Global identification of genes targeted by DNMT3b for epigenetic silencing in lung cancer.

The maintenance cytosine DNA methyltransferase DNMT1 and de novo methyltransferase DNMT3b cooperate to establish aberrant DNA methylation and chromatin complexes to repress gene transcription during cancer development. The expression of DNMT3b was constitutively increased 5-20-fold in hTERT/CDK4-immortalized human bronchial epithelial cells (HBECs) before treatment with low doses of tobacco carcinogens. Overexpression of DNMT3b increased and accelerated carcinogen-induced transformation. Genome-wide profiling of transformed HBECs identified 143 DNMT3b-target genes, many of which were transcriptionally regulated by the polycomb repressive complex 2 (PRC2) complex and silenced through aberrant methylation in non-small-cell lung cancer cell lines. Two genes studied in detail, MAL and OLIG2, were silenced during transformation, initially through enrichment for H3K27me3 and H3K9me2, commonly methylated in lung cancer, and exert tumor suppressor effects in vivo through modulating cancer-related pathways. Re-expression of MAL and OLIG2 to physiological levels dramatically reduced the growth of lung tumor xenografts. Our results identify a key role for DNMT3b in the earliest stages of initiation and provide a comprehensive catalog of genes targeted for silencing by this methyltransferase in non-small-cell lung cancer.

Aerosolised 5-azacytidine suppresses tumour growth and reprogrammes the epigenome in an orthotopic lung cancer model.

BACKGROUND: Epigenetic silencing by promoter methylation and chromatin remodelling affects hundreds of genes and is a causal event for lung cancer. Treatment of patients with low doses of the demethylating agent 5-azacytidine in combination with the histone deacetylase inhibitor entinostat has yielded clinical responses. The subcutaneous dosing route for consecutive days and reduced bioavailability of 5-azacytidine because of inactivation by cytidine deaminase may limit the expansion of epigenetic therapy into Phase III trials. To mitigate these barriers, an aerosol of 5-azacytidine was generated and characterised. METHODS: The effect of aerosol vs systemic delivery of 5-azacytidine on tumour burden and molecular response of engrafted lung tumours in the nude rat was compared. RESULTS: Pharmacokinetics revealed major improvement in the half-life of 5-azacytidine in lung tissue with aerosol delivery. Aerosolised 5-azacytidine significantly reduced lung tumour burden and induced global demethylation of the epigenome at one-third of the comparable effective systemic dose. High commonality for demethylation of genes was seen in tumours sampled throughout lung lobes and across treated animals receiving the aerosolised drug. CONCLUSION: Collectively, these findings show that aerosolised 5-azacytidine targets the lung, effectively reprogrammes the epigenome of tumours, and is a promising approach to combine with other drugs for treating lung cancer.

Health care utilization in chronic obstructive pulmonary disease. A case-control study in a health maintenance organization.

BACKGROUND: Information about health care utilization and costs among patients with chronic obstructive pulmonary disease (COPD) is needed to improve care and for appropriate allocation of resources for patients with COPD (COPD patients or cases) in managed care organizations. METHODS: Analysis of all inpatient, outpatient, and pharmacy utilization of 1522 COPD patients continuously enrolled during 1997 in a 172,484-member health maintenance organization. Each COPD case was matched with 3 controls (n = 4566) by age (+/-5 years) and sex. Information on tobacco use and comorbidities was obtained by chart review of 200 patients from each group. RESULTS: Patients with COPD were 2.3 times more likely to be admitted to the hospital at least once during the year, and those admitted had longer average lengths of stay (4.7 vs 3.9 days; P<.001). Mean costs per case and control were $5093 vs $2026 for inpatient services, $5042 vs $3050 for outpatient services, and $1545 vs $739 for outpatient pharmacy services, respectively (P<.001 for all differences). Patients with COPD had a longer smoking history (49.5 vs 34.9 pack-years; P =.002) and a higher prevalence of smoking-related comorbid conditions and were more likely to use cigarettes during the study period (46.0% vs 13.5%; P<.001). CONCLUSIONS: Health care utilization among COPD patients is approximately twice that of age- and sex-matched controls, with much of the difference attributable to smoking-related diseases. In this health maintenance organization, inpatient costs were similar to and outpatient costs were much higher than national averages for COPD patients covered by Medicare.

