Sorting, Identification And Enrichment Of Side Population Cells In Acute Monocytic Leukemia THP-1Cell Line

Background and Objective:Cancer stem cell (CSCs) theory demonstrate the origin of cancer from a new perspective.CSCs theory suggests that CSCs, generally stay in a quiescent state, are a small subset of cancer cells that possess stem cell-like properties-the ability to self-renew via asymmetric division and to produce differentiated progeny and it is this small population of cells within a cancer that are responsible for drug resistance and the recurrence of cancers. Hence, in order to eradicate the cancer and cure the patient, specificly targeting CSCs therapeutically must be explored in the treatment of many cancers. Leukemia is a hematopoietic malignancies and the existence of CSCs was first proposed in leukemia. The CSCs in leukemia patients also be called leukemia stem cells(LSCs).Much research of the medical field have focued on CSCs and people hope that it could provide new ideas for targeted therapy by studying CSCs. Therefor, sorting, identification and enrichment of CSCs become particularly important. Side popula-tions (SP) cells, with the capacity to efflux the dye Hoechst33342, represent only a small fraction of the whole cells population. SP cells possess some intrinsic stem cell properties and contain the tumor stem cell richly. The ways, combination of SP cells assay, analysis and fluorescence activated cells sorting is widely recognized as one of the best ways of separating CSCs. Leukemia cell lines possess the characteristics of the unlimited passage ability in vitro.The objective of this study is to assay and identify if the human acute monocytic leukemia cell lines THP-1includes side population (SP) cells or not. To identify SP cells subsets and study the differences between THP-1SP and non-SP(NSP) cells in leukocyte surface antigens,the cell cycle, proliferation and colony forming ability, the multidrug resistance gene and apoptosis gene expression. To enrich leukemia stem cells subpopulation in human acute monocytic leukemia cell lines THP-1with arabinosylcytosine (Ara-C) and study the relationship between SP cells and LSCs.Methods:1. Fluorescent microscope was employed for detecting the percentage and morphology of SP cells in THP-1cells. The percentage of SP cells in logarithmic growth period THP-1was detceted using flow cytometry, too.2. SP cells and NSP cells subpopulations were collected. Leukocyte surface antigens, cell cycle of two subpopulations were examined using flow cytometry. The proliferation and colony forming ability between SP cells and NSP subpopulations were compared in terms of colony assay and growth curve.The mRN A expression of multidrug resistance proteins (ABCG2,ABCB1) and apoptosis gene (Bcl-2, Bax) were examined by real time PCR and real time quantitative PCR.3. THP-1cells were incubated with different concentrations of arabinosylcytosine (Ara-C) for24h and detected the proportion of SP cells, respectively.Results:1. Fluorescent microscope analysis with Hoechst33342staining demonstrated that the side population cells in THP-1cells was accounted for (4.85■1.35)%.There was no significant difference between SP and NSP cells in morphology.2. Flow cytometry analysis with Hoechst33342staining demonstrated that the percentage of SP cells was (1.81±0.99)%in THP-1cells. These SP cells were practically diminished in the presence of Hoechst33342and verapamil, a calcium channel blocker. The SP and NSP cells in THP-1cells were sorted separately and the purity tests show that the SP cells tube were concentrated in the SP regional and NSP cells tube is still concentrated in sub-region of the main group of cells. 3. The CD34+CD38-cells expression in the SP cells subpopulation were higher than that in the NSP cells subpopulation (P<0.05).A majority of the SP cells keeps in the G0/G1Phase, and the NSP cells stays in the S/G2/M Phase. The proliferation and colony forming ability of SP cells subpopulation wre significantly higher than those of NSP cells (P<0.05). The mRNA expression of multidrug resistance gene (ABCG2、ABCB1) of SP cells were higher than that of NSP cells.There was no difference between SP cells and NSP cells in Bax expression (P>0.05), but the Bcl-2/Bax value were significantly higher than those of in the NSP cells(P<0.05) which were examined using PCR.4. After co-cultured with Ara-C, the proportion of SP cells improved significantly and the proportion of SP cells raised up with the Ara-C concentration.Conclusions:1.The human acute monocytic leukemia cell lines THP-1contains SP cells, and the proportion of SP cells is much lower. The SP cells possess some intrinsic stem cell properties and may contain the leukemia stem cells richly.2.The SP cells could be enriched after co-cultured with Ara-C and it can be applied to study leukemia stem cells.