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Mitochondria are involved in a variety of critical cellular functions, and their impairment drives cell injury. The mitochondrial ribosome (mitoribosome) is responsible for the protein synthesis of mitochondrial DNA-encoded genes. These proteins are involved in oxidative phosphorylation, respiration, and ATP production required in the cell. Mitoribosome components originate from both mitochondrial and nuclear genomes. Their dysfunction can be caused by impaired mitochondrial protein synthesis or mitoribosome misassembly, leading to a decline in mitochondrial translation. This decrease can trigger mitochondrial ribosomal stress and contribute to pulmonary cell injury, death, and diseases. This review focuses on the contribution of the impaired mitoribosome structural components and function to respiratory disease pathophysiology. We present recent findings in the fields of lung cancer, chronic obstructive pulmonary disease, interstitial lung disease, and asthma. We also include reports on the mitoribosome dysfunction in pulmonary hypertension, high-altitude pulmonary edema, and bacterial and viral infections. Studies of the mitoribosome alterations in respiratory diseases can lead to novel therapeutic targets.

The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed at high levels in human alveolar type II (ATII) cells in the lung. A common polymorphism causing an amino acid substitution (K333Q) was previously linked to a loss of LCAD antigen in the lung tissue in sudden infant death syndrome. However, the effects of the polymorphism on LCAD function has not been tested. The present work evaluated recombinant LCAD K333Q. Compared to wild-type LCAD protein, LCAD K333Q exhibited significantly reduced enzymatic activity. Molecular modeling suggested that K333 is within interacting distance of the essential FAD cofactor, and the K333Q protein showed a propensity to lose FAD. Exogenous FAD only partially rescued the activity of LCAD K333Q. LCAD K333Q protein was less stable than wild-type when incubated at physiological temperatures, likely explaining the observation of dramatically reduced LCAD antigen in primary ATII cells isolated from individuals homozygous for K333Q. Despite the effect of K333Q on activity, stability, and antigen levels, the frequency of the polymorphism was not increased among infants and children with lung disease.

The alveolus participates in gas exchange, which can be impaired by environmental factors and toxins. There is an increase in using electronic cigarettes (e-cigarettes); however, their effect on human primary alveolar epithelial cells is unknown. Human lungs were obtained from nonsmoker organ donors to isolate alveolar type II (ATII) cells. ATII cells produce and secrete pulmonary surfactant and restore the epithelium after damage, and mitochondrial function is important for their metabolism. Our data indicate that human ATII cell exposure to e-cigarette aerosol increased IL-8 levels and induced DNA damage and apoptosis. We also studied the cytoprotective effect of DJ-1 against ATII cell injury. DJ-1 knockdown in human primary ATII cells sensitized cells to mitochondrial dysfunction as detected by high mitochondrial superoxide production, decreased mitochondrial membrane potential, and calcium elevation. DJ-1 knockout (KO) mice were more susceptible to ATII cell apoptosis and lung injury induced by e-cigarette aerosol compared with wild-type mice. Regulation of the oxidative phosphorylation (OXPHOS) is important for mitochondrial function and protection against oxidative stress. Major subunits of the OXPHOS system are encoded by both nuclear and mitochondrial DNA. We found dysregulation of OXPHOS complexes in DJ-1 KO mice after exposure to e-cigarette aerosol, which could disrupt the nuclear/mitochondrial stoichiometry, resulting in mitochondrial dysfunction. Together, our results indicate that DJ-1 deficiency sensitizes ATII cells to damage induced by e-cigarette aerosol leading to lung injury.

