Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis.

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis.

Castration-resistant prostate cancer (PCa) is refractory to hormone therapy and new strategies for treatment are urgently needed. We found that androgen-insensitive (AI) PCa cells, LNCaP-AI, are reprogrammed to upregulate the mitotic kinase Plk1 (Polo-like kinase 1) and other M-phase cell-cycle proteins, which may underlie AI PCa growth. In androgen-depleted media, LNCaP-AI cells showed exquisite sensitivity to growth inhibition by subnanomolar concentrations of a small molecule inhibitor of Plk1, BI2536, suggesting that these cells are dependent on Plk1 for growth. In contrast, the androgen-responsive parental LNCaP cells showed negligible responses to BI2536 treatment under the same condition. BI2536 treatment of LNCaP-AI cells resulted in an increase in cell death marker PARP-1 (polymerase-1) but did not activate caspase-3, an apoptosis marker, suggesting that the observed cell death was caspase-independent. BI2536-treated LNCaP-AI cells formed multinucleated giant cells that contain clusters of nuclear vesicles indicative of mitotic catastrophe. Live-cell time-lapse imaging revealed that BI2536-treated giant LNCaP-AI cells underwent necroptosis, as evidenced by 'explosive' cell death and partial reversal of cell death by a necroptosis inhibitor. Our studies suggest that LNCaP-AI cells underwent reprogramming in both their cell growth and cell death pathways, rendering them highly sensitive to Plk1 inhibition that induces necroptosis. Harnessing necroptosis through Plk1 inhibition may be explored for therapeutic intervention of castration-resistant PCa.

Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions.

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H(2)O(2)-supported oxidation of N(omega)-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280-Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280-Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.

Flexible bronchoscopy in molecular biology.

Flexible fiberoptic bronchoscopy has allowed researchers to use the bench to bedside approach in the study and therapy of lung diseases. Through bronchoscopy, the lung is a relatively convenient source of samples for the direct evaluation of human gene expression and function. Sampling of respiratory epithelium is performed by brushing with a cytology brush, whereas the epithelial lining fluid and the inflammatory cells in the bronchoalveolar space are obtained by bronchoalveolar lavage. Furthermore, bronchoscopy has been a cornerstone essential to gene therapy trials for lung disease.

Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity.

Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway.

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, and N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited by N(omega)-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitors trans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH(4)Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-kappaB inhibitor IkappaB-alpha, thus preventing activation of NF-kappaB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.

Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis.

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Endothelial nitric oxide synthase as a potential susceptibility gene in the pathogenesis of emphysema in alpha1-antitrypsin deficiency.

A role for endothelial nitric oxide synthase (NOS3) in the susceptibility of individuals with alpha1-antitrypsin (alpha1AT) deficiency to destructive lung disease was evaluated. Six polymorphic sites were identified within the NOS3 gene (i.e., -924A/G, -788C/T, -691C/T, 774C/T, 894G/T, and 1998C/G). The genotype distribution was determined in 339 patients and 94 control individuals. Frequency of the 774T allele in severely affected individuals was 0.417 versus 0.269 in control subjects (P = 0.018), whereas the 894T allele frequency was 0.427 versus 0.280 in control subjects (P = 0.024). Patients with less severe lung disease had the 774T and 894T allele frequencies of 0.289 and 0.344, respectively, similar to frequencies in a control group (P > 0.3). No direct correlation between pulmonary function and five other NOS3 polymorphisms was observed. Thus, functional allelic variants that are in linkage disequilibrium with the 774C/T and 894G/T may be present in the specified genomic area. These data are consistent with a modulatory role for NOS3 in destructive lung disease associated with alpha1AT deficiency.

Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins.

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (DeltaE1) or lacking the Ad E3 region in addition to E1 sequences (DeltaE1DeltaE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34(cdc2) protein levels. In some instances, infection with DeltaE1 or DeltaE1 DeltaE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DeltaE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in DeltaE1 or DeltaE1 DeltaE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.

Gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemia-reperfusion lung injury.

Hyperoxia and ischemia-reperfusion cause profound lung cellular damage mediated, in part, by generation of oxygen radicals. We hypothesized that gene therapy can be used to overcome oxidant injury by augmenting intracellular antioxidant enzymes. Adult rats were injected intratracheally with an adenovirus (Ad) vector encoding human superoxide dismutase (CuZn-SOD) or catalase cDNA, a mixture of both Ad vectors, or a control Ad vector containing no exogenous gene. Expression of human catalase and CuZn-SOD was demonstrated 3 days later in distal lung epithelial cells and alveolar macrophages, using ELISA and immunochemistry. After exposure to 100% O2 for 62 hr, survival was greater in rats injected with the catalase and/or SOD Ad vectors than in control rats. Ischemia-reperfusion injury was evaluated in the isolated perfused lung model. Overexpression of SOD worsened ischemia-reperfusion injury. Interestingly, concomitant overexpression of catalase prevented this adverse effect, but did not protect against ischemia-reperfusion injury. We conclude that Ad-mediated transfer to lungs of both catalase and SOD cDNAs protects from pulmonary O2 toxicity. Absence of protection against ischemia-reperfusion using intratracheal Ad injections may be related to the lack of endothelial protection, despite epithelial expression of catalase and SOD.

Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization.

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

Superoxide-mediated actin response in post-hypoxic endothelial cells.

The mechanism leading to changes in the superstructure of endothelial cells exposed to ischemia and reperfusion remains uncharacterized. We show that in post-hypoxic endothelial cells, the simple re-addition of oxygen induces a profound reorganization of the actin cytoskeleton. The total filamentous actin pool increases by 41% and translocation of actin filaments to the submembranous network is observed. Concurrent with the actin polymerization, increased tyrosine phosphorylation of endothelial cell substrates is detected on Western blots. Overexpression of superoxide dismutase using replication incompetent adenovirus inhibits the actin and tyrosine phosphorylation responses to reoxygenation. Inhibition of tyrosine kinases with the isoflavone genistein also suppressed the actin polymerization response to reoxygenation, but unlike superoxide dismutase, genistein also induced the collapse of the superstructure of endothelial cells upon reoxygenation. These experiments support the concept that reoxygenation following a period of hypoxia can induce the remodeling of the actin cytoskeleton in endothelial cells. Such a response requires the intact coupling of superoxide producing pathway(s) with tyrosine kinase pathway(s).

Alternative splicing of human inducible nitric-oxide synthase mRNA. tissue-specific regulation and induction by cytokines.

Human inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells, and several human tissues was transcribed to cDNA and analyzed using polymerase chain reaction with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: (i) exon 5; (ii) exons 8 and 9; (iii) exons 9, 10, and 11; and (iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein (resulting from exon 5 deletion) and three iNOS proteins with in-frame deletions. Southern analyses of polymerase chain reaction products were consistent with tissue-specific regulation of alternative splicing. In cultured cells, iNOS induction by cytokines and lipopolysaccharide was associated with an increase in alternatively spliced mRNA transcripts. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of nitric oxide synthesis.

Structural diversity in the 5'-untranslated region of cytokine-stimulated human inducible nitric oxide synthase mRNA.

Inducible nitric oxide synthase, the critical enzyme responsible for the enhanced synthesis of nitric oxide in inflammatory states, is widely expressed in mammalian cells. To evaluate potential regulatory roles of the 5'-untranslated region (UTR) in the human inducible nitric oxide synthase gene, the transcription initiation sites and structure of the 5'-UTR of human inducible nitric oxide synthase were examined. Freshly isolated human alveolar macrophages, bronchial epithelial cells, and several types of cultured cells were evaluated following stimulation with cytokines (i.e. interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6). The mRNA was analyzed by reverse transcription-polymerase chain reaction. Northern analysis, and 5'-rapid amplification of cDNA ends. Despite the presence of a TATA box in the promoter region, multiple transcription initiation sites were observed, some extending several hundred base pairs upstream from the main TATA-directed initiation site. Alternative splicing in the 5'-UTR of human inducible nitric oxide synthase mRNA resulted in further diversity. The TATA-independent inducible nitric oxide synthase mRNA transcripts were up-regulated by cytokines. The long and complex 5'-UTRs contain eight partially overlapping open reading frames upstream of the putative inducible nitric oxide synthase ATG, which may have an important role in translational regulation of human inducible nitric oxide synthase mRNA.

Gene therapy for the respiratory manifestations of cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major manifestations are on the airway epithelial surface, with purulent mucus, recurrent infections, chronic inflammation, and loss of lung function. Consequent to mutations in both parental genes, airway epithelial cells have insufficient CFTR function. Because this can be corrected in vitro by transfer of the normal CFTR gene into airway epithelial cells, it is reasonable to hypothesize that the respiratory manifestations of CF could be prevented by transfer of the normal human CFTR cDNA to the airway epithelium in vivo. Over the past 6 years, our laboratory has developed a strategy to accomplish this goal using a replication deficient E1-E3- recombinant adenovirus (Ad) serotype 5 vector containing the normal human CFTR cDNA (AdCFTR). Studies with experimental animals demonstrate that with administration of such a vector to the airways, the human CFTR cDNA could be transferred to the airway epithelium, with expression of the human CFTR cDNA for at least 6 weeks. Extensive preclinical studies in vitro and in vivo demonstrated that the risks to humans were sufficiently low to initiate a Phase I trial using the AdCFTR vector to treat the respiratory manifestations of CF in humans. Following approval by the National Heart, Lung, and Blood Institute Institutional Review Board, the National Institutes of Health Biosafety Committee, the National Institutes of Health Recombinant DNA Advisory Committee, and the Food and Drug Administration, we initiated the first human trial of gene therapy for CF on April 17, 1993. The clinical study is still ongoing, with safety and efficacy data being evaluated, but there is clear evidence that it is feasible to transfer and express the normal CFTR cDNA to the airway epithelium in vivo in individuals with CF.

