Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Δ cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Δ dun1Δ cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin–Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint

Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

<div><p>Eosinophils are produced in the bone marrow from CD34⁺ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.</p></div

Modified LDBM culture yields functional eosinophils.

<p>(A) Total number of cultured cells (mean ± SD, <i>n</i> = 3 wells per condition) at Day 14 that migrated toward CCL11 (left panel, gray bars) or leukotriene B4 (LTB4, right panel, white bars) at the indicated doses is shown from a representative of 3 independent experiments. ***<i>P</i><0.001, ^#<i>P</i><0.0001 vs. cells that migrated to media alone. (B–C) Total number of wild-type (WT) and CCR3-deficient (CCR3KO) cultured cells (mean ± SD, <i>n</i> = 3 wells per condition) at Day 14 that migrated toward (B) CCL11 or (C) LTB4 at the indicated doses is shown from a representative of 2 independent experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. WT. (D) Fold change (mean ± SEM, <i>n</i> = 4 independent experiments with 3 wells per condition per experiment) in eosinophil peroxidase (EPO) activity detected in the supernatants after activation of eosinophils with phorbol 12-myristate 13-acetate (PMA) at the indicated doses. *<i>P</i><0.05, **<i>P</i><0.01 vs. unstimulated controls. (E) Cytokine levels (mean ± SD, representative of 3 independent experiments with 3 wells per experiment shown) in supernatant from Day 14 cells after 24 hours in culture. (F) Fold change (mean ± SD) in total polymerized actin in cultured eosinophils and peripheral blood eosinophils after stimulation with CCL11 or leukotriene B4 (LTB4) at the indicated time points (*<i>P</i><0.05 vs. cultured eosinophils, representative of 2 independent experiments is shown with <i>n</i> = 3 wells per condition and per time point). CCR3, C-C chemokine receptor type 3; gMFI, geometric mean fluorescence intensity; IL, interleukin.</p

Eosinophils can be cultured from frozen bone marrow cells and in a 96-well plate.

<p>(A) Histogram (left panel) of cultured eosinophils that had been frozen, thawed and then stained with viability dye is shown. Percentages of viable (negative for the dye) and dead (positive for the dye) eosinophils recovered from the frozen vial are indicated in the histogram. Surface expression of CCR3 and Siglec-F (right panel) on all cells recovered from the frozen vial is shown in a representative density plot from 4 independent experiments. (B) Total number of (mean ± SD, <i>n</i> = 3 wells per condition) Day 15 eosinophils (Fresh eosinophils) or Day 14 eosinophils that had been frozen and then thawed the next day (Frozen eosinophils) that migrated toward CCL11 at the indicated doses (a representative of 2 independent experiments is shown). ***<i>P</i><0.001, ^#<i>P</i><0.0001 vs. fresh eosinophils of the same treatment group. (C) Eosinophil yield (mean ± SEM, <i>n</i> = 3 independent experiments with 3 wells per condition per experiment) at Day 14 from cultures started with freshly harvested low-density bone marrow (Fresh LDBM, black bar) or frozen/thawed LDBM (Frozen LDBM, white bar). (D) Eosinophil yield (mean ± SD) at Day 14 from cultures started with LDBM that was freshly prepared (Fresh LDBM), was prepared from whole bone marrow (WBM) that had been frozen and thawed (Frozen WBM), was prepared from WBM and then frozen and thawed (Frozen LDBM), and was prepared from freeze-thawed WBM and then frozen and thawed again (Frozen WBM/LDBM) is shown. Data are a representative of 2 independent experiments with 3 wells per condition per experiment. (E) Total number of eosinophils (mean ± SD, <i>n</i> = 3 wells per condition) that migrated toward CCL11 at the indicated doses (a representative of 2 independent experiments is shown). **<i>P</i><0.01, ***<i>P</i><0.001, ^#<i>P</i><0.0001 when compared to eosinophils from Fresh LDBM of the same treatment group. (F) Eosinophil yield (mean ± SEM) at Day 14 in flat- or round-bottomed, 96-well plates from cultures started with LDBM cells at the indicated concentrations (<i>n</i> = 3 independent experiments with 3 wells per condition per experiment). *<i>P</i><0.05, ***<i>P</i><0.001. CCL11, C-C motif chemokine 11; CCR3, C-C chemokine receptor type 3; Siglec-F, sialic acid–binding immunoglobulin-like lectin F.</p

EoP identification via surface markers.

<p>Analysis of BALB/c murine whole bone marrow (WBM) by flow cytometry reveals an eosinophil lineage–committed progenitor (EoP) population; gating strategy to delineate EoPs from WBM starts with (A) excluding counting beads (upper left corner) that were added to the cell suspension and then including (B) only single cells that are (C) viable, (D) express CD34 but are negative for lineage markers (CD3, CD4, CD8, CD19, CD45R, Gr-1) and Sca-1 and (E) co-express CD117 (c-Kit) and CD125 (IL-5Rα). All gate frequencies are expressed as a percentage of the parent gate, except for panel A, which shows percentage within that gate. CD, cluster of differentiation; FSC, forward scatter; IR, infrared; Sca-1, stem cell antigen 1; SSC, side scatter.</p

Modified LDBM culture yields mature eosinophils.

<p>(A) Schematic of liquid culture for eosinophils is shown. (B–C) Absolute number (mean ± SD, <i>n</i> = 3 wells per time point, representative of 3 independent experiments is shown) of eosinophils (CCR3⁺Siglec-F⁺ cells) per well after indicated days of IL-5 stimulation is shown (Days 4–14 of culture schematic presented in panel A and extended to Day 16). (D) Two representative dot blots of Siglec-F and CCR3 expression on the surface of cultured cells at Day 14 are shown with percentage of gated live cells in the upper right corner. (E) Surface expression of CCR3 and Siglec-F by cultured eosinophils (red line) and native eosinophils (black line) compared to isotype control (shaded) is shown. Results are representative of 2 independent experiments. (F) Normalized gene expression (mean ± SD, n = 2 independent experiments) in cultured eosinophils at Day 14 is shown. (G) Light microscopic images of representative cytospin preparations from native eosinophils from naïve bone marrow and cultured eosinophils stained with a modified Giemsa protocol at Day 15. Magnification of 1000X. <i>Ccr3</i>, C-C chemokine receptor type 3; <i>Epx</i>, eosinophil peroxidase; IL-5, interleukin 5; <i>Mbp</i>, major basic protein; SCF, stem cell factor; Siglec-F, sialic acid–binding immunoglobulin-like lectin F.</p