The loss or decreased expression of beta 2m-microglobulin (beta2m) causes cellular resistance to chemotherapy. Moreover, exogenous beta 2m protein causes caspase-dependent apoptosis, but independently of caspases-3, -8, and -9, in drug sensitive and resistant variants. In this application, we propose to explore the molecular mechanism(s) by which beta2m triggers apoptosis and how it increases the efficacy of anticancer agents. Our long-term objectives are to unravel the molecular mechanism(s) of beta2m-induced apoptosis, and to apply this knowledge to identify and/or develop new therapeutic regimens.
The Specific Aims are to (1) restore beta2 expression in drug resistant cells and assess its influence on the cytotoxic effects of chemotherapeutic drugs, (2) identify specific caspases or non-caspase proteins involved in the execution of beta 2m-induced apoptosis, and (3) investigate the role of the tumor necrosis factor-alpha (TNF-alpha) receptor family and increased reactive oxygen species (ROS) in beta 2m-induced apoptosis.
Specific Aim 1 will (a) determine whether the increase in endogenous J32m in MCF-7 and MCF-7/Adr-5 cells increases the efficacy of DOX, VCR, Taxol and VP-16, (b) determine whether exogenous beta 2m synergistically enhances apoptosis induced by these anticancer agents in these cells, (c) evaluate whether [beta 2m alone or in combination with these drugs exerts similar apoptotic effects on the estrogen receptor-negative breast cancer cell line MDA-MB-231 and its DOX-resistant derivative MDA-MB-231/Adr, and (d) examine whether [beta 2m modulates the effects of anticancer drugs on caspases during apoptosis and affects the cell cycle in these cells, and ascertain biochemically how apoptosis-triggering by [beta 2m and anticancer agents differs.
Specific Aim 2 will (a) explore by Western blot analysis whether caspases-4, -7, -10, -11, -12 and -13 are activated during beta 2m-triggered apoptosis, (b) assess beta 2m-induced caspase activation by detecting cleavage of the caspase substrate Z-VAD-afc, (c) identify the proteins that bind specifically to the biotin-VAD-fmk and biotin-YVAD-cmk affinity probes and 125I-labeled YVAD-cmk in the cytosols from beta 2m-treated and untreated control cells, and (d) determine the identity of the proteins specifically labeled with these affinity probes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing.
Specific Aim 3 will (a) explore the mechanisms by which beta 2m induces apoptosis through death receptors, (b) determine whether [beta 2m induces increased ROS through the mitochondria, and (c) investigate whether cellular redox state plays a role in cytochrome c release by [beta 2m. These studies will aid in understanding the molecular mechanism(s) of beta 2m-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.
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