Evaluation of the respiratory epithelium of normals and individuals with cystic fibrosis for the presence of adenovirus E1a sequences relevant to the use of E1a- adenovirus vectors for gene therapy for the respiratory manifestations of cystic fibrosis.

Lung disease associated with disorders such as cystic fibrosis (CF) may be amenable to somatic gene therapy in which there is delivery of the normal gene directly to the respiratory epithelium using E1a- adenovirus (Ad) type 2- or 5-based vectors. For safety reasons, the Ad vectors are rendered replication deficient by deletion of the E1a region. Because there is the theoretical possibility of an E1a- replication-deficient vector replicating as a result of recombination or complementation with Ad 2/5 E1a sequences present in the target cell, this study is directed toward evaluating respiratory epithelium of normals and individuals with CF for the presence of E1a sequences. Using Ad 2/5 E1a-specific primers and the polymerase chain reaction to evaluate DNA recovered from freshly isolated nasal and bronchial epithelium recovered by brushing, E1a sequences were detected in respiratory epithelium of 19 of 91 normals (21%). In the E1a-positive samples, the average of E1a copy number was 55 +/- 18/10(3) recovered cells. In CF individuals, 7 of 52 (13%) had detectable E1a sequences in the respiratory epithelium, with E1a copy number in the positive samples of 80 +/- 21/10(3) recovered cells. These results demonstrate that there are detectable Ad 2/5 E1a sequences in the respiratory epithelium of a small percentage of normals and individuals with CF. Because of the theoretical potential of such sequences supporting replication of E1a- Ad vectors, human gene therapy protocols for CF utilizing such vectors should consider evaluating study individuals for the presence of Ad 2/5 E1a sequences in the respiratory epithelium.

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 x 10(9) pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 x 10(9) pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6-12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Partitioning of work of breathing in mechanically ventilated COPD patients.

In 10 sedated paralyzed mechanically ventilated chronic obstructive pulmonary disease (COPD) patients, we measured the inspiratory mechanical work done per breath on the respiratory system (WI,rs). We measured the tracheal and esophageal pressures to assess the lung (L) and chest wall (W) components of WI and used the technique of rapid airway occlusion during constant-flow inflation to partition WI into static work [Wst, including work due to intrinsic positive end-expiratory pressure (WPEEPi)], dynamic work due to airway resistance, and the additional resistance offered by the respiratory tissues. Although the patients were hyperinflated, the slope of the static volume-pressure relationships of the lung did not decrease with inflation volume up to 0.8 liter. WI,W was similar in COPD patients and normal subjects. All components of WI,L were higher in COPD patients. The increase in Wst,rs was due entirely to WPEEPi. Our data suggest that, during spontaneous breathing, COPD patients would probably develop inspiratory muscle fatigue, unless continuous positive airway pressure were applied to reduce WPEEPi.

Lung and chest wall mechanics in mechanically ventilated COPD patients.

By use of the technique of rapid airway occlusion, the effects of inspiratory flow, volume, and time on lung and chest wall mechanics were investigated in 10 chronic obstructive pulmonary disease (COPD) patients mechanically ventilated for acute respiratory failure. We measured the interrupter resistance (Rint), which in humans reflects airway resistance; the additional resistances due to time constant inequality and viscoelastic pressure dissipations within the lungs (delta RL) and the chest wall; and the static and dynamic elastances of lung and chest wall. We observed that 1) static elastances of lung and chest wall in COPD patients were similar to those of normal subjects; 2) Rint of the lung was markedly increased and flow dependent in COPD patients, whereas Rint of the chest wall was negligible as in normal subjects; and 3) in COPD patients, delta RL was markedly increased at all inflation flows and volumes, reflecting increased time constant inequalities within the lungs and/or altered viscoelastic behavior. The results imply increased dynamic work due to Rint and delta RL and marked time dependency of pulmonary resistance and elastance in COPD patients.