Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells.

We investigated how cystic fibrosis (CF) alters the relationship between Cl(-) and mucin secretion in cultures of non-CF and CF human tracheobronchial gland mucous (HTGM and CFTGM, respectively) cells. Biochemical studies showed that HTMG cells secreted typical airway mucins, and immunohistochemical studies showed that these cells expressed MUC1, MUC4, MUC5B, MUC8, MUC13, MUC16, and MUC20. Effects of cumulative doses of methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP on mucin and Cl(-) secretion were studied on HTGM and CFTGM cultures. Baseline mucin secretion was not significantly altered in CFTGM cells, and the increases in mucin secretion induced by mediators were unaltered (Iso, Phe) or slightly decreased (MCh, ATP). Across mediators, there was no correlation between the maximal increases in Cl(-) secretion and mucin secretion. In HTGM cells, the Cl(-) channel blocker, diphenylamine-2-carboxylic acid, greatly inhibited Cl(-) secretion but did not alter mucin release. In HTGM cells, mediators (10(-5) M) increased mucin secretion in the rank order ATP > Phe = Iso > MCh. They increased Cl(-) secretion in the sequence ATP > MCh ≈ Iso > Phe. The responses in Cl(-) secretion to MCh, ATP, and Phe were unaltered by CF, but the response to Iso was greatly reduced. We conclude that mucin secretion by cultures of human tracheobronchial gland cells is independent of Cl(-) secretion, at baseline, and is unaltered in CF; that the ratio of Cl(-) secretion to mucus secretion varies markedly depending on mediator; and that secretions induced by stimulation of ß-adrenergic receptors will be abnormally concentrated in CF.

Wnt and Hedgehog are critical mediators of cigarette smoke-induced lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype. CONCLUSIONS/SIGNIFICANCE: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies.

Tobacco smoke control of mucin production in lung cells requires oxygen radicals AP-1 and JNK.

In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFR-independent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK.

DNA methylation regulates the expression of Y chromosome specific genes in prostate cancer.

PURPOSE: We hypothesized that DNA methylation regulates the differential expression of Y chromosome specific genes in prostate cancer. To test this hypothesis we analyzed the expression of Y chromosome specific genes in 5-aza-2'-deoxycytidine (5-azaC) treated and untreated prostate cancer cell lines. MATERIALS AND METHODS: To test this hypothesis Y chromosome specific genes were analyzed in prostate cancer cells treated with the demethylation agent 5-azaC. Total RNA was extracted and reverse transcribed, and polymerase chain reaction was performed using gene specific primers. These primers were designed based on the sequence available in the public genome data bank. The 10 Y chromosome specific genes DAZ, CDY, SRY, RBMY1A, RBMY1H, RBMII, BPY1, BPY2, PRY and TSPY were analyzed in the PC3, ND1, DU145, LNCaP, TSUPr1 and DUPro prostate cancer cell lines by reverse transcriptase-polymerase chain reaction. Normal testis RNA was used as a positive control. RESULTS: Of the 10 Y chromosome specific genes DAZ gene expression was lacking in all prostate cancer cell lines but after demethylation treatment with 5-azaC DAZ expression was restored. The SRY gene was also lacking in all prostate cancer cell lines except LNCaP. After demethylation SRY gene expression was restored in PC3, ND-1, DU-145, TSUPr1 and DUPro. There was no expression of the CDY and BPY2 genes before and after 5-azaC treatment in all prostate cancer cell lines. Expression of the RBMY1A, RBMY1H and RBMII genes was lacking in all prostate cancer cell lines but after demethylation the expression of all 3 was restored in the ND1, DU-145 and LNCaP cell lines. The BPY1 gene was only expressed in LNCaP cells but after treatment with 5-azaC all other cell lines, namely PC3, ND1, DU145, LNCaP and DUPro, restored BPY gene expression. PRY gene expression was lacking in all prostate cancer cell lines but after demethylation only LNCaP restored expression of this gene. TSPY was expressed only in LNCaP but after demethylation ND-1 cells restored expression of the TSPY gene. CONCLUSIONS: To our knowledge we report the first study showing that expression of the Y chromosome specific genes DAZ, SRY, RBMY1A, RBMY1H, RBMII, BPY1, PRY and TSPY is regulated by DNA methylation in prostate cancer.