ELSEVIER
Effect of bursal anti-steroidogenic peptide (BASP) on cortisol biosynthesis in ACTH-stimulated canine adrenocortical carcinoma cells in vitro
J.A. Byrda, C.E. Deana, T.W. Fossumb, B.M. Hargisa,*
aDepartments of Poultry Science and Veterinary Pathobiology, College of Veterinary Medicine, Room 119 VMS, College Station, TX 77843, USA
Department of Veterinary Small Animal Medicine and Surgery, Texas Agricultural Experiment Station. Texas A&M University System, College Station. TX 77843, USA
Accepted 14 September 1994
Abstract
Previous studies from our laboratory have demonstrated that bursal anti-steroidogenic peptide (BASP) inhibits progesterone biosynthesis from ovine luteinizing hormone-stimulated chicken ovar- ian granulosa cells. In the present investigation, we evaluated the efficacy of BASP for reducing cortisol secretion from normal canine adrenocortical cells and neoplastic adrenocortical cells from a dog with Cushing’s syndrome.
Treatment of adrenocortical cells derived from either normal healthy dogs or a cushingoid dog with adrenocorticotropic hormone (ACTH; 0-10 nM) caused an approximately two-fold increase in cortisol production from both normal or tumor derived adrenocortical cells. Small but significant decreases (up to 34%) in cortisol production were observed from normal and tumor derived canine adrenocortical cells when exposed to increasing concentrations of BASP (0.0-0.15 bursal equivalents; BEQ). Incubation of adrenocortical carcinoma cells or normal adrenocortical cells with ACTH (0- 10 nM) and BASP (0.0-0.15 BEQ) increased cyclic AMP formation up to 2.5-fold. Interestingly, BASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells.
Data from the present studies indicate that BASP is capable of suppressing basal and ACTH- stimulated cortisol production from normal or tumor derived adrenocortical cells in vitro. The possible clinical efficacy of homologous canine BASP on caninc adrenal function or chicken BASP in other species of animals remains to be evaluated.
Abbreviations
ACTH, adrenocorticotropic hormone; BASP, bursal anti-steroidogenic peptide; BEQ, bursal equiv- alents; cAMP, cyclic AMP; RIA, radioimmunoassay; SE, standard error.
* Corresponding author.
1. Introduction
Cushing’s syndrome was first described by Harvey Cushing (1932) as a disorder caused by ‘pituitary basophilism’. Cushing’s syndrome is a term used to describe all aspects of the clinical and chemical abnormalities resulting from chronic overproduction of glucocorti- coids. It is recognized as one of the most common endocrine disorders in dogs with adrenal tumors accounting for 7-15% of the spontaneous cases of hyperadrenocorticism. In almost all cases, adrenal tumors are unilateral in dogs.
Chronically elevated endogenous secretion of corticosteroids from dogs with hyperad- renocorticism can cause a number of clinical signs including polydipsia, polyuria, polypha- gia, abdominal distention, muscle weakness, lethargy, alopecia, obesity and increased panting (Feldman, 1983). A serious consequence of hyperadrenocorticism is an impairment of normal immune function. Small elevations of circulating endogenous glucocorticoids have been reported to stimulate an increase in antibody formation with a decrease in T- lymphocyte suppressor cells in vitro (Orson et al., 1983). However, high concentrations of circulating cortisol have been shown to suppress antibody synthesis and kill B-lymphocytes both in vitro and in vivo (Settipane et al., 1978; Grayson et al., 1981). While the negative effects of adrenal corticosteroids on immune function are well described (Warnes et al., 1960; Grayson et al., 1981; Tizard, 1992) a similar ability of the immune system to modulate adrenal corticosteroid biosynthesis has not yet been demonstrated.
