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PubMed1998

Chimeric scFv/gamma receptor-mediated T-cell lysis of tumor cells is coregulated by adhesion and accessory molecules.

Adhesion and accessory molecules play a critical role in T-cell activation and effector function in general and in tumor cell recognition and lysis in particular. We investigated the contribution of CD2, CD3, CD11a/CD18, CD54 and CD58 molecules in T lymphocyte-tumor cell interactions mediated by chimeric immunoglobulin receptors. The chimeric receptor is composed of a single chain antibody binding site and a gamma-chain signal transducing molecule (scFv/gamma). T lymphocytes expressing such scFv/gamma receptors recognize the G250 Ag on renal cell carcinoma (RCC) in an major histocompatibility complex (MHC)-unrestricted manner and exert RCC selective cytolysis. A coregulatory role for CD2, CD3 and CD11a/CD18 molecules in scFv/gamma-mediated cytolysis was demonstrated using monoclonal antibody (MAb)-induced inhibition of scFv/gamma-mediated cytolysis. The inhibition of lysis was not due to inhibition of cytotoxic T lymphocyte (CTL)-target cell conjugation but rather to a post-conjugate signaling event. Binding of CD54 and CD58 MAbs to the RCC did not inhibit cytolysis of RCC cells that expressed high levels of both CD54 and the G250 antigen (Ag) (A75), whereas cytolysis of RCC expressing intermediate levels of CD54 and G250 Ag (SK-RC-17 cl.4) was partly inhibited by the CD54 MAb. Binding of low concentrations of G250 MAb to RCC (A75) rendered these cells sensitive to CD54 MAb inhibition, demonstrating a direct functional relation between G250 Ag expression level and adhesion molecules. Taken together, our findings indicate a coregulatory role for CD2, CD3 and CD11a/CD18 molecules in the scFv/gamma-mediated cytolysis of tumor cells and show that the requirement of CD11a/CD18-CD54 interactions is dependent on the level of free Ag. This make these gene-transduced T lymphocytes attractive tools for adoptive immunogene therapy of cancer.

Antibodies, Monoclonal↗
PubMed1996

Inhibition of human complement-dependent cell lysis by bovine aortic endothelial cells transfected with membrane-bound complement-regulatory factor (DAF and HRF20) gene using a retroviral vector.

The endothelial cells (EC) of xenografts are the target of hyperacute rejection induced by complement activation via the classical and/or the alternative pathway. To protect these cells from the attack of human complement, decay-accelerating factor (DAF, CD55) and homologous restriction factor 20 (HRF20, CD59), which belong to human complement regulatory factors, were transfected into bovine aortic EC (BAEC) using retroviral vector. Cell surface expression of DAF and HRF20 on BAEC transfectants (BAEC/DAF, BAEC/HRF20) is comparable to that on human umbilical vein EC. Phosphatidyl inositol-phospholipase C treatment diminished or abolished cell surface expression of DAF and HRF20 on BAEC. The addition of human serum to BAEC led to complement-dependent cell lysis, whereas practically no lysis was observed after addition of human serum to BAEC/DAF and BAEC/HRF20. The addition of human serum plus rabbit complement to BAEC/DAF and BAEC/HRF20 caused complement-dependent cell lysis that was comparable to that observed for BAEC. These data demonstrate that xenograft EC transfected with DAF or HRF20 cDNA using retroviral vector are protected from complement-dependent cell lysis.

Animals↗
PubMed2006

Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis.

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

Buffers↗
PubMed1996

Expression of CD46, CD55, and CD59 on renal tumor cell lines and their role in preventing complement-mediated tumor cell lysis.

Nucleated cells are protected from complement-mediated injury by the expression of membrane-bound regulators of complement activation (mRCA) CD46, CD55, and CD59. Increased expression of these mRCA may be a mechanism by which tumor cells protect themselves from complement-mediated injury and prevent an inflammatory response. In the present study, we have investigated whether human renal tumor cell lines and cultured proximal tubular epithelial cells express CD46, CD55, and CD59 and whether these mRCA influence complement-mediated lysis of these cells. The expression of CD46, CD55, and CD59 was measured by flow cytometry. To determine the effect of mRCA on lysis, tumor cells were opsonized with complement activating anti-HLA class l mAb. Lysis was measured in the presence or absence of anti-CD46, anti-CD55 or anti-CD59 mAb and serum as a source of complement, using a 51Cr release assay. Flow cytometric analysis revealed that renal tumor cell lines and proximal tubular epithelial cells all express CD46, CD55, and CD59. Lysis of renal tumor cell lines in the presence of rabbit serum depended on the number of HLA class I molecules expressed by the tumor cells. Using human serum, complement-mediated lysis was decreased by at least one-third as compared with rabbit serum. The susceptibility of renal tumor cells for complement-mediated lysis could be increased up to the level observed with rabbit serum by inhibiting the function of CD59. Inhibition of the function of CD46 or CD55 with mAb directed against these mRCA had no substantial effect on lysis. We conclude from this work that renal tumor cells and proximal tubular epithelial cells express CD46, CD55, and CD59. Of these mRCA, CD59 is most efficient in preventing complement-mediated lysis of these cells. Expression of mRCA on tumor cells may influence the effectiveness of immunotherapy with tumor-associated mAb.

