EFFECT OF CD44 EXPRESSION ON T CELL ACUTE LYMPHOCYTIC LEUKEMIA

Purpose: Our objective was to determine the impact of CD44 expression on T-ALL tumorigenesis and metastasis using Jurkat cells engineered to express surface CD44. Methods: The E6.1 Jurkat cells were engineered to express the CD44 standard isoform. DNA content and aneuploidy was assessed by propidium iodide staining and karyotyping. Myristoylated-Akt and LY294002 were used to increase or decrease Akt activation in an attempt to rescue cells from aneuploidy or to induce aneuploidy. Propidium iodide and BrdU incorporation was used to investigate the restriction checkpoint regulation. RNA was isolated from CD44 expressing cells and the vector control and assessed using a Cell Cycle RT2-qPCR array and a Human Signal Transduction Pathway Finder RT2-qPCR array. Results: Flow cytometry confirmed the expression of CD44 on the transfected cell line but not the open vector control. DNA content analysis showed that CD44 expressing cells were aneuploid and had a higher number of chromosomes when compared to the open vector control. Myr-Akt did not rescue CD44 expressing cells from aneuploidy. LY294002, an inhibitor of Akt activation, was used to treat the open vector control, and there was no change in aneuploidy when Akt was inhibited in control cells. The restriction point of the cell cycle was determined to be defective in CD44 expressing cells, preventing the cells from undergoing apoptosis due to the detection of aneuploidy. The two PCR arrays contained significant downregulation in gene areas that focused on aneuploidy, proliferation, NOTCH signaling, and immune function. Detailed array analysis using IPA software provided Akt, EGR-1, NOTCH, and p53 as potential upstream effectors of the observed aneuploidy and potential decreased invasiveness of CD44 expressing cells. Conclusions: Our results show that expression of CD44 induces hypophosphorylation of Akt and aneuploidy. CD44 expression alters mRNA levels of several key proteins involved in invasion such as Serpin E1 and NOTCH. These results point us towards future experiments to investigate the invasiveness and metastatic potential of our cells in vivo. CD44 expressing tumor cells in patients show decreased NOTCH signaling and responsiveness to therapy, and our results are in agreement with clinical findings. Future work will focus on determining the link between aneuploidy, proliferation, and signaling pathway modifications due to CD44 expression within T-ALL cells and will pave the way towards using CD44 expression as a prognostic marker for T-ALL.