Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Incidence and clinicobiologic characteristics of leukemic B-cell chronic lymphoproliferative disorders with more than one B-cell clone.

Leukemic B-chronic lymphoproliferative disorders (B-CLPDs) are generally believed to derive from a monoclonal B cell; biclonality has only occasionally been reported. In this study, we have explored the incidence of B-CLPD cases with 2 or more B-cell clones and established both the phenotypic differences between the coexisting clones and the clinicobiologic features of these patients. In total, 53 B-CLPD cases with 2 or more B-cell clones were studied. Presence of 2 or more B-cell clones was suspected by immunophenotype and confirmed by molecular/genetic techniques in leukemic samples (n = 42) and purified B-cell subpopulations (n = 10). Overall, 4.8% of 477 consecutive B-CLPDs had 2 or more B-cell clones, their incidence being especially higher among hairy cell leukemia (3 of 13), large cell lymphoma (2 of 10), and atypical chronic lymphocytic leukemia (CLL) (4 of 29). In most cases the 2 B-cell subsets displayed either different surface immunoglobulin (sIg) light chain (n = 37 of 53) or different levels of the same sIg (n = 9 of 53), usually associated with other phenotypic differences. Compared with monoclonal cases, B-CLL patients with 2 or more clones had lower white blood cell (WBC) and lymphocyte counts, more frequently displayed splenomegaly, and required early treatment. Among these, the cases in which a CLL clone coexisted with a non-CLL clone were older and more often displayed B symptoms, a monoclonal component, and diffuse infiltration of bone marrow and required early treatment more frequently than cases with monoclonal CLL or 2 CLL clones.

Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry.

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) studies are useful in multiple myeloma (MM). However, the definition of the best technique and clinical utility are still unresolved issues. The aim of this study was to analyze and compare the clinical utility of MRD studies in MM with two different techniques: allelic-specific oligonucleotide real-time quantitative PCR (ASO-RQ-PCR), and flow cytometry (FCM). DESIGN AND METHODS: Bone marrow samples from 32 MM patients who had achieved complete response after transplantation were evaluated by ASO-RQ-PCR, using TaqMan technology, and multiparametric FCM. RESULTS: ASO-RQ-PCR was only applicable in 75% of patients for a variety of technical reasons, while FCM was applicable in up to 90%. Therefore, simultaneous PCR/FCM analysis was possible in only 24 patients. The number of residual tumor cells identified by both techniques was very similar (mean=0.29%, range=0.001-1.61%, correlation coefficient=0.861). However, RQ-PCR was able to detect residual myelomatous cells in 17 patients while FCM only did so in 11; thus, 6 cases were FCM negative but PCR positive, all of them displaying a very low number of clonal cells (median=0.014%, range=0.001-0.11). Using an MRD threshold of 0.01% (10(-4)) two risk groups with significantly different progression-free survival could be identified by either PCR (34 vs. 15m, p=0.04) or FCM (27 vs. 10m, p=0.05). INTERPRETATION AND CONCLUSIONS: Although MRD evaluation by ASO-RQ-PCR is slightly more sensitive and specific than FCM, it is applicable in a lower proportion of MM patients and is more time-consuming, while both techniques provide similar prognostic information.