Arthur S. Tischler

PC12 Pheochromocytoma Cultures in Neurobiological Research

Publisher Summary This chapter presents an overview of PC12 pheochromocytoma cultures in neurobiological research. The PC12 line of rat pheochromocytoma cells promises to be highly useful for studying both chromaffin cells and neurons and, consequently, has been employed in an increasing number of laboratories. PC12 cells propagated in vivo or in culture without nerve-growth factor are readily classifiable as pheochromocytomas by current morphological and chemical criteria. They have no processes and are characterized by numerous secretory granules ranging in diameter up to 350 nm. They also contain total catecholamine stores comparable to those in rat adrenal glands and show intense formaldehyde-induced fluorescence. In addition to catecholamines, PC12 cells contain several other secretory products, some of which have also been reported in human pheochromocytomas. The morphology of the cells and their synthesis, storage, release, and uptake of neurohumoral or neurotransmitter substances can be modulated in a number of ways within the overall chromaffin cell-like phenotype. This chapter reviews the currently known properties of the PC12 line. It also discusses the ways in which it has been and could be exploited to increase the knowledge of neuronal and neurosecretory cells.

Pheochromocytoma: Recommendations for clinical practice from the First International Symposium

The First International Symposium on Pheochromocytoma, held in October 2005, included discussions about developments concerning these rare catecholamine-producing tumors. Recommendations were made during the symposium for biochemical diagnosis, localization, genetics, and treatment. Measurement of plasma or urinary fractionated metanephrines, the most accurate screening approach, was recommended as the first-line test for diagnosis; reference intervals should favor sensitivity over specificity. Localization studies should only follow reasonable clinical evidence of a tumor. Preoperative pharmacologic blockade of circulatory responses to catecholamines is mandatory. Because approximately a quarter of tumors develop secondary to germ-line mutations in any one of five genes, mutation testing should be considered; however, it is not currently cost effective to test every gene in every patient. Consideration of tumor location, presence of multiple tumors, presence of metastases, and type of catecholamine produced is useful in deciding which genes to test. Inadequate methods to distinguish malignant from benign tumors and a lack of effective treatments for malignancy are important problems requiring further resolution.

Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells

NERVE growth factor (NGF) is a chemically characterised polypeptide which affects the development of the sensory and sympathetic nervous systems and is required for maintenance of sympathetic neurones1–3. Its mechanism of action and its precise role in embryogenesis are unknown.

Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice

Brain (CATH.a) and adrenal (PATH.1 and PATH.2) cell lines have been established that synthesize abundant dopamine and norepinephrine and express the appropriate catecholaminergic biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase. The lines were derived from TH-positive tumors in transgenic mice carrying the SV40 T antigen oncogene under the transcriptional control of 773 base pairs of 5′ flanking sequences from the rat TH gene. Although the lines continue to express T antigen, they exhibit neuronal properties such as neurofilaments and synaptophysin and lack glial intermediate filaments. Although in vivo TH is only expressed in postmitotic neurons in the CNS, the CATH.a line demonstrates that TH expression and continued cell division are not incompatible after oncogenic transformation.

Glucocorticoids Increase Catecholamine Synthesis and Storage in PC 12 Pheochromocytoma Cell Cultures

Abstract: Glucocorticoids, cholera toxin and high plating density all increase the activity of tyrosine 3‐monooxygenase (TH) in cultured PC12 pheochromocytoma cells. Glucocorticoids increase enzyme activity in cells treated with cholera toxin and in cells grown at high plating density. Glucocorticoids also increase the content of stored catecholamines in the cells. In cells cultured under routine conditions, glucocorticoids primarily increase the stores of dopamine. The addition of ascorbate to the culture medium increases the storage of norepinephrine in both steroid‐treated and untreated cells. Incubation of the cells in media containing 56 nM K+ causes the release of the same percentage of stored dopamine from steroid‐treated as from untreated cells. Steroid‐treated cells contain more dopamine than do untreated cells and therefore, in response to high K+, the steroid‐treated cells secrete more dopamine than do untreated cells. We conclude that the activity of tyrosine 3‐monooxygenase in PC12 cells can be regulated by several distinct mechanisms; that glucocorticoids cause a coordinate increase in TH activity and in catecholamine storage; that steroids increase the storage of catecholamines in a releasable pool; and that the steroid‐induced increase in catecholamine storage may result in increased secretion of catecholamines from steroid‐treated cells.

C‐cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B

Dominantly inherited multiple endocrine neoplasia type 2B (MEN2B) is characterized by tumors of the thyroid C‐cells and adrenal chromaffin cells, together with ganglioneuromas of the gastrointestinal tract and other developmental abnormalities. Most cases are caused by substitution of threonine for Met918 in the RET receptor tyrosine kinase, which is believed to convert the RET gene to an oncogene by altering the enzymes substrate specificity. We report the production of a mouse model of MEN2B by introduction of the corresponding mutation into the ret gene. Mutant mice displayed C‐cell hyperplasia and chromaffin cell hyperplasia progressing to pheochromocytoma. Homozygotes did not develop gastrointestinal ganglioneuromas, but displayed ganglioneuromas of the adrenal medulla, enlargement of the associated sympathetic ganglia and a male reproductive defect. Surprisingly, homozygotes did not display any developmental defects attributable to a loss‐of‐function mutation. Thus, while our results support the conclusion that the Met918Thr substitution is responsible for MEN2B, they suggest that the substrate specificity of the RET kinase does not interfere with its normal role in the development of the kidneys and enteric nervous system.

Electrical excitability of cultured adrenal chromaffin cells.

1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro‐electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded ‐50mV. 3. Chromaffin cells were capable of generating all‐or‐nothing over‐shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(‐6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae.

Attenuation of the media of coronary arteries in advanced atherosclerosis.

Human coronary artery wall architecture was analyzed in detail in 127 histologic sections with varying degrees of narrowing due to atherosclerotic plaque. A planimetry-microscope system was used to morphometrically determine percent luminal cross-sectional area narrowing due to atherosclerotic plaque, absolute area of the coronary artery media and total cross-sectional area of the coronary artery section. In 65 sections in which the native coronary artery lumen was narrowed less than 75%, the area of the coronary artery media corrected for total coronary cross-sectional area (Mc) was 0.244 +/- 0.055 mm2. In contrast, among 62 sections in which the coronary artery lumen was narrowed more than 75% in cross-sectional area, Mc measured 0.180 +/- 0.078 (p less than 0.001). Thus, in coronary artery segments with advanced atherosclerosis, there is substantial attenuation of the media, normally the principal component of the coronary artery wall.

Multidirectional differentiation in neuroendocrine neoplasms.

Histopathological and experimental observations indicate that tumors composed wholly or in part of neuroendocrine elements may arise in tissues derived from ectoderm (including neuroectoderm), mesoderm, and endoderm. These tumors frequently exhibit multidirectional differentiation as manifested by multihormonality and by the presence of morphological features indicative of divergent differentiation both in vivo and in vitro. The existence of stem cells, plasticity of differentiated cells, microenvironmental influences, and random events are factors which might all interact to determine the characteristics of any particular tumor. The production of characteristic regulatory peptide products in association with tumors of specific histological subtypes and with other neuroendocrine markers suggests mechanisms for nonrandom activation of multiple genes common to neuroendocrine-programmed cells. Future studies applying new molecular biological techniques to intact tissues and to in vitro models may help to clarify the mechanisms that regulate the expression of the neuroendocrine phenotype in normal and neoplastic states.

Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice

Abstract. Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.