Typical three-domain cry proteins of Bacillus thuringiensis strain A1462 exhibit cytocidal activity on limited human cancer cells

Bacillus thuringiensis strain A1462 produced two parasporal inclusion proteins with a molecular mass of 88 kDa that were converted to 64-kDa toxins when activated by proteinase K digestion. Both toxins exhibited strong cytocidal activity against two human cancer cell lines, HL60 (myeloid leukemia cells) and HepG2 (liver cancer cells), while low or no toxicities were observed against 11 human and three mammalian cell lines, including four non-cancer cell lines. The cytotoxicity of both toxins on susceptible cells was characterized by rapid cell swelling. Gene cloning experiments provided two novel genes encoding 88-kDa Cry proteins, Cry41Aa and Cry41Ab. The amino acid sequences of the two proteins contain five block regions commonly conserved in B. thuringiensis insecticidal Cry proteins. This is the first report of the occurrence of typical three-domain Cry proteins with cytocidal activity preferential for cancer cells.     

B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens

Macrophages are activated by lymphokines (LK) to kill tumor cell and microbial targets. Interferon-gamma (IFN) is the major LK activity in conventional, antigen or mitogen-stimulated spleen cell culture fluids for induction of these macrophage effector functions. In view of the recent demonstration that murine macrophage-like cell lines have receptors for B cell stimulatory factor-1/interleukin 4 (BSF-1), a possible role for BSF-1 in regulation of macrophage function was considered. In this communication, thioglycollate-elicited murine peritoneal macrophages were shown to express about 2300 high affinity (Ka approximately 2 X 10(10) M-1) BSF-1 receptors/cell. Peritoneal macrophages treated with purified, T cell-derived BSF-1 developed potent tumoricidal activity against fibrosarcoma target cells. The concentration of BSF-1 that induced 50% of maximal tumor cytotoxicity was 38 +/- 4 U/ml for seven experiments; similar dose-responses were observed with recombinant BSF-1. That BSF-1 dose-responses for induction of macrophage-mediated tumor cytotoxicity were not affected by 5 micrograms/ml polymyxin B suggested that contaminant endotoxins played little or no role in cytotoxic activity. BSF-1 alone (less than or equal to 500 U/ml) was not directly toxic to tumor cells or macrophages. Macrophage tumoricidal activity induced by BSF-1 but not by IFN was inhibited greater than or equal to 90% with monoclonal anti-BSF-1 antibody. BSF-1 induced Ia antigen expression on peritoneal macrophages and increased (twofold to threefold) FcR(II)-dependent binding of murine IgG immune complexes to bone marrow-derived macrophages (greater than 98% macrophages). Based on these findings, it was concluded that BSF-1 is a potent macrophage activation factor.     

嵌合型E1B 55-kDa蛋白缺陷型腺病毒载体治疗肿瘤的评价

ONYX-015和H101为可复制E1B 55-kDa蛋白缺陷的C族腺病毒,它们正作为抗癌药物进行临床研究. 然而它们在癌症基因治疗中的应用却受到了C族腺病毒天然特性的制约,部分原因是由于在恶性肿瘤中C族腺病毒受体(coxsackievirus-adenovirus receptor,CAR) 的表达量较低. 构建了一个以H101为骨架的含有编码35型腺病毒鞭毛区域的基因,替代5型腺病毒鞭毛基因的嵌合型腺病毒载体. 这一改动使得腺病毒载体可以通过一种在肿瘤中高表达的膜蛋白CD46感染肿瘤细胞. 应用RT-PCR方法检测不同肿瘤细胞株中CAR和CD46表达量的区别. 在CAR受体低表达的细胞株中(MDA-MB-435和MCF-7),H101-F35表现出比H101和ONYX-015更强的细胞杀伤效果;在CAR受体高表达的细胞株中(A549,NCI-H446,Hep3B,LNCaP,ZR-75-30和Bcap-37),H101-F35、 H101和ONYX-015的细胞杀伤效果则相似. 在荷MDA-MB-435肿瘤的裸鼠模型中,注射H101-F35的抑瘤效果比注射H101的抑瘤效果更明显. 这些结果表明嵌合型溶瘤腺病毒载体H101-F35在肿瘤基因治疗中将有很好的应用前景.     