Amino Acid Sequence↗
PubMed1989

Effector-target co-aggregation as a crucial step in the neutrophil-mediated tumour cell lysis.

The Daudi cell lysis by human neutrophils, incubated with phorbol myristate acetate (PMA), was inhibited by amino acids (taurine, methionine), consistent with the involvement of hypoclorous acid (HOCl) in the lytic process. Also, the lysis was inhibited by a monoclonal antibody (mAb J-90) directed against the leukocyte function-associated antigen-1 (LFA-1). The inhibition of the target cell lysis by mAb J-90 is not due to a HOCl-scavenging mechanism, as suggested by the use of control mAb Dako M-1 (anti CD-15). As detected by the microscopic examination of samples from test tubes and measured by monitoring the light transmission from the cell suspensions, neutrophils and Daudi cells were found to co-aggregate during the lytic reaction. Co-aggregation was efficiently inhibited by the mAb J-90. The results suggest that tumour cell lysis by PMA-triggered human neutrophils involves at least two events: production of HOCl and LFA-1-mediated effector-target adhesion.

Antibodies, Monoclonal↗
PubMed1986

Susceptibility to NK cell lysis is abolished in tumor cells by a factor which restores their contact inhibited growth.

It is well recognized that physical contact between natural killer (NK) cells and tumor targets is necessary for cell lysis. Therefore, any modulation of the tumor cell surface that alters intercellular contact could affect NK cell cytotoxicity. To examine this hypothesis, a contact inhibitory factor (CIF), which had been shown to restore contact inhibition of growth to several malignant cell lines was tested for its ability to render such cells immune to recognition by NK cells. When three NK-sensitive melanoma and two NK-sensitive colon carcinoma targets were cultured with CIF, they did not only change morphologically, but also showed a 70% to 95% reduction in their sensitivity to lysis by NK cells. In addition, K562 cells, which grow in suspension and do not permit a morphologic evaluation of the CIF effect, also became resistant to lysis by NK cells after culture with CIF. CIF did not reduce the viability nor the cytotoxicity of NK cells. CIF did not contain interferon nor did the CIF-treated targets induce the production of interferon during the cytotoxicity assay. It is concluded that restoration of contact inhibition of growth and resistance to NK cell lysis are cell surface phenomena that may run in parallel.

Cell Line↗
PubMed1990

Surfactant-induced cell toxicity and cell lysis. A study using B16 melanoma cells.

The effects of a variety of detergents (non-ionic, ionic and bile derivatives) on B16 melanoma cells have been examined. Two main effects can be clearly differentiated: loss of cell viability and cell lysis. Under our conditions, cell-surfactant interaction is highly dependent on the nature of the amphiphile (more specifically, on its critical micellar concentration). Loss of cell viability occurs at surfactant concentrations below the critical micellar concentration, i.e. the incorporation of detergent monomers into the cell membranes is enough to impair their barrier function, so that Trypan Blue is no longer actively secreted outside the cell. On the other hand, cell lysis only occurs at or near the critical micellar concentration of the detergent, i.e. when the bilayer-micelle transition may take place. Comparative studies using B16 cells and phospholipid vesicles indicate that the amount of detergent required to induce cell lysis is the same that produces disruption of the lipid bilayer. Thus, our results suggest that membranes are the primary target for the toxicologic effects of surfactants on cells. Moreover, they provide a rationale for the interpretation of other studies in this field: previous results from different laboratories are shown to fit very well our data.

Animals↗
PubMed1989

Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities.

Acquired Immunodeficiency Syndrome↗
PubMed1992

[Observation of the in vivo tumor cell lysis mediated by NK cells].

