Theret-activating ligand GDNF is differentiative and not mitogenic for normal and neoplastic human chromaffin cells in vitro

Activating mutations of the receptor tyrosine kinase,ret, are associated with multiple endocrine neoplasia type 2A (MEN-2A). However, the mechanisms leading to tumor development are unclear. Glial-derived neurotrophic factor (GDNF) activates wild-typeret via interaction with a second receptor, GFR α-1. We have utilized GDNF to stimulate normal and neoplastic chromaffin cells in order to ask whetherret activation is mitogenic. Cells from three normal adult adrenal medullas, one sporadic pheochromocytoma, and three MEN-2A pheochromocytomas were labeled with bromodeoxyuridine (BrdU) for 12 d in the presence or absence of GDNF or nerve growth factor (NGF), which is known to stimulate neurite outgrowth, but not proliferation in human chromaffin and pheochromocytoma cell cultures. Responses to GDNF and NGF were comparable, except for two MEN-2A pheochromocytomas that responded minimally to GDNF and robustly to NGF. These tumors responded to GDNF biochemically, as measured by phosphorylation of mitogen-activated protein kineses, despite their weak morphological responses. Our findings suggest that activation ofret may not be sufficient to produce chromaffin cell hyperplasia or neoplasia directly by stimulating cell proliferation. However, the possibility that altered cell-cell or cell-substrate interactions might cause responses to become differentiative rather than proliferative in vitro has not been ruled out. We also demonstrate, for the first time, that at least some human pheochromocytomas with an MEN-2Aret mutation respond to a normalret ligand. This responsiveness could be mediated by a remaining normalret allele or by other mechanisms.