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  • 1
    ISSN: 1573-8280
    Keywords: monocyte ; cytotoxicity ; monokine ; tumor cytotoxic factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Human blood monocytes activated to the tumoricidal state were previously found to release a factor(s) responsible for tumor cell killing. The activity of the tumor cytotoxic factor(s) (TCF) was determined by release assay of radioactivity from human A375 melanoma cells. On fractionation of the supernatant of activated monocytes by Ultrogel AcA34 and TSK-G3000SW gel chromatographies two major peaks of the material with TCF activity with MWs of 30,000 and 15,000, called TCF-I and TCF-11, respectively were obtained. TCF-II could be neutralized by polyclonal anti-IL-1β antiserum, but anti-IL-1α antiserum did not neutralize either factor. TCF-I was separated by ampholine column electrofocusing into three major fractions with TCF activity at pI 5, 6 and 6.8, named TCF-1α, TCF-1β and TCF-1γ, respectively. The cytotoxic and IL-1 activities of TCF-1α were neutralized by anti-IL-1α serum, whereas those of TCF-1β and TCF-1γ were not completely neutralized by anti-IL-1α or anti-IL-1β antiserum. On DEAE ion-exchange chromatography (TSK DEAE 5PW) TCF-Iβ gave two peaks with TCF activity (TCF-Iβ1 and TCF-Iβ2). TCF-Iβ1 was slightly neutralized by anti-TNFα antibody, but TCF-Iβ2 was not affected by antisera against IL-1α and IL-1β, or anti-TNFα antibody, thus ruling out the possibility that tumor necrosis factor (TNFα) might be involved in tumor cell killing mediated by TCF-Iβ2. These results indicate that human monocyte-mediated cytotoxicity against human A375 melanoma cells is mediated in part by a tumor cytotoxic factor (TCF; MW, 30,000; pI 6), differing from IL-1 and TNFα.
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  • 2
    ISSN: 1573-8280
    Keywords: intrapleural RIL-2 ; lung cancer ; malignant pleurisy ; recombinant interleukin-2 (RIL-2)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Forty-three patients with malignant pleurisy due to lung cancer were entered into the trial to evaluate clinical efficacy of intrapleural instillation of recombinant interleukin-2 (RIL-2). Among 35 evaluable patients, serial cytological examinations of pleural effusion following the start of the treatment revealed disappearance of malignant cells in 26 (74%). Malignant cells were detected again in 7 of the 26, however, cytology remained negative in the other 19 patients for longer than 4 weeks. Pleural effusion disappeared roentogenographically in 13 of 35 evaluable patients. Additional 8 patients demonstrated marked decrease of pleural effusion. Complete response (CR) which means disappearance of both malignant cells and pleural effusion for longer than 4 weeks was obtained in 13 of the 35 patients (37%). No serious side effects were experienced in this trial. These results indicate that intrapleural RIL-2 is one of candidates to control intractable malignant pleurisy due to lung cancer.
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  • 3
    ISSN: 1573-8280
    Keywords: IL-1 ; Monocyte ; N-CWS ; TNF-α
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Human blood monocytes were obtained from peripheral blood of healthy donors by counter-flow centrifugal elutriation. Functional integrity of monocytes for production of interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) in response toNocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 μg/ml produced IL-1 and TNF-α extracellularly. Extracellular TNF activity appeared within 4 h, and maximally, 16 h after N-CWS stimulation, whereas longer time was needed for IL-1 activity to appear, the peak production being at 24 h. The neutralizing experiment also showed that anti TNF-α antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independen cy of IL-1 production of TNF-α. These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.
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  • 4
    ISSN: 1432-0851
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon-γ (rIFN-γ), but not other cytokines [rIFN-αA, rIFN-β, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN-γ and then with FK-565. FK-565 also acted synergistically with rIFN-γ to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1α) antiserum, but not by a specific anti-(IL-1β) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1α of human blood monocytes can be induced by two activation signals (rIFN-γ then FK-565) at their suboptimal concentrations.
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  • 5
    ISSN: 1432-0851
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Human blood monocytes freshly isolated by centrifugal elutriation from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the tumoricidal state by incubation in vitro with FK-565, (heptanoyl-γ-D-Glu-(L)-meso-α,ε-A2pm(L)-D-AlaOH), which is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Among 11 different derivatives of FK-565, 7 analogs were more potent activators of monocytes for tumor cell killing than FK-565. The maximal expression of tumoricidal nonocytes was dependent on the concentration of FK-565 or its analogs added and the ratio of monocytes to target tumor cells. In a parallel experiment, a combination of a subthreshold concentration of FK-565 or its analogs (FR-42148 and FR-42149) and recombinant interferon γ (rIFN-γ) induced significant monocyte-mediated tumorcell killing, indicating that the effects of rIFN-γ and acyltripeptide or its analogs in monocyte activation are synergistic. In contrast to rIFN-γ, recombinant rIFN-αA and rIFN-β had additive effects with acyltripeptide or its analogs in human monocyte activation. These results suggested that synthetic acyltripeptide and its analogs combined with rIFN-γ could be of clinical value for in situ activation of the tumoricidal activity of human blood monocytes responsible for eradication of cancer metastases.
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  • 6
    ISSN: 1432-0851
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.