Pulmonary emphysema is characterized by airspace enlargement and the destruction of alveoli. Alveolar type II (ATII) cells are very abundant in mitochondria. OXPHOS complexes are composed of proteins encoded by the mitochondrial and nuclear genomes. Mitochondrial 12S and 16S rRNAs are required to assemble the small and large subunits of the mitoribosome, respectively. We aimed to determine the mechanism of mitoribosome dysfunction in ATII cells in emphysema. ATII cells were isolated from control nonsmokers and smokers, and emphysema patients. Mitochondrial transcription and translation were analyzed. We also determined the miRNA expression. Decreases in ND1 and UQCRC2 expression levels were found in ATII cells in emphysema. Moreover, nuclear NDUFS1 and SDHB levels increased, and mitochondrial transcribed ND1 protein expression decreased. These results suggest an impairment of the nuclear and mitochondrial stoichiometry in this disease. We also detected low levels of the mitoribosome structural protein MRPL48 in ATII cells in emphysema. Decreased 16S rRNA expression and increased 12S rRNA levels were observed. Moreover, we analyzed miR4485-3p levels in this disease. Our results suggest a negative feedback loop between miR-4485-3p and 16S rRNA. The obtained results provide molecular mechanisms of mitoribosome dysfunction in ATII cells in emphysema.

DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO2−) and sulfonate (-SO3−) forms. DJ-1 with Cys106-SO2− has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO3−. We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO3− was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO3− DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H2O2 in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H2O2 as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO3− DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond–nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO3− form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.

Background Cigarette smoke is the main risk factor of pulmonary emphysema development, which is characterized by alveolar wall destruction. Mitochondria are important for alveolar type II (ATII) cell metabolism due to ATP generation. Methods We isolated ATII cells from control non-smoker and smoker organ donors, and after lung transplant of patients with emphysema to determine mitochondrial function, dynamics and mitochondrial (mt) DNA damage. Findings We found high mitochondrial superoxide generation and mtDNA damage in ATII cells in emphysema. This correlated with decreased mtDNA amount. We also detected high TOP1-cc and low TDP1 levels in mitochondria in ATII cells in emphysema. This contributed to the decreased resolution of TOP1-cc leading to accumulation of mtDNA damage and mitochondrial dysfunction. Moreover, we used lung tissue obtained from areas with mild and severe emphysema from the same patients. We found a correlation between the impaired fusion and fission as indicated by low MFN1, OPA1, FIS1, and p-DRP1 levels and this disease severity. We detected lower TDP1 expression in severe compared to mild emphysema. Interpretation We found high DNA damage and impairment of DNA damage repair in mitochondria in ATII cells isolated from emphysema patients, which contribute to abnormal mitochondrial dynamics. Our findings provide molecular mechanisms of mitochondrial dysfunction in this disease. Fund This work was supported by National Institutes of Health (NIH) grant R01 HL118171 (B.K.) and the Catalyst Award from the American Lung Association (K.B.).

Emphysema is characterized by alveolar wall destruction induced mainly by cigarette smoke. Oxidative damage of DNA may contribute to the pathophysiology of this disease. We studied the impairment of the non-homologous end joining (NHEJ) repair pathway and DNA damage in alveolar type II (ATII) cells and emphysema development. We isolated primary ATII cells from control smokers, nonsmokers, and patients with emphysema to determine DNA damage and repair. We found higher reactive oxygen species generation and DNA damage in ATII cells obtained from individuals with this disease in comparison with controls. We also observed low phosphorylation of H2AX, which activates DSBs repair signaling, in emphysema. Our results indicate the impairement of NHEJ, as detected by low XLF expression. We also analyzed the role of DJ-1, which has a cytoprotective activity. We detected DJ-1 and XLF interaction in ATII cells in emphysema, which suggests the impairment of their function. Moreover, we found that DJ-1 KO mice are more susceptible to DNA damage induced by cigarette smoke. Our results suggest that oxidative DNA damage and ineffective the DSBs repair via the impaired NHEJ may contribute to ATII cell death in emphysema.

Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1β, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.

