Deregulation of translational control is an obligatory step in carcinogenesis;however, diagnostic tools to examine the activity of the translational machinery in human cells and tissue are lacking. This has hampered studies to link prognosis and therapeutic response to translational activity as a function of cancer type and stage, and has precluded large scale drug discovery efforts to modulate the translation initiation machinery despite its potential as a therapeutic target. In response to NIH Program Announcement RFA-RR-06-004, here we request support through the R21 mechanism to develop an assay system to quantify the translational state of human cells and tissues that is simple, rapid, accurate and high throughput-compatible. Our approach is based on the well established rabbit reticulocyte cell free translation system, except we substitute standardized cell lysates prepared directly from the cells or tissue of interest. To the extract we add a bicistronic reporter RNA encoding 2 forms of luciferase, one translated in a cap-dependent manner and the other via an internal ribosomal entry site (IRES);and quantify the 2 forms of luminescence as a read out of the intrinsic cap-dependent and cap- independent translational activity of the cell or tissue of origin. We propose to develop this assay through 4 Specific Aims:
Aim 1) Develop and optimize protocols to generate and quantify translational activity in lysates from primary human cell lines;
Aim 2) Define receiver operating characteristic (ROC) curves for the assay using agonists and antagonists of translation with well-defined mechanisms of action;
Aim 3) Develop and optimize protocols to generate and quantify translational activity in lysates from breast and lung tissue specimens;
Aim 4) Compare translational activity in lysates from breast and lung tissue specimens between cells and their corresponding tissue of origin;control cells and their corresponding diseased counterparts;control tissue and their corresponding diseased counterparts. If we are successful, this would represent the first step in developing medical diagnostics to differentiate healthy and pathological tissues according to their translational properties, to link prognosis and therapeutic response to translational activity as a function of cancer type and stage and provide a powerful tool for emerging drug discovery efforts to develop translational modifiers. Pathological activation of translation, the process of converting mRNA template into protein, is on the causal pathway to cancer;however, diagnostic tools to quantify this process in human cells and tissue are lacking. This has hampered studies to link prognosis and therapeutic response to the activity of the translational machinery as a function of cancer type and stage, and has precluded large-scale drug discovery efforts to modulate the translational machinery despite its potential as a therapeutic target. Here we propose to develop an assay system to quantify the translational state of human cells and tissues that is simple, rapid, accurate and high throughput-compatible. If successful, this would represent the first step in developing medical diagnostics to differentiate healthy and pathological tissues according to their translational properties, to link prognosis and therapeutic response to translational activity as a function of cancer type and stage and provide a powerful tool for emerging drug discovery efforts to develop translational modifiers.
| Ghosh, Brahma; Benyumov, Alexey O; Ghosh, Phalguni et al. (2009) Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation. ACS Chem Biol 4:367-77 |
