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).