Molecular studies of apoptosis induced by antiresorfitve bisphosphonate drugs. Michael T. Rogers
Chilton KM., Luckman SP., Graham R., Russell G., Coxon FP.
Bisphosphonates have become the most important class of drugs used clinically to inhibit osteoclast-mediated bone résorption. The exact molecular mechanisms by which BPs inhibit bone résorption have not been identified (due to the difficulty in isolating large numbers of osteoclasts) but appear to result in osteoclast apoptosis. We have found that BPs also induce apoptosis of J774 macrophages, identified on the basis of characteristic morphological changes as well as internucleosomal DNA fragmentation. Since the structure-activity relationships for induction of J774 apoptosis in vitro by a wide range of BPs matches the structure-activity relationships for inhibition of bone résorption in vivo, this suggests that apoptosis in J774s and osteoclasts occurs by a similar mechanism. We are therefore using J774 cells as surrogate osteoclasts in order to clarify the molecular events that lead to BP-induced apoptosis. Apoptosis occurs after approximately 24 hours of treatment with 100μlM BP. However, a shorter (4 hour) exposure to BP also results in apoptosis, which still occurs about 20 hours later. This suggests that BPs do not trigger apoptosis directly. Indeed, BPs do not cause DNA fragmentation when incubated with isolated nuclei. DNA fragmentation and changes in nuclear morphology in J774 cells can be inhibited in the presence of SOOuM aurintricarboxylic acid, although loss of cell viability still occurs in the absence of these changes. Apoptosis appears to be dependent on de novo protein synthesis since coincubation with BP and cycloheximide (CHX) or actinomycin D prevents apoptosis. CHX appears to inhibit the final stages of BP-induced apoptosis, since delaying the addition of CHX up to 14 hours after the onset of BP treatment also inhibits apoptosis. Although BPs are effective metal ion chelators, apoptosis does not appear to be due to depletion of intracellular zinc. The specific intracellular zinc chelator TPEN, by contrast with BPs, causes apoptosis without DNA fragmentation. In addition, the effect of TPEN is more rapid and occurs at lower concentrations (≥4μM) than those of BPs required to cause apoptosis. These observations are beginning to shed new light on the intracellular events caused by BP drugs that may lead to apoptosis.