Dielectric spectroscopy of mammalian cells

The capacitance of suspensions of CHO and HeLa cells (0.5–3×10⁶ cells/ml) has been measured between 0.2 and 10 MHz. As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes. At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only. The intensity of this signal varies linearly with the biomass and cell size.At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5×10⁶ cells/ml can be accurately measured.Suggestions are made how to make these measures on-line, non-invasive and in real time.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

Evaluation of the function of human invariant NKT cells from cancer patients using alpha-galactosylceramide-loaded murine dendritic cells

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-gamma, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-gamma. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated alpha-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.     

Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.     

Dielectric characterization of bacterial cells using dielectrophoresis

Measurements of dielectrophoretic collection spectra of Escherichia coli and Staphylococcus aureus suspensions are used for obtaining dielectric characteristics of both types of bacteria. The experiments are interpreted using a numerical method that models the cells as compartmented spherical or rod-like particles. We show the usefulness of this simple method to extract significant information about the electrical properties of Gram-negative and -positive bacteria.     

褪黑素对小鼠MFC前胃癌细胞的survivin、caspase-3表达的影响

目的探讨褪黑素（Melatonin,MLT）对小鼠前胃癌细胞（murine foregastric carcinoma,MFC）的生存素survivin和半胱天冬酶-3（cysteinyl aspartate-specific protease-3,caspase-3）表达的影响。方法使用不同浓度褪黑素处理培养的胃癌细胞,运用细胞免疫组化、实时荧光定量PCR、免疫印迹法、分光光度法等方法检测褪黑素在抑制小鼠MFC前胃癌细胞增殖过程中对细胞survivin、caspase-3的表达影响。结果与空白对照组相比,6mmol/L浓度褪黑素干预组的胃癌细胞survivin表达下调,caspase-3蛋白表达上调,并呈剂量依赖性。结论褪黑素能够降低胃癌细胞survivin的表达,进而激活caspase-3的活性,是褪黑素体外抑制肿瘤细胞增殖的作用机制之一。     

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.     

Functional changes in murine mammary cancer cells elicited by CoCl2-induced hypoxia

Low O₂ levels in solid tumors are associated with increase in hypoxia-inducible factor 1α (HIF-1α). The present study examines functional changes involved in adaptation to hypoxia of the LMM3 mammary tumor cell line, using CoCl₂ as hypoxic mimetic. Our results showed that LMM3 cells were not only tolerant to 150 μM CoCl₂ but they can overgrowth in vitro respect to untreated cells. Hypoxia inhibited cell invasion, migration, MMP-9 activity and NO levels. Macrophage cytotoxicity augmented under hypoxia but was blunted by conditioned media from tumor cells. In vivo tumorigenicity of CoCl₂-treated cells was greater than controls. Our results show stabilization of HIF-1α in LMM3 cells under CoCl₂ and functional changes associated with enhanced cell survival and growth but not with tumor dissemination.     

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.     

Apoptotic sensitivity of murine IAP-deficient cells

Although numerous studies have implicated the IAPs (inhibitor of apoptosis proteins) in the control of apoptotic cell death, analyses of murine Iap-targeted cells have not revealed significant differences in their susceptibility to apoptosis. In the present study, we show that, under defined conditions, murine cells lacking XIAP (X-linked inhibitor of apoptosis) and c-IAP (cellular IAP) 2, but not c-IAP1, exhibit heightened apoptotic sensitivity to both intrinsic and extrinsic apoptotic stimuli.     

COX and LOX eicosanoids modulate platelet activation and procoagulation induced by two murine cancer cells

Involvement of arachidonic acid cyclooxygenase (COX) and lipoxygenase (LOX) metabolites in platelet aggregation and coagulation induced by two varieties of cancer cells of murine transplantable tumors was studied. A lung alveolar carcinoma (LAC) and a fibrosarcoma (FS), induced platelet aggregation and plasma coagulation (P<0.05). Pretreatment of both tumor lines with a COX inhibitor did not block the tumor cell induced platelet aggregation (TCIPA). COX [12(S)-HTT] and LOX [12(S)-HETE], metabolites of washed platelets (WP), alone or co-incubated with LAC or FS cells, were analyzed. We observed higher 12(S)-HETE release with respect to 12(S)HHT when WP were co-incubated with LAC cells. With both neoplastic cell (NC) lines prothrombin time (PT) was shortened. Pretreatment of NC with iodoacetic acid, soybean trypsin inhibitor or Factor X-deficient plasma increased the PT. These results indicate that AA metabolites play a role on the procoagulation and platelet aggregation induced by mesenchymal and epithelial murine cancers.