Loss of caveolin-1 from bronchial epithelial cells and monocytes in human subjects with asthma.

BACKGROUND: Caveolin-1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation. METHODS: Therefore, we investigated whether caveolin-1 is deficient in asthmatic patients and in a murine model of asthma. RESULTS: Immunohistochemical analyses of endobronchial biopsies showed a remarkable loss of caveolin-1 in the lungs of asthmatic patients compared with controls. This loss was most evident in bronchial epithelial cells and associated with an increase in the expression of extracellular matrix proteins: collagen I, tenascin, and periostin. Cultured primary bronchial epithelial cells of asthmatics had lower caveolin-1 expression compared with control cells. In addition, caveolin-1 expression was significantly decreased in peripheral blood monocytes from asthma patients. The loss of caveolin-1 was also observed in a mouse model for asthma (mice sensitized and challenged with aspergillus fumigatus). CONCLUSIONS: To our knowledge, this is the first demonstration that the regulatory protein caveolin-1 is reduced in patients with asthma.

Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells.

The present results demonstrate that the exposure of human myeloid leukemia HL-60 and KG-1 cells to clinically achievable concentrations of taxol produced internucleosomal DNA fragmentation of approximately 200 base-pair multiples, and the morphologic changes characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Taxol-induced PCD was associated with a marked inhibition of suspension culture growth and clonogenic survival of HL-60 cells. In addition, taxol treatment decreased BCL-2 oncogene expression, which is known to block PCD. The exposure to taxol moderately decreased c-myc expression, but did not induce c-jun expression--which has been previously noted for a variety of DNA interactive, antileukemic drugs. These findings indicate that taxol may induce leukemic cell death partly by the alternative but gene-directed and active mechanism of PCD.

Characterization of a human myeloid leukemia cell line highly resistant to taxol.

Taxol-resistant sublines of HL-60 myeloid leukemia cells (HL-60/TAX100 and HL-60/TAX1000) have been isolated in vitro by subculturing in progressively higher concentrations of taxol. HL-60/TAX100 and HL-60/TAX1000 cells are capable of continuous growth in the presence of 0.1 microM and 1.0 microM taxol, respectively, and the IC50 (50% growth inhibitory dose) values for taxol for the two sublines are 0.34 and 2.44 microM as compared to 3.1 nM for the parent HL-60 cells. HL-60/TAX100 and HL-60/TAX1000 cells display a variable degree of cross-resistance to taxotere, vincristine and doxorubicin, but are sensitive to the antimetabolite Ara-C. Both HL-60/TAX100 and HL-60/TAX1000 cells over-express MDR-1 m-RNA and the membrane efflux multidrug transporter P-glycoprotein (PGP), as determined by Western blot and immunofluorescence labeling with anti-PGP antibodies. Consequently, exposure of the taxol-resistant cells to [3H]taxol or daunomycin results in the accumulation of significantly lower levels of the two drugs. Co-treatment with cyclosporine (0.5 microgram/ml) or verapamil (10 microM) partially overcomes taxol resistance in HL-60/TAX1000 cells. Following treatment with clinically relevant concentration of taxol (1.0 microM for 24 h), HL-60 but not HL-60/TAX1000 cells display intracellular microtubular bundling, markedly enhanced accumulation of the cells in G2/M phase of cell-cycle and internucleosomal DNA fragmentation associated with apoptosis which is independent of bcl-2 gene expression. These taxol-resistant myeloid leukemia cells may serve as in vitro experimental models for examinating strategies which may have potential applicability for overcoming taxol resistance.

Effect of combined treatment with interleukin-3 and interleukin-6 on 4-hydroperoxycyclo-phosphamide-induced programmed cell death or apoptosis in human myeloid leukemia cells.

