Nuclear mechanisms in cell death induced by HAMLET (human alpha-lactalbumin made lethal to tumour cells)

HAMLET (human alpha-lactalbumin made lethal to tumour cells), a protein-lipid complex originally isolated from human milk, induces programmed cell death selectively in tumour cells. It consists of partially unfolded alpha-lactalbumin in complex with oleic acid. It was previously not known if the unfolding of alpha-lactalbumin alone accounts for the activity of HAMLET or if the lipid adds biological activity. This was investigated using alpha-lactalbumin mutants that are present in the unfolded state at physiological conditions. These proteins were not active in cell death assays but could be converted to biologically active complexes with oleic acid, showing that unfolding of alpha-lactalbumin is not sufficient and that lipid structures are required for the cell death-inducing activity of HAMLET. HAMLET targets and accumulates in nuclei of sensitive cells but does not reach the nuclei of resistant cells, suggesting that a nuclear effect may be important for the cell death induction. We identified histones as nuclear targets for HAMLET, and HAMLET interacted with both denatured and native histones, as well as with histones in nucleosomes. In vivo, in tumour cell nuclei, HAMLET colocalised with histones and perturbed the global chromatin structure. The interaction with histones required partial unfolding of alpha-lactalbumin, achieved either by removal of calcium from the protein or conversion to HAMLET-like complexes with lipids. The lipid stabilised a histone-binding conformation of alpha-lactalbumin, but did not contribute to the binding specificity. Although there was a species variation in the ability of alpha-lactalbumin to bind histones, all but one of the biologically active HAMLET-like complexes tested were able to interact with histones. The HAMLET-histone interaction may induce damage to the chromatin, and cell death resulting from nuclear damage is often signalled through the p53 system. However, using cells with defined p53 status, we found no correlation between p53 and sensitivity of cells to HAMLET. We propose that the interaction of HAMLET with histones in the nuclei of tumour cells may lead to cell death in a p53-independent manner by disrupting the structure and function of chromatin.