Abstract 5125: Defining the minimal ING1b-derived peptide that is pro-apoptotic

The Inhibitor of Growth 1b (ING1b) is a type II tumor suppressor and a core, stoichiometric member of HDAC-containing chromatin-modifying complexes. ING1b contributes to regulation of gene expression, DNA damage repair and stress signalling, cell growth and senescence, tumorigenesis and apoptosis. Mislocalization of ING1b and decreased levels of ING1b are commonly found in human tumors and cancer cell lines, suggesting the possibility of using ING1b levels as a prognostic marker in clinics. Consistent with a pro-apoptotic function, ING1b protein positively regulates p21 and bax gene expression, and ING1b overexpression promotes apoptosis in p53-dependent and p53-independent manners. The inactivation of apoptosis pathways that is frequently observed in cancer cells causes a dramatic decrease in the efficiency of many cancer treatments. The inactivation of ING1b and suppression of apoptosis in tumors and a greater sensitivity of cancer vs. normal cells to elevated levels of ING1b suggest that modulation of ING1b expression in tumors may serve as a viable approach for cancer therapy. Our aim is to expand understanding of the molecular mechanism(s) by which ING1b promotes apoptosis in cancer cells. We strive to define ING1b regions that are necessary for its apoptotic function in order to design minimal recombinant peptides with potent apoptosis-inducing properties. We report that peptides containing the third alpha helix (A3H) and NLS/NTS domains of the ING1b protein are able to induce apoptosis at levels comparable with those seen for full length ING1b protein. Cells overexpressing full length ING1b protein or the A3H-NLS/NTS peptide show similar changes in cell morphology characteristic of apoptosis, exhibit increased levels of PARP cleavage, and display similar levels of apoptosis as determined by FACS analysis using an Annexin V assay. While the A3H region is necessary but not sufficient, the NLS/NTS domain is required, and partially sufficient, for induction of apoptosis. The A3H-NLS peptide exhibited strong nucleolar localization characteristic of full length ING1b. Consistently, overexpression of ING1b deletion mutants lacking the A3H-NLS/NTS sequence failed to induce apoptosis. Substituting ING1b's NLS/NTS domain with the short conventional NLS and NTS sequences dramatically reduced pro-apoptotic properties of recombinant peptides, thus indicating a novel molecular function of NLS/NTS domain in ING1b-mediated apoptosis. Currently, we assess the effica