Phenotypic development of neonatal rat chromaffin cells in response to adrenal growth factors and glucocorticoids: focus on pituitary adenylate cyclase activating polypeptide

We have investigated the regulation of the morphological phenotype of chromaffin cells cultured from 6-day-old rat adrenal glands. We show that pituitary adenylate cyclase activating polypeptide (PACAP), which is present in and released from nerves innervating chromaffin cells, rapidly induces neuritic growth, affecting 25% of tyrosine hydroxylase-positive chromaffin cells after 3 days at an optimal concentration of about 20 nM. PACAP does not synergistically act with other factors known to promote neurite growth, including nerve growth factor (NGF), basic fibroblast growth factor (bFGF, FGF-2), and ciliary neurotrophic factor (CNTF). The neurite promoting effect of PACAP and FGF-2 is entirely overridden by dexamethasone (2 × 10⁻⁸ M) suggesting that, despite the presence of these promoting factors in the adrenal medulla, glucocorticoids from the adrenal cortex are probably sufficient to prevent the development of neuronal traits in adrenal chromaffin cells.     

Human metalloprotease-disintegrin Kuzbanian regulates sympathoadrenal cell fate in development and neoplasia

The development of the sympathetic nervous system involves cell-cell interactions that regulate the fate and migration of progenitor neural cells. Recent evidence shows that focal membrane-bound protease activity is critical for such interactions. The Drosophila kuzbanian (kuz) gene is required in neurogenesis and encodes a highly conserved, membrane-bound metalloprotease- disintegrin closley related to theTNF- alphaconvertingenzyme (TACE). We have characterized the human and mouse kuz homologs and mapped human kuz to chromosome 15q22. During mouse embryonic development Kuz is expressed mainly in the sympathoadrenal and olfactory neural precursors. Once sympathoadrenal cells differentiate into chromaffin cells in the adult adrenal medulla, they no longer express Kuz. However, we found that tumors of sympathoadrenal origin, such as pheochromocytomas and neuroblastomas, overexpress Kuz. Further, transfection of a kuz construct lacking the protease domain, but not the full-length construct, induces neurite formation in PC12 chromaffin tumor cells. Taken together our results suggest a critical role for Kuz in regulation of sympathoadrenal cell fate.      

Adrenal Medullary Nodules in Beckwith-Wiedemann Syndrome Resemble Extra-Adrenal Paraganglia

Clues to mechanisms regulating development and tumorigenesis may be provided by studies of unusual diseases. Beckwith-Wiedemann syndrome (BWS) is a rare congenital disorder apparently related to abnormal regulation of insulin-like growth factor-2 (IGF-2) production. IGF-2 mRNA has been previously localized to the chief cells of extra-adrenal paraganglia and to adult, but not fetal, adrenal medulla. Expression of IGF-2 by neuroblastomas has been hypothesized to reflect extra-adrenal paraganglionic differentiation. In the adrenals of a fetus with BWS, we have observed both increased numbers of chromaffin cells and organoid nodules resembling extra-adrenal paraganglia. Immunoreactive IGF-2 was observed in both cell types, but was also observed in chromaffin cells in the normal fetal adrenal. The findings suggest autocrine or paracrine influences of IGF-2 in regulating the number and phenotype of cells derived from sympathoadrenal precursors in the developing adrenal medulla as well as in extra-adrenal paraganglia. These results have implications for the interpretation of data from neuroblastoma studies.     

Distinctive Effects of Rat Fibroblast Growth Factor-2 Isoforms on PC12 and Schwann Cells

Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during postnatal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC 12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC 12 cells.     

Sympathoadrenal progenitors in embryonic chick sympathetic ganglia show distinct responses to glucocorticoid hormones

