Progenitor Cell Line (hPheo1) Derived from a Human Pheochromocytoma Tumor

<div><p>Background</p><p>Pheochromocytomas are rare tumors generally arising in the medullary region of the adrenal gland. These tumors release excessive epinephrine and norepinephrine resulting in hypertension and cardiovascular crises for which surgery is the only definitive treatment. Molecular mechanisms that control tumor development and hormone production are poorly understood, and progress has been hampered by the lack of human cellular model systems. To study pheochromocytomas, we developed a stable progenitor pheochromocytoma cell line derived from a primary human tumor.</p><p>Methods</p><p>After IRB approval and written informed consent, human pheochromocytoma tissue was excised, minced, dispersed enzymatically, and cultured in vitro. Primary pheochromocytoma cells were infected with a lentivirus vector carrying the catalytic subunit of human telomerase reverse transcriptase (hTERT). The hTERT immortalized cells (hPheo1) have been passaged >300 population doublings. The resulting cell line was characterized morphologically, biochemically and for expression of neuroendocrine properties. The expression of marker enzymes and proteins was assessed by immunofluorescence staining and immunoblotting. Telomerase activity was determined by using the telomeric repeat amplification protocol (TRAP) assay.</p><p>Results</p><p>We have established a human pheochromocytoma precursor cell line that expresses the neuroendocrine marker, chromogranin A, when differentiated in the presence of bone morphogenic protein 4 (BMP4), nerve growth factor (NGF), and dexamethasone. Phenylethanolamine N-methyltransferase (PNMT) expression is also detected with this differentiation regimen. CD-56 (also known as NCAM, neural cell adhesion molecule) is expressed in these cells, but CD31 (also known as PECAM-1, a marker of endothelial cells) is negative.</p><p>Conclusions</p><p>We have maintained hTERT-immortalized progenitor cells derived from a pheochromocytoma (hPheo1) in culture for over 300 population doublings. This progenitor human cell line is normal diploid except for a deletion in the p16 region and has inducible neuroendocrine biomarkers. These cells should be a valuable reagent for studying mechanisms of tumor development and for testing novel therapeutic approaches.</p></div

Characterization of hPheo1 cells.

<p>A. Hematoxylin and eosin staining shows plump polygonal cells with eosinophilic cytoplasm. B. Karyotypic analysis shows a female diploid pattern with a single cytogenetic abnormality, del9p22 on one chromosome, near its telomeric end (arrow). C. SNP analysis confirms the deletion of 9p, which encompasses the <i>CDKN2A</i> (p16 gene).</p

Differentiation of hPheo1 cells.

<p>A. Western blot for chromogranin A shows expression more prominent after differentiation treatment. B. Western blot for PNMT shows expression more prominent after differentiation treatment. C. Presence of COMT in hPheo1 cells is seen, whether treated with differentiating factors or not. D. Neurite formation in hPheo1 cells occurs after treatment with Nerve Growth Factor (NGF).</p

Immunofluorescence reveals CD56 is positive in hPheo1 and PC-12 cells.

<p><b>CD31 is negative in hPheo1 but positive in HUVEC cells.</b></p