PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

Abstract

Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2'-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.

Differential gene expression analysis by RNA-seq reveals the importance of actin cytoskeletal proteins in erythroleukemia cells

“…We found that the pattern of the major cell cycle phases, G1 vs S vs G2/M, was similar between MEL-R cells and undifferentiated MEL progenitors (MEL-0 h). By contrast, differentiated MEL cells (MEL-96 h) accumulated at G1, a phenomenon that has been previously observed during MEL cell differentiation (Kiyokawa et al, 1993; Vanegas et al, 2003; Fernández-Nestosa et al, 2008). Nevertheless, we observed that in terms of DNA content, MEL-R cells acquired a tetraploid phenotype as revealed by the shift in DNA content to the right (Fig.…”

“…1B. Sfpi1/PU.1 was one of the selected genes that, as we demonstrated previously (Fernández-Nestosa et al, 2008), is not expressed in the resistant cell line and served in this case as a positive control for the RNA-seq efficiency. We observed that the majority of the differentially expressed genes are up-regulated in MEL compare to MEL-R cells (Fig.…”

“…MEL-resistant cell line (hereafter called MEL-R) derived from MEL-DS19 was previously established in our lab by growing MEL cells continuously in the presence of 5 mM HMBA (Fernández-Nestosa et al, 2008). 3T3-Swiss albino fibroblasts cells were obtained from the Animal Cell Culture Facility from the Centro de Investigaciones Biológicas, CSIC.…”

“…Insertion of the Friend spleen focus-forming virus (SFFV) genome occurs several kilobases upstream of the Sfpi1/PU.1 locus initiation start site (Fernández-Nestosa et al, 2008). This causes the constitutive activation of Sfpi/PU.1 resulting in the blocking of erythroid differentiation and the development of erythroleukemia (reviewed in Ruscetti, 1999).…”

“…This causes the constitutive activation of Sfpi/PU.1 resulting in the blocking of erythroid differentiation and the development of erythroleukemia (reviewed in Ruscetti, 1999). MEL cells can be induced to reinitiate the differentiation program by the addition of chemical agents such as hexamethylene bisacetamide (HMBA) (Fernández-Nestosa et al, 2008). We have previously reported the establishment of an HMBA-resistant cell line (MEL-R) before.…”