Recently, our laboratory has partially isolated a low molecular weight ( =3000-5000) peptide-bursal anti-steroidogenic peptide (BASP)-from the chicken bursa of Fabricius that inhibits the production of chicken gonadal and adrenal steroids to basal levels in vitro (Byrd et al., 1993, 1994). The low molecular weight of BASP suggests that the peptide may be teleologically conserved between species, and this peptide has been postulated to function as a natural suppressor of steroid producing cells (i.e. gonadal and adrenal organs) (Byrd et al., 1993, 1994). However, possible therapeutic efficacy of BASP for hyperadren- ocorticism has not been evaluated. The present study was conducted to evaluate the efficacy of BASP (isolated from prepubertal chickens) for reduction of cortisol secretion from canine adrenocortical tumor cells surgically obtained from a dog with a steroid-producing adrenocortical tumor.
2. Materials and methods
2.1. Hormone and reagents
Complete culture medium consisted of Eagle’s minimum essential medium, containing 25 mM HEPES buffer and included 0.1% bovine serum albumin, Fraction V, 50 IU penicillin ml-1 and 0.05 mg streptomycin ml -1. The final pH of the medium was adjusted to 7.4 with 1 N NaOH. Adrenocorticotropic hormone (ACTH; human fragment 1-24) was obtained from Sigma Chemical Co. and diluted immediately before use with cell culture medium. Partially purified BASP was extracted from chicken bursa of Fabricius as previously described (Byrd et al., 1993).
2.2. Collection of tissue
Adrenal tissues were collected from a dog with Cushing’s syndrome and four normal dogs. A 9-year-old female spayed Belgian Sheepdog was referred for evaluation of poly- phagia, polydipsia, polyuria, and diarrhea, alopecia, and muscle atrophy. Cushing’s syn- drome was suspected and plasma cortisol concentrations were measured. A tentative diagnosis of functional adrenocortical tumor was made. Baseline plasma cortisol concen- tration was 9.9 µg dl-1; 8 h following administration of 0.1 mg dexamethasone kg-1, cortisol levels were essentially unchanged (11.4 µg dl-1).
Abdominal ultrasonography revealed a hypoechoic soft tissue mass located cranial to the left kidney. At surgery, a 3.5 cm × 3.5 cm encapsulated mass was found associated with the left adrenal gland. The mass was excised, trimmed free of connective tissue, and diced into small pieces (approximately 2 mm) in ice-cold culture medium for cell isolation. A section was taken for histological examination which confirmed the diagnosis of adrenocortical carcinoma.
Adrenal glands were collected from four healthy anesthetized male and female adult dogs, trimmed free of connective tissue, and diced into small pieces (about 2 mm) in ice- cold culture medium for cell isolation.
2.3. Cell isolation and incubation
The adrenocortical carcinoma cells from a dog with Cushing’s syndrome and adrenals removed from the four healthy dogs were isolated according to procedures that were previously described (Carsia et al., 1987). Briefly, adrenal tissues were decapsulated, minced with scissors, washed three times with equal volumes of cell culture medium, and recovered by centrifugation (220×g). Adrenal pieces were enzymatically dispersed at 37℃ for 1 h with gentle shaking in 50 ml of cell culture medium containing 1 mg ml -1 of collagenase. Following a 1 h incubation, cells were washed three times with cell culture medium. Cells were filtered through nylon mesh to remove any large cell clumps or cellular debris. Viability was determined using trypan blue dye exclusion and cells were counted on a live/dead cell basis (Freshney, 1983). Cells were diluted to a concentration of 100 000 viable cells per 250 ul and directly added to borosilicate culture tubes (12 mm × 75 mm). Treatment groups consisted of 0.0, 0.03, 0.075 or 0.15 bursal equivalents (BEQ) of BASP. For each concentration of the test compound, five replicates of 0.0, 0.01, 0.1, 1.0, 10 nM ACTH or 1 mM dibutyryl cAMP were added to the appropriate tubes. Treatments were added to the culture tubes in a total volume of 250 pl so that the final volume was 0.5 ml. Cultures were incubated at 37℃, saturated humidity, with 5% CO2 for 2 h. Following the incubation, 0.1 mM 3-isobutyl-1-methylxanthine was added to each culture tube and the tubes were then immediately frozen at - 20℃ prior to radioimmunoassay (RIA ) for cortisol and cAMP. Cortisol (content of cells plus medium) was determined by a commercially available RIA kit. The sensitivity for the cortisol assay was 1 ng ml-1 with an interassay coefficient of variation of 7.1%. Cellular cAMP content of succinylated samples was meas- ured by a highly sensitive and specific RIA. The sensitivity for the cAMP assay was 800 fmol ml -1 with an interassay coefficient of variation of 7.4%. Each experiment was repeated twice using two separate cell dispersions.