Summary The sympathoadrenal cell lineage originates from the neural crest and comprises the neurons of sympathetic ganglia, adrenal and extra-adrenal chromaffin cells, and the so-called small intensely fluorescent cells.In vitro studies using mammalian immature chromaffin cells, adrenal or sympathetic ganglionic progenitors, or ganglionic small intensely fluorescent cells, have suggested that glucocorticoid hormones are essential for inhibiting neuronal differentiation of sympathoadrenal progenitors and promoting the chromaffin cell phenotype. In avian systems, however, the distinct cellular phenotypes in this lineage and the molecular cues underlying their differentiation have not been fully explored. In the chick embryo, early sympathetic ganglion anlagen are populated by granule-containing cells that morphologically resemble small intensely fluorescent cells and chromaffin cell progenitors. These cells subsequently disappear from the ganglia, by death and by transition into fully differentiated sympathetic neurons, as indicated by the appearance of cells that are ultrastructurally intermediate between granule-containing cells and fully differentiated neurons (granule-containing cells in transition). In the present study, we show that treatment of cultured sympathetic cells dissociated from embryonic day (E) 7, 9, or 11 lumbar sympathetic ganglia with the glucocorticoid hormones hydrocortisone or corticosterone has neither an inhibitory nor an inductive effect on phenotypes of granule-containing cells or granule-containing cells in transition. In cell cultures of E15 ganglia, however, glucocorticoid treatment induces a granule-containing cell resembling the granule-containing phenotype. These results suggest that the early granule-containing cells and granule-containing cells in transition in chick sympathetic ganglia are not the counterparts of glucocorticoid-responsive mammalian small intensely fluorescent or chromaffin progenitor cells, despite their morphological similarity. However, E15 sympathetic ganglia apparently contain a glucocorticoid-responsive progenitor population that can differentiate into chromaffin-like cells. These progenitors seem to require a systemic or intraganglionic developmental signal or undergo a temporal switch that renders them susceptible to glucocorticoids.     

Differential subcellular distribution of PC1, PC2 and furin in bovine adrenal medulla and secretion of PC1 and PC2 from this tissue.

The subcellular distribution of PC1, PC2 and furin was determined in bovine adrenal medulla by immunoblotting of fractions obtained by density gradient centrifugation. PC1 and PC2 were found to be confined to chromaffin granules whereas furin (C-terminal-peptide) was absent from these organelles. Stimulation of bovine adrenal medulla by carbamoylcholine chloride induced the secretion of PC1 and PC2. The secreted enzymes had the same molecular size as PC1 and PC2 present in chromaffin granules.     

Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF

Summary In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C₆ glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C₆ glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64–82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histofluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.     

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the alpha1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.     

Cortical-chromaffin cell interactions in the adrenal gland

Adrenal catecholamines and steroids are important regulators of the stress response, immune function, blood pressure, and energy homeostasis. Historically, the two cell populations within the adrenal gland—the steroid-producing adrenocortical cells and the catecholamine-producing chromaffin cells—have been regarded as two independent endocrine systems. Research on adrenal physiology and pathophysiology has therefore largely focused on the individual understanding of each cell type. However, adrenal cortex and medulla appear to be interwoven and show multiple contact zones without separation by connective tissue or interstitial membranes. In vitro studies, animal models, and the analysis of human adrenal pathophysiology have demonstrated critical importance of cortical-chromaffin crosstalk for adrenal function and disease. Thus, chromaffin cells regulate steroid-hormone release by the adrenal cortex and steroids induce catecholamine production in the medulla. Consequently, disorders of the adrenal cortex have been shown to affect chromaffin cell function and vice versa. Mouse models of adrenal cortical dysfunction, such as the targeted disruption of the 21-hydroxylase- or the CRHR1 genes, show alterations in chromaffin cell function, while disruption of tyrosine hydroxylase, a key enzyme in catecholamine synthesis, impairs adrenal cortical function. Accordingly, patients with congenital adrenal hyperplasia (CAH) and Addison’s disease show reduced catecholamine biosynthesis. Immense progress in characterizing the mechanisms of chromaffin-cortical interactions has been achieved in recent years. Here, we summarize the current view on intraadrenal communication with respect to adrenal pathophysiology.     

Cografts of adrenal medulla with C6 glioma cells in rats with 6-hydroxydopamine-induced lesions

Amitotic [3H]thymidine-labeled C6 glioma cells, which are known to produce neurotrophic factor(s), were grafted alone and with adrenal chromaffin cells in an attempt to improve chromaffin cell survival and phenotypic differentiation. Long-Evans rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway were divided into four groups: (1) those receiving adrenal medullary cells co-transplanted with C6 glioma cells; (2) those receiving adrenal medullary graft alone; (3) those receiving C6 glioma grafts alone; and (4) those serving as a vehicle control group. All rats were killed one month after transplantation. Immunohistochemical, neurochemical, and autoradiographic methods were used to identify and characterize the grafted cells. Tyrosine hydroxylase-immunoreactive cells were found in all animals that received grafts of the adrenal medulla alone or of adrenal medulla co-transplanted with C6 glioma cells. The cograft recipients had more tyrosine hydroxylase-immunoreactive cells than the hosts receiving just adrenal chromaffin cells (P less than 0.05). Additionally, more grafted chromaffin cells formed processes in the former group. All three tissue recipient groups (adrenal medullary, C6 glioma cell, and cografted animals) had a significant reduction (P less than 0.05) in ipsilateral rotations after amphetamine (0.5 mg/kg i.p.) injections as compared to the control vehicle recipient group. Moreover, the reduction in rotation was more marked in the cografted hosts than in the other two implanted groups (P less than 0.05). Significantly higher dopamine levels were found in the transplant sites of both cograft and adrenal medullary graft recipients than in sham grafted control animals.     