Background Infection with the novel severe acute respiratory syndrome coronavirus 2 has been associated with a hypercoagulable state. Emerging data from China and Europe have consistently shown an increased incidence of venous thromboembolism (VTE). We aimed to identify the VTE incidence and early predictors of VTE at our high-volume tertiary care center. Methods We performed a retrospective cohort study of 147 patients who had been admitted to Temple University Hospital with coronavirus disease 2019 (COVID-19) from April 1, 2020 to April 27, 2020. We first identified the VTE (pulmonary embolism [PE] and deep vein thrombosis [DVT]) incidence in our cohort. The VTE and no-VTE groups were compared by univariable analysis for demographics, comorbidities, laboratory data, and treatment outcomes. Subsequently, multivariable logistic regression analysis was performed to identify the early predictors of VTE. Results The 147 patients (20.9% of all admissions) admitted to a designated COVID-19 unit at Temple University Hospital with a high clinical suspicion of acute VTE had undergone testing for VTE using computed tomography pulmonary angiography and/or extremity venous duplex ultrasonography. The overall incidence of VTE was 17% (25 of 147). Of the 25 patients, 16 had had acute PE, 14 had had acute DVT, and 5 had had both PE and DVT. The need for invasive mechanical ventilation (adjusted odds ratio, 3.19; 95% confidence interval, 1.07-9.55) and the admission D-dimer level ≥1500 ng/mL (adjusted odds ratio, 3.55; 95% confidence interval, 1.29-9.78) were independent markers associated with VTE. The all-cause mortality in the VTE group was greater than that in the non-VTE group (48% vs 22%; P = .007). Conclusions Our study represents one of the earliest reported from the United States on the incidence rate of VTE in patients with COVID-19. Patients with a high clinical suspicion and the identified risk factors (invasive mechanical ventilation, admission D-dimer level ≥1500 ng/mL) should be considered for early VTE testing. We did not screen all patients admitted for VTE; therefore, the true incidence of VTE could have been underestimated. Our findings require confirmation in future prospective studies.

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 μg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo. Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3′UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression.

Pulmonary emphysema is characterized by alveolar type II (ATII) cell death, destruction of alveolar wall septa, and irreversible airflow limitation. Cigarette smoke induces oxidative stress and is the main risk factor for this disease development. ATII cells isolated from nonsmokers, smokers, and patients with emphysema were used for this study. ATII cell apoptosis in individuals with this disease was detected. DJ-1 and S100A8 have cytoprotective functions against oxidative stress-induced cell injury. Reduced DJ-1 and S100A8 interaction was found in ATII cells in patients with emphysema. The molecular function of S100A8 was determined by an analysis of the oxidation status of its cysteine residues using chemoselective probes. Decreased S100A8 sulfination was observed in emphysema patients. In addition, its lower levels correlated with higher cell apoptosis induced by cigarette smoke extract in vitro. Cysteine at position 106 within DJ-1 is a central redox-sensitive residue. DJ-1 C106A mutant construct abolished the cytoprotective activity of DJ-1 against cell injury induced by cigarette smoke extract. Furthermore, a molecular and complementary relationship between DJ-1 and S100A8 was detected using gain- and loss-of-function studies. DJ-1 knockdown sensitized cells to apoptosis induced by cigarette smoke extract, and S100A8 overexpression provided cytoprotection in the absence of DJ-1. DJ-1 knockout mice were more susceptible to ATII cell apoptosis induced by cigarette smoke compared with wild-type mice. Our results indicate that the impairment of DJ-1 and S100A8 function may contribute to cigarette smoke-induced ATII cell injury and emphysema pathogenesis.

Rationale Alveolar type II (ATII) cells act as adult stem cells contributing to alveolar type I (ATI) cell renewal and play a major role in idiopathic pulmonary fibrosis (IPF), as supported by familial cases harbouring mutations in genes specifically expressed by these cells. During IPF, ATII cells lose their regenerative potential and aberrantly express pathways contributing to epithelial–mesenchymal transition (EMT). The microRNA miR-200 family is downregulated in IPF, but its effect on human IPF ATII cells remains unproven. We wanted to 1) evaluate the characteristics and transdifferentiating ability of IPF ATII cells, and 2) test whether miR-200 family members can rescue the regenerative potential of fibrotic ATII cells. Methods ATII cells were isolated from control or IPF lungs and cultured in conditions promoting their transdifferentiation into ATI cells. Cells were either phenotypically monitored over time or transfected with miR-200 family members to evaluate the microRNA effect on the expression of transdifferentiation, senescence and EMT markers. Results IPF ATII cells show a senescent phenotype (p16 and p21), overexpression of EMT (ZEB1/2) and impaired expression of ATI cell markers (AQP5 and HOPX) after 6 days of culture in differentiating medium. Transfection with certain miR-200 family members (particularly miR-200b-3p and miR-200c-3p) reduced senescence marker expression and restored the ability to transdifferentiate into ATI cells. Conclusions We demonstrated that ATII cells from IPF patients express senescence and EMT markers, and display a reduced ability to transdifferentiate into ATI cells. Transfection with certain miR-200 family members rescues this phenotype, reducing senescence and restoring transdifferentiation marker expression., Idiopathic pulmonary fibrosis alveolar epithelial type II cells show senescence and EMT features, but miR-200b and miR-200c can restore the ability of type II cells to transdifferentiate in vitro into type I alveolar epithelial cells http://bit.ly/359tlit