Natural killer cells/large granular lymphocytes (NK/LGL) separated on discontinuous Percoll gradient from rat spleen cells were injected iv to rats (7 x 10(7) cells/rat) 3 days following iv inoculation of 2 x 10(6) Walker-256 cells. Two and 4 hr after NK/LCL injection, animals were sacrificed and the lungs examined by light and immunoelectron microscopy. The latter was done using colloidal gold-labelled polyclonal antibody against purified rat LGL cytoplasmic granules. At 2 hr following iv NK/LGL, in addition to the scattered individual tumor cells and minute tumor foci, many lymphocytes were seen accumulating in the small pulmonary vessels and capillaries. This was not observed in tumor-inoculated control rats. At 4 hr, many extravasated lymphocytes reached the lung parenchyma, some of which had attached to the tumor cells. Immunoelectromicroscopically, lymphocytes were found in intimate contact with the tumor cells with the cytoplasmic gold particles clustering at the cell contact site. Gold particles could also be seen closely adherent to the plasma membrane of degenerating tumor cells. This is the first in vivo demonstration of the role of cytotoxic granules of NK cells in tumor cell lysis.

Animals↗
PubMed1981

Oncogenic transformation and cell lysis in C3H/10T 1/2 cells and increased sister chromatid exchange in human lymphocytes by nickel subsulfide.

We have scored frequency of transformation and appearance of the oncogenic marker "long microvilli" in mouse embryo fibroblast C3H/10T 1/2 cells in culture for the compound Ni3S2. Furthermore, we have scored for Ni3S2-induced sister chromatid exchange in cultured human lymphocytes. Nickel subsulfide in moderate doses caused morphological transformation to type I, II, and III foci and induced long microvilli on the cells in the transformed cultures, demonstrating oncogenic transforming ability. Higher doses led to cell lysis after a lag period. The carcinogenic potency of Ni3S2 in this system was not as strong as that of methylcholanthrene. Ni3S2 increased the sister chromatid exchange frequency in human lymphocytes in a marginal, not dose-dependent way. The increased sister chromatid exchange may indicate that the carcinogenic effect of Ni3S2 is genetic rather than epigenetic.

Animals↗
PubMed1988

Early events in target-cell lysis by cytotoxic T cells.

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

Animals↗
PubMed1992

Apparent contribution of respiratory gas exchange to the in vitro "cell density effect" in ultrasonic cell lysis.

Ultrasonic damage to cells in vitro has been reported to vary dramatically with cell density; the particle density per se has been proposed to be responsible for this effect via interference with gas body activity. Reported here are the results of experiments designed to test the general postulate that the "cell density effect" can be explained in part by cellular modification of the suspension medium by respiratory gas exchange. Ultrasonic cell lysis is shown to occur at a much higher level in suspensions supplemented with the respiratory inhibitor NaCN than in control suspensions when suspension densities exceed 2 x 10(7) cells/mL. Moreover, at constant cell density, cell lysis diminishes with increasing pre-insonation incubation time at 37 degrees C. The rate of change in cell lysis with incubation time was diminished significantly by cyanide treatment. These observations are consistent with the postulate that respiratory O2:CO2 exchange enriches the medium in CO2 while depleting the medium of oxygen, which cavitates more readily than CO2, thereby diminishing the potential for cavitation-related cellular damage. However, this is only a partial explanation of the cell concentration dependence of cell lysis; cell density per se is an important factor in the "cell density effect."

Animals↗
PubMed1975

Lysis of tumor cells by antibody and complement. II. Lack of correlation between amount of C4 and C3 fixed and cell lysis.

Two antigenically distinct diethylnitrosamine-induced guinea pig hepatoma cell lines, line-1 and line-10, sensitized with rabbit anti-Forssman or with tumor-specific antibody, were more susceptible to killing by human complement (HuC) than by guinea pig complement (GPC). This difference could not be ascribed to differences in the amount of C1, C4, and C3 fixed: millions of C4 and hundreds of thousands of C3 were detected on cells whether they were killed or not killed by the C sources. Tumor cells sensitized with anti-Forssman IgM antibody generally had more GP C4 and C3 than Hu C4 and C3 bound to their surfaces. Cells sensitized with anti-tumor antibody generally had more Hu C4 and C3 than GP C4 and C3 bound to their surfaces. The resistance to killing of nucleated cells by antibody and C may be due in part to intrinisic properties of the cell.

Animals↗
PubMed1985

Role of superoxide and hydrogen peroxide in cell lysis during irradiation in vitro of Ehrlich ascitic carcinoma cells in the presence of melanin.