Natural and antibody-dependent cell-mediated activity against Salmonella typhimurium by peripheral and intestinal lymphoid cells in mice

Cell-mediated immune responses were assessed employing a 2-hr in vitro cytotoxicity assay against S. typhimurium. It was observed that lymphocytes from GALT as well as from peripheral lymphoid organs possessed natural antibacterial activity, whereas macrophages were devoid of this spontaneous activity. The distribution of this newly described natural activity was PPL greater than MnL greater than IEL = SpL = PBL greater than PoL; this did not correlate with the organ distribution of NK activity against YAC-1 tumor cells, which was PBL greater than SpL = IEL greater than MnL = PoL = PPL. Moreover, the phenotype of the splenic effector cell of the natural activity against S. typhimurium showed some differences from that of NK activity. In fact, both these cells were asialo GM1+, Fc-receptor+, nonadherent, and nonphagocytic, but the former was Thy-1.2- and the latter Thy-1.2+. The effector cell of the natural antibacterial activity in the Peyer's patches had the same phenotype as the splenic one. It was then observed that the antibacterial activity could be augmented by the addition of immune antibodies against S. typhimurium. This was particularly evident employing IEL, SpL, and PBL as effector cells, whereas PPL and MnL did not show any antibody-dependent antibacterial activity. Furthermore, these last two populations could not mediate ADCC against CRBC. Employing selective methods to deplete cell populations, we observed that, at least at the splenic level, there is also a cell that differs in its phenotypic characteristics from that mediating natural antibacterial activity but that plays a role in the antibody-dependent reactions. In conclusion, these results suggest that natural and antibody-dependent antibacterial mechanisms might be important in defense against S. typhimurium, particularly at the gastrointestinal level, where many bacterial infections first take place and begin to interact with the host immune system.     

Tumor-specific targeting of a cell line with natural killer cell activity by asialoglycoprotein receptor gene transfer

Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

No effects of pulsed electromagnetic fields on expression of cell adhesion molecules (integrin, CD44) and matrix metalloproteinase-2/9 in osteosarcoma cell lines

Pulsed electromagnetic fields (PEMF) could enhance the cytocidal effects of chemotherapeutic drugs on malignant tumor cell lines, but metastasis effects of PEMF on tumor cells have not been investigated. We investigated the effects of PEMF exposure on the expression levels of some metastasis-related molecules, including integrin α subunits (α1, α2, α3, α4, α5, α6, αv), integrin β subunits (β1, β2, β3, β4), CD44, and matrix metalloproteinase-2/9 (MMP-2/9) in four human osteosarcoma cell lines (HOS, MG-63, SAOS-2, NY) and two mouse osteosarcoma cell lines (DOS, LM8) by using FACScan analysis, gelatin zymography, and Western blot analysis. Our results indicate that PEMF exposure has no effect on the expression of some molecules that are associated with tumor cell invasion and metastasis, and therefore suggest that PEMF exposure may be safely applied to chemotherapy for osteosarcoma.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.     

Immunoregulation in cancer-bearing hosts. Down-regulation of gene expression and cytotoxic function in CD8+ T cells.

