OVERALL LACK OF REGULATED SECRETION IN A PC12 VARIANT CELL CLONE

A stable clone of PC12 neuroendocrine cells, named 27, known from prev ious studies to exhibit a defect of regulated secretion (lack of regul ated secretory proteins, of synaptophysin, of dense granules and of ca techolamine uptake and release; Clementi, E., Racchetti, G., Zacchetti , D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 94 4-953) was characterized in detail to clarify the nature of its phenot ype and the mechanisms of its establishment, The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsin s, neurofilament subunit H, neuronal kinesin, and alpha-latrotoxin rec eptor. Moreover, various intracellular membrane systems of PC12-27, in cluding the endoplasmic reticulum and the Golgi complex, appeared simi lar to control PC12 in both morphology and marker expression. In contr ast, all the investigated markers located either in dense granules (do pamine-beta-hydroxylase), in synaptic-like microvesicles (the acetylch oline transporter) or in both these regulated secretory organelles (VA MP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins inv olved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist i n a protein synthesis blockade that mRNA studies (reverse transcriptio n-polymerase chain reaction, Northern blotting and actinomycin D exper iments) 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 secretory proteins, chromo granin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 ce lls are fully incompetent for both branches of regulated secretion, th ose of dense granules and synaptic-like microvesicles, possibly becaus e of the impairment of a general expression control system that appear s to operate independently of neuroendocrine cell differentiation.