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Rumours, Untruths In Addition To The Dasatinib

We focused on CD82 because this protein functions as an adhesion molecule that is important to maintain the character of LSCs. We attempted to validate these results Afatinib in other CD34+/CD38? AML cells isolated from patients (11 from BM, 5 from PB, cases 1�C14, 17, 18) by FACS. In 15 of 16 cases (94%), the relative expression levels of CD82 were significantly higher in CD34+/CD38? AML cells (68 �� 27%) as compared with their CD34+/CD38+ counterparts (30 �� 19%) (p < 0.01, Fig. 1a, Supporting Information Fig. S2a). On the other hand, a mean 35 �� 19% of CD34+ hematopoietic stem/progenitor cells isolated from healthy volunteers (n = 6) were positive for CD82 staining (Supporting Information Fig. S2b). In addition, we found that imatinib-resistant EOL-1R cells which stayed on a dormant state and possessed the immature character with aberrant expression of CD34 (data not shown) expressed a greater amount Dasatinib of CD82 on their cell surface (96 �� 1%) than parental EOL-1 cells (47 �� 4%) (Fig. 1a). Aberrant expression of CD82 was associated with inactivation of matrix metalloproteinase 9 (MMP9) in the H1299 human lung carcinoma cells.14 We therefore examined the relationship between CD82 and MMPs in CD34+/CD38? AML cells. Real-time RT-PCR found that the levels of MMP9 were significantly lower in CD82 over-expressed CD34+/CD38? AML cells than their CD34+/CD38+ counterparts (n = 7, p < 0.01) (Fig. 1b). On the other hand, levels of MMP2 in CD34+/CD38? AML cells were almost identical to those in CD34+/CD38+ counterparts (Fig. 1b). We also found that the levels of both MMP-9 and -2 were down-regulated in imatinib-resistant EOL-1R cells as compared with parental EOL-1 cells (Fig. 1b). To explore a potential link between CD82 and MMPs in leukemia cells, EOL-1R cells were transiently transfected Dasatinib order with either scrambled control or CD82 siRNA (Fig. 1c), which efficiently decreased levels of CD82 in these cells (from 96 �� 1% to 41 �� 1%, Fig. 1c). The MMPs enzymatic activity in these cells was determined by performing gelatin zymography with the culture supernatant as well as whole cell proteins extracted from EOL-1 and EOL-1R cells (Fig. 1d). Interestingly, when CD82 was down-regulated in EOL-1R cells by an siRNA, enzymatic activity of MMP9 was dramatically increased (Fig. 1d), suggesting that CD82 negatively regulated MMP9. Real-time RT-PCR found that down-regulation of CD82 by an siRNA increased levels of MMP9 by nearly twofold in EOL-1R cells (Fig. 1e). On the other hand, levels of MMP2 were not affected by down-regulation of CD82 (Fig. 1e). Moreover, to explore the function of CD82 in freshly isolated CD34+/CD38? AML cells, we genetically down-regulated CD82 in these cells. An shRNA targeting CD82 decreased expression of CD82 in four cells (case 1, from 85 �� 2% to 47 �� 3%; case 2, from 75 �� 1% to 13 �� 2%; case 6, from 47 �� 7% to 21 �� 3%, p = 0.066; case 14, from 43 �� 1% to 18 �� 1%, Fig. 1f).
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