Pulmonary emphysema is characterized by alveolar wall destruction, and cigarette smoking is the main risk factor in this disease development. S100A8 is a member of the S100 protein family, with an oxidative stress-related and antiinflammatory role. The mechanisms of human alveolar type II (ATII) cell injury contributing to emphysema pathophysiology are not completely understood. We wanted to determine whether S100A8 can protect ATII cells against injury induced by cigarette smoke and this disease development. We used freshly isolated ATII cells from nonsmoking and smoking organ donors, as well as patients with emphysema to determine S100A8 function. S100A8 protein and mRNA levels were low in individuals with this disease and correlated with its severity as determined by using lung tissue from areas with mild and severe emphysema obtained from the same patient. Its expression negatively correlated with high oxidative stress as observed by 4-hydroxynonenal levels. We also detected decreased serine phosphorylation within S100A8 by PKAα in this disease. This correlated with increased S100A8 ubiquitination by SYVN1. Moreover, we cultured ATII cells isolated from nonsmokers followed by treatment with cigarette smoke extract. We found that this exposure upregulated S100A8 expression. We also confirmed the cytoprotective role of S100A8 against cell injury using gain- and loss-of-function approaches in vitro. S100A8 knockdown sensitized cells to apoptosis induced by cigarette smoke. In contrast, S100A8 overexpression rescued cell injury. Our results suggest that S100A8 protects ATII cells against injury and cigarette smoke-induced emphysema. Targeting S100A8 may provide a potential therapeutic strategy for this disease.

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2–related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

Alveolar type II (ATII) cells synthesize, store, and secrete pulmonary surfactant and restore the epithelium after damage to the alveolar epithelium. Isolation of human ATII cells provides a valuable tool to study their function under normal and pathophysiological conditions. Moreover, maintenance of their differentiated phenotype in vitro allows further study of their function. Here we describe a protocol for efficient ATII cell isolation, characterization, and culture.

Surfactant, a phospholipoprotein produced and stored by alveolar type II (ATII) cells, lowers surface tension in the alveolus and distal airways and is essential for normal lung mechanics and gas exchange.1 Decreases in surfactant production induced by prematurity or disease cause alveolar collapse, hypoxemia and reductions in lung compliance, thereby requiring greater distending pressures to inflate the lung and increases in the work of breathing.2 Prior studies dating back >30 years have shown that alveolar hypocapnia causes depletion of lamellar bodies in ATII cells and decreases surfactant production.3 However, the mechanism(s) underlying the effect(s) of hypocapnia on surfactant secretion by ATII cells are not well understood. The paper by Kiefmann et al 4 in Thorax sheds light on molecular mechanisms which may contribute to the effects of alveolar hypocapnia on surfactant production in vivo. The authors examined surfactant secretion, cytosolic and mitochondrial calcium and alveolar volume and compliance in two rodent preparations: isolated, blood-perfused rat lungs …

Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.

