ING3 protein expression profiling in normal human tissues suggest its role in cellular growth and self-renewal.

Members of the INhibitor of Growth (ING) family of proteins act as readers of the epigenetic code through specific recognition of the trimethylated form of lysine 4 of histone H3 (H3K4Me3) by their plant homeodomains. The founding member of the family, ING1, was initially identified as a tumor suppressor with altered regulation in a variety of cancer types. While alterations in ING1 and ING4 levels have been reported in a variety of cancer types, little is known regarding ING3 protein levels in normal or transformed cells due to a lack of reliable immunological tools. In this study we present the characterization of a new monoclonal antibody we have developed against ING3 that specifically recognizes human and mouse ING3. The antibody works in western blots, immunofluorescence, immunoprecipitation and immunohistochemistry. Using this antibody we show that ING3 is most highly expressed in small intestine, bone marrow and epidermis, tissues in which cells undergo rapid proliferation and renewal. Consistent with this observation, we show that ING3 is expressed at significantly higher levels in proliferating versus quiescent epithelial cells. These data suggest that ING3 levels may serve as a surrogate for growth rate, and suggest possible roles for ING3 in growth and self renewal and related diseases such as cancer.

Domain recognition of the ING1 tumor suppressor by a panel of monoclonal antibodies.

The inhibitor of growth (ING) family of proteins play key roles in cell cycle arrest, apoptosis, cell aging, and the DNA damage response. To date, several domains including the plant homeodomain (PHD), lamin interacting domain (LID), and nuclear localization sequence (NLS) have been identified in the ING family of proteins that contribute to their function. To better understand the functional attributes of the ING proteins, we have developed and further characterized a panel of monoclonal IgGs that we call CAbs 1-9 based on their recognition sites, strength of binding affinity, and their specificity for ING1. All of the nine CAbs recognize the C-terminal half of the p33(ING1b) protein, which is fully conserved among all ING1 isoforms, being encoded by a common exon. Two of the nine CAbs bind a fragment that includes the PHD, which is the most conserved domain among ING family proteins (ING1-5), and one CAb cross-reacts with all ING family proteins that are encoded by different genes. Five of the nine CAbs recognized a fragment of ING1, which includes the NLS. Another two, CAb3 and CAb9, show affinity against an inter-domain sequence between the LID and the NLS. The sequence between the LID and NLS is less conserved among the ING proteins and, as expected, CAbs 3 and 9 were completely specific for ING1. Understanding the domains recognized by the different CAbs should further the functional analysis of the ING proteins that are known to participate in a wide variety of protein complexes, both in the cytoplasm and in the nucleus where they bind epigenetic histone marks via their PHD regions and lamin A via their LID domains.

Evaluating the outcomes of a distance-accessible PhD program.

The Doctor of Nursing Science (DNS) Program at Indiana University School of Nursing matriculated the first students in 1978. In 1996, the DNS program was phased out and the PhD in Nursing Science program was approved. Given advances in technology, market demand, and faculty expertise, a decision was made in 2002 to revise the PhD program to make it "distance-accessible" with courses and other learning experiences designed to be delivered using both synchronous and asynchronous Web-based technologies. As part of the newly designed program, distance-accessible courses were paired with an annual 2-week summer residential intensive learning experience on the Indiana University-Purdue University at Indianapolis (IUPUI) campus. This article discusses the process and outcome evaluation for this innovative program, with a particular focus on pedagogies faculty have found effective for scholarly mentoring and on best practices for distance-accessible doctoral education. Evaluation of program outcomes indicates graduates are well positioned to provide leadership in the areas of knowledge development for nursing science, practice, and education.

ING1 isoforms differentially affect apoptosis in a cell age-dependent manner.

Recently, several novel human ING1 isoforms have been cloned. However,the biochemical functions and the involvement of these proteins in apoptosis remain uncharacterized. We have examined the apoptotic effects and biochemical functions of the two major human ING1 isoforms p47(ING1a) and p33(ING1b) in young and senescent human diploid fibroblasts induced to enter into apoptosis by diverse treatments. We have found that ING1 displayed isoform-, stimulus- and cell age-dependent apoptotic properties. We present evidence indicating that ING1 proteins bind to chromatin and are regulated in a manner related to their apoptotic properties. In agreement with previous reports, we have found that only young but not senescent fibroblasts were able to enter into apoptosis induced by growth factor deprivation. This effect was accompanied by up-regulation of endogenous p33(ING1b). Ectopic up-regulation of p33(ING1b), but not p47(ING1a), also induced apoptosis and sensitized young but not senescent cells to UV irradiation and hydrogen peroxide-mediated apoptosis. Cotransfection of p33(ING1b) and the tumor suppressor p53 increased the percentage of apoptotic cells yielded by either of these two proteins alone, in agreement with data from tumor cell models. Finally, we found that the chromatin binding affinity of p33(ING1b) was increased in senescent cells, which were resistant to apoptosis. Together, these data support the idea that the apoptotic functions of ING1 may be exerted by chromatin-related functions that are subject to cell age-dependent mechanisms of regulation.