Apoptosis-induced concomitant release of cytosolic proteins and factors which prevent cell death.

In the course of the apoptotic cell death, cells fragment into apoptotic bodies, the elimination of which by phagocytosis is thought to avoid the release of cytosolic constituents whose occurrence is indicative for necrotic cell death. Confluent cultures of chicken embryo fibroblasts, however, show a different behaviour. After serum deprivation, they transiently released with the same time course mitogenic activity, lactate dehydrogenase and cytosolic peptidyl prolyl cis-trans isomerases into the serum-free culture medium. The release correlated in time with a decrease of the cell number which started approximately 3 h after serum removal and ceased within approximately 10 h at about half of the initial cell density. Morphological features like cell shrinkage, membrane blebbing and cell fragmentation as well as internucleosomal DNA fragmentation indicated apoptotic cell death whereas necrotic cell death could be excluded. Conditioned medium (M(r) > or = 30 kDa) from serum-deprived cultures of chicken embryo fibroblasts completely prevented chicken embryo fibroblasts to undergo apoptosis as did phorbol 12-myristate, 13-acetate and, to -60%, L-cysteine. Cycloheximide had no effect on serum deprivation-induced apoptosis. From the present results it can be concluded that chicken embryo fibroblasts and possibly other cells undergoing apoptosis release cytosolic components and endogenous survival factor(s) which prevent apoptosis.

CDGF (chicken embryo fibroblast-derived growth factor) is mitogenically related to TGF-beta and modulates PDGF, bFGF, and IGF-I action on sparse NIH/3T3 cells.

Chicken embryo fibroblast (CEF)-derived growth factor (CDGF), which was recently isolated from serum-free conditioned medium (SFCM) of confluent primary cultures of CEF (A. Geistlich and H. Gehring, Eur. J. Biochem. 207, 147-153, 1992), exhibited a strong mitogenic activity on sparse cultures of NIH/3T3 cells. The activity of CDGF was different from that of SFCM; i.e., the onset of DNA synthesis was delayed for about 7 h. CDGF induced maximally 25% of the activity of serum or SFCM if the activity was measured with a 2-h[3H]thymidine pulse starting 15 h after stimulation of the cells, indicating loss of a protein which modulated the mitogenic activity of CDGF. However, [3H]-thymidine incorporation of cells stimulated with approximately 50 pM CDGF reached serum values after prolongation of the thymidine pulse to 24 h. PDGF, at a concentration of approximately 300 pM, and bFGF (approximately 10 pM) exhibited strong activities in the 2-h pulse, whereas TGF-beta behaved like CDGF. IGF-I induced [3H]thymidine incorporation only weakly and only in the 2-h pulse. EGF did not induce any [3H]thymidine incorporation at all. CDGF together with small concentrations of bFGF (3.5 pM), higher concentrations of PDGF (300 pM), or IGF-I (1 nM) increased synergistically thymidine incorporation in the 2-h pulse, exceeding in the case of PDGF and bFGF the values obtained with 10% serum. Such a synergism could not be demonstrated with alpha-fetoprotein or fetuin, two serum proteins which have been reported to cooperate with growth factors. Regarding induction of cellular growth, only PDGF proved similar to serum, whereas cells stimulated with CDGF or TGF-beta showed a decreased rate of multiplication during the first day after stimulation. After this lag, however, CDGF- and TGF-beta-stimulated cells grew also with a rate similar to that obtained with serum, indicating the induction of an autocrine mitogen by CDGF or TGF-beta. FGF, IGF-I, and PDGF all enhanced CDGF-induced cell growth during the first day, whereas an additive stimulation over at least 2 days was observed with PDGF. CDGF behaved similar to TGF-beta in the synergism with IGF-I.

Isolation and characterization of a novel type of growth factor derived from serum-free conditioned medium of chicken embryo fibroblasts.

A strong mitogenic activity for fibroblastic cells has been found in serum-free medium of growth-arrested primary cultures of chicken embryo fibroblasts (CEF). This serum-free conditioned medium promoted growth of NIH/3T3 cells and primary as well as secondary cultures of CEF. The mitogenic activity was as potent as 5% serum. Half-maximum stimulation was obtained with 20% of the initial concentration of the conditioned medium. The activity eluted at high M(r) (1-2 x 10(5)) from a gel-filtration column under nondenaturing conditions and was trypsin insensitive and thiol insensitive. Treatment with acid or urea converted the mitogen to a low-molecular-mass form, which showed a delayed induction of DNA synthesis. Purification of this factor (10000-fold) to apparent homogeneity was achieved by preparative isoelectric focusing, gel filtration, reverse-phase HPLC and nonreducing SDS/PAGE. The factor, termed CEF-derived growth factor (CDGF) was a 32-kDa, disulfide-linked heterodimer of a 15-kDa and a 17-kDa subunit as judged by SDS/PAGE, with a pI of approximately 7 in 8 M urea. It exhibited partial stability towards heat treatment and was trypsin sensitive. CDGF was active only in its dimeric form and half-maximum stimulation of NIH/3T3 cells was obtained at approximately 10 pM. The mitogenic activity was not suppressible by an antibody neutralizing the activity of transforming growth factor beta 1, 2 and 3 (TGF-beta). The physico-chemical properties suggest that CDGF is not identical with one of the common growth factors like fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, or TGF-beta but rather represents a novel type of growth factor.