Effect Of ABCG2 Expression On Cell Proliferation, Differentiation And Apoptosis

The ability of early hematopoietic progenitors to efflux certain fluorescent dyes such as rhodamine 123 and Hoechst 33342 has long been appreciated and exploited for the isolation of these cells by fluorescence-activated cell sorting (FACS). Fluorescent dye Hoechst 33342 staining and subsequent FACS analysis of dual-wavelength Hoechst fluorescence revealed a specific side population displaying low red and low blue fluorescence. Hence, the isolated cells are termed side-population (SP) cells. Recently, ABCG2, a member of the ABC transporter family, was found to be responsible for the Hoechst dye efflux in these cells. Furthermore, expression of ABCG2 is restricted to the most immature hematopoietic progenitors in human bone marrow and is sharply down-regulated at the committed progenitor level, consistent with an in vivo role for this transporter in immature cells. But what physiologic roles does this transporter play in stem cells is still unknown.To explore the possible role it may paly in stem cells, ABCG2 was forced to express in K562 cells, which originally do not express this gene. Results showed that expression of ABCG2 did not cause an observable morphological change in K562 cells, suggested that it was not involved in the regulation of cell skeleton. Furthermore, we focused on its effect on the three basic characters of cells: proliferation, differentiation and apoptosis. Viable cell counts were determined using trypan-blue dye exclusion test, and results showed a relatively slower expansion rate in ABCG2 transfected K562 cells compared with control vector transfected cells. Following 72h culture, pcDNA3.1 control vector transfected cells gave out a nearly 3-fold expansion while ABCG2 transfected cells only expanded about2.5-fold. Correspondingly, ABCG2 transfection caused a GO/G1 cell cycle arrest in K562 cells (G0/G1%: 43% versus 31%). All the above results suggested that ABCG2 might play an important role in quiescence maintenance of hematopoietic stem cells through regulation of cell cycle.Additionally, differentiation of K562 cells was examined. Results showed that ABCG2 expression increased megakaryocytic differentiation of K562 cells induced by PMA. We speculated that it was the cell cycle arrest caused by ABCG2 that promoted differentiation of K562 cells.To investigate the underlying mechanisms how ABCG2 transports the substrates, we constructed the EGFP-ABCG2 fusion vector. After transfection into MCF-7 cells, the transport ability was examined in EGFP expressing cells. As shown in the results, in pEGFP-Cl/ABCG2 transfected cells, which EGFP expression indicated the expression of ABCG2, the green fluorescence positive cells were resistant to Hochest33342 staining, while those negative cells were not. In pEGFP-Cl control vector transfected cells, though some were also green fluorescence positive, they did not efflux Hochest33342. The cells that express ABCG2 are functionally effective in substrate efflux render us the important tool to study on the underlying mechanism.