Expression of cross-tolerance to a wide range of conditions in a human lung cancer cell line after adaptation to nitric oxide.

Previously, we have shown that A549, a human lung adenocarcinoma, can be adapted to nitric oxide (NO●). NO● is a nitrogen-based free radical that is synthesized by a family of enzymes known as nitric oxide synthases. NO● has been shown to be overexpressed in patient populations of different cancers. In addition, it has been observed that patients who express high levels of nitric oxide synthases tend to have poorer clinical outcomes than those with low levels of expression. The original cell line A549 (parent) and the adapted A549-HNO (high nitric oxide) cell line serve as a useful model system to investigate the role of NO● in tumor progression and prognosis. We have previously shown that the A549-HNO-adapted cells grow aggressively when compared to A549-parent cells. Furthermore, we have shown that the A549-HNO-adapted cells exhibit a higher percentage of cell viability when exposed to ultraviolet and X-ray radiation than the A549-parent cells. Cancer patients who develop resistance to one treatment often become resistant to other previously unencountered forms of treatment. This phenomenon is known as cross-tolerance. To determine whether NO● is a potential cross-tolerance causing agent, we have expanded our research by conducting parallel studies to a variety of other agents and conditions beyond radiation and ultraviolet exposure. We exposed both cell lines to varying levels of chemotherapeutic drugs (taxol and doxorubicin), temperature, pH, calcium chloride, cadmium chloride, copper chloride, sodium chloride, ferrous chloride, and sodium-R-lipoic acid. Our results show that the A549-HNO cells exhibit greater viability than the A549-parent cells when exposed to each of the various conditions. Therefore, NO● is one potential driving force that can make tumor cells exhibit cross-tolerance.

Transcriptomic Analysis of mRNAs in Human Monocytic Cells Expressing the HIV-1 Nef Protein and Their Exosomes.

The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes from Nef-expressing U937 cells and their corresponding miRNAs being preferentially retained in cells. These are AATK, SLC27A1, and CDKAL and are important in apoptosis and fatty acid transport. Thus, our study identifies selectively expressed mRNAs in Nef-expressing U937 cells and their exosomes and supports a new mode on intercellular regulation by the HIV-1 Nef protein.

A549 cells adapted to high nitric oxide show reduced surface CEACAM expression and altered adhesion and migration properties.

The migration and adhesion properties of tumors affect their metastatic rate. In the present study, we investigated carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1, 5, and 6 expression in high nitric oxide (HNO)-adapted lung cancer cells compared to parent cells. We observed high transcript levels of CEACAM 1 (4S, 4L), CEACAM 5, and CEACAM 6 in HNO cells compared to parent cells. However, the surface expression was low in HNO cells. Interestingly, the intracellular protein levels were high for these three CEACAMs. We confirmed these results with immunohistochemical experiments. Further, the adhesion and migration assays showed reduced clumping in HNO-adapted A549 (A549-HNO) cells and faster migration rates, respectively. These results document the altered adhesion and migration properties of cells adapted to HNO. Further, our studies also indicate a dynamic regulation of CEACAM protein expression and surface transport in HNO cells.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells.

INTRODUCTION: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. METHOD: Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. RESULTS: Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. CONCLUSIONS: This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Part I-mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways.

Our previous studies demonstrate that A549, a human lung adenocarcinoma line, could be adapted to the free radical nitric oxide (NO([Symbol: see text])). NO([Symbol: see text]) has been shown to be overexpressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serves as a useful model system to study the role of NO([Symbol: see text]) in tumor biology. It is well known that DNA damage response (DDR) is altered in cancer cells and NO([Symbol: see text]) is known to cause DNA damage. Modulations in molecular mechanisms involved in DNA damage response in A549-HNO cells can provide better insights into the enhanced growth behavior of these cells. Thus, here, we carried out a series of time course experiments by treating A549 and A549-HNO cells with NO([Symbol: see text]) donor and examining levels of proteins involved in the DDR pathway. We observed induced expression of key components of DDR pathway in A549-HNO cells. The HNO cells showed sustained expression of key proteins involved in both nonhomologous end joining (NHEJ) and homologous recombination pathways, whereas parent cells only expressed low levels of NHEJ pathway proteins. Further with prolonged NO([Symbol: see text]) exposure, ATR, Chk1, and p53 were activated and upregulated in HNO cells. Activation of p53 results in inhibition of apoptosis through induced Mcl1 expression. It also leads to cell cycle modulation. Interestingly, several reports show that cancer stem cells have enhanced expression of proteins involved in DNA damage response and also activated an antiapoptotic response. Our results here suggest that our HNO adapted A549 cells have increased activation of DNA damage response pathway proteins which can lead to better DNA repair function. Enhanced DDR leads to activation of antiapoptosis response and modulation in the cell cycle which may lead to better survival of these cells under harsh conditions. Thus, our present investigation further supports the hypothesis that HNO exposure leads to survival of these cells.

Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher than those of the parent cells. This suggests an initial bypass of cell cycle checkpoints as p21CIP1 can inhibit the activity of all cyclin/Cdk complexes. p21CIP1 is also known to inhibit p53-induced apoptosis. This could be important during later phases of the cell cycle to allow time for repair of damaged DNA and thus better survival of HNO cells.

The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference.

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR).