07-623 Sigma-AldrichAnti-Clara Cell Secretory Protein Antibody

Lung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied.Viral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.Here we show that the use of a recombinant adenovirus expressing Cre-recombinase ("AdenoCre") to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well.Our findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.

Antenatal stimulation of lung growth is a reasonable approach to treat congenital diaphragmatic hernia (CDH), a disease characterized by pulmonary hypoplasia and hypertension. Several evidences from the literature demonstrated a possible involvement of renin-angiotensin system (RAS) during fetal lung development. Thus, the expression pattern of renin, angiotensin-converting enzyme, angiotensinogen, type 1 (AT₁) and type 2 (AT₂) receptors of angiotensin II (ANGII) was assessed by immunohisto-chemistry throughout gestation, whereas the function of RAS in the fetal lung was evaluated using fetal rat lung explants. These were morphometrically analyzed and intracellular pathway alterations assessed by Western blot. In nitrofen-induced CDH model, pregnant rats were treated with saline or PD-123319. In pups, lung growth, protein/DNA ratio, radial saccular count, epithelial differentiation and lung maturation, vascular morphometry, right ventricular hypertrophy and overload molecular markers, gasometry and survival time were evaluated. Results demonstrated that all RAS components were constitutively expressed in the lung during gestation and that ANGII had a stimulatory effect on lung branching, mediated by AT₁ receptor, through p44/42 and Akt phosphorylation. This stimulatory effect on lung growth was mimicked by AT₂-antagonist (PD-123319) treatment. In vivo antenatal PD-123319 treatment increased lung growth, ameliorated indirect parameters of pulmonary hypertension, improved lung function and survival time in nonventilated CDH pups, without maternal or fetal deleterious effects. Therefore, this study demonstrated a local and physiologically active RAS during lung morphogenesis. Moreover, selective inhibition of AT₂ receptor is presented as a putative antenatal therapy for CDH.

Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE) and mass-spectrometry- (MS-) based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11) and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE). These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

We previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen's protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment.Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3 h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation.Pretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes.Donor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen's protective effects against I/R injury during transplantation.

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.

The role of umbilical cord blood (CB)-derived stem cell therapy in neonatal lung injury remains undetermined. We investigated the capacity of human CB-derived CD34(+) hematopoietic progenitor cells to regenerate injured alveolar epithelium in newborn mice. Double-transgenic mice with doxycycline (Dox)-dependent lung-specific Fas ligand (FasL) overexpression, treated with Dox between embryonal day 15 and postnatal day 3, served as a model of neonatal lung injury. Single-transgenic non-Dox-responsive littermates were controls. CD34(+) cells (1 × 10(5) to 5 × 10(5)) were administered at postnatal day 5 by intranasal inoculation. Engraftment, respiratory epithelial differentiation, proliferation, and cell fusion were studied at 8 weeks after inoculation. Engrafted cells were readily detected in all recipients and showed a higher incidence of surfactant immunoreactivity and proliferative activity in FasL-overexpressing animals compared with non-FasL-injured littermates. Cord blood-derived cells surrounding surfactant-immunoreactive type II-like cells frequently showed a transitional phenotype between type II and type I cells and/or type I cell-specific podoplanin immunoreactivity. Lack of nuclear colocalization of human and murine genomic material suggested the absence of fusion. In conclusion, human CB-derived CD34(+) cells are capable of long-term pulmonary engraftment, replication, clonal expansion, and reconstitution of injured respiratory epithelium by fusion-independent mechanisms. Cord blood-derived surfactant-positive epithelial cells appear to act as progenitors of the distal respiratory unit, analogous to resident type II cells. Graft proliferation and alveolar epithelial differentiation are promoted by lung injury.

Runx3 is essential for normal murine lung development, and Runx3 knockout (KO) mice, which die soon after birth, exhibit alveolar hyperplasia. Wound healing, tissue repair, and regeneration mechanisms are necessary in humans for proper early lung development. Previous studies have reported that various signaling molecules, such as pErk, Tgf-beta1, CCSP, pJnk, Smad3, and HSP70 are closely related to wound healing. In order to confirm the relationship between lung defects caused by the loss of function of Runx3 and wound healing, we have localized various wound-healing markers after laser irradiation in wild-type and in Runx3 KO mouse lungs at post-natal day 1. Our results indicate that pERK, Tgf-beta1, CCSP, pJnk, and HSP70 are dramatically down-regulated by loss of Runx3 during lung wound healing. However, Smad3 is up-regulated in the Runx3 KO laser-irradiated lung region. Therefore, the lung wound-healing mechanism is inhibited in the Runx3 KO mouse, which shows abnormal lung architecture, by reduced pErk, Tgf-beta1, CCSP, pJnk, and HSP70 and by induced Smad3.