CELLULAR RESISTANCE TO THE CYTOTOXIC EFFECTS OF OXYGENATED STEROLS AND ANTIESTROGENS

Abstract

Oxygenated derivatives of cholesterol (oxysterols) have long been known to exhibit cytotoxic properties against many different cell types both in vitro and in vivo, The mechanism of this cytotoxic effect, often seen at micromolar concentrations, is unknown but may be of considerable importance in view of studies suggesting that oxysterols may be pathogenetically involved in diseases such as atherosclerosis. In an attempt to explore the cellular mechanisms of oxysterol cytotoxicity, we have developed cell lines which are specifically resistant to the cytotoxic effects of oxysterols. By exposing the murine lymphoma cell line EL4 and the leukaemia cell line K36 to incremental concentrations of 7-ketocholestanol, we have succeeded in establishing cell lines whose LD50 values for 7-ketocholestanol were up to 100 times that of the parental cell lines. Characterisation studies revealed that (i) the daughter cell lines, selected for their resistance against 7-ketocholestanol, were also resistant against the cytotoxic effects of other oxysterols such as 25-hydroxycholesterol; (ii) the daughter cell lines were, however, as sensitive as their parental counter-parts to the cytotoxic effects of "non-specific" toxic agents such as azide, ethanol, Triton X-100, and heat; and (iii) surprisingly, the oxysterol-resistant daughter cell lines also showed resistance against the cytotoxic effect of a completely different class of compounds, namely the nonsteroidal antiestrogens, such as tamoxifen and clomiphene. The last observation is intriguing because earlier studies indicated that both oxysterols and nonsteroidal antiestrogens bind with high affinity to a specific microsomal protein called the antiestrogen-binding site (AEBS), raising the possibility that oxysterols and antiestrogens may share, at least in part, a similar mechanism of cytotoxic action. Further studies on the possible biochemical basis of oxysterol/antiestrogen cytotoxicity revealed that the resistance against the cytotoxic action of these agents could not be explained by reduced cellular uptake of the cytotoxic substance or by a change in the binding affinity and binding capacity of AEBS. Studies with anti-phosphotyrosine antibodies revealed diminished quantities of a 120 kDa protein and increased amounts of a 70 kDa protein in the resistant daughter cell lines as compared to the parental cell lines. Whether these proteins play a role in the resistance against or susceptibility to the cytotoxic effects of oxysterols and antiestrogens is a question which requires further study.