Clarification of PC12 Cell Growth Inhibition by a Sialoglycopeptide from Bovine Brain under Low Serum Condition.

Abstract

ABSTRACT. To study the effect of a bovine brain sialoglycopeptide (SGP) on the nerve cell growth, PC12 cells were used as a model system. Though the inhibition of cell growth, measured by the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, by SGP was slight under the serum concentration of 10%, it was clearly detected under that of 1 to 2%. For the preparation of SGP, filtration through a nitrocellulose membranewas useful.A sialoglycopeptide (SGP) from bovine brain (molecular weight of 18 kDa) was reported to inhibit cell growth ofafibroblast cell line, NIH 3T3 cells (4, 13). Inhibition of DNAsynthesis was shown in various cells; fibroblast cell, tumor cell, transformed cell and also primary cultured cell (4). The presence of the SGP receptors in 3T3 cells was shown by use of the 125I-labeled SGP (2, 14). The native form of SGP with molecular weight of 66,000 was isolated from the cell membrane in bovine brain (5). The above data suggest that the SGPmight participate in the contact inhibition in these cells.In this paper, the PC12 cell, rat pheochromocytoma, was used as a model of premature neuron, having the potential of a neuron-like outgrowth whenexposed to NGF(6). The effect of SGP on the PC12 cell growth was analyzed using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay (1, ll). The MTT assay is based on the dehydrogenase activity in mitochondria of living cells. Therefore, this assay is used to determine the living cell number. The inhibitory effect of SGPunder 10%serum condition was not clearly detected. Whenthe serum concentration in the media was decreased to the range of 1 to 2%, the effect was clearly detected. This dependency on the serum concentration probably originated from the presence of cell growth factors (12) and also inhibitory factors (3, 7) in the serum. MATERIALS AND METHODSChemicals. Materials were obtained from the following sources: Dulbecco's modified Eagle's medium(DMEM) and F12 nutrient mixtures (Ham (F12)) (Gibco); fetal bovine serum (FBS) and horse serum (HS) (Flow Laboratory); bovine serum albumin, pronase E (from Streptomyces griseus), MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) and polyethylenimine (Sigma); SDS-PAGElow molecular weight marker (Pharmacia); nitrocellulose membrane filter (pore size 0.2 //m) for the sterilization of the culture medium or SGP solution, plates and dishes for cell culture (Corning). All other chemicals were reagent-grade materials from commercial sources.