Subcellular redistribution of BAX during apoptosis induced by anticancer drugs

BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malign ant cells to anticancer drugs. In spite of many studies, the molecular mech anism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to vari ous anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (S TP), 2-chloro-2'-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or w ere stimulated to apoptosis with the drugs examined. Laser scanning cytomet ry was applied for the quantitative analysis of BAX expression, and distrib ution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pi xel (BAX MP), the parameter corresponding to aggregation of BAX in the cell , was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The mos t pronounced effect was In the case of 2CdA, CPT and STP. The increase In B AX MP was observed only when antibody recognizing the 43-61 amino acid sequ ence was used. When antibody binding the N-terminal epitope (11-30 amino ac id sequence) was applied, the number of cells expressing high BAX MP signif icantly decreased. These results Indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX In cancer cells, which is dep endent on BAX activation by Its cleavage at the N-terminal epitope and expo sure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. T he increase in BAX Nf and, occurring in most treatments, the increase in th e BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activa ted form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BA X from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, o ccurring within 60-180 min of cell exposure to the drug. The subcellular tr anslocations of BAX preceded in time the appearance of morphological sympto ms of apoptosis. In conclusion, (I) in spite of different molecular mechani sms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX a ctivation and subcellular translocations from the cytosol to organellar mem branes and nucleus are key cellular responses to drugs bearing proapoptotic properties. [(C) 2001 Lippincott Williams & Wilkins.].