Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the U.S. Because cigarette smoking is so importantly implicated in the pathogenesis of COPD and because mucus hypersecretion plays such an important role in COPD, understanding of the mechanisms of smoking-induced mucus hypersecretion could lead to new therapies for COPD. Cigarette smoke causes mucin overproduction via EGF receptor (EGFR) in airway epithelial cells, but the cellular mechanism remains unknown. Airway epithelial cells contain EGFR proligands on their surfaces, which can be cleaved by metalloprotease and subsequently bind to EGFR resulting in mucin production. We hypothesize that TNF-alpha-converting enzyme (TACE) is activated by cigarette smoke, resulting in increased shedding of EGFR proligand, leading to EGFR phosphorylation and mucin induction in human airway epithelial (NCI-H292) cells. Here we show that cigarette smoke increases MUC5AC production in NCI-H292 cells, an effect that is prevented by an EGFR-neutralizing antibody and by specific knockdown of transforming growth factor-alpha (TGF-alpha) using small interfering RNA (siRNA) for TGF-alpha, implicating TGF-alpha-dependent EGFR activation in the responses. Cigarette smoke increases TGF-alpha shedding, EGFR phosphorylation, and mucin production, which are prevented by metalloprotease inhibitors (GM-6001 and TNF-alpha protease inhibitor-1) and by specific knockdown of TACE with TACE siRNA, implicating TACE in smoking-induced responses. Furthermore, pretreatment with antioxidants prevents smoking-induced TGF-alpha shedding and mucin production, suggesting that reactive oxygen species is involved in TACE activation. These results implicate TACE in smoking-induced mucin overproduction via the TACE-proligand-EGFR signal pathway in NCI-H292 cells.     

CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.     

Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation.

Eosinophil recruitment and mucus hypersecretion are characteristic of asthmatic airway inflammation, but eosinophils have not been shown to induce mucin production. Because an epidermal growth factor receptor (EGFR) cascade induces MUC5AC mucin in airways, and because EGFR is up-regulated in asthmatic airways, we examined the effect of eosinophils on MUC5AC mucin production in NCI-H292 cells (a human airway epithelial cell line that produces mucins). Eosinophils were isolated from the peripheral blood of allergic patients, and their effects on MUC5AC mucin gene and protein synthesis were assessed using in situ hybridization and ELISAs. When IL-3 plus GM-CSF or IL-3 plus IL-5 were added to eosinophils cultured with NCI-H292 cells, MUC5AC mucin production increased; eosinophils or cytokines alone had no effect. Eosinophil supernatant obtained by culturing eosinophils with IL-3 plus GM-CSF or IL-3 plus IL-5 also increased MUC5AC synthesis in NCI-H292 cells, an effect that was prevented by selective EGFR inhibitors (AG1478, BIBX1522). Supernatant of activated eosinophils induced EGFR phosphorylation in NCI-H292 cells. Supernatant of activated eosinophils contained increased concentrations of TGF-alpha protein (an EGFR ligand) and induced up-regulation of TGF-alpha expression and release in NCI-H292 cells. A blocking Ab to TGF-alpha reduced activated eosinophil-induced MUC5AC synthesis in NCI-H292 cells. These results show that activated eosinophils induce mucin synthesis in human airway epithelial cells via EGFR activation, and they implicate TGF-alpha produced by eosinophils and epithelial cells in the EGFR activation that results in mucin production in human airway epithelium.     

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

Amphiregulin potentiates airway inflammation and mucus hypersecretion induced by urban particulate matter via the EGFR-PI3Kα-AKT/ERK pathway

