The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.

Purpose: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis.Experimental Design: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated.Results: In histologically normal squamous mucosa, no to low-level methylation (0 -22%) was noted in some specimens at all genes except RAR␤2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RAR␤2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RAR␤, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas.Conclusions: Methylation of p16, RAR␤, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.

Purpose: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. Experimental Design: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARb2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. Results: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK , or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8 %) adenoid cystic carcinomas, 6 (33.3 %) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0 %) salivary duct carcinomas. RASSF1 and RARb2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). Conclusions: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARb2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.Salivary gland neoplasms are composed of histopathologically and clinically diverse entities of disputed histogenesis and unpredictable behavior (1 -3). Efforts to identify biomarkers that assist in diagnosing these tumors and in explaining their evolution and progression have been unrewarding (4 -6). The underlying reasons for such slow progress in understanding these tumors include rarity, failure to grow cell lines, clinical and methodologic interstudy differences, and the lack of a phenotypic progression model of their evolution. However, recent molecular genetic studies have provided important information on common chromosomal loci and genes alterations in a spectrum of the tumors (7 -15). These data are critical in advancing our understanding of their biology and identifying new targets of potential therapeutic applications.Epigenetic alterations may also play a role in the development of these tumors. A particular advantage of these alterations is that, unlike genetic alterations, they are reversible and can be restored in vitro and in vivo by using demethylating agents in cell lines and tumors (16 -19). Methylation is one of the epigenetic modifications that play an important role in the transcriptional inactivation of tumor suppressor genes in human cancer (20). Several m...

The N-formyl peptide chemoattractant receptor (fMLF-R) is a cell-surface, G-protein-coupled glycoprotein that mediates the directed locomotion of neutrophils upon binding N-formylated peptides. The fMLF-R is encoded primarily by a 1.6-kb mRNA in differentiated HL-60 and U937 cells, although larger less abundant transcripts are present. To study the origin of different fMLF-R transcripts, the genetic linkage of chemotactic receptor genes, and the regulation of fMLF-R gene expression, we determined the copy number, chromosomal location, structural organization, and 5'-flanking sequence of the human fMLF-R gene. BamHI restriction fragments derived from a human fMLF-R genomic cosmid clone were isolated, subcloned, and sequenced. These data indicate that the fMLF-R structural gene is approximately 7.5 kb in length and is comprised of two exons separated by an approximately 5.0-kb intron. The first exon encodes 66 bp of the 5'-untranslated sequence, while exon 2 encodes the coding and 3'-untranslated sequences. The genomic organization of the fMLF-R gene is similar to that of the adrenergic beta-1 and beta-2 G-protein-coupled receptor genes in that the coding sequence is contained in a single exon. The different 3'-untranslated sequences observed in fMLF-R cDNA clones are contiguous in the genomic structure, thereby indicating that these clones are derived in part by alternative polyadenylation. Southern blot analysis using human X hamster somatic cell hybrids and in situ hybridization indicated that the h-fMLF-R gene is located on chromosome 19q13.3. Primer extension experiments using dbcAMP-differentiated U937 RNA indicated a single transcriptional initiation site. Sequence analysis 5' of the transcriptional initiation site indicated possible cis-acting motifs that may regulate fMLF-R gene expression. These included AP-1 and CK-2 consensus sequences that bind nuclear factors of the Fos/Jun family and NF-GMb, respectively.

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