Human Neonatal Foreskin Fibroblasts Research Articles

Cytotoxicity, anchorage‐independent growth, and DNA adduct formation in human neonatal fibroblasts by 1, 2, 3, 4‐tetrahydro‐7, 12‐dimethylbenz(a)‐anthracene (TH‐DMBA), its six aryl fluoro regioisomers, and an exo methylene tautomer

1,2,3,4-Tetrahydro-7,12-dimethylbenz(a)anthracene (TH-DMBA), its six possible fluorosubstituted regioisomers, and the C-7 exo methylene tautomer of the 11F derivative have been investigated for their cytotoxicity and for their ability to induce anchorage-independent growth and to form adducts in human neonatal foreskin fibroblasts. All compounds tested exhibited a low level of cytotoxicity, determined as percent cloning efficiency, up to a final concentration of 30 micrograms/ml. Except for 5F-TH-DMBA and the C-7 exo methylene tautomer, all compounds induced anchorage-independent growth of neonatal foreskin fibroblasts in soft agar at all concentrations tested (1, 3, 10 and 30 micrograms/ml). The C-7 exo methylene tautomer induced anchorage-independent growth only at a concentration of 10 micrograms/ml. Among the compounds tested the 6F derivative was the most effective compound at 1 microgram/ml. The D-ring fluoro isomers induced anchorage-independent growth at a frequency comparable to TH-DMBA itself, with the 11F derivative being the least effective of the four D-ring regioisomers. All compounds except 5F-TH-DMBA formed detectable adducts with cellular DNA as determined by 32P postlabeling procedures, when the cells were treated at 1 microgram/ml. Two adducts were detected in cells treated with TH-DMBA and four adducts were detected in DNA obtained from cells treated with 6F-TH-DMBA. The level of bonding for the D-ring fluoro isomers was quantitatively less and sometimes qualitatively different than that for TH-DMBA. For the D-ring compounds, the ability to induce anchorage-independent growth frequency correlated with the total quantity of adduct formed. The C-7 exo methylene tautomer formed a single adduct and the level of bonding was less than one adduct per 10(9) nucleotides. Analysis of these results led to the proposal that the planar anthracene ring structure (rings B, C, and D) of TH-DMBA and possibly oxidative metabolism at benzylic carbon 4 of the A-ring are important to DNA bonding and initiation of induction of anchorage-independent growth.

Cultured endothelial cells produce a platelet-derived growth factor-like protein.

The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4 degrees C. The (125)I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serum-free conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4 degrees C binding of either (125)I-labeled insulin to 3T3 cells or (125)I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of (125)I-labeled PDGF to its receptor ( approximately 30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, approximately 7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of (125)I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.