cis-DDP (cis-diamminedichloroplatinu-m(II), CAS 15663-27-1) is widely used in chemotherapy of many types of cancer. However, besides effectiveness, it gives many side effects which limit its clinical application. Therefore, nowadays studies are focused on searching for novel analogs of cis-DDP, at least equally effective in chemotherapy but less toxic. One of them might be cis-BAFDP (cis-bis(3-aminoflavone)dichloroplatinum(II)) with one of the hydrogen atoms of the amino group of cis-DDP replaced by a flavone ring. 3Aminoflavone (AF) which posseses the desired NH2 group has been used as non-leaving ligand. The complex has been obtained in the reaction of AF and K2PtCl4. There are no data concerning evaluation of structural studies of cis-BAFDP, the beneficial anticancer properties of which were proved in vitro and in vivo. Therefore it was worthwhile to undertake a confirmation of the chemical structure of this compound by applying various spectroscopic techniques especially because of its potential pharmacological application. With this aim in mind this compound was characterized by: IR, 1H NMR, 195Pt NMR, UV absorption and fluorescence spectroscopy. Moreover, stronger apoptosis induction by cis-BAFDP than cis-DDP in the human non-small cancer cell line A549 was observed using Hoechst 33258/propidium iodide double staining.

Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5′ extensions showed a high frequency of misrepair in Δ tdp 1 cells. The joining errors in Δ tdp 1 cells were predominantly 2-4 nucleotide insertions. Ends with 3′ extensions or blunt ends did not show enhanced frequencies of errors, although Δ tdp 1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3′ nucleosidase activity regulates the processing of DNA ends by generating a 3′ phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3′ phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3′ phosphate is a key step in Tdp1-mediated error prevention during NHEJ.

Amphotericin B (AmB) is the major antifungal drug used for the treatment of opportunistic fungal pathogens. In this study, we investigate another more subtle effect of this antifungal than ergosterol interaction, the effect of AmB pre-treatment of several fungi on cell surface charge. Pre-treatment of the yeasts with sub-inhibitory concentrations (SIC) of AmB also affects the phagocytic killing of yeasts. Phagocytosis of yeast takes into account the cell surface properties and was greater in yeast treated by AmB than controls. In order to explain this phenomenon, the cell wall charged components were quantified and compared to the control. Results show an increase of Glc-N in cell wall treated cells and reduction of amino acids and phosphorus. In addition, the variation of cell wall components with cell surface properties and the consequences which can be generated on the phagocytosis of the yeasts have been correlated. Results show a significant difference of the zeta-potential, which is a measure of the net distribution of electrical charges of the fungal cell surface between the treated cells and the controls. Pre-treatment of the yeast by AmB produces variations of cell wall component. These events on cell wall composition are correlated to cell surface status.

Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls. A small amount of a beta(1--4) linear homopolymer of GlcNAc in the cell wall forms chitin. To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied. Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions. The results were compared to chemical assays. IR spectra allow identifying the structural types of polysaccharides in yeast walls. Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods. The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.

Yeast strains belonging to the genera Candida, Kluyveromyces and Schizosaccharomyces were tested for their susceptibility (or resistance) to amphotericin B (AmB) in relation to their cell wall chitin content. Results showed that membrane sterol contents did not enable us to explain resistance or susceptibility of these yeasts to AmB. Indeed, we noted that resistant strains were as rich in ergosterol as sensitive strains. The suppression of the wall of yeasts induced an increase in susceptibility to AmB. Strains with high cell wall chitin content were more sensitive to this polyenic antifungal agent than strains with low chitin content. Growth of the yeasts in the presence of chitin induced a resistance of the yeasts to AmB. Similar results were obtained after treatment of the cells by chitinase. In contrast, growth of the yeasts in the presence of chitin synthase activators induced high susceptibility to AmB. Yeast cell wall chitin is an aminopolysaccharide, usually at low concentrations. In Schizosaccharomyces pombe its presence was not established. This polymer is associated with glucans in the wall matrix of the lateral wall and in the budding scars. Even at low content, this polymer seems to play an essential role in the sensitivity (or resistance) of yeast cells to AmB.

