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Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 μM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.

Recent advances in our understanding of the molecular biology of lymphatic endothelial cells have revealed that these vessels, besides their known function in tissue homeostasis and immunity, constitute conduits for the tumor cells to metastasize. One of the factors that contribute to tumor spread is the acquisition of an angiogenic phenotype as a response to the onset of tumor hypoxia. To our knowledge, little is known about the effects of low oxygen levels on the lymphatic vasculature. Therefore, we used cDNA microarrays to study the transcriptional changes occurring in hypoxia exposed lymphatic endothelial cells. Our analysis was then complemented by functional assays showing that these cells responded with increased attachment to the extracellular matrix, delayed proliferation and production of reactive oxygen species. Differential expression of genes involved in these processes such as NADPH oxidase 4, the tissue inhibitor of metalloproteinase 3, and TGFβ induced protein I, was found. Hypoxia was also found to increase mRNA levels of the cytokine CXCL-12 and its receptor CXCR4. Moreover, adhesion experiments revealed that hypoxia increased the binding of non-small cell lung carcinoma cells to this endothelium in a CXCR4 dependent way. We thus illustrate the response of lymphatic endothelial cells to hypoxia and suggest targets to study tumor metastasis through these vessels.

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.