The causes of the decreased immune responsiveness in tumor-bearing hosts are incompletely understood. The impact of a decreased immune response in cancer patients on the clinical response in immunotherapy trials has not been evaluated. The present report demonstrates a marked decrease in the therapeutic efficacy of adoptively transferred T lymphocytes obtained from murine hosts bearing tumor for greater than 30 days [late tumor-bearing mice (TBM)] as compared with normal mice and mice bearing tumor for less than 21 days (early TBM). In vitro analysis of the functions of the T lymphocytes from late TBM showed an apparently normal proliferative response to anti-CD3 and IL-2 with adequate lymphokine production from CD4+ cells, but a significant decrease in the cytotoxic function of CD8+ cells. The decreased cytotoxicity was not because of cell-mediated suppression. The expression of granzyme B mRNA was significantly delayed and decreased in magnitude in CD8+ cells from late TBM. Culture supernatants from two unrelated tumor cell lines were able to inhibit the cytotoxic activity of normal CD8+ cells in vitro. The tumor-derived suppressive factor is not transforming growth factor-beta (TGF-beta), but it has not been further characterized. The data suggest that one potential mechanism responsible for immunologic defects in patients with large tumor burdens is a tumor-induced defect that compromises the function of CD8+ effector T cells.     

Growth inhibition of human nasopharyngeal carcinoma in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies

Monoclonal antibodies (MoAbs) were developed against epidermal growth factor (EGF) receptor on the human epidermoid carcinoma cell line A431. The A431 antigen recognized by the MoAbs has an apparent molecular weight of approximately 170,000, with the same molecular weight as the CNE-2 cell line (poorly differentiated nasopharyngeal carcinoma). Administration of anti-EGF receptor MoAbs inhibited tumor formation, caused by the CNE-2 and A431 cell lines, in athymic mice. When the same MoAbs were used in therapy against Tca8113 (a human tongue carcinoma) and HeLa cells (a human cervical carcinoma), tumor growth was not affected. The number of EGF receptors and the apparent dissociation constants for 125I-EGF on CNE-2 and A431 were 1.3 x 10(5)/cell (Kd 7.7 x 10(-8) M) and 1.4 x 10(6)/cell (Kd 2.4 x 10(-9) M), respectively. Three anti-EGF receptor MoAbs were used in these studies. MoAbs 3 and 176, capable of competing with EGF for receptor binding, showed significant tumor growth inhibition. MoAb 101 was incapable of blocking the binding of EGF to its receptor and was not as effective as MoAbs 3 and 176 in tumor growth inhibition. Our observation is that in vitro, MoAb anti-EGF receptor is cytostatic, rather than cytocidal, against CNE-2 and A431.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

Antigen distribution drives programmed antitumor CD8 cell migration and determines its efficiency

Understanding both the role of tumor Ag in CD8 cell differentiation and the reasons that CD8 cells may work inefficiently is crucial for therapeutic approaches in cancer. We studied OT-1 CD8 cell responses in vivo in a differential Ag-distribution model that used EG-7, the EL-4 thymoma transfected with OVA. On their initial Ag encounter, OT-1 CD8 cells underwent programmed expansion in the lymph nodes, where they acquired the ability to migrate to the encapsulated tumor site after > or =4 divisions, without continuous antigenic stimulation. This short antigenic stimulation was sufficient to induce the migration differentiation program, which included modulation of chemokine receptor mRNA expression and down-regulation of CD62L. Moreover, Ag quantity determined the behavior of the OT-1 CD8 cells, including their effector functions and sensitivity to apoptosis. Thus, the initial Ag encounter drives the programmed cell migration potencies, but neither effector functions nor cell death can occur without continuous TCR triggering.     

Novel mast cell lines with enhanced proliferative and degranulative abilities established from temperature-sensitive SV40 large T antigen transgenic mice