Rationale Emphysema is caused by the destruction of alveolar wall septa. The major risk factor for this disease is cigarette smoke and effective therapies are very limited. Alveolar type II (ATII) cells are in the gas exchange portion of the lung. They make and secrete pulmonary surfactant, and proliferate to restore the epithelium after damage to the more sensitive alveolar type I cells. Methods Control ATII cells were isolated from deidentified control non-smoker and smoker organ donors whose lungs were not suitable for transplantation and donated for medical research. Furthermore, as a unique approach, we have developed a new method on how to isolate ATII cells from excess tissue from lung transplants obtained from patients with emphysema using magnetic microbeads. We determined DNA damage, DNA repair, oxidative stress, injury, and inflammation in human primary ATII cells isolated from these individuals in comparison with controls. Results Our data indicates high oxidative stress in human ATII cells induced by cigarette smoke extract in vitro as measured by 4-HNE staining by immunocytofluorescence. We also observed DNA double strand breaks, high DNA damage, and low DNA repair in these cells. Moreover, we found greater proinflammatory response as determined by IL-8 and IL-6 levels by ELISA. Furthermore, ATII cells obtained from patients with emphysema had greater injury compared to cells obtained from control non-smokers and smokers. Conclusion Our results indicate oxidative ATII cell damage induced by cigarette smoke and in emphysema. Observed DNA damage/repair imbalance may contribute to cell death in this disease. The study of ATII cell injury may improve our knowledge on this disease pathogenesis and may lead to novel therapeutic strategies to slow the progression of emphysema. Note: This abstract was not presented at the meeting. Citation Format: Beata Kosmider, Liudmila Vlasenko, Nathaniel Marchetti, Sudhir Bolla, Chenna Mandapati, Nathaniel Xander, Gerard Criner, Karim Bahmed. Impairment of DNA double strand break repair in human primary alveolar type II cells in emphysema [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2017-500

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.

DNA topoisomerase II (Top2) is the target of a chemically diverse set of compounds with substantial antitumor activity. Agents such as doxorubicin and etoposide are FDA approved for the treatment of both leukemias and solid tumors. Most clinically active drugs targeting Top2 lead to elevated levels of covalent Top2-cleaved DNA complexes and exert their antitumor effect through enzyme-mediated DNA damage. Drugs targeting Top2 are accompanied by substantial toxicities including cardiomyopathy following treatment with anthracyclines, and the generation of secondary leukemias. Biochemical and structural studies have illuminated many aspects of the action of drugs targeting topoisomerase II, although the precise molecular details of enzyme inhibition remain poorly understood. This chapter surveys some of the approved agents that target topoisomerase II, as well as some of the newer compounds that may have clinical promise. Although currently used Top2 targeting drugs act by generating enzyme-mediated DNA damage, catalytic inhibition remains a tantalizing possibility. Since current results suggest that topoisomerase IIβ, one of the two mammalian isoforms of topoisomerase II, is important for some of the deleterious effects of Top2 agents, the possibility of isoform-specific inhibitors is also considered.

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5′-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.

Growth of the yeast Kluyveromyces bulgaricus in a glucose peptone liquid medium supplemented or not with calcium led to flocculent and weakly flocculent cells, respectively. Phosphopeptidomannans (PPM), which are cell wall peripheral components, are involved in recognition phenomena as lectin ligands. PPM were extracted from whole yeasts, their chemical composition as well as their molecular mass were determined. The PPM chemical composition was different between flocculent and weakly flocculent cells. Treatment of the PPM with imidazol buffer and/or mercaptoethanol, showed differences of stability. The PPM of weakly flocculating cells were much less stable than that of the high flocculent ones. Infrared spectra of the polymers reinforced that the change of stability may result from an alteration of the glucidic moiety with respect to the peptidic moiety.

The culture of two Kluyveromyces species, Kluyveromyces lactis (ATCC 96897) and Kluyveromyces bulgaricus (ATCC 96631), in the presence of subinhibitory doses of amphotericin B leads to the selection of mutants which are resistant to this polyene. The mutants show an alteration of their cell wall composition with the main change corresponding to an increase of chitin. The enzyme activities involved in the metabolism of this polymer, i.e. chitin synthases and chitinase, were measured. The results demonstrate that in both mutants the activity of chitinase was drastically decreased by 99% in comparison with the activity measured in the corresponding wild-type strain while no significant change of the chitin synthase I, II and III activities could be detected.

