The tripeptide H-
Gly-His-Lys-
OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition
metal ions Cu++ and Fe++ in vivo and may exert its
biological effects as a
peptide-
metal chelate. At physiological pH in vitro, GHL associates with ionic
copper,
cobalt,
iron,
molybdenum,
manganese,
nickel, and
zinc, but has no affinity for
calcium,
manganese,
potassium, and
sodium. GHL acts synergistically with
copper,
iron,
cobalt, and
zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic
hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit
DNA synthesis and
lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating
peptide factors in plasma or medium, may provide a mechanism for expression and regulation of
biological activities influenced by transition metals and
polypeptide growth factors. The observed effects of GHL-
metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of
hepatoma cells in monolayer culture.