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    It should be noted that patch clamp and other types of experiments with isotonic concentrations of HCO 3 - are not trivial and may be compromised by pH fluctuations Kunzelmann et al. Quinton and others provided further evidence that bicarbonate transport is essential for proper mucus release and viscosity Choi et al. In fact, HCO 3 - transport is impaired in a number of different epithelial tissues derived from CF patients Kunzelmann et al. Importantly, human lung pathology was brilliantly reproduced in a CF pig model. Using this CF pig model, reduced airway surface liquid pH, impaired bacterial killing, and mucus abnormalities were demonstrated Pedersen et al.

    Along this line it appears noteworthy that treatment with the anti-inflammatory drug ibuprofen was able to rescue trafficking mutant Fdel-CFTR Carlile et al. Large phase 3 studies demonstrated a moderate improvement in the percentage of predicted FEV1 between 2.

    Studies with next generation compounds, e. Recent clinical trials reported great therapeutic success with triple combinations of the corrector VX with VX or VX, together with the potentiator VX Despite such great success in correcting mutant CFTR, critical voices were raised regarding a combinatorial drug treatment.

    Finally, the costs for treatment by these drugs are often prohibitive Ferkol and Quinton, Thus, there is a need for alternative drug treatments. A number of other strategies to correct biosynthesis of misfolded CFTR are currently under investigation Recent screening efforts identified the translation initiation factor 3a eIF3a as a potentially druggable central hub for the biogenesis of CFTR Hutt et al.

    TMEM16A is upregulated in secretory cells strongly contributing to mucus secretion. The majority of these double-barreled channels are operating as phospholipid scramblases, i.

    TMEM16A is typically localized in the apical plasma membrane of epithelial cells. However, it is also found to be expressed basolateral and in intracellular compartments Schreiber et al.

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    Although endogenous TMEM16 proteins are mostly localized intracellularly, overexpression of these proteins together with purinergic receptors allows partial trafficking to the plasma membrane Tian et al. Upregulation of TMEM16A is predominant in mucus producing cells and to a much lesser degree in ciliated epithelial cells Huang et al.

    While this may be explained by the limited sensitivity of the available antibodies, it also raises questions as to what degree other members of the TMEM16 family might participate in CaCC. As mentioned above, expression of TMEM16A in normal adult airways is hardly detectable by immunohistochemistry Ousingsawat et al.

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    Ciliated epithelial cells, and particularly the recently identified ionocytes express CFTR and are in charge of fluid secretion Montoro et al. Expression of TMEM16A is low in ciliated cells when compared to mucus producing club and goblet cells.

    The contribution of TMEM16A to overall fluid secretion by the airway epithelium might therefore be limited, while it plays a central role for basal mucus secretion Benedetto et al. This will be further outlined below. Distal airways of a control mouse and an ovalbumin-sensitized asthmatic mouse. Blue, DAPI staining of nuclei. Analysis of freshly isolated human nasal epithelial cells demonstrates ATP-induced steady-state secretion only in non-CF cells, but not in CF nasal cells.

    This has already been described in a number of previous studies Huang et al. Airway inflammation is well-known to induce hyperresponsiveness of ASM Brightling et al. A similar exocytic mechanism may apply to the process of mucus secretion by club and goblet cells. In inflammatory CF airway disease, the function of TMEM16A may be marginal in ciliated cells and ionocytes, but may be pronounced in secretory cells due to strong upregulation of expression. According to this, pharmacological activation of TMEM16A in CF and asthma patients could have adverse effects on lung function due to its prosecretory effect on mucus release.

    Airway narrowing was confirmed by analysis of the airway cross section Benedetto et al.

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    Control mouse airways show very little mucus alcian blue. In contrast, airways from ovalbumin-sensitized mice show pronounced mucus accumulation. Denufusol was developed by Inspire pharmaceuticals later taken over by Merck. Cells were treated with different doses of dacarbazine DTIC for 1, 2 or 3 days, and cell viability relative to control analyzed using the 4-methylumbelliferyl heptanoate MUH assay.

    Figure S4.

    Side population SP cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases.

    In addition, the SP reconstituted the heterogeneous composition of the human A melanoma cell line, and its clonogenic activity was 2.

    Gene-expression analysis revealed upregulated expression in the melanoma SP versus the MP of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors.

    Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC.

    The melanoma SP may therefore represent an interesting therapeutic target. Resistance to chemotherapy is a major cause of treatment failure, and better knowledge of the melanoma chemoresistant cells can open the way to more efficient therapies.

    In several types of cancer, the so-called side population SP enriches for chemoresistant cells [ 2 — 4 ]. Because of this dye expulsion, the SP is portrayed as a side-branch of Hoechstlow cells in dual-wavelength flow cytometry FACS [ 2 — 6 ]. Moreover, SP phenotype and chemoresistance are proposed characteristics of cancer stem cells CSC , a subpopulation within the tumor that holds the highest capacity to drive growth and progression with invasion and metastasis of the tumor [ 2 — 4 ] and reviewed in 11 — Recent studies clearly support the existence of CSC in cancers, their chemoresistant nature and functional relevance [ 14 — 17 ].

    Very recently, Luo et al. In the present study, we analyzed a larger series of human melanoma specimens covering a wide range of progression phases, explored its prognostic potential, determined genome-wide expression in primary melanomas directly from the patient, and tested resistance to dacarbazine, still the most commonly used single-agent chemotherapeutic in advanced-melanoma therapy [ 23 ].

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    In addition, resistance to hypoxia, and tumorigenic and clonogenic potential were investigated. Together, our analyses point toward enrichment of the human melanoma SP in chemoresistant and tumorigenic activity. Results Human melanoma contains a side population In a recent study by Luo et al.

    A SP was detected in all melanoma samples analyzed, representing 0.

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