The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADPribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis. Cell Death and Differentiation (2000) 7, 102 ± 111.
We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue. In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS). Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression. Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls. Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid. Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death. © 2001 Cancer Research Campaign http://www.bjcancer.com
The molecular basis for the clinical presentation of broad-range drug resistance in childhood ALL is poorly understood. In this study, high level cross-resistance to the glucocorticoid dexamethasone was encountered in a childhood ALL cell line selected for resistance to methotrexate (CEM MTX-R3). Compared with wild-type (WT) CEM cells, MTX-R3 cells had significantly fewer glucocorticoid binding sites, as well as reduced glucocorticoid receptor protein and mRNA levels. DNA sequencing and restriction fragment-length polymorphism (RFLP) analysis showed that WT cells expressed both a wild-type and a mutant (GR753F) glucocorticoid receptor allele, while MTX-R3 cells expressed only the GR753F allele. Therefore, the crossresistance of MTX-R3 cells to dexamethasone appeared due to loss of expression of the wild-type glucocorticoid receptor allele. In an effort to gain insight into the underlying basis for the development of cross-resistance to methotrexate and glucocorticoids, glucocorticoid receptor nuclear translocation experiments were carried out. Exposure of WT cells to either dexamethasone or the cytotoxic agents cytarabine and methotrexate caused translocation of the glucocorticoid receptor from the cytoplasm into the nucleus. These data indicate that exposure of childhood ALL cells to cytotoxic agents may result in ligand-independent glucocorticoid receptor activation which, in the context of the outgrowth of drug-resistant cells, could lead to the co-selection of glucocorticoid resistance. Leukemia (2001) 15, 929-935.
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