Identification and initial characterization of stathmin by the differential display method in nerve growth factor-treated PC12 cells

Abstract

The differential display of mRNA is a new strategy to identify genes that are differentially expressed under altered conditions. We applied this method to determine differential gene expression in the rat pheochromocytoma cell line during differentiation induced by nerve growth factor (NGF).Three different mRNA species were isolated, and their differential expression was confirmed by RT-PCR. One of the mRNA species was identified as stathmin, a 19 kDa cytosolic protein attracting increasing interest for its role in signal transduction. In the NGF-treated PC12 cells, the expression of stathmin mRNA increased in a time-dependent manner, as assessed by northern blot analysis and RT-PCR. We also assessed by northern blot analysis how the expression of stathmin mRNA was altered in human pheochromocytomas (n ¼ 5) compared with that in normal adrenal medulla tissue (n ¼ 5). The mRNA concentrations were found to be significantly greater in the pheochromocytomas than in the normal tissues. It has been shown that stathmin mRNA concentrations are increased in various tumor cells. As pheochromocytomas are well-differentiated tumors of neural origin, it is not unexpected that stathmin mRNA is overexpressed in these tumors.Stathmin was isolated and identified as a differentially expressed gene by the differential display method in PC12 cells during differentiation induced by NGF. In addition, stathmin mRNA was found to be overexpressed in human pheochromocytomas. The mechanisms responsible for the up-regulation of stathmin mRNA during differentiation of PC12 cells and the significance of its overexpression in human pheochromocytomas remain to be determined.

Stathmin Expression in Pheochromocytomas, Paragangliomas, and in other Endocrine Tumors

“…Stathmin expression was described previously in PC12 pheochromocytoma cells where its expression was increased after nerve growth factor treatment which stimulated phosphorylation of stathmin [11,12]. A stathmin-like gene, which belongs to the stathmin family of phosphoproteins, RB3, was also reported in the rat hypothalamus [13].…”

Expression of stathmin in human uterus and decidualizing endometrial stromal cells

“…Stathmin expression in decidualization (Puissant et al 1995), pheochromocytoma cells (Takekoshi et al 1998), and the gonadotrope (Drouva et al 1995); it might be associated with glandular proliferation or morphological changes. Interestingly, we found in our present study that the stathmin signals in human endometrial tissue were distinctly localized to the stromal cells of the functional area rather than the endometrium close to the myometrium (the stratum basale).…”

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

“…Total RNA was extracted as described previously (Takekoshi et al, 1998). Semi-quantitative PCR was performed according to a described method (Wizigmann-Voos et al, 1995;Meister et al, 1999).…”

“…The reaction was carried out in a Program Temp Control System, Gene Amp PCR System 9600 (Perkin-Elmer-Cetus, CA). A program of 30 cycles was chosen on the basis of previous results and preliminary experiments (Wizigmann-Voos et al, 1995;Takekoshi et al, 1998) that demonstrated a linear relationship between the input DNA and the RT-PCR product obtained under these conditions. Human GAPDH sense (5V-CGA TGC TGG CGC TGA GTA C) and antisense (5V-CGT TCA GCT CAG GGA TGA CC) oligonucleotides were used as an internal standard.…”