HEPARIN AND ITS NON-ANTICOAGULANT ANALOGS INHIBIT HUMAN KERATINOCYTE GROWTH WITHOUT INDUCING DIFFERENTIATION

In addition to its anti-coagulant effect, heparin inhibits the growth of several types of cells. Recent studies suggest that heparin inhibit ion of proliferation of cultured human keratinocytes results primarily from interaction with keratinocyte-generated, heparin-binding autocri ne growth factors. In this study, we evaluated whether non-anticoagula nt heparin analogs, and oligosaccharide fragments of heparin, retain t he growth-inhibitory properties of whole heparin on human keratinocyte s. Second-passage neonatal keratinocytes were grown in serum-free kera tinocyte growth medium, and the effect of heparin analogs was studied in the absence of exogenous growth factors using keratinocyte-conditio ned medium. Cell proliferation was assessed by measurement of both DNA content and [H-3]-thymidine incorporation. The addition of heparin in hibited the conditioned medium-stimulated keratinocyte proliferation i n a dose-dependent manner, with 80% inhibition at or above 10 mu g/ml. Moreover, heparin was not toxic to keratinocytes (as detected by prop idium-iodide fluorescence and by retention of normal protein synthetic rate) and it did not induce terminal differentiation (as measured by cornified envelope formation). Furthermore, heparin stimulated protein secretion by keratinocytes without altering rates of protein synthesi s. The growth-inhibitory effects of heparin oligosaccharides were dire ctly proportional to their chain length. The hexasaccharide unit repre sented the minimum requirement for inhibition, whereas decasaccharide units demonstrated nearly equivalent growth inhibition to native hepar in. Finally, two non-anticoagulant heparin analogs were equipotent wit h heparin in inhibiting autocrine-induced keratinocyte growth. These s tudies show that the growth-inhibitory activities of heparin are indep endent of the anticoagulant effects and that decasaccarides contain th e optimal oligosaccaride chain length for the antiproliferative effect in human keratinocytes.