2.4. Statistical analysis
Data obtained from each experiment were subjected to analysis of variance using the SAS Program (Statistical Analytical Systems Institute Inc., 1988). Significantly different means were separated using Duncan’s multiple range test (Duncan, 1955). Significance is reported at P < 0.05.
3. Results
3.1. Normal adrenocortical cell cortisol biosynthesis
In Fig. 1, BASP significantly suppressed cortisol production (up to 23.8%) as compared to unstimulated canine adrenal cells. The highest concentration of BASP evaluated (0.15 BEQ) significantly suppressed (up to 18%) cortisol production from adrenocortical cells derived from healthy dogs at all doses of ACTH. Similarly, BASP (0.075 BEQ) suppressed cortisol biosynthesis (up to 13.7%) from normal adrenocortical cells stimulated with high concentrations of ACTH (0.1-10 nM). The lowest dose of BASP evaluated (0.03 BEQ) was only effective in attenuating cortisol secretion from either unstimulated (0 nM ACTH) or the highest concentration of ACTH (10 nM). The test compound BASP did not affect normal cortisol synthesis in adrenal cells stimulated with 1 mM dibutyryl cAMP.
3.2. Adrenocortical carcinoma cell cortisol biosynthesis
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Fig. 1. Effects of increasing concentrations of partially purified BASP (0.03-0.15 BEQ) on cortisol production from 200 000 normal canine adrenocortical cells ml-1 in the absence or presence of ACTH (0-10 nM) or 1 mM dibutyryl cAMP (DB-CAMP) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P <0.05) from the control within each secretagogue concentration.
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production from unstimulated (0 nM ACTH) adrenocortical carcinoma cells (Fig. 2) was observed to be more than ten-fold greater than basal secretion from cells derived from normal dogs (Fig. 1). Stimulation of adrenocortical carcinoma cells with ACTH alone further increased cortisol production approximately two-fold at all concentrations evaluated (Fig. 2). All concentrations of BASP evaluated (0.03, 0.075 and 0.15 BEQ) significantly inhibited (up to 34%) basal (unstimulated) cortisol biosynthesis. ACTH-stimulated cortisol production was suppressed by inclusion of 0.075 BEQ BASP (0.01 nM ACTH) or 0.015 BEQ BASP (0.1 nM ACTH) (Fig.2). Similarly, all doses of BASP significantly suppressed cortisol production when combined with 1 nM ACTH. Furthermore, only 0.075 or 0.15 BEQ significantly suppressed both 10 nM ACTH- and 1 mM dibutyryl cAMP-stimulated adrenocortical carcinoma cell cortisol biosynthesis (Fig. 2).
3.3. Normal adrenocortical cell cAMP formation
Fig. 3 illustrates cAMP production from ACTH-stimulated (0-10 nM) normal adreno- cortical cells in the presence of increasing concentrations of BASP (0.03-0.15 BEQ). Regardless of the concentration of ACTH (0-10 nM) evaluated, BASP significantly stim- ulated cAMP formation in a dose dependent manner (Fig. 3).
