Flow Cytometric Measure of Phosphorylated Histones as a Novel, More Sensitive, Means to Determine Mitotic Index in Canine Tumor Cells
Fellow: Daniela Hernandez Muguiro
Mentor: Erica Behling-Kelly
DESCRIPTION (provided by applicant):
An important aspect of the grading scheme of many neoplasms is assessment of the mitotic index (MI). Current methodologies for MI assessment, such as microscopic enumeration of mitoses, flow cytometric analysis of DNA content and immune staining with Ki67 and cellular cycle proteins (e.g. cyclins) are limited by various factors that decrease their diagnostic value. Recent studies performed on human neoplastic cells and neoplasms have shown that flow cytometric detection of phosphorylated histone H3 (PHH3) is a specific and accurate marker of mitosis, which provides a rapid and unbiased assessment of the MI. Site specific phosphorylation of this nuclear protein occurs almost exclusively in mitotic mammalian cells, and it is rapidly dephosphorylated after completion of mitosis. Unlike Ki67, this mitotic marker is not detected in apoptotic cells, and compared to cyclin B1, PHH3 provides a broader window of detection of proliferating cells. We hypothesize, therefore, that PHH3 will be a sensitive and specific marker of mitosis in canine cells. We will test this hypothesis via flow cytometric quantification of its expression in canine neoplastic cells exposed to different treatments that will either increase the proportion of mitotic cells or that will inhibit histone H3 phosphorylation. Results will be compared to previously established methods of cell cycle analysis and across treatments using a two-way ANOVA with a Dunn’s post-test. If the generated data validates the use of PHH3 as a proliferation marker, future studies would then evaluate PHH3’s prognostic value in different canine neoplasms (e.g., lymphoma), and correlate its expression with other validated markers of cellular proliferation.