(each 40 bp) from ABCA3 promoter region (Figure 4C) and determined which probe could form complex with SALL4B (Figure 4D). As shown in Figure 4D, probe #1 formed a strong complex with SALL4B nuclear extract (lane 2), and the binding was completely ablated with unlabelled oligonucleotide probe #1 (lane 3). In addition, this specific band was ablated after addition of a SALL4 antibody (Figure 4E, lane 4), but not with addition of a Tubulin antibody (Figure 4E, lane 5), IgG (Figure 4E, lane 6 and 7) was used as a negative control. The ablation of a specific band has been reported when an antibody being added to a protein- DNA interaction in an EMSA experiment, and is due to fact that certain antibodies can compete or interrupt the protein-DNA interaction. Though no supershift band was observed with our SALL4 antibody, we interpreted the ablation of a specific band by our SALL4 antibody but not the controls (IgG or Tubulin antibodies) a consequence of competition or interruption of SALL4 protein-DNA interaction by this antibody. Transcriptional activation of ABCA3 gene by SALL4 We then sought to investigate whether SALL4 can regulate the ABCA3 promoter by analyzing the human ABCA3 promoter activity. A 59 upstream of the transcription start site of ABCA3, which contained the SALL4 binding site, was subcloned into the 59 end of a pGL4.2 basic luciferase reporter plasmid (Figure S6). The SALL4 responsiveness of the ABCA3 promoter was then evaluated through cotransfection of the ABCA3-luciferase con- struct and Renilla luciferase plasmid together with increasing ratios of the SALL4A or SALL4B expression constructs relative to the ABCA3 promoter construct. As we increased the molar excess of the SALL4A or B constructs, the ABCA3 promoter was activated in a dose-dependent manner (Figure S6). SALL4 is able to affect the levels of endogenous expressions of ABCA3 and ABCG2 To determine the regulation of ABCG2 and ABCA3 genes by SALL4, we next examined the expression levels of these two genes in 293 cells overexpressing SALL4A and SALL4B. 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. By qRT-PCR, we found that the expression level of ABCA3 in SALL4 overexpressing cells was increased by 33% compared to those of the controls (Figure 5A). Surprisingly, the expression of ABCG2 was increased even more, up to 74%. We next measured the expressions of ABCA3 and ABCG2 in SALL4-knockdown cells. qRT-PCR analysis showed that retrovi- ral infection with shRNA for SALL4 markedly repressed SALL4 expression in CD34+ cells by 70% (Figure S7). Down-regulation of SALL4 repressed the expression of ABCA3 gene by 80% in CD34+ cells, and similarly, decreased expression level of ABCG2 was observed as well. Similar results were observed for KG1a cells (Figure 5B and data not shown). We further investigated the correlation of SALL4 and ABCA4 and ABCG2 expression in blood cell lines. SALL4 expression in HL60 was relatively lower than that in KG1a and RPMI8226. Similar to the expression of SALL4, the expression of ABCA3 and ABCG2 was lower in HL60 and higher in KG1a and RPMI8226 cell lines (Figure 5C). Having established the correlation of SALL4 and the ABC genes in cell lines and CD34+ cells, we next investigated its clinical association in primary AML patients. Using the AML cohort first described by Roel et al [36], we stratified these 461 AML samples into SALL4-high and SALL4-low, according to mean intensity of SALL4. The expression of ABCA3 and ABCG2 was then examined in these two groups. Interestingly, the expression of SALL4 Affects the Side Population both genes was significantly higher in the SALL4 high group (P = 0.0001, Figure 5D, and P = 0.02 Figure 5E respectively) compared to SALL4 low group, showing a positive correlation of ABCA3 and ABCG2 with SALL4 in primary AML samples. To validate, we performed real-time PCR on 34 primary samples from a separate cohort. These samples were stratified with the mean value of SALL4 to SALL4 high and SALL4 low groups. It was found that ABCG and ABCA3 are significantly higher in the SALL4 high population (P = 0.001, P = 0.0001 respectively), and this correlates well with the Roel data set (Figure 5F and G). Taken together, these results suggest that ABCA3 is a direct downstream target gene of SALL4, and the expressions of two ABC genes, ABCA3 and ABCG2, are positively regulated by SALL4. Discussion The concept of cancer stem/initiating cells is designed to define a cancer subpopulation that has the potential to propagate the tumor and may be responsible for driving tumor development, progression, metastasis and drug resistance or relapse. Xenograft transplantations have been used to test the tumorigenic ability of subsets of cancer cells in immune-compromised mice. It was initially observed that only a very small percentage of tumor cells with unique surface markers had this property, and the bulk of the cells in the tumor lacked this capacity. With the availability of more immune-compromised mouse strains, the spectrum of cancer cells that has this tumorigenic ability has grown broader. In addition, the drug resistance or relapse properties of these cancer initiating cells have not been vigorously tested in this transplan- tation model using cells isolated by surface markers. Parallel to this approach, the ability of a subgroup of cells to efflux lipophilic fluorescent Hoechst dyes to produce a characteristic side population (SP) profile based on fluorescence-activated flow cytometric analysis has proven valuable as a marker to identify multipotent stem cells in a variety of tissues, including ES cells and various adult stem cells and cancer stem/initiation cells. Therefore, it is more reasonable to combine the transplantation and side population assays to define the cancer initiating cells. The best demonstrated tumor model is from the SP from acute myeloid leukemia samples, which have been shown to have leukemogenic properties in a xenotransplantation model, as well as being resistant to drug treatment [12,17]. These data indicate that the ability of malignant SP cells to expel anticancer drugs may directly improve their survival and sustain their clonogenicity during exposure to cytostatic drugs, allowing disease recurrence when therapy is withdrawn. The expressions of several ATP binding cassette (ABC) transporters, including breast cancer resistance protein (BCRP or ABCG2) and ABCA3, are thought to be responsible for expulsion or sequestration of the cytotoxic drugs [23,24,12,13]. These genes are highly expressed in normal hematopoietic CD34+ stem/progenitor cells and SP cells in AML samples [12,13]. The mechanism(s) involved in the regulation of the expressions of these genes remain under investigation. SALL4 is a newly discovered pluripotency stem cell factor in murine ES cells. Our group has also shown that it is involved in the self-renewal of leukemic initiation and hematopoietic stem/ progenitor cells (HSCs). It is a unique gene that has important functional role(s) in maintaining all three types of stem cells, such as ES cells, HSCs and LSCs. We have shown that SALL4 plays an essential role in myeloid leukemogenesis. It is aberrantly expressed in human primary acute myeloid leukemia [27]. Overexpression of SALL4 in a murine model leads to myeloid leukemic PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18372