Flow Cytometric Measurement of Mutation Frequency in a Murine Model of Acute Promyelocytic Leukemia
Ben Buelow, Sushma Umesh, Elaine Garcia, Scott Kogan. University of California, San Francisco, CA
Background: Sequential acquisition of mutations is generally believed to underlie tumorigenesis. Studies in murine leukemia models indirectly support this hypothesis based on a long latency of cancer onset despite constitutive expression of oncogenic fusion proteins. Notably, mice which express the PML-RARα fusion protein present in >95% of human acute promyelocytic leukemias (APL) develop a mimic of APL with a median latency of 8.5 months. Strikingly, PML-RARα has been shown to interfere with DNA repair machinery, suggesting a causal link between the initiating translocation and subsequent cancer-promoting mutations. Nevertheless, whether PML-RARα expression affects mutation frequency in precancerous cells remains unanswered because current methods are not well suited to identifying mutations in individual cells. Adaptation of flow cytometry (FC) to the study of mutation frequency is therefore desireable, but the need for corresponding phenotypic alterations is prohibitive because the second (unmutated) copy of a mutated gene masks any detectable phenotype. However, it has been shown that red blood cells with an inactivating mutation of the single expressed copy of the X-linked PIG-a gene (single copy in females as well because of X-inactivation) show loss of the GPI membrane anchor that is the product of this enzyme and can thus be isolated by FC.
Design: We investigated if this one to one correlation between mutation events and phenotypic alterations detectable by FC can be used to measure mutation frequency in murine myeloid cells. To this end we are sorting neutrophils (PMNs) based on light scatter and cell surface marker expression and examining these cells for GPI as well as other X-linked markers such as gp91phox. We are also using each system to generate estimates of mutation frequency in wild type and PML-RARα expressing PMNs, and comparing these results to previous data generated in murine erythrocytes.
Results: We here present an evaluation of flow cytometry for the quantification of mutation frequency in murine myeloid cells and show data demonstrating a remarkably high mutation rate in both wild type and PML-RARα expressing PMNs compared to erythroid cells.
Conclusions: To our knowledge, this work represents a novel application of flow cytometry to investigate mutation frequency in a transgenic murine leukemia model and provides a broadly applicable technique for the characterization of mutation frequency during the pre-cancerous phase of hematopoietic, and, with a variation in method, solid organ tumors.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 205, Monday Morning