SALL4 Affects the Side Population Figure 5. SALL4 affects the levels of endogenous expression of ABCA3 and ABCG2. (A) 293 cells were transiently transfected with SALL4A or SALL4B or pcDNA as a control. At 48 hr after transfection, RNA was isolated from transfected cells. Transfected cells were subjected to qRT-PCR to measure the mRNA expressions of ABCA3 and ABCG2. The mRNA expressions of ABCA3 and ABCG2 were significantly increased by SALL4. Data are represented as mean 6 S.D. from three independent experiments. *** denotes p,0.001, ** p,0.01, *p,0.05. (B) CD34+ cells were transduced with retrovirus expressing shRNA against SALL4 and subjected to qRT-PCR analysis to measure the expression levels of ABCA3 and ABCG2. The expression level of ABCA3 was reduced by 80% and the expression level of ABCG2 was reduced by 74% in CD34+ cells compared to those of the controls. The mRNA level was normalized with the internal control GAPDH. (C) qRT-PCR analysis of SALL4A and B, ABCG2, and ABCA3 in HL-60, KG1a, and RPMI8226 cells. For quantification of gene expression, amplification of GAPDH was performed as an endogenous control to standardize the amount of sample. (D and E) SALL4 and ABC gene expressions were correlated in primary AML samples. 461 primary AML samples (GSE6891) were stratified into SALL4 high and SALL4 low using the mean intensity of SALL4 as a threshold. (F and G) qRT-PCR was performed on 34 primary samples with primers for SALL4, ABCG2 and ABCA3. Mean value of SALL4 was used to stratify the samples to SALL4 high or SALL4 low groups. Data are represented as mean 6 S.D. from three independent experiments. Y axis: Relative SALL4 expression when compared to controls. doi:10.1371/journal.pone.0018372.g005 development. Moreover, down-regulation of SALL4 in leukemic cell lines triggers apoptosis [28]. In this study, we correlated the expression of SALL4 with primary AML patient diagnosis and treatment status. We found that AML patients who responded to the treatment had decreasing SALL4 expression throughout the treatment course; while AML patients with disease relapse or drug resistance had increasing SALL4 expression that was correlated to the disease progression. We furthered our study by investigating whether change of SALL4 expression affect chemoresistance (drug responses) of leukemic cells. We found that increased SALL4 expression in leukemic KG1a and 293 cells was associated with chemoresistance, and conversely, knocking down of SALL4 led to a more sensitive response to drug treatment. We then propose that SALL4, a stem cell factor gene, is responsible for the SP phenotype and drug resistance by regulating the ABC genes. The expression of SALL4 in SP and non-SP cells was investigated and we found that SALL4 was enriched in SP leukemic cells. The preferential expression of SALL4 in SP cells is not limited to the hematopoietic system. In addition to AML, SALL4 has been reported to be expressed in solid tumors such as germ cell tumors (GCTs), gastric cancer, and breast cancer [34,35]. We have also observed the expression of SALL4 in breast cancer as reported. Moreover, SALL4 is enriched in SP in breast cancer cell line MCF7 along with ABCA3 and ABCG2, and increased SALL4 expression led to an expansion of SP in MCF7 cells (Figure S8–10). As part of a global SALL4 gene target investigation, we performed ChIP-on-chip on the leukemic cell line NB4, 293 cells, as well as primary CD34+ cells. In all three data sets, we found that SALL4 had potential binding sites for one ABC gene, ABCA3. We further demonstrated that SALL4 could bind to and activate the promoter of ABCA3. Therefore, ABCA3 is a direct target of SALL4. Interestingly, SALL4 could affect the expression levels of both ABCA3 and ABCG2. While overexpression of SALL4 led to increased ABCA3 and ABCG2 expression, knocking down of SALL4 in CD34+ cells and KG1a resulted in decreased expression of ABCA3 and ABCG2, as well as the frequency of SP cells. Most importantly, the expression of SALL4 was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Though there is no evidence to support that ABCG2 is a direct target of SALL4, at least by our study of ChIP-qPCR around its promoter region, it is possible that SALL4 can regulate the expression of ABCG2 through some distal regions which were not investigated by our current study. Alternatively, we have shown that SALL4 represses PTEN expression through recruiting the NuRD complex [30]. The PTEN and Akt pathway has recently been implicated in maintaining the SP phenotype in glioma stem cells and breast cancer stem cells [37,38], probably through affecting the activity of ABCG2. It is possible that SALL4 affects the expression of ABCG2 (BCRP) indirectly through its regulation of PTEN. In summary, we are the first to connect a stem cell factor with ABC genes that are responsible for the side population property, a property shared by various normal and cancer stem cells. While the biological implication of SALL4 and ABC transporters in ES cells and other tissue stem cells remain a great interest for further investigation, in this study, we focused on the functional role of SALL4 in the SP of leukemic cells. The side population has been proposed to be a tumor population with intrinsic mechanisms for cytostatic drug resistance that might provide clues for improved therapeutic intervention. Our results suggest that SALL4 plays a role in drug sensitivity, at least in part, through the regulation of ABC genes and maintenance of SP cells, and may be responsible for drug-resistance in leukemia. Further studies will determine whether SALL4 is a novel therapeutic target for leukemic stem/ initiation cells in primary AML patients. Supporting Information Figure S1 SP cells has significantly higher SALL4 protein expression than that of Non-SP cell. (A) Sorted Non-SP cells (upper panel) or SP cells (lower panel) from KG1a cells were immunostained with SALL4 antibody (Green for Sall4, Blue for DAPI). (B) SALL4 fluorescence signal per cell was quantified using Image J software. (TIF) Figure S2 Non-SP cells do not give rise to SP cells. (A) KG1a cells were incubated with Hoechst 33342 dye as previously described and analyzed by flow cytometry. Non-SP cells were sorted and cultured with RPMI1640 media for 3 days. (B) Cultured Non-SP cells were re-analyzed by flow cytometry and no SP population was identified. (TIF) Figure S3 Increased leukemic engraftment of KG1a SP in vivo in a xenotransplant mouse model. Four months after injection of 16105 of SP or non-SP cells into sub-lethally irradiated NOD-SCID mice, the SP recipients had 37% of human CD45 cells detected in their bone marrows which were derived from the SP cells, while the non-SP recipients had only 0.1% of human CD45 cells by FACS analysis. X axis: mouse CD45 expression, Y axis: human CD45 expression. (TIF) Figure S4 Knocking down of SALL4 leads to reduced frequency of the side population. (A) Freshly sorted SP cells from KG1a were counted, plated in six-well plates and cultured with RPMI 1640 for a week. Expanded SP cells were transduced with retrovirus expressing SALL4-specific shRNA for knocking down of SALL4 (lower panel) or scramble shRNA as a control (upper panel) and re-analyzed for side population as previously PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18372