Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.
Cells from the acini of human tracheal glands were grown in culture to produce confluent cell sheets of mucous or mixed seromucous phenotype. Levels of mediator-induced Cl secretion in mucous cells were 2-18% those of seromucous cells. Levels of the cystic fibrosis transmembrane conductance regulator (an apical membrane Cl channel) were also much less in mucous than in seromucous cells. These results suggest that serous cells are more important than mucous cells in providing the fluid component of gland secretions.
Hepatocyte growth factor (HGF) can influence epithelial cell growth and differentiation. We examined the actions of recombinant human HGF (rhHGF) on the differentiation of human primary tracheal epithelial (HTE) cells cultured in vitro for up to 96 h. Basolateral, but not apical, treatment of confluent HTE cell sheets for 48 h with rhHGF led to increases in cell height, cell volume, cilia, and total protein content. Basolateral rhHGF treatment produced a decrease in HGF receptor (c-met) expression but had no effect on c-met mRNA levels. HTE cell sheets treated with rhHGF for 48 h showed a significant increase in mediator-induced Cl- secretion and a decrease in amiloride-sensitive sodium absorption. No effect on transepithelial resistance was observed with rhHGF treatment. The enhancement of short-circuit responses by basolateral rhHGF was dose dependent. Our results demonstrate that rhHGF has hypertrophic actions on, and can influence the differentiation of, human airway epithelia in vitro, presumably through the activation of c-met at the basolateral surface of these cells.
We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising trastuzumab, a stable thioether linker, and the maytansine derivative DM1, a microtubule inhibitor. T-DM1 has been evaluated for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer in several phase 2 trials. Unexpectedly, the dose-limiting toxicity in patients treated with T-DM1 was dose- and duration-dependent thrombocytopenia, even though HER2 is not detected on platelets. To understand the mechanism of T-DM1-induced thrombocytopenia, we evaluated the effect of T-DM1 on platelet (PLT) function and formation from hematopoietic stem cells (HSCs). Using PLT-rich plasma (PRP) or washed PLT (WP) from healthy human donors, PLT function was assessed by aggregometry and flow cytometry to evaluate activation markers (PAC1; CD62P) in the presence of T-DM1, trastuzumab, control ADC (anti-CD22 conjugated to DM1), free DM1, or vehicle. Human HSCs (CD133+/CD34+) were purified from healthy donor bone marrow, expanded in hematopoietic expansion media, and transferred to megakaryocyte (Mk) differentiation media for 14 days to obtain mature Mks, in the presence of T-DM1, trastuzumab, ADC control (5B6, anti-gD conjugated to DM1), and vehicle. T-DM1 uptake and catabolism in Mks were evaluated by incubating the cells with T-[3H]DM1. Incubation with T-DM1, free DM1, or any controls at clinically relevant concentrations had no observable effect on platelet aggregation in PRP or WP and no direct impact on PLT activation (PAC1 binding or CD62P expression). Neither T-DM1 nor free DM1 inhibited PLT aggregation and activation induced by the PLT agonists collagen and TRAP6. Treatment of mature Mks with T-DM1 and control ADC resulted in a decrease in Mk number and a significant decrease (∼3-fold) in Mk production from HSC precursors, as measured by a reduction in CD41+/CD61+ cells. Incubation of Alexa488-conjugated trastuzumab and T-DM1 with Mks resulted in surface binding and internalization as assessed by immunofluorescence and flow cytometry. Preincubation with anti-CD32 decreased Alexa488-conjugated T-DM1 antibody binding and uptake (∼2-fold), suggesting that FcR IIb contributes to this process. To determine the fate of the internalized antibody, we quantified the uptake and catabolism of T-DM1 in Mks using T-[3H]DM1. Total cellular T-[3H]DM1 increased over the 3-day incubation with peak concentrations occurring during the first 8 hours of incubation, and a peak total uptake in the Mks of 0.4% of the total dose. The metabolized intracellular⌝-free [3H]DM1 levels were 0.1% of the total dose. By quantification of the HSCs and various ploidy populations, T-DM1 was found to primarily inhibit Mk production from HSCs rather than directly impact mature Mks. Furthermore, the similarity of effects between T-DM1 and 5B6-DM1 suggests that the DM1 moiety is responsible for the observed platelet reduction. Our experiments show that T-DM1 did not have a direct effect on PLT function but did impair Mk and PLT production. DM1-conjugated antibodies were internalized in a target-independent, partially Fc-dependent manner into Mks, and intracellular release of DM1 resulted in a reduction of the stem cell population. These data support the hypothesis that the thrombocytopenia observed in clinical trials may be a consequence of impaired PLT production from Mks in the bone marrow. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A135.
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