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  • 7
    ISSN: 1432-1335
    Keywords: Matrigel ; ECM ; Lung cancer ; Colony assay ; Anchorage-independent growth ; SCLC ; NSCLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Lung cancers have been distinguished into small-cell lung cancer (SCLC) and non-small cell-lung cancer (NSCLC) types on the basis of their clinical behaviors and their responses to treatment. Moreover, growth of most SCLC cell lines in liquid culture medium is nonadherent, while that of most NSCLC cell lines is adherent. In this study, we examined the effect of matrigel (reconstituted basement membrane components), which is known to have growth-stimulatory activity on various human tumor cell lines in immunodeficient mice, on soft-agar colony formation of a panel of SCLC and NSCLC cell lines to clarify its mechanism of growth stimulation of cancer cells. Matrigel enhanced colony formation of all 9 NSCLC cell lines and 4 of 9 SCLC cell lines. There was a statistically significant difference (P〈0.01) between colony formations with and without matrigel of NSCLC cell lines, but not for SCLC cell lines. In liquid culture medium, all 9 NSCLC lines and 3 of 9 SCLC lines adhered to plastic dishes, whereas the other SCLC lines did not. Matrigel enhanced colony formation of all 3 adherent-type SCLC lines and 1 of 6 nonadherent-type NSCLC lines. Matrigel enhanced colony formation of both of 2 adherent-type non-lung cancer cell lines and 1 of 2 nonadherent-type leukemia cell lines. Neither transforming growth factor β, collagen type IV, fibronectin, nor laminin, which are components of matrigel, enhanced colony formation of an NSCLC cell line in soft agar. The increase in the colony number of the NSCLC cell line by matrigel was abrogated by the protein kinase inhibitors staurosporine and UCN-01.
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  • 8
    ISSN: 1432-1335
    Keywords: Key words Matrigel  ;  ECM  ;  Lung cancer  ;  Colony assay  ;  Anchorage-independent growth  ;   SCLC  ;  NSCLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Lung cancers have been distinguished into small-cell lung cancer (SCLC) and non-small cell-lung cancer (NSCLC) types on the basis of their clinical behaviors and their responses to treatment. Moreover, growth of most SCLC cell lines in liquid culture medium is nonadherent, while that of most NSCLC cell lines is adherent. In this study, we examined the effect of matrigel (reconstituted basement membrane components), which is known to have growth-stimulatory activity on various human tumor cell lines in immunodeficient mice, on soft-agar colony formation of a panel of SCLC and NSCLC cell lines to clarify its mechanism of growth stimulation of cancer cells. Matrigel enhanced colony formation of all 9 NSCLC cell lines and 4 of 9 SCLC cell lines. There was a statistically significant difference (P 〈 0.01) between colony formations with and without matrigel of NSCLC cell lines, but not for SCLC cell lines. In liquid culture medium, all 9 NSCLC lines and 3 of 9 SCLC lines adhered to plastic dishes, whereas the other SCLC lines did not. Matrigel enhanced colony formation of all 3 adherent-type SCLC lines and 1 of 6 nonadherent-type NSCLC lines. Matrigel enhanced colony formation of both of 2 adherent-type non-lung cancer cell lines and 1 of 2 nonadherent-type leukemia cell lines. Neither transforming growth factor β, collagen type IV, fibronectin, nor laminin, which are components of matrigel, enhanced colony formation of an NSCLC cell line in soft agar. The increase in the colony number of the NSCLC cell line by matrigel was abrogated by the protein kinase inhibitors staurosporine and UCN-01.
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  • 9
    ISSN: 1432-1335
    Keywords: CMK ; SCF ; RB protein ; Cyclin A ; gpIIb/IIIa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The biological effects of c-kit ligand (stem-cell factor: SCF) on an immortalized human megakaryocytic cell line (CMK) was evaluated using methods including the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, surface marker analysis, DNA cell-cycle analysis and immunoblotting. SCF stimulated the growth of CMK cells. Incubation with SCF resulted in increased expression of IIb/IIIa platelet-related glycoprotein (gpIIb/IIIa), indicating enhanced differentiation of CMK cells. Treatment of CMK cells with SCF resulted in a decrease in the subpopulation in the G1 phase, with a reciprocal increase in those in the S phase and the G2+M phase. Moreover, SCF significantly increased cellular expression of cyclin A, a regulatory subunit of cyclin-dependent protein kinase (CDK), and the ratio of phosphorylated/dephosphorylated retinoblastoma gene product (RB protein). These results suggest that SCF stimulates the growth and differentiation of megakaryocytic cells possibly through mechanisms related to the activation of cell-cycle-dependent serine/threonine kinase and inactivation of the nuclear tumor-suppressor gene product.
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  • 10
    ISSN: 1432-1335
    Keywords: Key words Lung cancer ; NSCLC ; SCLC ; Cyclin A ; Retinoblastoma gene ; RB ; Immunoblotting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cell-cycle-dependent phosphorylation of the tumor-suppressor protein product of the retinoblastoma gene (RB) is mediated by a family of cyclin-dependent kinases and cyclins. We examined the expressions of RB protein and cyclin A protein in 13 small-cell lung cancer (SCLC) lines and 14 non-small-cell lung cancer (NSCLC) lines by immunoblotting. RB protein was not present or was of a mutant type in 77% of the SCLC lines (10/13) but was present in all the NSCLC lines. Cyclin A was expressed in 38% of the SCLC lines (5/13) and in 86% of the NSCLC lines (12/14). A positive correlation (P = 0.0034) between expression of cyclin A and wild-type RB protein was found by Fisher's exact probability test. Densitometric analysis of the expression of RB protein in RB(+) lung cancer lines showed that the phosphorylated form was predominant in 2/3 of the SCLC and 8/14 of the NSCLC lines. The positive correlation between the expressions of RB protein and cyclin A suggests that RB protein in most RB(+) lung cancer cell lines is a target of cyclin-A-dependent kinase and that the tumor-suppressor function may be inactivated by phosphorylation.
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