Transforming growth factor-beta, but not ciliary neurotrophic factor, inhibits DNA synthesis of adrenal medullary cells in vitro

Transforming growth factor-betas are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor-betas are synthesized, stored and released by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase immunocytochemistry as a marker for young postnatal rat chromaffin cells, we show that treatment with fibroblast growth factor-2 (1 nM) and insulin-like growth factor-II (10 nM) increased the fraction of 5-bromo-2'-deoxyuridine-labeled nuclei from 1% to about 40% of the cells in the absence of serum. In the presence of fibroblast growth factor-2 and insulin-like growth factor-II, transforming growth factor-beta1 (0.08 nM) reduced 5-bromo-2'-deoxyuridine labeling by about 50%, without interfering with chromaffin cell survival or death. Doses lower and higher than 0.08 nM were less effective. Similar effects were seen with transforming growth factor-beta3. In contrast to transforming growth factor-beta, ciliary neurotrophic factor, which inhibits proliferation of sympathetic progenitor cells, was not effective on rat chromaffin cells from postnatal day 6. Glucocorticoids also suppress DNA synthesis in fibroblast growth factor-2/insulin-like growth factor-II-treated chromaffin cells. This effect was not mediated by chromaffin cell-derived transforming growth factor-beta, as shown by addition of neutralizing antibodies. We conclude that one function of adrenal medullary transforming growth factor-beta may be to act as a negative regulator of chromaffin cell division.     

Nerve growth factor receptor expression in peripheral and central neuroectodermal tumors, other pediatric brain tumors, and during development of the adrenal gland.

Nerve growth factor (NGF) is important to the survival, development, and differentiation of neurons. Its action is mediated by a specific cell surface transmembrane glycoprotein, nerve growth factor receptor (NGFR). In this study, NGFR expression by human fetal and adult adrenal medullary tissue, peripheral nervous system (PNS) neuroectodermal tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), and CNS gliomas was examined by an immunohistochemical technique. Sixty-nine tumors in total were probed in this manner. Nerve growth factor receptor immunoreactivity was confined to nerve fibers and clusters of primitive-appearing cells in the fetal adrenal, and to nerve fibers and ganglion cells of the adult adrenal medulla; adrenal chromaffin cells were negative. In PNS neuroectodermal tumors, there was NGFR expression in tumor cells of 6 of 11 neuroblastomas and 6 of 6 ganglioneuroblastomas or ganglioneuromas. Thirteen of thirty-five CNS PNETs showed NGFR positivity. In most CNS PNETs, NGFR was restricted to scattered single or small groups of cells, but two tumors with astroglial differentiation showed much more extensive immunoreactivity. Most astrocytomas (11 of 14) and all ependymomas (3 of 3) were intensely NGFR positive.     

Regulation of neurotensin content in adrenal medullary cells: comparison of PC12 cells to normal rat chromaffin cells in vitro

Radioimmunoassay studies of cultures of PC12 pheochromocytoma cells have shown progressive increments in content and release of neurotensin in response to combinations of dexamethasone, nerve growth factor, activators of adenylate cyclase and lithium. We have studied the distribution of immunoreactive neurotensin by immunocytochemistry in cultures of PC12 cells and normal rat chromaffin cells, with two objectives: (i) to determine how changes measured by radioimmunoassay in extracts of PC12 cell populations are manifested at the level of individual cells and (ii) to determine whether normal chromaffin cells respond to combinations of agents similarly to PC12 cells. Staining for immunoreactive neurotensin is not identifiable in PC12 cells maintained in control medium or with any of the medium supplements alone. Approximately 3% of cells are stained after maintenance with dexamethasone plus nerve growth factor, verus 17% with dexamethasone plus nerve growth factor plus forskolin, and 33% with all four agents. This heterogeneity does not appear to result from clonal diversity, or to be cell cycle-dependent. Individual PC12 cells recruited to produce neurotensin in response to particular signals may, however, have passed a critical stage of differentiation toward a chromaffin cell, rather than neuronal phenotype before exposure to those signals. Staining for immunoreactive neurotensin is observed in up to 18% of normal chromaffin cells maintained with dexamethasone plus nerve growth factor, up to 45% with dexamethasone plus nerve growth factor plus forskolin, and up to 54% with all four agents. Proportions of cells stained under the various culture conditions are established before birth and in fetal cultures staining is confined for the most part to cells which do not undergo neuronal differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of basic fibroblast growth factor (FGF-2)-associated with tumour proliferation in human pancreatic carcinoma

