Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (−) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER− and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER− and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER− cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER− human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF–EGFR interaction is mediated via activation of nuclear factor κB (NF-κB). The basal level of active NF-κB in ER− cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-κB activation factor. NF-κB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER− cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294–002, blocked this event, suggesting a role of the former in the activation of NF-κB by EGF. Go6976, a well-characterized NF-κB inhibitor, blocked EGF-induced NF-κB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER− cells. Thus Go6976 and similar NF-κB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER− breast cancer patients.

Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1.

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G1 phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G1 phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A- and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is required for DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G1 and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells.

Total glycans from the cell layer and the culture medium of human vascular smooth muscle cells (VSMC) that had been cultivated in the presence of platelet-derived growth factor (PDGF) were isolated and purified by gel filtration after Pronase and DNase digestion and alkaliborohydride treatment. Measurements of the content of neutral hexoses and uronic acids revealed that PDGF stimulates total glycan synthesis by proliferating VSMC in a linear fashion from 24 h to 72 h of incubation. In contrast, total glycan synthesis by human fibroblasts, epithelial cells, or endothelial cells was not affected by PDGF, indicating cell-type specificity. Chemical, biochemical, and enzymological characterization of the total glycans synthesized by VSMC showed that PDGF stimulates the secretion of a 340-kDa glycan molecule in a time-dependent manner from 24 h to 72 h. This molecule is highly acidic, shares a common structure with hyaluronic acid, and exhibits a potent antiproliferative activity on VSMC. These results suggest that VSMC in response to PDGF are capable of controlling their own growth and migration by the synthesis of a specific form of hyaluronic acid with antiproliferative potency, which may be involved in the regulation of the local inflammatory responses associated with atherosclerosis.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study

BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study

Abstract BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays. (C) 2005 Elsevier SAS. All rights reserved.

Toward the discovery of vaccine adjuvants:coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Antigen Drives Regulatory B10 Cell Development and Function

B cells mediate immune responses via the secretion of antibody and interactions with other immune cell populations through antigen presentation, costimulation, and cytokine secretion. Although B cells are primarily believed to promote immune responses using the mechanisms described above, some unique regulatory B cell populations that negatively influence inflammation have also been described. Among these is a rare interleukin (IL)-10-producing B lymphocyte subset termed “B10 cells.” B cell-derived IL-10 can inhibit various arms of the immune system, including polarization of Th1/Th2 cell subsets, antigen presentation and cytokine production by monocytes and macrophages, and activation of regulatory T cells. Further studies in numerous autoimmune and inflammatory models of disease have confirmed the ability of B10 cells to negatively regulate inflammation in an IL-10-dependent manner. Although IL-10 is indispensable to the effector functions of B10 cells, how this specialized B cell population is selected in vivo to produce IL-10 is unknown. Some studies have demonstrated a link between B cell receptor (BCR)-derived signals and the acquisition of IL-10 competence. Additionally, whether antigen-BCR interactions are required for B cell IL-10 production during homeostasis as well as active immune responses is a matter of debate. Therefore, the goal of this thesis is to determine the importance of antigen-driven signals during B10 cell development in vivo and during B10 cell-mediated immunosuppression.

Chapter 3 of the dissertation explored the BCR repertoire of spleen and peritoneal cavity B10 cells using single-cell sequencing to lay the foundation for studies to understand the full range of antigens that may be involved in B10 cell selection. In both the spleen and peritoneal cavity B10 cells studied, BCR gene utilization was diverse, and the expressed BCR transcripts were largely unmutated. Thus, B10 cells are likely capable of responding to a wide range of foreign and self-antigens in vivo.

Studies in Chapter 4 determined the predominant antigens that drive B cell IL-10 secretion during homeostasis. A novel in vitro B cell expansion system was used to isolate B cells actively expressing IL-10 in vivo and probe the reactivities of their secreted monoclonal antibodies. B10 cells were found to produce polyreactive antibodies that bound multiple self-antigens. Therefore, in the absence of overarching active immune responses, B cell IL-10 is secreted following interactions with self-antigens.

Chapter 5 of this dissertation investigated whether foreign antigens are capable of driving B10 cell expansion and effector activity during an active immune response. In a model of contact-induced hypersensitivity, in vitro B cell expansion was again used to isolate antigen-specific B10 clones, which were required for optimal immunosuppression.

The studies described in this dissertation shed light on the relative contributions of BCR-derived signals during B10 cell development and effector function. Furthermore, these investigations demonstrate that B10 cells respond to both foreign and self-antigens, which has important implications for the potential manipulation of B10 cells for human therapy. Therefore, B10 cells represent a polyreactive B cell population that provides antigen-specific regulation of immune responses via the production of IL-10.