Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to 1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; 2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and 4) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakarycotyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA123006-02
Application #
7282643
Study Section
Special Emphasis Panel (ZCA1-SRRB-E (M2))
Program Officer
Lubensky, Irina
Project Start
2006-09-01
Project End
2008-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
2
Fiscal Year
2007
Total Cost
$168,773
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Pathology
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195