Ambient particulate matter (PM) promotes the development and exacerbation of chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma, by increasing inflammation and mucus hypersecretion. However, the biological mechanisms underlying PM-induced airway inflammation and mucus hypersecretion remain unclear. Amphiregulin (AREG) is an important ligand for epidermal growth factor receptor (EGFR) and participates in the regulation of several biological functions. Here, the PM-exposed human bronchial epithelial cell (HBEC) model was used to define the role of AREG in PM-induced inflammation and mucus hypersecretion and its related signaling pathways. The expression of AREG was significantly increased in a dose-dependent manner in HBECs subjected to PM exposure. Moreover, PM could induce inflammation and mucus hypersecretion by upregulating the expression of IL-1α, IL-1β, and Muc-5ac in HBECs. The EGFR, AKT, and ERK signaling pathways were also activated in a time- and dose-dependent manner. The AREG siRNA markedly attenuated PM-induced inflammation and mucus hypersecretion, and activation of the EGFR-AKT/ERK pathway. Exogenous AREG significantly increased the expression of IL-1α, IL-1β, and Muc-5ac, and induced activation of the EGFR-AKT/ERK pathway in HBECs. Further, under PM exposure, exogenous AREG significantly potentiated PM-induced inflammation and mucus hypersecretion, and activation of the EGFR-AKT/ERK pathway. Tumor-necrosis factor-alpha converting enzyme (TACE) and EGFR specific inhibitor pretreatment showed that AREG was secreted by TACE-mediated cleavage to regulate PM-induced inflammation and mucus hypersecretion by binding to the EGFR. Moreover, according to the inhibitory effect of specific inhibitors of the class I PI3K isoforms, AKT and ERK, PM-induced inflammation and mucus hypersecretion was regulated by PI3Kα activation and its downstream AKT and ERK pathways. This study strongly suggests the adverse effect of AREG in PM-induced inflammation and mucus hypersecretion via the EGFR-PI3Kα-AKT/ERK pathway. These findings contribute to a better understanding of the biological mechanisms underlying exacerbation of chronic respiratory diseases induced by PM exposure.     

LPS-stimulated MUC5AC production involves Rac1-dependent MMP-9 secretion and activation in NCI-H292 cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients’ serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.     

IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils

Mucus hypersecretion contributes to the morbidity and mortality in acute asthma. Both T helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR) signaling have been implicated in allergen-induced goblet cell (GC) metaplasia. Present results show that a cascade of EGFR involving neutrophils is implicated in interleukin (IL)-13-induced mucin expression in GC. Treatment with a selective EGFR tyrosine kinase inhibitor prevented IL-13-induced GC metaplasia dose dependently and completely. Instillation of IL-13 also induced tumor necrosis factor-alpha protein expression, mainly in infiltrating neutrophils. Control airway epithelium contained few leukocytes, but intratracheal instillation of IL-13 resulted in time-dependent leukocyte recruitment by IL-13-induced IL-8-like chemoattractant expression in airway epithelium. Pretreatment with an inhibitor of leukocytes in the bone marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented both IL-13-induced leukocyte recruitment and GC metaplasia. These findings indicate that EGFR signaling is involved in IL-13-induced mucin production. They suggest a potential therapeutic role for inhibitors of the EGFR cascade in the hypersecretion that occurs in acute asthma.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death

In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.     

Stretch-induced fetal type II cell differentiation is mediated via ErbB1-ErbB4 interactions

Stretch-induced differentiation of lung fetal type II epithelial cells is mediated through EGFR (ErbB1) via release of HB-EGF and TGF-α ligands. Employing an EGFR knock-out mice model, we further investigated the role of the ErbB family of receptors in mechanotranduction during lung development. Deletion of EGFR prevented endogenous and mechanical stretch-induced type II cell differentiation via the ERK pathway, which was rescued by overexpression of a constitutively active MEK. Interestingly, the expression of ErbB4, the only ErbB receptor that EGFR co-precipitates in wild-type cells, was decreased in EGFR-deficient type II cells. Similar to EGFR, ErbB4 was activated by stretch and participated in ERK phosphorylation and type II cell differentiation. However, neuregulin (NRG) or stretch-induced ErbB4 activation were blunted in EGFR-deficient cells and not rescued after ErbB4 overexpression, suggesting that induction of ErbB4 phosphorylation is EGFR-dependent. Finally, we addressed how shedding of ligands is regulated by EGFR. In knock-out cells, TGF-α, a ligand for EGFR, was not released by stretch, while HB-EGF, a ligand for EGFR and ErbB4, was shed by stretch although to a lower magnitude than in normal cells. Release of these ligands was inhibited by blocking EGFR and ERK pathway. In conclusion, our studies show that EGFR and ErbB4 regulate stretch-induced type II cell differentiation via ERK pathway. Interactions between these two receptors are important for mechanical signals in lung fetal type II cells. These studies provide novel insights into the cell signaling mechanisms regulating ErbB family receptors in lung cell differentiation.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization

Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin (E-cad). Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Here, we showed that p120ctn modulates smoke-induced cell migration via the EGFR/Src-P pathway. Chemical blockade of EGFR/Src signaling inhibited smoke-induced activation of cofilin (an actin severing protein) and promoted cell migration in the presence of p120ctn but had little effect on blocking migration in the absence of p120ctn. These data suggested that smoke-induced cell migration was mediated via an EGFR/Src-dependent signaling pathway in cells that expressed p120ctn, but upon loss of p120ctn, migration continued to occur via an alternative, EGFR/Src-independent pathway. Thus, gradual loss of membrane p120ctn with lung cancer progression may contribute to reduced effectiveness of conventional chemotherapies, such as those directed against EGFR.     