 Basic fibroblast growth factor (FGF-2) is one of the mitogens that facilitate epithelial proliferation and angiogenesis. We analysed the expression of FGF-2 and type I fibroblast growth factor receptor (FGFR1) in 20 selected cases of human pancreatic carcinoma (PC) in connection with proliferation of tumour cells and intratumour endothelial cells (ECs), using immunohistochemistry and in situ hybridization (ISH). By FGF-2 immunostaining, tumour cells were strongly positive in 10 cases (50%). By FGFR1 immunostaining, stromal fibroblasts and ECs occasionally showed positive staining. Tumour cells in 12 cases (60%) were strongly positive. Expression of FGF-2 mRNA, as examined by ISH, was detected in 12 cases (60%) of PC, and its distribution pattern was similar to that of FGF-2 immunostaining. We divided these cases into two groups according to the result of FGF-2 immunostaining, and examined the Ki67 labelling indices of tumour cells and ECs between these two groups. These two proliferative indices were significantly higher in FGF-2-positive than in FGF-2-negative cases (P<0.05, P<0.05, respectively). These findings suggest that the expression of FGF-2 in PC is strongly associated with the proliferation of tumour cells and ECs and its increased expression may give tumour a growth advantage. Received: 13 November 1996 / Accepted: 12 March 1997     

D2-protein in PC12 pheochromocytoma cells after nerve growth factor stimulation

The rat brain D2-protein has previously been shown to play a role in interneuronal adhesion Monospecific antisera against this protein reacted with adrenal medulla cells and with PC12 pheochromocytoma cells as demonstrated by indirect immunofluorescence. Upon stimulation by nerve growth factor the PC12 cells extend neurites. These neurites were shown to contain D2-protein both by immunofluorescence and by crossed immunoelectrophoresis. The findings substantiate the close relationship between neurons from the central nervous system and both the PC12 cells line, as well as adrenal medulla cells.     

Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress

We reported that plasma brain-derived neurotrophic factor (BDNF) was maximally elevated following a 60-min period of acute immobilization stress and that salivary glands were the main source of plasma BDNF under this stress condition. However, the expression pattern of the BDNF receptor, Tyrosine receptor kinase B (TrkB), under this condition has yet to be determined. We therefore investigated the effect of this stress on the expression level of TrkB in various rat organs using real-time PCR. No significant differences were found between controls and 60 min-stressed rats with respect to TrkB level in various organs. Only adrenal glands showed significantly increased TrkB mRNA levels after 60 min of stress. TrkB mRNA and protein were observed to localize in chromaffin cells. In addition, we investigated whether BDNF-TrkB interaction influences the release of stress hormones from PC12 cells, derived from chromaffin cells. Truncated receptor, TrkB-T1, was identified in PC12 cells using RT-PCR. Exposure of PC12 cells to BDNF induced the release of catecholamine. This BDNF-evoked release was totally blocked by administration of the K252a in which an inhibitor of Trk receptors. Thus, BDNF-TrkB interactions may modulate catecholamine release from adrenal chromaffin cells under acute stress conditions.     

Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia

Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.     

First continuous human pheochromocytoma cell line: KNA. Biological, cytogenetic and molecular characterization of KNA cells

Pheochromocytomas are rare tumours, with an incidence of 1–2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line (KNA) was established from a sporadic pheochromocytoma of the right adrenal gland of a 73-year-old woman. The KNA cells grow as suspensions of spheroids and show the morphological and immunocytochemical characteristics of neuronal chromaffin cells, such as neuroendocrine granules, and positive reactions to chromogranin- and related peptide-, neuron specific enolase and vasoactive intestinal peptide antibodies. Neurite-like processes are formed after addition of nerve growth factor. Chromosomal analyses revealed a diploid (46,XX, n=50) to hypodiploid (43–45,XX, n=15) karyotype. In hypodiploid metaphases most frequently #19, #17, #21 and #22 were missing. Chromosome arms 1p and 4q showed apparently consistent interstitial deletions: 6q, 8q, 13q and 22q showed clonal interstitial deletions. The cell line shows a heterozygous sequence variant TGC (cysteine) to TGG (tryptophan) in codon 611 in exon 10 of the RET proto-oncogene. So far, PC-12, a rat adrenal pheochromocytoma, has been the only continuous pheochromocytoma cell line available. KNA represents the first report on a human continuous pheochromocytoma cell line, the first report of structural chromosome aberrations in pheochromocytomas and the first report of a RET mutation TGC to TGG in exon 10 of the RET proto-oncogene in a sporadic pheochromocytoma. © 1998 Chapman and Hall