In autologous bone marrow transplantation in patients with acute myeloid leukemia (AML), 4-hydroperoxycyclophosphamide (4-HC) is a commonly used ex vivo purging agent for leukemic blasts. In the present report, we demonstrate that exposure to high concentrations of 4-HC for 1 hour, as used in ex vivo bone marrow purging, produces internucleosomal DNA fragmentation characteristic of apoptosis, or programmed cell death (PCD), in human myeloid leukemia HL60 cells. Lower concentrations of 4-HC (10, 20, or 50 microM/L) failed to cause this effect, while higher concentrations (> or = 200 microM/L) produced random DNA fragmentation. 4-HC-mediated internucleosomal DNA fragmentation was associated with a marked induction in c-jun and significant reductions in bcl-2 and c-myc oncogene expressions. A combined treatment with interleukin-3 (IL-3) plus IL-6 for 18 hours before an additional, 1-hour concurrent treatment with 4-HC (100 microM/L) significantly increased 4-HC-induced DNA fragmentation as well as colony growth inhibition of HL60 cells. The effects of cotreatment with IL-3 plus IL-6 were also associated with a further, modest decrease in bcl-2 and c-myc and augmentation of c-jun expression. These findings highlight an alternative mechanism of 4-HC-induced leukemic cell death that can be potentially enhanced by cotreatment with IL-3 plus IL-6.

Effect of hemopoietic growth factors G-CSF and pIXY 321 on the activity of high dose Ara-C in human myeloid leukemia cells.

Recently, high dose Ara-C (HIDAC) has been shown to induce leukemic cell death in vitro by the alternative process of programmed cell death (PCD) or apoptosis which correlates with the inhibition of their clonogenic survival. Since co-treatment with hemopoietic growth facts (HGFs) GM-CSF and IL-3 have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells, we examined the effect of HGFs pIXY 321 (a GM-CSF/IL3 fusion protein) and G-CSF on HIDAC induced PCD and related gene expressions as well as HIDAC mediated colony growth inhibition of human myeloid leukemia cells. Treatment with G-CSF or pIXY 321 alone for up to 24 hours neither suppressed nor induced PCD in HL-60 or KG-1 cells. However, exposure to either of the HGFs for 20 hours followed by a combined treatment for 4 hours with HIDAC plus either of the HGFs versus HIDAC alone significantly enhanced the intracellular Ara-CTP accumulation and the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of C-jun expression but a significant repression in BCL-2 and c-myc expressions. In addition, the treatment with either of the HGFs plus HIDAC versus HIDAC alone produced a significantly greater inhibition of the clonogenic survival of the myeloid leukemia cells. These findings underscore an additional mechanism of leukemic cell death induced by HIDAC which can be modulated by the HGFs to improve the antileukemic activity of HIDAC.

Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway.

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.

A contaminant of L-tryptophan enhances expression of dermal collagen in a murine model of eosinophilia myalgia syndrome.

The eosinophilia-myalgia syndrome was associated with the ingestion of L-tryptophan products containing a number of contaminants, one of which has been identified as 1,1'-ethylidene-bis-(L-tryptophan) (EBT), also known as peak E or peak 97. In earlier studies, we demonstrated that EBT induces inflammation and fibrosis in dermal and subcutaneous tissue of C57BL/6 mice. Others have shown EBT to be a potent stimulus for fibroblast activation and collagen synthesis in vitro, and dermal tissue from EMS patients reveals evidence of enhanced collagen gene expression. In the present study using Northern blot analysis and in situ hybridization, we demonstrate enhanced expression of genes for types I, III, and VI collagen in the dermis and subcutis of C57BL/6 mice treated with EBT for 3-21 days. Increased type I procollagen mRNA was noted on day 6 of EBT treatment and was followed by enhanced expression of type III and VI procollagen mRNA at day 21. L-Tryptophan, free of contaminants associated with the eosinophilia-myalgia syndrome epidemic, increased dermal collagen mRNA to a lesser extent than did EBT. Increased procollagen gene expression was accompanied by evidence of enhanced TGF-beta 1 expression in the dermis and subcutis. This animal model provides additional evidence for EBT as a causal agent of the eosinophilia-myalgia syndrome and should prove useful in the study of the pathogenesis of that syndrome.

Depletion of protein kinase Cepsilon in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression.