In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

Signaling via the type 1 insulin-like growth factor receptor (IGF1R) confers resistance to EGF receptor (EGFR) inhibitors. It is plausible that reciprocal EGFR compensation could mediate resistance to IGF1R inhibition, prompting us to investigate effects of IGF1R depletion on EGFR signaling in breast cancer cells expressing relatively high (MDA-MB-468) or low (MCF7) EGFR. Transient IGF1R knockdown induced enhanced phosphorylation of the EGFR and its effectors JNK, ERKs and STAT5, but this did not prevent apoptosis induction and inhibition of clonogenic survival following IGF1R knockdown. We used IGF1R shRNA to induce chronic IGF1R depletion, and achieved stable gene silencing in MCF-7 cells; here, EGFR overexpression led to EGFR hyperphosphorylation, again without abrogating survival inhibition after IGF1R knockdown. In both cell lines, dual receptor knockdown prevented EGFR hyperphosphorylation, but induced no greater inhibition of clonogenic survival than IGF1R knockdown alone. These results suggest that the EGFR cannot compensate for IGF1R depletion, and are encouraging for the strategy of IGF1R targeting.     

Dual inhibition of focal adhesion kinase and epidermal growth factor receptor pathways cooperatively induces death receptor-mediated apoptosis in human breast cancer cells

The focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) are protein-tyrosine kinases that are overexpressed and activated in human breast cancer. To determine the role of EGFR and FAK survival signaling in breast cancer, EGFR was stably overexpressed in BT474 breast cancer cells, and each signaling pathway was specifically targeted for inhibition. FAK and EGFR constitutively co-immunoprecipitated in EGFR-overexpressing BT474 cells. In low EGFR-expressing BT474-pcDNA3 vector control cells, inhibition of FAK by the FAK C-terminal domain caused detachment and apoptosis via pathways involving activation of caspase-3 and -8, cleavage of poly(ADP-ribose) polymerase, and caspase-3-dependent degradation of AKT. This apoptosis could be rescued by the dominant-negative Fas-associated death domain, indicating involvement of the death receptor pathway. EGFR overexpression did not inhibit detachment induced by the FAK C-terminal domain, but did suppress apoptosis, activating AKT and ERK1/2 survival pathways and inhibiting cleavage of FAK, caspase-3 and -8, and poly(ADP-ribose) polymerase. Furthermore, this protective effect of EGFR signaling was reversed by EGFR kinase inhibition with AG1478. In addition, inhibition of FAK and EGFR in another breast cancer cell line (BT20) endogenously overexpressing these kinases also induced apoptosis via the same mechanism as in the EGFR-overexpressing BT474 cells. The results of this study indicate that dual inhibition of FAK and EGFR signaling pathways can cooperatively enhance apoptosis in breast cancers.     

Measurement of proliferation nuclear and membrane markers in tumor cells by flow cytometry

Nuclear and membrane markers that have been related to proliferative activity were measured by flow cytometry. The markers studied were transferrin receptor (TR), Ki-67 antigen, and epidermal growth factor receptor (EGFR). Two-color analysis for DNA via propidium iodide binding and for antigen expression via either a direct or indirect immunofluorescence assay was performed on three different cell lines and a solid human tumor model. The three cell lines tested were MCF-7 (breast), K-562 (leukemia), and A431 (a squamous cell). The solid tumor was obtained by subcutaneous injection of A431 cells into an athymic nude mouse. Our results demonstrate that TR are cell-cycle specific and can be readily measured in the cell lines. Ki-67 antigen is also cell-cycle specific in the cell lines tested, but the mean channel specific fluorescence uptake varies in the cell types. Finally, the EGFR was observed only in the A431 cell line, with most cells equally expressing this receptor. A bimodal distribution of EGFR was observed in A431 cells obtained from a solid tumor grown in an athymic nude mouse system. This suggests that cell line analysis may not always represent what might be observed under in vivo conditions. There are advantages to flow cytometry measurements of these factors which might be useful in predicting how patients should be treated and possibly the prognosis of cancer patients.