Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

Zinc-finger E-box-binding homeobox 1 (ZEB1) plays a crucial role in the maintenance of lung cancer stem cells resistant to gefitinib.

BACKGROUND: Zinc-finger E-box-binding homeobox 1 (ZEB1) is an important regulator of epithelial-mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR-200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT-mediated acquired resistance to gefitinib in EGFR-mutant non-small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. METHODS: PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as "gefitinib-resistant persisters" (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. RESULTS: GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR-200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR-200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133- and BMI1-positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR-mutant NSCLC patients with gefitinib resistance. CONCLUSIONS: ZEB1 plays an important role in gefitinib-resistant lung CSCs with EMT features via regulation of miR-200c and BMI1.

Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal neoplasms. Pulmonary manifestations frequently develop earlier than other organ involvements, prompting a diagnosis of BHDS However, the mechanism of lung cyst formation and pathogenesis of pneumothorax have not yet been clarified. Fibroblasts were isolated from lung tissues obtained from patients with BHDS (n = 12) and lung cancer (n = 10) as controls. The functional abilities of these lung fibroblasts were evaluated by the tests for chemotaxis to fibronectin and three-dimensional (3-D) gel contraction. Fibroblasts from BHDS patients showed diminished chemotaxis as compared with fibroblasts from controls. Expression of fibronectin and TGF-ß1 was significantly reduced in BHDS fibroblasts when assessed by qPCR Addition of TGF-ß1 in culture medium of BHDS lung fibroblasts significantly restored these cells' abilities of chemotaxis and gel contraction. Human fetal lung fibroblasts (HFL-1) exhibited reduced chemotaxis and 3-D gel contraction when FLCN expression was knocked down. To the contrary, a significant increase in chemotactic activity toward to fibronectin was demonstrated when wild-type FLCN was overexpressed, whereas transduction of mutant FLCN showed no effect on chemotaxis. Our results suggest that FLCN is associated with chemotaxis in lung fibroblasts. Together with reduced TGF-ß1 expression by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung fibroblast dysfunction, thereby impairing tissue repair. These may reveal one mechanism of lung cyst formation and pneumothorax in BHDS patients.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14-17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.

Treatment with insulin-like growth factor 1 receptor inhibitor reverses hypoxia-induced epithelial-mesenchymal transition in non-small cell lung cancer.

Insulin-like growth factor 1 receptor (IGF1R) is expressed in many types of solid tumors including non-small cell lung cancer (NSCLC), and enhanced activation of IGF1R is thought to reflect cancer progression. Epithelial-mesenchymal transition (EMT) has been established as one of the mechanisms responsible for cancer progression and metastasis, and microenvironment conditions, such as hypoxia, have been shown to induce EMT. The purposes of this study were to address the role of IGF1R activation in hypoxia-induced EMT in NSCLC and to determine whether inhibition of IGF1R might reverse hypoxia-induced EMT. Human NSCLC cell lines A549 and HCC2935 were exposed to hypoxia to investigate the expression of EMT-related genes and phenotypes. Gene expression analysis was performed by quantitative real-time PCR and cell phenotypes were studied by morphology assessment, scratch wound assay, and immunofluorescence. Hypoxia-exposed cells exhibited a spindle-shaped morphology with increased cell motility reminiscent of EMT, and demonstrated the loss of E-cadherin and increased expression of fibronectin and vimentin. Hypoxia also led to increased expression of IGF1, IGF binding protein-3 (IGFBP3), and IGF1R, but not transforming growth factor ß1 (TGFß1). Inhibition of hypoxia-inducible factor 1α (HIF1α) with YC-1 abrogated activation of IGF1R, and reduced IGF1 and IGFBP3 expression in hypoxic cells. Furthermore, inhibition of IGF1R using AEW541 in hypoxic condition restored E-cadherin expression, and reduced expression of fibronectin and vimentin. Finally, IGF1 stimulation of normoxic cells induced EMT. Our findings indicated that hypoxia induced EMT in NSCLC cells through activation of IGF1R, and that IGF1R inhibition reversed these phenomena. These results suggest a potential role for targeting IGF1R in the prevention of hypoxia-induced cancer progression and metastasis mediated by EMT.

Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor.

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as "gefitinib-resistant persisters" (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1-all genes involved in stemness-were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.

Hypoxia induces gefitinib resistance in non-small-cell lung cancer with both mutant and wild-type epidermal growth factor receptors.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non-small-cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild-type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor-α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia-inducible factor-1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild-type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR-mutant NSCLC through the activation of wild-type EGFR mediated by the upregulation of TGFα. The presence of wild-type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.

Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm.

The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

Sex hormones alter Th1 responses and enhance granuloma formation in the lung.

BACKGROUND: The lung is one of the sites of granulomatous responses, which are characterized by the recruitment and organization of activated macrophages and lymphocytes. There have been several reports that have shown that some pulmonary granulomatous diseases, such as sarcoidosis and nontuberculous mycobacterial disease, are likely to be characterized by a preponderance in postmenopausal females. Although sex hormones have been shown to play an important role in the regulation of the immune system, the influence of sex hormones on pulmonary granuloma formation is still unclear. OBJECTIVES: The purpose of this study was to assess whether sex hormones are involved in granulomatous inflammation and to evaluate how sex hormones modulate this response in the lung. METHODS: Ovariectomized rats were used as an experimental postmenopausal model in which chronic pulmonary granulomatous inflammation was induced by intravenous injection of complete Freund's adjuvant. RESULTS: Histological analysis of lung tissues demonstrated enhancement of granuloma formation in the ovariectomized group. Such enhanced granuloma formation was significantly associated with generalized Th1-biased cytokine production in the bronchoalveolar lavage fluid. CONCLUSION: These results indicate that sex hormones play an important role in pulmonary granuloma formation by altering the Th1 responses.

Secretory protein with RING finger domain (SPRING) specific to Trypanosoma cruzi is directed, as a ubiquitin ligase related protein, to the nucleus of host cells.

While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi. This gene was specific to T. cruzi and was designated spring (secretory protein with RING finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro. These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells.

Protein kinase A regulatory subunit interacts with P-Type ATPases in Trypanosoma cruzi.

Cyclic AMP-protein kinase A (PKA) signaling is important for the growth and differentiation of Trypanosoma cruzi. Immunofluorescence suggests that PKA can associate with the plasma membrane of trypomastigotes. We found that the PKA regulatory subunit interacts with several P-type ATPases. These P-type ATPases may play a role in anchoring PKA to the plasma membrane in T. cruzi.

Interaction between cFLIP and Itch, a ubiquitin ligase, is obstructed in Trypanosoma cruzi-infected human cells.

Death receptor-mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite-infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP(L), a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP(L,) leading to proteasomal degradation, is inhibited in parasite-infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP(L), was found to be equivalent in T. cruzi-infected and in uninfected cells. However, co-immunoprecipitation analysis showed that the interaction between cFLIP(L) and Itch is strongly inhibited in T. cruzi-infected cells. This unique parasite strategy, which has not been reported in any other pathogen-infected cells, may allow the host cell to accumulate cFLIP(L), eventually resulting in the inhibition of apoptosis of T. cruzi-infected cells.

Molecular cloning and characterization of ouabain-insensitive Na(+)-ATPase in the parasitic protist, Trypanosoma cruzi.

Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na(+) efflux is generally governed by P-type ATPases, Na(+)/K(+)-ATPase in animals and Na(+)-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na(+) concentrations outside and low Na(+) concentrations inside host cells. However, T. cruzi Na(+) efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na(+)-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na(+) concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na(+) and K(+) and is insensitive to ouabain, a specific inhibitor of Na(+)/K(+)-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na(+) stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist.

Gene expression profiles in response to Fas stimulation in Trypanosoma cruzi-infected host cells.

To determine the molecular mechanism by which apoptosis is inhibited in Trypanosoma cruzi-infected host cells, we used human cDNA apoptosis chips to compare the gene expression profiles in response with 'death ligands target' (Fas) stimulation in infected and uninfected cells. Of the 164 apoptosis-related genes examined, 20, including those encoding both pro- and anti-apoptotic proteins, were highly up-regulated in the infected group. Genes encoding caspases and apoptosis inhibitors were optimally expressed 10-30 min after induction of apoptosis, whereas genes involved in transcriptional regulation and cell proliferation were up-regulated after 2-24 h. These results suggest that host anti-apoptotic gene(s) may play a crucial role in the inhibition of Fas-mediated apoptosis in T. cruzi-infected cells.

Trypanosoma cruzi posttranscriptionally up-regulates and exploits cellular FLIP for inhibition of death-inducing signal.

Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically up-regulates cellular FLICE inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, posttranscriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T. cruzi-infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T. cruzi posttranscriptionally up-regulates and exploits host c-FLIP for the inhibition of death-inducing signal, a mechanism that may allow parasites to persist in host cells.

Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli.

The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.