal tumor specimens, they
were greatly reduced compared to the numbers seen in the normal adrenal
zona fasciculata. A strong relationship between gap junction protein
plaque numbers at the cell surface and cellular differentiation of the
tumor is seen. Specifically, steroid-producing adenomas, which are more
differentiated than the cells of the carcinoma, have more gap junctions
than the carcinoma but fewer than those in the normal adrenal gland
(P < 0.05). Large structures positive for
1 connexin 43 were seen within the cytoplasm
of the adenomas. We have not to date attempted to characterize the
nature of the cytoplasmic staining seen in the adenoma tissue. This
staining appears to be within the cytoplasm and not at areas of
cell-cell contact, where it would be if the 1
connexin 43 proteins were participating in gap junction channels
between cells. This pattern of large cytoplasmic fluorescence staining
is thus not consistent with the presence of functional gap junctions.
The cytoplasmic staining observed in the adenoma is interesting, in
that it may be indicative of improper 1
connexin 43 protein trafficking to the cell membrane, thus impairing
functional gap junction formation.
Only additional studies will elucidate the mechanism of gap junction
loss in tumor cell populations and reveal the nature of the large
cytoplasmic fluorescent structures seen. Evaluation in future studies
of the amounts of protein by Western blot analysis will help to
elucidate the mechanism for loss of gap junctions in tumor tissue.
Western blot analysis in the present study, however, would not have
helped to detect changes in the distribution of gap junctions in the
three zones of the adrenal gland, nor would it be useful in
distinguishing between cytoplasmic protein structures and cell surface
gap junctions. In this study we found a strong correlation between gap
junction protein plaque numbers at the cell surface and cellular
differentiation of tumors. Taken together, the data suggest that
1 connexin 43 gap junctions play a role in
regulating the growth and differentiation of adrenal cells.
In addition to ACTH and angiotensin II, the integrated control of adrenocortical function involves direct innervation, regulation of the blood supply to the gland, and intraglandular cell-cell interactions (39, 40, 41, 42). These systems not only affect the regulation of moment to moment function, but also participate in the development and differentiation of the adrenal gland and in tumorigenesis (42, 43). Alterations in intercellular communication, local production of growth factors and cytokines, and aberrant expression of ectopic receptors on adrenal tumor cells have been implicated in adrenal cell growth, hyperplasia, tumor formation, and autonomous hormone production.
In summary, the decrease in gap junctions in the malignant adrenal tumors examined coincides with a decline in cellular differentiation and the loss of normal function. The development of treatments of adrenal cancer based on these differences in gap junction communication may be possible. In addition, the prediction of adrenal malignancy based on the lack of gap junctions might serve as a very useful diagnostic marker. Measurements of gap junction number, combined with other markers, might distinguish malignant from nonmalignant tumors and might provide the basis for unique and effective treatments.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received April 20, 1999.
Revised October 18, 1999.
Accepted October 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glycerrhetinic acid (GA) affects the steroidogenic response of
bovine adrenal cells to ACTH. Proc of the 79th Annual Meet of The
Endocrine Soc. 1997; 317.
This article has been cited by other articles:
 |
|
 |
|
  A.-M. Lefrancois-Martinez, J. Bertherat, P. Val, C. Tournaire, N. Gallo-Payet, D. Hyndman, G. Veyssiere, X. Bertagna, C. Jean, and A. Martinez Decreased Expression of Cyclic Adenosine Monophosphate-Regulated Aldose Reductase (AKR1B1) Is Associated with Malignancy in Human Sporadic Adrenocortical Tumors J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 3010 - 3019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. King and P. D. Lampe Mice deficient for the gap junction protein Connexin32 exhibit increased radiation-induced tumorigenesis associated with elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation Carcinogenesis, May 1, 2004; 25(5): 669 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Mansmann, J. Lau, E. Balk, M. Rothberg, Y. Miyachi, and S. R. Bornstein The Clinically Inapparent Adrenal Mass: Update in Diagnosis and Management Endocr. Rev., April 1, 2004; 25(2): 309 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-W. Zhang, K. Nakayama, K.-I. Nakayama, and I. Morita A Novel Route for Connexin 43 to Inhibit Cell Proliferation: Negative Regulation of S-Phase Kinase-associated Protein (Skp 2) Cancer Res., April 1, 2003; 63(7): 1623 - 1630. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
