A systems biology approach reveals the dose- and time-dependent effect of primary human airway epithelium tissue culture after exposure to cigarette smoke in vitro.

To establish a relevant in vitro model for systems toxicology-based mechanistic assessment of environmental stressors such as cigarette smoke (CS), we exposed human organotypic bronchial epithelial tissue cultures at the air liquid interface (ALI) to various CS doses. Previously, we compared in vitro gene expression changes with published human airway epithelia in vivo data to assess their similarities. Here, we present a follow-up evaluation of these in vitro transcriptomics data, using complementary computational approaches and an integrated mRNA-microRNA (miRNA) analysis.

Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression.

Background: High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression.

A modular cell-type focused inflammatory process network model for non-diseased pulmonary tissue.

Exposure to environmental stressors such as cigarette smoke (CS) elicits a variety of biological responses in humans, including the induction of inflammatory responses. These responses are especially pronounced in the lung, where pulmonary cells sit at the interface between the body's internal and external environments. We combined a literature survey with a computational analysis of multiple transcriptomic data sets to construct a computable causal network model (the Inflammatory Process Network (IPN)) of the main pulmonary inflammatory processes.

Construction of a computable network model for DNA damage, autophagy, cell death, and senescence.

Towards the development of a systems biology-based risk assessment approach for environmental toxicants, including tobacco products in a systems toxicology setting such as the "21st Century Toxicology", we are building a series of computable biological network models specific to non-diseased pulmonary and cardiovascular cells/tissues which capture the molecular events that can be activated following exposure to environmental toxicants. Here we extend on previous work and report on the construction and evaluation of a mechanistic network model focused on DNA damage response and the four main cellular fates induced by stress: autophagy, apoptosis, necroptosis, and senescence. In total, the network consists of 34 sub-models containing 1052 unique nodes and 1538 unique edges which are supported by 1231 PubMed-referenced literature citations.

Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers.

Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.

Nrf2: friend and foe in preventing cigarette smoking-dependent lung disease.

Chronic exposure to cigarette smoke (CS) generally confronts cellular defense systems with one of the strongest known environmental challenges. In particular, the continuous exposure of tissues of the respiratory tract to abundant concentrations of radicals; volatile compounds of the gas phase, mainly reactive oxygen and nitrogen species; and CS condensate deposits trigger a pleiotropic adaptive response, generally aimed at restoring tissue homeostasis. As documented by numerous studies published over the past decade, a hallmark of this defense system is the activation of the transcription factor NF-E2-related factor 2 (Nrf2), which, consequent to its established role as master regulator of the cellular antioxidant response, has been shown to orchestrate the first line of defense against cell- and tissue-damaging components present in CS.

A computable cellular stress network model for non-diseased pulmonary and cardiovascular tissue.

Background: Humans and other organisms are equipped with a set of responses that can prevent damage from exposure to a multitude of endogenous and environmental stressors. If these stress responses are overwhelmed, this can result in pathogenesis of diseases, which is reflected by an increased development of, e.g.

Construction of a computable cell proliferation network focused on non-diseased lung cells.

Background: Critical to advancing the systems-level evaluation of complex biological processes is the development of comprehensive networks and computational methods to apply to the analysis of systems biology data (transcriptomics, proteomics/phosphoproteomics, metabolomics, etc.). Ideally, these networks will be specifically designed to capture the normal, non-diseased biology of the tissue or cell types under investigation, and can be used with experimentally generated systems biology data to assess the biological impact of perturbations like xenobiotics and other cellular stresses.

Endoplasmic reticulum stress induced by aqueous extracts of cigarette smoke in 3T3 cells activates the unfolded-protein-response-dependent PERK pathway of cell survival.

Cigarette smoke (CS) generally places severe stress on cells, as reflected by gene expression profiling and pathway analysis, which, among other effects, also suggested activation of the unfolded protein response pathway triggered by the stressed endoplasmic reticulum (ER stress). Here, we present data indicating that noncytotoxic concentrations of aqueous extracts of CS induce a distinct ER stress response in immortalized nontransformed Swiss 3T3 cells, primarily by activating the PERK pathway of global protein synthesis inhibition. Activation of PERK and PERK-dependent signaling by aqueous extracts of CS was demonstrated by (i) the inhibition of protein synthesis, (ii) the phosphorylation of PERK and its substrate eIF2alpha, (iii) the activation of ATF4, and (iv) the expression of ATF4-dependent target genes chop, gadd34, BiP, and atf3.

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts.

Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53.

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells.

Involvement of the DNA repair protein hHR23 in p53 degradation.

The stability of the tumor suppressor protein p53 is regulated via the ubiquitin-proteasome-dependent proteolytic pathway. Like most substrates of this pathway, p53 is modified by the attachment of polyubiquitin chains prior to proteasome-mediated degradation. However, the mechanism(s) involved in the delivery of polyubiquitylated p53 molecules to the proteasome are currently unclear.

HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53.

The RING finger proteins HdmX and Hdm2 share significant structural and functional similarity. Hdm2 is a member of the RING finger family of ubiquitin-protein ligases E3 and targets the tumor suppressor protein p53 for degradation. Although HdmX also binds to p53, HdmX does not induce p53 degradation.

siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.

The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6.