34. KOSTECKÁ,Zuzana, BLAHOVEC,J., MESTER,J., CAVAILLE,F.: Peptic components of amniotic fluid regulate cell proliferation. (Az amnion-folyadédk peptidjei szabályozzák a sejtproliferációt)
34. Kostecká
érk: 97.11.25.
UEVM, 04001 Kosice, Hlinkova 1/A, Slovak Republic (first author's address)
Dept.Chem.Biochem.Biophys. Univ.Vet.Med., Kosice, Slovakia
INSERM U-55, Paris, France
INTRODUCTION
Growth factors, their receptors and binding proteins participate in cell proliferation and differentiation during embryonal and foetal development. Human amniotic fluid con-tains not only IGF-I, IGF-II, EGF, NGF and HGF, but also high concentration of IGFBPs. The IGFs binding proteins and IGFs molecules associate in amniotic fluid to form complex. The situation appears to be controversial because IGF-IGFBP complex inhibits cell proliferation in some cases or potentiates it in other ones.
In this investigation we were interested in actual mitogenic activity of peptide substances in sheep amniotic fluid. Chemically transformed mouse fibroblasts BP-A31 were used for indication of cell proliferation. They are able to enter quiescence without presence of growth factors and exogenously adding some mitogen starts all biochemical processes leading to cell division. Our results show that sheep amniotic fluid contains not only growth factor like IGF-I, but also some mitogen with higher molecular weight which probably represents IGFBPs or other similar proteins.
MATERIAL AND METHODS
The amniotic fluid was obtained from ewes at 10 weeks of gestation by laparothomy, hysterothomy and punction of amniotic sac. Delipidated and concentrated amniotic fluid was filtered through 17 G 4 filter and loaded into Sephadex G-10 column (5x70 cm). Elution was performed using a 0.04 mol.l-1 NH4HCO3 at a flow rate 12 ml.h-1. The content of low molecular weight peptides was measured by ninhydrin reaction and mitogenic activity of two lyophilisated fractions was tested. Benzo-d-pyrene-trans-formed BALB/c 3T3 mouse fibroblasts (BP-A31 cells) were cultured in d-MEM supplemented with 6% fetal calf serum in a humidified atmosphere containing 5 % CO2. For the study of mitogenic effects the cells were seeded in 24-well boxes (40 000 cells per well). After 24 hours the medium was replaced with 1 ml serum free d-MEM plus 2.5 TM FeSO4 and the cells were allowed to enter quiescence during the next 72 hours. Cells were incubated with 2 TCi 3H-thymidine (Amersham, Les Ulis, France) and fractions of sheep amniotic fluid for 24 hours. The incorporation of 3H-thymidine was terminated by acidification with 1 mol.l-1 ascorbic acid. The cells were fixed with 5% trichloracetic acid, solubilized in 0.1 mol.l-1 NaOH and the incorporated radioactivity was determined by liquid scintillation counting. Data shown are means of triplicates SEM. The differences were evaluated by Student's t test.
The void volume fraction after gel filtration on Sephadex G-10 was resolved in 10 % acetic acid and loaded into Sephadex G-50 column (3x90 cm). Elution was performed by 10 % acetic acid at a flow rate 9.5 ml.h-1. The content of peptides in fractions obtained after gel chromatography was determined by the measurement of absorbancy at wave length of 280 nm. All fractions were lyophilisated and tested with BP-A31 cells.
For testing of mechanisms of signal transduction we used next effectors:
- - insulin as activator of tyrosine kinase receptors
- - 2.10-7 mol.l-1 (Boehringer, Mannheim, BRD),
- - phorbol-12-myristate-13-acetate (PMA) as protein kinase C activator - 100 ng.ml-1,
- - 3 -isobutyl -1 -methyl xanthine (IBMX) as cAMP- phosphodiesterase inhibitor
- - 5.10-4 mol.l-1 (Sigma, St. Louis, MO, USA).
- Other compounds were from usual commercial sources.
RESULTS
By using of gel chromatography on Sephadex G-10 we separated amniotic fluid into two ninhydrin positive peaks (Fig. 1) with different molecular weight. Elution of first peak (A) coincided with void volume of column and the second one (B) contained small molecular weight peptides (Ã 0.7 kDa). Mitogenic activity of both fractions tested on chemically transformed BALB/c 3T3 mouse fibroblasts is shown in figure 2. Positive mitogenic effect of A fraction (p < 0.01) means that this fraction contains substances important in mitogenic signal transduction. On the other hand the non-separated sample of amniotic fluid has no mitogenic influence on the cells and B fraction contains some cell proliferation inhibitors (p < 0.05).
By further gel chromatography of A fraction at acidic condition on Sephadex G-50, two components with mitogenic activity were separated. One component (I) was eluted immediately after void volume of the column, the other one (II) was coeluted with 125I-IGF-I (Fig.3). The largest part of material (oligopeptides with molecular weight lower than 7 kDa) was mitogenic inactive.
For testing of mechanism of signal transduction leading to cell division we used PMA, insulin and IBMX. Both active fractions showed additive effect in presence of PMA (p < 0.05 in fraction II and no-significance increasing in fraction I). On the other hand we observed inhibition of 3H-thymidine incorporation into DNA by coincubation BP-A31 cells with IBMX (p < 0.05 in fraction I and p < 0.001 in fraction II).
CONCLUSION
Ovine amniotic fluid contains mitogenic active proteins or polypeptides. We suppose that a component with higher molecular weight eluted in the vicinity of the void volume of Sephadex G-50 represents probably IGFBPs or other similar proteins.
Figure 1.
Gel chromatography of ovine amniotic fluid on Sephadex G-10 column
Figure 2.
Mitogenic activtiy of amniotic fluid fractions
C - only cells, AF - nonseparated amniotic fluid, A,B - fractions obtained by gel chromatography, * = p < 0.05, ** = p < 0.01
Figure 3.
Chromatography of fraction A on Sephadex G-50. Fractions were measured at 280 nm (A) and tested for mitogenic activity (cpm).