Total RNA (5?ng) from each sample was used for cDNA synthesis using the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA, USA). the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins. Erythropoiesis is a complex multistage process in which multipotential hematopoietic stem cells give rise to mature red blood cells (RBCs) through coordinated programs of gene expression, proliferation and differentiation.1 The earliest committed erythroid progenitor is the erythroid burst-forming unit (BFU-E) that further differentiates through erythroid colony-forming unit (CFU-E), proerythroblasts, basophilic and polychromatophilic erythroblasts and orthochromatic erythroblasts, giving rise to reticulocytes by enucleation. Reticulocytes are then released into the circulation, where they shed their RNA and degrade mitochondria to become mature RBCs.2, 3 Erythropoiesis must be tightly regulated to maintain the production of RBCs under both normal and stress conditions. This complex process is controlled intricately at each Hoechst 33258 analog 2 stage of the production of RBCs by multiple proteins, RNAs, DNA and chromatin modifications.1, 4, 5, 6, 7 Impairment of these regulatory pathways can lead to anemia, which has been estimated to affect nearly a quarter of the world’s population.8 Moreover, defective erythropoiesis can be fatal under conditions of stress such as acute Rabbit Polyclonal to NCAPG2 blood loss, chronic infections and myeloablative therapeutic procedures used to treat malignant diseases. Therefore, a better understanding of the molecular mechanisms controlling the production of RBCs are important to control anemia and may lead to avenues for developing new therapeutic strategies. Poly(ADP-ribose) polymerase-2 (PARP-2) belongs to a family of enzymes that catalytically cleave (and studies that proposed a role for PARP-2 in chromosome segregation during cell division.21, 46 For instance, Menissier de Murcia and to what extent this replication-linked role of PARP had in the context of a living organism remained unknown. Our results provided strong evidence that PARP-2, but not PARP-1, is required for efficient erythropoiesis in mice by limiting RS on erythroid progenitors, a function that becomes essential in the context of an enhanced demand for the production of Hoechst 33258 analog 2 RBCs. Nowadays, there is considerable excitement about the prospect of anticancer compounds that act through targeting PARP proteins, although non-isoform-selective PARP inhibitors are available.14 Our observations reveal potential off-target effects that may arise from the use of such compounds.62 In addition, the specific role of PARP-2 in RS, a major driver of genomic instability,63, 64, 65 may have implications in the design and use of drugs targeting PARP proteins. Materials and Methods Animals and treatments Parp-1?/?, Parp-2?/?, Puma?/? and p21?/? mice have been described previously.46, 66, 67, 68 P21?/? mice were bred with Parp-1?/? or Parp-2?/? mice, whereas Puma?/? mice were bred with Parp-2?/? mice, to generate heterozygous mice, which were then bred to generate all genotypes. Genotyping was performed by PCR analysis using tail DNA as described.46, 68, 69, 70 To induce acute anemia, mice were injected intraperitoneally with PHZ (Sigma-Aldrich, St. Louis, MO, USA) at 40?mg/kg on day 0, 1 and 3. All mice were in C57BL6 background and were kept under pathogen-free conditions in the animal house facility at the Barcelona Biomedical Research Park (Barcelona, Spain). Animal studies were approved by the Institutional Animal Care and Use Committee. Blood analysis For cell analysis, peripheral blood from 10- to 12-week-old mice was collected in EDTA-coated vials. Blood cell parameters were measured immediately after sample collection using an Abacus Junior Vet Hematology Blood Analyzer (Diatron, Hialeah, FL, USA). Blood smears were air dried and stained with MayCGrnwald Giemsa for routine analysis. Differential cell counts were made blind on more than 1000 cells on random areas of slides. EPO levels in plasma were determined by ELISA Assay Hoechst 33258 analog 2 Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. Haptoglobin and hemoglobin levels in serum were determined by ELISA Assay Kits (Abcam, Cambridge, UK).