Figure 1. SALL4 expression in AML patients correlated with treatment status. SALL4 had the highest expression level in acute untreated AML patients (N = 36), low expression level in partial remission (PR) patients (N = 7), lower expression level in complete remission (CR) patients (N = 25) and healthy controls (N = 30) (A). The level of SALL4 expression declined at remission phase after chemo- therapy with decreased blast count in five AML patients in the CR group (B, n = 5), but increased in the PR group with drug resistance (C, n = 3). Each line represents one patient. Y axis: relative SALL4 expression level reported as 2-DDCt relative quantitative method. doi:10.1371/journal.pone.0018372.g001 SALL4 Affects the Side Population Furthermore, we followed up with eight patients to monitor the changes of SALL4 expression during the course of AML treatment at three time points, which included the untreated acute phase, the improving phase (whereby the patient was responding to the chemotherapy as indicated by decreased blast counts, and was at the mid-point of their treatment cycles), and the end of the treatment cycle (of eight patients, five in complete remission (CR) phase, and the other three were in relapse phase). The level of SALL4 expression declined throughout the treatment process for the drug responsive group (Figure 1B) and increased in the drug- resistant group (Figure 1C). Overexpression of SALL4 is associated with drug resistance in cell lines To assess whether the increased expression of SALL4 in AML relapse patients is the consequence or the cause of drug resistance in AML, we compared the proliferation state of a stable cell line with SALL4 overexpression to that of the controls after exposure to two clinically common active chemotherapy agents: doxorubi- cin and daunorubicin. To determine the effect of SALL4 in drug resistance, we choose to use 293 cells for the following reasons. First, since SALL4 is expressed in solid tumors such as germ cell tumors (GCTs), gastric cancer, and breast cancer [34,35] in addition to AML, we hypothesized that SALL4 can confer a general drug resistant phenotype. Second, the 293 cells have relatively lower SALL4 expression compared to blood cell lines, which is better to demonstrate the gain-of functional study. 293 cells overexpressing SALL4B or vector control were plated in a 96-well plate and treated with various concentrations of doxorubicin or daunorubi- cin. At 48 hours after drug treatment, cell proliferation was measured using the MTS reagent (Promega). Similar to their clinical activity, both agents significantly inhibited the proliferation of 293 cells. As shown in Figure 2A and B, overexpression of SALL4 resulted in a significant reduction in cytotoxicity observed following exposure to doxorubicin (2.0-fold) and daunorubicin (1.5-fold). We next performed the loss-of-function analysis of SALL4. Knocking down of SALL4 in a myeloid leukemic line, KG1a cells, was achieved by retrovirus-mediated delivery of shRNA against SALL4 as we previously described [28,31]. qRT- PCR analysis showed that retroviral infection markedly repressed SALL4 expression in KG1a cells by 60%. As shown in Figure 2C, SALL4 knock-down cells displayed more sensitivity to doxorubicin than parental cells. These data suggest that SALL4 plays an important role in drug sensitivity. Preferential Expression of SALL4 in the SP cells SP cells are defined as cells that show higher efflux of chemicals including DNA-binding dye Hoechst 33342. Therefore, it is not surprising that SP cells are resistant to multiple chemotherapy drugs. Since increased SALL4 expression is associated with drug resistance, we hypothesized that SALL4 might mediate drug resistance at least in part, through the maintenance of SP cells. We first examined the endogenous expression level of SALL4 in SP cells in several hematological cell lines. The SP, as defined by low Hoechst 33342 blue/red fluorescence intensity after incubation with Hoechst 33342, was detected in all cell lines tested at a frequency of 0.5–5%. As a control, this side population disappeared after treatment with verapamil (50 mM) which inhibited the function of the ABC transporter (Figure 3A, right panel). A representative SP profile from KG1a cell line is shown in Figure 3A. We then sorted SP and non-SP cells from four different blood cancer cell lines, HL-60, RPMI8226, NB-4 and KG1a cells, and measured the expression of SALL4 in SP cells and non-SP PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18372