Pancreatic cancer is the fourth leading cause of cancer mortality in the U.S. with only a 25% one-year survival rate and 100% mortality rate. No favorable therapeutic strategy has been established in recurrent pancreatic cancer. The cell surface protein CD147 is a glycoprotein and plays a central role in chemoresistance of pancreatic cancer and its upregulation indicates poor prognosis. However, the mechanism by which this protein is involved in pancreatic cancer is still poorly understood. Here we show that knockdown of CD147 in pancreatic cancer cells decelerates tumor growth and sensitizes cells to chemotherapeutic drugs. To identify the mechanisms involved in the slow growth, we screened several genes using a luciferase method. We found that knockdown of CD147 leads to decreased transcription of several genes involved in cancer progression such as telomerase and VEGFC. Knockdown of CD147 leads to high sensitivity of pancreatic cancer cells to chemotherapeutics such as gemcitabine and doxorubicin. Interestingly, the metastatic cell line, L3.6pL, is more sensitive to both chemotherapeutic drugs than the non-metastatic cell line, Panc-1. CD147 knockdown also leads to less drug efflux than control as detected by rhodamine assay. Our hypothesis is that CD147 may interact and block some membrane transporters, which lead to an increase of intracellular drug and therefore to chemosensitivity. We analyzed the transcriptional activity of several membrane transporters including ABCC5, ABCC10 and ABCA2. We found that there is a decrease in their transcription levels of approximately 50% compared to the control. This may suggest that CD147 is involved in drug transport through the cytoplasmic membrane. We have also determined that depleting CD147 leads to an increase of sensitivity in pancreatic cancer cells to chemotherapeutic drugs such as doxorubicin and gemcitabine. Gemcitabine can create DNA damage such as double stand breaks in cells, followed rapidly by histone H2AX phosphorylation (𝛄H2AX), with foci observed within the first minute. Using CD147 knockdown cells, we showed that knockdown of CD147 leads to the depletion of H2AX phosphorylation and to an increase of apoptosis, in agreement with high drug accumulation. We found high ROS levels in these cells as measured by the DCF using flow cytometry. This can explain an increase of apoptosis in cells with CD147 knockdown. Our results indicate that CD147 acts to enhance tumor growth in several ways and contributes to chemoresistance in pancreatic cancer. We explored the mechanism of action of CD147, which can be a potential novel therapeutic target in pancreatic cancer. Citation Format: Karim Bahmed, Michael Holliday, Agnieszka Kendrick, Elan Eisenmesser. Novel role of CD147 in chemoresistance in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 887. doi:10.1158/1538-7445.AM2013-887

The generation of elevated levels of enzyme: DNA covalent complexes is the key event in cell killing by many drugs targeting DNA topoisomerases. These agents, termed topoisomerase poisons generate protein linked DNA strand that block transcription and replication, leading to cell death. A critical step in the repair of topoisomerase mediated DNA damage is the removal of protein that is covalently attached to DNA. Several specialized repair enzymes, including Tdp1 (tyrosyl DNA phosphodiesterase I) and TTRAP (TRAF and TNF receptor-associated protein) can hydrolyze phosphotyrosyl: DNA linkages. We previously reported that yeast Tdp1 could hydrolyze 5′ as well as 3′ phosphotyrosyl linkages. Human Tdp1 can also hydrolyze 5′ phosphotyrosyl linkages, although the efficiency of the reaction with the human enzyme is much less than that seen with the yeast enzyme. Interestingly, the human enzyme processes adducts bearing a seven amino acid peptide linked to an oligonucleotide with greater efficiency than a 5′ biotin linked oligonucleotide, suggesting that the nature of the adduct at DNA ends influences Tdp1 reaction kinetics. We also used substrates derived from Top2 trapped covalent complexes to assess the ability of other DNA repair enzymes to remove peptides covalently bound to DNA. We found that the heterodimeric nuclease Slx1/Slx4 is able to remove Top2 peptides that are covalently bound to DNA. This result is consistent with our genetic data from yeast that a mutation in the subunit that includes the nuclease (Slx1) is hypersensitive to Top2 poisons, but not sensitive to other DNA damaging agents. Our results indicate that there are multiple pathways for repairing Top2 covalent complexes and suggest that the Slx1/Slx4 dependent pathway may be particularly relevant to repairing topoisomerase mediated damage at replication forks. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3500.

Background Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. Methods We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. Results Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. Conclusions We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific.