@1 Connexin 43 Gap Junctions Are Decreased in Human Adrenocortical Tumors*
S. A. MURRAY, K. DAVIS, L. M. FISHMAN, AND S. R. BORNSTEIN
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
ABSTRACT
Gap junctional communication disorders have been implicated in the etiology of benign and malignant tumors. Understanding the type, distribution, and frequency of gap junctions in adrenal disorders should provide insight into the role of gap junctions in adrenal car- cinogenesis as well as information that may be useful in developing improved diagnosis and treatment of adrenal diseases. Using immu- nocytochemical techniques, we have characterized and compared @1 connexins 43 gap junction protein levels in normal adrenal glands to those in benign and malignant adrenocortical human tumors. In ad- dition, 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 B2 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 com- pared 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. (J Clin En- docrinol Metab 85: 890-895, 2000)
A DRENAL TUMORS are fairly common (1, 2). Approx- imately 4% of all computed tomography scans reveal the presence of an adrenal tumor (3). The differentiation between malignant and benign adrenocortical tumor is an important, but often difficult, distinction in the early diag- nosis and treatment of adrenal diseases. Today, tumor size is used as a preoperative indicator of malignant potential for adrenal tumors (4); however, a better diagnostic marker is needed. At present, there are no reliable histological markers justifying needle biopsy.
Loss of intercellular communication through gap junctions is thought to be involved in the metastatic events character- istic of carcinogenesis in many tissues (5-8). These gap junc- tions are membrane specializations that provide low resis- tance pathways for direct intercellular exchange of molecules smaller than 1000 Da (9-11). The molecules transferred have been postulated to include signals that modulate growth, differentiation, and various functions in recipient cells (12- 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 (5-8), 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 sup- ports the role of gap junctions in the loss of regulated growth and in neoplastic development. In contrast, reducing @1 con- nexin 43 gap junctions by transfecting cells with antisense com- plementary DNA against @1 connexin 43 has been shown to increase adrenal cortical cell proliferation (17).
Although the distribution of connexin in the human ad- renal 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 (18-29). For example, the highest amounts of @1 connexin 43 gap junction protein were found in the inner, slower growing, cortical zones (zona fasciculata/zona re- ticularis) (18-20). In contrast, the cells of the outer, more rapidly dividing, cortical layer (zona glomerulosa) (18) had relatively few gap junctions (18-20). 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 de- crease in proliferation (21). It can be suggested from these types of experiments that gap junction channels are impor- tant in regulation of normal adrenal homeostasis and growth. Loss of growth control in the adrenal cortex may
Received April 20, 1999. Revision received October 18, 1999. Accepted October 22, 1999.
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.
* This work was supported by NSF Grant IBN-98-08428 and the Pilot Project Program of the Prostate and Urologic Cancer Center of the University of Pittsburgh Cancer Institute.
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 tu- mor cell growth can be inhibited via gap junction-mediated communication with cells containing adenoviral tumor- restricted promoter-toxic gene constructs (22-24). The pos- sibility therefore exists that adrenal tumors in the future could be treated via gap junction communication with bio- engineered 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 charac- terize 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 spec- imens removed from patients with adrenal tumors.
Materials and Methods
Tissue preparation and type
The adrenals were removed from the donors and processed for pro- tein analysis by immunohistochemistry (18, 19).
Patients
Of the nine tumor specimens examined, three were adenoma with autonomous cortisol secretion, and six were carcinomas (malignant tu- mors) 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 immuno- histochemical 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 ve- nous structures, invasion of sinusoidal structures, invasion of the cap- sule 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% CO2 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 characteriza- tion of antibodies directed against synthetic peptides corresponding to
cytoplasmic domains between transmembranes 2 and 3 or the carboxyl end of the connexin (29-33) of the following three different gap junction proteins were performed: @1 connexin 43 peptide extending from res- idues 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 B2 (connexin 26) (33). The preparation and characterization of these anti- bodies have been previously described, and immunocytochemical stain- ing with these antibodies has been demonstrated in skin (ß1 connexin 32 and ß2 connexin 26) (33,34) or in adrenal cell populations (œ] connexin 43) (18-21).
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 Labo- ratories, 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 stan- dard immunocytochemical protocol (18). At the end of the 1-h incuba- tion, the cell and gland preparations were rinsed and incubated on a drop of Cy3 affinity-purified goat antirabbit IgG (Jackson ImmunoRe- search 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 ad- dition to observations on adjacent sections, after the immunocytochem- ical 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 spec- imen, 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 pos- sibility 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 pro- tein 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 ex- pression 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 num- ber 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
Description of the normal human adrenal gland
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 (a1 connexin 43, ß1 connexin 32, and ß2 connexin 26) found in the adrenal tissues or adrenal cell line. The mor- phology of the gap junctions observed with immunohisto- chemical techniques was that of single fluorescent puncta. No staining characteristic of @1 connexin 43 gap junctions was observed with preimmune incubated sections or sec- tions 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 junc- tion 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 re- ticularis regions were more abundant than those gap junc- tions 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
A
B
D
NORMAL
CARCINOMA
ADENOMA
n
n
n
A
B
C
CARCINOMA TISSUE
Gap Junction
CARCINOMA CELL LINE (H295)
n
n
D
E
average gap junction plaque size (+SD was 2. 3 ± 0.93 pm2. @1 connexin 43 gap junctions were not observed in the medulla.
Description of the human adrenal tumors
Adenoma. Three different glucocorticoid-producing adeno- mas 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 tu- mor 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 junc- tion plaque sizes in the adenoma populations (mean ± SD, 2.3 ± 0.9 pm2) 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 cyto- plasmic 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 ade- noma, 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 com- pared to the zona fasciculata from the normal adrenal glands. The average size (±SD) of gap junction plaques (2.3 ± 0.8 um2) 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 col- leagues (35) from an invasive primary adrenocortical car- cinoma of a patient who showed symptoms of mineralo- corticoid, 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. B1 and ß2 connexin protein plaques were not found in H295 populations, and there was no dye communication in this cell line (data not shown).
20
GAP JUNCTION NUMBER/CELL
10
*
*
0
NORMAL
ADENOMA
CARCINOMA
Discussion
In this study we demonstrated @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 func- tional 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 com- munication of regulatory molecules, gap junctions may serve to hold cells together, and the loss of gap junctions may facilitate both migration and metastatic behavior. Glomeru- losa cell migration into the inner cortex may be facilitated by the lack of gap junctions between the cells in the zona glo- merulosa, 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 ex- pression 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 miner- alocorticoid-producing outer zone has few gap junctions, grows rapidly, and shows little responsiveness to ACTH (18-20). In support of a relationship between ACTH re- sponse 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 junc- tions and decrease proliferation in response to ACTH stim- ulation (38). In vivo measurements of changes in gap junc-
tions 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 (18-20), the possibility of a relationship between proliferation and gap junctions might be anticipated in neo- plasms. There is extensive evidence that tumor cells exhibit uncontrolled growth as a result of diminished ability to com- municate with and respond to metabolic signals from sur- rounding 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 reg- ulatory 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 num- bers 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 charac- terize the nature of the cytoplasmic staining seen in the adenoma tissue. This staining appears to be within the cy- toplasm 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 cyto- plasmic fluorescence staining is thus not consistent with the presence of functional gap junctions. The cytoplasmic stain- ing 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 anal- ysis 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 innerva- tion, regulation of the blood supply to the gland, and in- traglandular cell-cell interactions (39-42). 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 au- tonomous 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 devel- opment of treatments of adrenal cancer based on these dif- ferences 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 non- malignant tumors and might provide the basis for unique and effective treatments.
Acknowledgments
We are grateful to Drs. Norton Gilula and Nalin Kumar for providing the antibodies used in this study, and to Uzma Shah for work with the H295 cells.
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