Exposure of hematopoietic cells to DNAdamaging agents induces cell-cycle arrest at G1 and G2/M checkpoints. Previously, it was shown that DNA damageinduced growth arrest of hematopoietic cells can be overridden by treatment with cytokine growth factors, such as erythropoietin (EPO) or interleukin-3 (IL-3). Here, the cytokine-activated signaling pathways required to override G1 and G2/M checkpoints induced by ␥-irradiation (␥-IR) are characterized. Using factordependent myeloid cells stably expressing EPO receptor (EPO-R) mutants, it is shown that removal of a minimal domain required for PI-3K signaling abrogated the ability of EPO to override ␥-IRinduced cell-cycle arrest. Similarly, the ability of cytokines to override ␥-IR- IntroductionExposure of mammalian cells to DNA-damaging agents triggers a variety of responses that include apoptotic cell death and cell-cycle arrest. 1 Failure of DNA-damaged cells to commit to either of these pathways, before the restoration of DNA integrity, may contribute to tumorigenic development by allowing the accumulation of new mutations. 2,3 Cellular determinants that predict whether cells will arrest or undergo apoptosis in the presence of genotoxic stress are not completely defined. Protection from DNA damage-induced apoptosis may be conferred by the expression of antiapoptotic proteins, such as Bcl-2 and Bcl-X L . 4 Various hematopoietic cytokines induce the expression of antiapoptotic proteins, and this coincides with their ability to protect against DNA damageinduced cell death. [5][6][7] In addition to the inhibition of apoptosis, growth factors acting through the hematopoietic cytokine receptor superfamily have also been shown to override the cell-cycle arrest response to DNA damage in hematopoietic cell lines. 7 However, the signaling pathways responsible for this ability to override DNA damage-induced cell-cycle checkpoints have not been defined.The classical pathway coupling DNA damage with cell-cycle arrest involves up-regulation of p53 and its transcriptional targets. In response to DNA damage, p53 induces the expression of growth inhibitory genes, such as p21 Cip1 and GADD45. 8 However, the existence of p53-independent mechanisms resulting in cell-cycle arrest has been demonstrated in lymphoid cells derived from p53 Ϫ/Ϫ mice. 9 Similarly, hematopoietic cell lines lacking p53 protein arrest in the G1 and G2/M phases after treatment with ionizing irradiation. 7 Thus, it remains unclear what mechanisms regulate the cell-cycle arrest response to DNA damage in hematopoietic cells. Further, it is not obvious how signaling pathways regulated by growth-promoting cytokines influence these G1 and G2/M cell-cycle checkpoints.Members of the hematopoietic cytokine receptor superfamily include the receptors for erythropoietin (EPO) and interleukin-3 (IL-3). 10 These receptors regulate a number of cellular functions in hematopoietic cell lineages, including growth and development. Receptors of this family function by activating one or more members of the Jak family of tyrosine k...

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G 1 checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/ Akt-dependent signaling pathway. Here, we show that ␥-irradiated murine myeloid 32D cells arrest in G 1 with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21Cip1 and p27 Kip1 , yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G 1 arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.

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Background. Graft-versus-host disease (GVHD) after liver transplantation (LT) is a rare but serious complication. The aim of this study is to identify risk factors, including immunosuppressive regimens, for mortality due to GVHD (fatal GVHD). Methods. Using data from the Organ Procurement and Transplantation Network and United Network for Organ Sharing registry, 77 416 adult patients who underwent LT between 2003 and 2018 were assessed. Risk factors for fatal GVHD were analyzed by focusing on induction and maintenance immunosuppression regimens. Results. The incidence of fatal GVHD was 0.2% (121 of 77 416), of whom 105 (87%) died within 180 d and 13 (11%) died between 181 d and 1 y. Median survival after LT was 68.0 (49.5–125.5) d. Recipient age minus donor age >20 y (hazard ratio [HR], 2.57; P < 0.001) and basiliximab induction (HR, 1.69; P = 0.018) were independent risk factors for fatal GVHD. Maintenance therapy with mycophenolate mofetil (MMF) was associated with a decrease in fatal GVHD (HR, 0.51; P = 0.001). In an increased risk cohort of patients with recipient-donor age discrepancy >20 y, MMF use was associated with a 50% decline in fatal GVHD (HR, 0.50; P < 0.001). Conclusions. Recipient age minus donor age >20 y remains a significant risk factor for fatal GVHD. The risk of fatal GVHD significantly increases in association with basiliximab induction and decreases with MMF maintenance. These associations were pronounced in patients with recipient minus donor age >20 y. These results emphasize the importance of donor age and individualized immunosuppression regimens on the risk of fatal GVHD.

Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43.