OBJECTIVE: To determine whether the extracellular matrix protein tenascin-C (TN-C) is overexpressed in lung fibroblasts from systemic sclerosis (SSc) patients, the molecular mechanisms regulating TN-C secretion in SSc and normal lung fibroblasts, and how these processes might contribute to lung fibrosis in SSc patients. METHODS: TN-C secretion by SSc and normal fibroblasts was compared in vivo (in bronchoalveolar lavage [BAL] fluid) and in vitro (in culture medium). The ability of thrombin to induce TN-C was confirmed at both the protein and the messenger RNA (mRNA) level. The role of protein kinase Cepsilon (PKCepsilon) in the expression of TN-C was evaluated by determining the effects of thrombin on PKCepsilon levels and by directly manipulating PKCepsilon levels via the use of antisense oligonucleotides. RESULTS: BAL fluid from SSc patients contained high levels of TN-C, whereas that from normal subjects contained little or no TN-C. In vitro, SSc lung fibroblasts expressed much higher amounts of TN-C than did normal lung fibroblasts. Consistent with the idea that thrombin is a physiologic inducer of TN-C, thrombin stimulated TN-C mRNA and protein expression in both SSc and normal lung fibroblasts by a mechanism that required proteolytic cleavage of the thrombin receptor. Surprisingly, thrombin treatment and antisense oligonucleotide-mediated depletion of PKCepsilon indicated that TN-C expression is regulated via opposite signaling mechanisms in SSc and normal cells. In SSc lung fibroblasts, thrombin decreased PKCepsilon levels, and the decreased PKCepsilon induced TN-C secretion; in normal fibroblasts, thrombin increased PKCepsilon levels, and the increased PKCepsilon induced TN-C secretion. Normal and SSc lung fibroblasts also differed in the subcellular localization of PKCepsilon, both before and after thrombin treatment. CONCLUSION: These studies are the first to demonstrate that thrombin is a potent simulator of TN-C in lung fibroblasts and that PKCepsilon is a critical regulator of TN-C protein levels in these cells. Furthermore, our results indicate that both the regulation of PKCepsilon levels by thrombin and the regulation of TN-C levels by PKCepsilon are defective in SSc lung fibroblasts.

High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells.

Mitoxantrone has been shown in vitro to exhibit a steep dose-response relationship with respect to the clonogenic survival of acute myeloid leukemia cells. In this report, we show that 1-hour exposure of human myeloid leukemia HL-60 and KG-1 cells to mitoxantrone concentrations ranging between 0.1 and 10.0 mumol/L induced internucleosomal DNA fragmentation of approximately 200-bp integer multiples, characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Mitoxantrone-mediated PCD was associated with a steep inhibition of the clonogenic survival of the leukemic cells. In addition, intracellularly, mitoxantrone-induced PCD was associated with a marked induction of c-jun and significant repression of c-myc and BCL-2 oncogenes. Pretreatment with the protein kinase C stimulator phorbol myristate acetate enhanced mitoxantrone-induced internucleosomal DNA fragmentation, whereas protein kinase C inhibitors staurosporine and H7 had no effect. These findings suggest that PCD is a potential mechanism underlying the steep dose-response relationship of mitoxantrone to the inhibition of clonogenic survival of acute myeloid leukemia cells.

Thrombin upregulates interleukin-8 in lung fibroblasts via cleavage of proteolytically activated receptor-I and protein kinase C-gamma activation.

Acute and chronic interstitial lung diseases are accompanied by evidence of inflammation and vascular injury. Thrombin activity in bronchoalveolar lavage fluid from such conditions is often increased, as well as interleukin (IL)-8. We observed that conditioned medium from lung fibroblasts exposed to thrombin has chemotactic activity for polymorphonuclear cells, and that this activity can be abolished by antibody to IL-8. We report that thrombin stimulates expression of IL-8 in human lung fibroblasts on both the messenger RNA and protein levels in a time- and dose-dependent manner. Stimulation of IL-8 expression by thrombin is inhibited by specific thrombin inhibitors. Synthetic thrombin receptor agonist peptide-14 mimics thrombin's stimulation of IL-8 expression in a dose-dependent manner consistent with the idea that upregulation of IL-8 by thrombin in human lung fibroblasts requires cleavage of proteolytically activated receptor-I. We demonstrate further that thrombin-induced IL-8 synthesis is regulated by protein kinase (PK) C. PKC-gamma may be involved in the upregulation of lung fibroblast IL-8 by thrombin because stimulation of lung fibroblasts with thrombin caused significant upregulation of PKC-gamma and because PKC-gamma antisense oligonucleotides inhibited the accumulation of PKC-gamma protein and IL-8 protein. Our data suggest that the PKC-gamma isoform increase observed after thrombin stimulation is required for thrombin-induced IL-8 formation by human lung fibroblasts.

Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells.

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.