Abstract
High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.
Highlights
High doses of bleomycin administered in the 1980s–1990s to patients with lymphomas and other tumors were associated with significant lung toxicity in general and apoptosis of epithelial cells in particular in 2–40% of patients, with up to 83% mortality in patients who developed lung fibrosis secondary to chemotherapy [1]
We have previously demonstrated in bleomycin-treated mouse lungs that, even when telomere length remains constant, telomerase is detected at levels that are inversely correlated with the level of lung epithelial cell apoptosis, and inhibition of telomerase with TMPYP4 increases cell death and apoptosis during evolution of lung fibrosis [23]
We found (Fig 1, table and inserts) that bleomycin upregulates genes that are known to appear after cell exposure to oxidative stress and DNA damage such as GADD45, mdm-2, and bcl-2; genes involved in the intrinsic apoptotic pathway such as caspase-9; genes involved in the extrinsic pathway from the TNF receptor (TNFr) family; and associated molecules such as TRAIL, TRAFs, TNFsf, and FADD
Summary
High doses of bleomycin administered in the 1980s–1990s to patients with lymphomas and other tumors were associated with significant lung toxicity in general and apoptosis of epithelial cells in particular in 2–40% of patients, with up to 83% mortality in patients who developed lung fibrosis secondary to chemotherapy [1]. Lung toxicity has been greatly reduced in more recent reports, albeit at the cost of a reduction in cumulative dose by 75% or more, from levels >100 mg/sqm to a practical limit of approximately 25 mg/sqm today. This ceiling on cumulative dose limits the effectiveness of an important chemotherapeutic agent. Intratracheal administration of bleomycin in mice has been widely used as an animal model mimicking side effects from treatment in lymphoma patients to study the mechanisms of lung injury, including the cycle of inflammation, and repair, and lung fibrosis [2, 3]. We have shown that following bleomycin treatment of murine lung epithelial (MLE)-cells in vitro [8,9,10], and following in vivo treatment of C57BL/6 mice [8], both primary epithelial cells and those from a cell line become more sensitive to Fas-induced apoptosis exerted either by Fas-agonists or by activated myofibroblasts [8]
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