The reactive species involved in the cell lysis during ultraviolet irradiation of Ehrlich ascitic carcinoma cells in the presence of red hair melanin (RHM) were investigated by determining 51Cr release from labeled cells. Cysteine at 1 mM in the presence of RHM increased the cell lysis during the incubation in the dark as well as during irradiation; this lysis was enhanced by superoxide dismutase (SOD). Catalase abolished the dark reaction and inhibited the cysteine-induced increase of cell lysis during irradiation. The cell lysis by the superoxide-generating xanthine oxidase system was not significantly increased by SOD, but was significantly decreased by nitroblue tetrazolium and completely abolished by catalase. The cell lysis induced by the supernatants obtained from the suspensions of RHM either irradiated alone or with cysteine was abolished by catalase. Sediments of irradiated RHM when incubated in the dark with the cells did not release 51Cr. Irradiation of the cells in the presence of the same sediments produced lysis which was not inhibited by catalase. These studies suggest that superoxide per se is not toxic to the cells, but the H2O2 formed by dismutation of superoxide produces cell lysis either directly or by generating OH through Fenton-type reactions. A large part of the cell lysis seen during irradiation of cells in the presence of RHM is not due to H2O2, but may possibly be due to the melanin free radicals formed during irradiation.

Animals↗
PubMed2002

Development of a laser-induced cell lysis system.

A novel cell lysis system was developed that is based on laser-induced disruption of bacterial and yeast cells. It will find application as a rapid, efficient and clean sample preparation step in bioanalytical detection systems. Using E. coli as our model analyte, we optimized cell lysis with respect to optimal laser wavelength, lowest energy input requirements, RNA release from the cells, and potential protein damage. The optimized system was finally applied to the lysis of four additional microorganisms. All experiments were carried out with about 2000 cells per sample or less. Initially, lysis was determined by the detection of cell survival after laser treatment using standard microbiological techniques, (i.e., cells were grown on nutrient agar plates). Then, actual release of mRNA from the cells was proven. Wavelengths investigated ranged from 500 nm to 1550 nm. An average power of 100 mW for the lasers was shown to be sufficient to obtain cell lysis at wavelengths above 1000 nm, with optimal wavelengths between 1250 nm and 1550 nm. Since water absorbs energy at those wavelengths, it is assumed that laser exposure results in an instantaneous increase of the cell temperature, which causes rupture of the cell membrane. Second, damage to protein solutions treated under optimized laser-lysis conditions was also studied. Using a pure solution of horseradish peroxidase as a model protein, no loss in enzyme activity was observed. Thus, it was concluded that damage to intracellular proteins is unlikely. Third, RNA release was tested using an E. coli specific RNA biosensor. Release of RNA was not detected from untreated cells, but laser-treated E. coli cells displayed significant RNA release due to laser-induced cell lysis. Finally, lysis of M. luteus, B. subtilis, B. cereus, and S. cerevisiae were investigated under optimized conditions. In all cases, laser-induced lysis of the cells was confirmed by determination of cell survival. Hence, laser-induced cell lysis is an efficient procedure that can be used for sample preparation, without damage to macromolecules, in bioanalytical detection systems for microorganisms. Miniaturized lasers and miniaturized cell-lysis chambers will create a simple, field-usable cell lysis system and allow the application of laser-induced cell lysis in micro Total Analysis Systems.

Bacillus cereus↗
PubMed1988

Properties and characterization of a rat spleen cell-derived factor that induces resistance to natural killer cell lysis in YAC lymphoma cells.

Supernatants of Con A-stimulated rat spleen cell cultures contain a factor that induces relative resistance to NK cell-mediated cytotoxicity in the YAC cell line, a line that is otherwise highly susceptible to murine NK cell-mediated lysis. This NK-lysis resistance-inducing factor (LRIF) has a Mr of 12,600 Da, as determined by gel filtration chromatography, and an isoelectric pH of 4.8. NK-LRIF is heat labile and is de-activated by treatment with proteolytic enzymes. Unlike immune-IFN (IFN-gamma), NK-LRIF is not inactivated by pH 2 treatment, and antibodies capable of neutralizing IFN-alpha and IFN-gamma do not abrogate the effect of NK-LRIF. Highly purified IL-2 preparations lack NK-LRIF activity. NK-LRIF does not induce a general resistance to lysis in YAC cells, because control and NK-LRIF-treated YAC cells were equally susceptible to alloimmune cytotoxic T cells. YAC cells treated with NK-LRIF showed a marked enhancement (5- to 10-fold) in the expression of class I MHC Ag. This observation supports the proposition that the NK susceptibility of target cells could be inversely related to the expression of class I MHC Ag.

Animals↗