3.4. Adrenocortical carcinoma cell cAMP formation
Fig. 4 shows cAMP production by isolated ACTH-stimulated and unstimulated canine adrenocortical carcinoma cells in response to 0.75 or 0.15 BEQ BASP. ACTH alone stimulated cAMP formation over two-fold in a dose dependent manner. Both concentrations of BASP evaluated (0.075 or 0.15 BEQ) significantly increased (up to two-fold) ACTH-
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stimulated canine adrenocortical carcinoma cell cAMP formation in a dose dependent manner (Fig. 4).
4. Discussion
We have evaluated the in vitro efficacy of bursal anti-steroidogenic peptide (BASP), a peptide isolated from the bursa of Fabricius of chickens, for ability to attenuate cortisol
production from both canine adrenocortical carcinoma cells and normal adrenocortical cells. BASP, derived from the humoral immune system, is a potent and highly efficacious sup- pessor of in vitro gonadal and adrenal steroid hormone production in birds (Byrd et al., 1993, 1994). Extensive evaluations of the function of this peptide in gonadal tissue have indicated that progesterone biosynthesis is blocked independent of the gonadotrophin recep- tor (Byrd et al., 1993). Progesterone is a known precursor for most endproduct steroids (Fantl et al., 1973), and several laboratories have reported that many tumors secrete only precursors for endproduct steroids (Fukushima and Gallagher, 1963; Fantl et al., 1973).
In the present studies, incubation of canine adrenocortical cells with BASP caused sig- nificant suppression of cortisol production in both normal and carcinoma cells. Previous investigations have indicated that BASP is capable of total suppression of ACTH-stimulated corticosteroid production in a homologous chicken derived adrenocortical cell culture (Tizard, 1992). Similar to previously reported findings, BASP was only partially efficacious in the suppression of ACTH-stimulated cortisol production from normal canine adreno- cortical cells in the present study (Fig. 1) (Byrd et al., 1994). Consistent with BASP- mediated effects on normal adrenocortical cells, BASP was observed to only partially attenuate either basal (unstimulated) or ACTH-stimulated cortisol production from canine adrenal carcinoma cells in vitro (Fig. 2). Interestingly, BASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells.
While many small bioactive peptides have been observed to be highly conserved between animal classes with regard to sequence and structure, some divergence of peptide and/or receptor structure may be anticipated with this 3000-5000 molecular weight peptide (Byrd et al., 1993). It is possible that this type of evolutionary divergence, and resultant incomplete agonist activity, is responsible for the incomplete suppression of cortisol secretion in the heterologous system presently investigated. Alternatively, the mammalian homologue of BASP, if such a peptide exists, may modulate corticosteroid production in a less marked fashion than in birds (Byrd et al., 1994). In support of the latter hypothesis, BASP has been shown to markedly increase cAMP levels in normal chicken, porcine and canine adreno- cortical cells (Byrd et al., 1994) and BASP induced cAMP elevations in excess of two-fold in both normal adrenal and carcinoma derived cells in the present experiments (Figs. 3 and 4). Approximately two- to three-fold elevations of cAMP have been observed in chicken adrenocortical cells in which complete suppression of ACTH-stimulated corticosteroid production was noted (Byrd et al., 1994). Definitive elucidation of differences in BASP- mediated attenuation of avian and mammalian adrenocortical cell function must await molecular characterization of the peptide in each animal class.
The present experiments indicate that avian derived BASP is capable of significant suppression of basal and ACTH-stimulated cortisol production from both normal canine adrenocortical cells and canine adrenocortical carcinoma cells in vitro. However, due to the incomplete suppression of cortisol production observed in both types of cells, anticipation of clinical utility of BASP or BASP analogs for treatment of canine Cushing’s disease must be viewed with some skepticism.
Acknowledgments
The authors would also like to acknowledge support for this research from the Texas Advanced Technology Program. The authors thank Dr. Roberta L. Relford for professional assistance.
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