The Chemokine CCL3 Regulates Myeloid Differentiation and Hematopoietic Stem Cell Numbers.

The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM.

DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction.

Epithelial ablation of Bcl-XL increases sensitivity to oxygen without disrupting lung development.

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium.

Hyperoxia augments ER-stress-induced cell death independent of BiP loss.

Cytotoxic reactive oxygen species are constantly formed as a by-product of aerobic respiration and are thought to contribute to aging and disease. Cells respond to oxidative stress by activating various pathways, whose balance is important for adaptation or induction of cell death. Our lab recently reported that BiP (GRP78), a proposed negative regulator of the unfolded protein response (UPR), declines during hyperoxia, a model of chronic oxidative stress.

p21(Cip1) protects against oxidative stress by suppressing ER-dependent activation of mitochondrial death pathways.

Although it is well established that the cell cycle inhibitor p21 protects against genotoxic stress by preventing the replication of damaged DNA, recent studies have shown that the cytoplasmic form can also protect. It protects by delaying the loss of the antiapoptotic proteins Mcl-1 and Bcl-X(L); however, the mechanism of regulation is unknown. Utilizing hyperoxia as a model of chronic oxidative stress and DNA damage, p21 was detected in the nucleus and cytoplasm and cytoplasmic expression of p21 was sufficient for cytoprotection.

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death.

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L).

p21(Cip1/Waf1/Sdi1) protects against hyperoxia by maintaining expression of Bcl-X(L).

p21(Cip1/WAF1/Sdi1) is a major transcriptional target of p53 that promotes survival of cells exposed to continuous oxidative stress caused by hyperoxia. Because p21 can protect against genotoxic stress by reducing p53-dependent transcription of the proapoptotic proteins PUMA and Bax, the current study uses genetically modified lines of HCT116 colon carcinoma cells to investigate whether p21-mediated protection against hyperoxia involves attenuation of the p53 apoptotic pathway. Hyperoxia stimulated p53-dependent expression of p21 and Bax.

Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development.

Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair.

p21Cip1 protection against hyperoxia requires Bcl-XL and is uncoupled from its ability to suppress growth.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211-82NLS) domain or its carboxy-terminal PCNA-binding (p2176-164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective.

Hyperoxic ventilated premature baboons have increased p53, oxidant DNA damage and decreased VEGF expression.

Hyperoxia is implicated in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. High levels of supplemental oxygen can result in microvascular endothelial cell death and may disrupt lung development. In postnatal animals, hyperoxia inhibits expression of vascular endothelial growth factor (VEGF), which is required for normal vascular development.

p21(Cip1/WAF1/Sdi1) does not affect expression of base excision DNA repair enzymes during chronic oxidative stress.

Exposure to chronic oxidative stress during elevated oxygen (hyperoxia) damages DNA and inhibits cell proliferation in G(1) through induction of the cyclin-dependent kinase inhibitor p21. Cells that fail to express p21 growth-arrest in S phase. The observation that growth arrest in G(1) is associated with reduced DNA damage and enhanced survival suggests that p21 may affect expression of base excision repair (BER) enzymes used to repair oxidized DNA.

Loss of Gadd45a does not modify the pulmonary response to oxidative stress.

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia.

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen.

Induced p21Cip1 in premature baboons with CLD: implications for alveolar hypoplasia.

Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells.

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein.

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter.

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia.

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint.

Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1).

Alveolar cells of the lung are injured and killed when exposed to elevated levels of inspired oxygen. Damaged tissue architecture and pulmonary function is restored during recovery in room air as endothelial and type II epithelial cells proliferate. Although excessive fibroblast proliferation and inflammation occur when abnormal remodeling occurs, genes that regulate repair remain unknown.