Utilization in COPD: patient characteristics and diagnostic evaluation.

STUDY OBJECTIVES: Information on current practices of COPD diagnosis and treatment is needed to identify opportunities for improving care. This study describes the clinical characteristics and diagnostic evaluations of COPD patients in a health maintenance organization (HMO) and a university-affiliated county medical center (UMC). DESIGN: Cross-sectional survey performed in a 174,484-member regional HMO and in The University of New Mexico Hospitals and Clinics (UNMH). PATIENTS: Two hundred COPD patients from each system randomly selected from administrative databases based on discharge diagnoses. RESULTS: COPD patients in the UMC, compared to those in the HMO, were younger (mean age, 59.3 vs 66.9 years, respectively), were more likely to be using home oxygen (33% vs 20%, respectively), and had fewer chronic medical conditions (mean number of conditions, 3.1 vs 3.7, respectively) (p < 0.01 for all differences). Approximately half of the COPD patients in both groups continued to smoke cigarettes during the study year. Only 38% of patients in the HMO and 42% in the UNMH system had spirometry results documented in their medical records. CONCLUSIONS: The demographic and clinical characteristics of the COPD patients in these two health-care systems were very different, but smoking status and utilization of diagnostic tests were similar. The diagnosis of COPD in most patients was based only on a history of chronic respiratory symptoms and smoking; spirometry often was not used to confirm the diagnosis. An increased emphasis on smoking cessation and more effective utilization of spirometry are needed to improve the management of COPD in these health-care systems.

Asthma in a Vietnamese refugee population.

Asians and Pacific Islanders comprise a large and growing minority group in the United States, yet data on health status specific to these populations are scant. We conducted an epidemiologic study of asthma in a Vietnamese refugee population to estimate prevalence, evaluate risk factors, and better understand treatments of asthma among Vietnamese individuals. One hundred twenty-four asthma cases were identified from a population of 2,536 new Vietnamese refugees in San Diego (prevalence = 49 per 1,000; 4.9%). Two nonasthmatic control groups of Vietnamese refugees, matched for age and gender with the asthma cases, were recruited for a case-control study, using a questionnaire administered in Vietnamese. Vietnamese asthmatic individuals used both Western and non-Western therapies. Most subjects used traditional health practices, such as coining, cupping, and oil inhalation. As compared with current-refugee controls, the asthmatic subjects used significantly more bleeding (OR: 3.40; 95% CI: 1.06 to 10.80) and herbal ingestion (OR: 1.87; 95% CI: 1.08 to 3.19). As compared with former-refugee controls, the asthmatic subjects used significantly more oil inhalation (OR: 2.58; 95% CI: 1.45 to 4.85), bleeding (OR: 8.64, 95% CI: 1.02 to 73.70), and herbal ingestion (OR: 1.93; 95% CI: 1.02 to 3.67). The presentation and recognition of asthma among the Vietnamese subjects were similar to those in other populations. This information may be helpful in designing culture-specific health-education programs.

Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.

Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.

Transfected cDNA directs expression of hemolytically active murine C4 in cultured mouse and monkey cells.

Previously cloned and sequenced full-length cDNAs for murine C4 and the closely related sex-limited protein (Slp) have been placed into an eucaryotic expression vector. Transfer of these DNA constructs transiently into monkey COS cells or stably into mouse L cells results in the expression and secretion of hemolytically active mouse C4 and mature Slp. We estimate from hemolytic activities that COS and L cells secrete 0.04 and 3%, respectively, of the C4 level found in mouse plasma. Slp expression is consistently only 10-20% that of C4 although the identical expression system is used for both. Our results show subtle but reproducible cell-type-specific differences in C4 maturation; they also indicate that surprisingly large shifts in electrophoretic mobility on SDS-polyacrylamide gels are induced by a small number of amino acid substitutions. The expression of C4 from cDNAs of known sequence provides a starting point for studies of structure/function relationships in C4 employing site-specific mutagenesis and gene transfer.