Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond–blackfan anemia using inducible expression of siRNA against RPS19

Abstract

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.

PIM1 kinase is destabilized by ribosomal stress causing inhibition of cell cycle progression

“…TF-1C and K562C cells, expressing inducible siRNA targeting RPS19 mRNA, were prepared in Karlsson's laboratory (Miyake et al, 2005). Cells (5 Â 10 6 ) were transiently transfected using 15 mg of plasmid DNA and 30 ml of JetPei transfection reagent (Polyplus transfection), or with 100-200 nM siRNA and 10 ml of Interferin transfection reagent (Polyplus transfection) according to the manufacturer's protocol.…”

“…To study the effect of alteration of ribosome synthesis on PIM1 expression, we used two modified cell lines, TF-1C and K562C, both infected with a lentiviral vector inducible for the expression of small interfering RNA specific for RPS19 mRNA (Miyake et al, 2005). The two parental cell lines differ in p53 expression: TF-1 expresses a wild-type p53 (Urashima et al, 1998), whereas K562 does not show detectable levels of p53.…”

Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia

“…The cytokine-dependent TF-1 cells, TF-1-B cells, (an inducible RPS19-deficient DBA model cell line) and TF-1-S control cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in the presence of 5 ng/ml GM-CSF (a gift from Novartis International, Basel, Switzerland, http:// www.novartis.com) as described previously [15]. To differentiate into the erythroid lineage, these cells were cultured with 5 U/ml recombinant EPO (Janssen-Cilag, Sollentuna, Sweden, http://www.…”

“…Construction of lentiviral packaging (pCMV-⌬R8.91 and pMD.G) and vector (pLV-TH-B, pLV-TH-S, and pLV-mRIY) plasmids has been described previously [14,15]. All recombinant lentivirus vectors were produced by transient transfection of 293T cells as previously described [14,15].…”

“…Therefore, we developed cellular models for RPS19-deficient DBA using inducible expression of siRNA against RPS19 in TF-1 and UT-7 cells [15]. Upon RPS19 silencing, these cell lines present a phenotype similar to that of progenitor cells from RPS19-deficient DBA patients, including reduced erythroid differentiation and a reduction in proliferative capacity.…”