Mast cells (MCs) play crucial roles in innate immunity to parasitic and bacterial infections as well as in hypersensitivity, such as the induction and exacerbation of allergy and autoimmune diseases. The regulatory mechanisms for MC development and effector functions are of great interest for developing novel therapeutic strategies against such disorders. Here we report the establishment of novel, immortalized MC lines from bone marrow (BM) cells of a temperature-sensitive mutant of SV40 large T antigen-transgenic mice (termed SVMCs). BM cells from tsSV40LT mice were cultured in the presence of interleukin (IL)-3 for 3 weeks, and then subjected to limiting dilution and single-cell cloning, yielding 27 independent MC clones, three of which were subjected to further analysis. On culture with nerve growth factor, stem cell factor and IL-3, these SVMC clones showed morphologic and biochemical changes from mucosal MC-like to connective-tissue MC-like phenotypes. These SVMC lines exhibited a significantly enhanced proliferation rate, and a higher responsiveness to the high affinity Fc receptor for IgE-mediated intracellular calcium mobilization and degranulation than those of BM-derived cultured MCs. These cell lines should facilitate studies on the mechanisms for the development, differentiation and effector functions of MCs in health and diseases.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on itsin vitro andin vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulating factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GMCSF-dependent cell line TF1 and was chemotactic for monocytes. Thein vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combination of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. Thein vivo antitumor effect indicated that rhGM-CSF/MCAF had marked antitumor effect against A549 tumor in nude mice and even completely suppressed tumor formation. rhGM-CSF/MCAF was significantly more effective in inhibiting tumor growth than rhGM-CSF. Histological analysis showed that tumor site injected with rhGM-CSF/MCAF was infiltrated by a large number of monocytes while a sparse infiltration of monocytes was observed at the tumor site injected with rhGM-CSF or normal saline, suggesting that the antitumor effect of rhGM-CSF/MCAF was mediated by the recruitment of a large number of monocytes to the tumor site.     

Gene expression in antigen-specific CD8+ T cells during viral infection

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.     

Osteosarcoma cell line growth inhibition by zoledronate-stimulated effector cells

Bisphosphonates have a profound effect on bone resorption and are widely used in the treatment of osteoclast-mediated bone diseases. Zoledronic acid (ZA), a third-generation biphosphonate, has a potent antitumor activity and expands gammadelta (γδ) T cells endowed of major histocompatibility complex-unrestricted lytic activity. Many solid tumors express tumor-specific antigens on their surface, representing targets for immune effector T cells. Nevertheless, the immune surveillance against clinically manifested tumors is relatively inefficient. Therefore, we investigated the hitherto unknown effects of ZA activated γδ T cells of normal donors on osteosarcoma cell lines. γδ T cells were stimulated with ZA and low doses of interleukin-2, and then analyzed for proliferation and generation of effector activity against osteosarcoma cell lines. Our results show the potent anti-tumor activity of ZA-stimulated γδ T cells and the enhanced immunosensitivity of osteosarcoma cell lines to γδ T cells suggesting that osteosarcoma is another γδ T cell susceptible tumor type.     

Changes in the Balance between Membrane-Bound and Soluble Forms of CD95 (Fas) during Selection of Tumor Cells in vivo

Studies concerning the expression of the receptor CD95 (Fas) by tumor cells and the role of this protein in apoptosis induced by the effector host cells that bear Fas-ligand are mainly focused on the membrane-bound form of Fas. There are only a few data about the production of the soluble form of Fas by the tumor cells, its role in the interaction with the effector host cells, and the possible changes in the synthesis of this protein during tumor progression. In the present work, three in vivo transformed parental cell lines of different origin and 24 of their variants isolated after a short cycle of natural selection in vivo were studied. It was demonstrated for the first time that: 1) production of the soluble Fas by all selected in vivo variant tumor cell lines increased significantly (2-10-fold) in comparison to the initial (parental) cell lines and did not depend on the origin of the parental lines. At the same time, the expression of the membrane bound form of Fas decreased considerably; 2) variations of the balance between membrane-bound and soluble forms of Fas in selected in vivo variant cells and the expression of the [H₂O₂ ^CA + PGE^S]-phenotype by these cells (this phenotype determines one of the essential mechanisms of the protection of a tumor cell in vivo) possibly represent independent secondary changes acquired during tumor progression in vivo.