Overall lack of regulated secretion in a PC12 variant cell clone

A stable clone of PC 12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretary proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and α-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-β-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription- polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretary proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation.