These observations prompted us to investigate in greater detail whether protein synthesis inhibitors induce death inside a wider range of cell types and evaluate the role of the Bax/Bak-mediated apoptotic pathway in mediating the cytotoxicity of these compounds. Open in a separate window Figure 1 Human being CLL cells and many leukemia or lymphoma-derived cell lines are highly sensitive to protein synthesis inhibitors. translation inhibitors, which result in the Bax/Bak-mediated apoptotic pathway. However, contrary to anticipations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal part and cautions that strong correlations do not usually signify causality. On the other hand, the killing of T lymphocytes was less Lomifyllin dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via option mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis completely. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells. mouse lymphoma model when combined with doxorubicin.14, 15, 16, 17 Although inhibitors of translation elongation, such as HHT, inhibit global protein synthesis, targeting the eIF4F complex has been proposed to be more selective, because the translation of certain mRNAs is thought to be particularly dependent on eIF4F.18 These eIF4F-dependent mRNAs often have highly structured 5 untranslated regions and many of them encode proteins involved in controlling cellular Lomifyllin proliferation, survival (e.g., Mcl-1) and/or oncogenesis.19, 20 Taken together, these observations have motivated the development of translation initiation inhibitors as cancer therapeutics.14, 17, 21 Although the mechanisms by which HHT and silvestrol inhibit protein synthesis are well characterized, precisely how they kill cells is unclear. It has been hypothesized that reduction of the anti-apoptotic Bcl-2 family member Mcl-1 constitutes the major, possibly even the Lomifyllin sole, driver of cell death.16, 17, 22, 23 Nevertheless, decreased levels of Bcl-2 have also been reported.15, 21 These pro-survival proteins act to restrain Bax and Bak, the two pro-apoptotic multi-BH domain name Bcl-2 family members that are essential for mitochondrial outer membrane permeabilization, an fundamental step in the so-called Bcl-2 family regulated’ (also called intrinsic’ or mitochondrial’) apoptotic pathway.24, 25 Once the mitochondrial barrier is breached, cytochrome and other apoptogenic factors are released into SP-II the cytosol to activate caspases, thereby driving cellular demolition. Other cell death pathways have also been implicated because translation inhibitors reduce the levels of cyclin D1, c-Myc, XIAP and cFlip.15, 22, 23, 26 However, most attempts to determine the mechanisms by which translation inhibitors cause cell death are based on observational and correlative data (e.g., reduction of Mcl-1 levels)16, 17, 22 and the relative impact of blocking a specific target has not been established. We, therefore, decided to use genetic tools to determine the of components of the apoptosis machinery in the cytotoxicity induced by translation inhibition by studying the effects of two promising but divergent inhibitors of protein synthesis: the translation elongation inhibitor HHT and the translation initiation inhibitor silvestrol. The hematopoietic system was our major focus as leukemias and lymphomas appear to be promising targets for these compounds.12, 16, 17 We surveyed a wide range of normal and transformed hematopoietic cells to establish the potential indications and determine the likely therapeutic windows. In addition to malignant cells, we found that non-transformed B lymphoid cells from many differentiation stages were highly sensitive to translation inhibition. Terminally differentiated non-cycling cells, such as neutrophils, were also sensitive. Unexpectedly, we found that Mcl-1 reduction was not the major contributor to death in a variety of cells and that cell killing did not usually occur solely via Bax/Bak-mediated apoptosis. Indeed, we found that long-term clonogenic potential after treatment with protein synthesis inhibitors can be independent of the Bcl-2 regulated pathway altogether. Our studies therefore provide critical information to guide the development and clinical application of these compounds Lomifyllin as well as anticipate potential side effects associated with their use. Results Many human leukemia-derived cells are highly sensitive to inhibitors of protein synthesis As cell lines derived from several.