沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.     

Toxoplasma gondii: virulence of tachyzoites in serum free media at different temperatures

Highly virulent Toxoplasma gondii tachyzoite multiplication was recorded on the 4th and 5th days post cultivation (dpc) in seven selected cell lines either with or without fetal calf serum (FCS) in the maintenance media. The multiplication rate was slightly lower in the absence of FCS. The cell line mono-layers collapsed dying by the 6th day of infection both in presence or absence of FCS at 37 degrees C. Carcinoma of human larynx (Hep2) and Madian Darby Bovine Kidney (MDBK) cell lines were the most suitable for in vitro multiplication, followed by that of African green monkey kidney cells (VERO), pooled kidney from 1-day-old hamster (BHK), rabbit kidney cells (RK13) and human rhabdomyosarcoma (RDA), while Chicken embryo cells (CER) were the least suitable. In absence of FCS, CER, BHK, Hep2, RDA and MDBK were able to maintain virulent tachyzoites at +4 degrees C for 14 days. The infectivity of the tachyzoites was however lower, killing 40% of the inoculated mice. Tachyzoites survived at room temperature, in the dark, for 14 days in Hep2, RDA and MDBK. However, Hep2 was the only one able to keep virulent tachyzoites until 21 dpc at room temperature and at +4 degrees C. Hep2 propagated tachyzoites were still alive but with low infectivity up to 28 dpc. The cell-lines failed to support the development of tachyzoites after 7 dpc at 37 degrees C and after the 35 dpc at lower temperatures.     

Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.     

The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.)

American ginseng root (Panax quinquefolius) has a number of purported therapeutic effects, including inhibition of cancer cell proliferation. The ability of environmentally relevant heavy metals to alter ginseng effects on cancer cell growth was the subject of this study. A water extract of American ginseng root was applied alone or in combination with physiologically relevant doses of either lead (Pb) or arsenite to MCF-7 breast cancer cells in vitro and effects on cell proliferation were determined. Ginseng alone produced a significant dose-dependent inhibition of MCF-7 cell proliferation starting at 0.5 mg ml(-1). Treatment of MCF-7 cells with 2.5 microM arsenite significantly decreased MCF-7 cell proliferation (p < 0.01). When cells were treated with arsenite (1.25 or 2.5 microM) in combination with ginseng extract (0.5 mg ml(-1)), there was an apparent synergistic inhibition of cell proliferation. Treatment of MCF-7 breast cancer cells with 50 microM Pb significantly decreased cell proliferation relative to control (p < 0.01), and concomitant ginseng and Pb treatment did not lead to a further decrease. These results suggest that contaminant heavy metals, some of which have been detected in ginseng root extracts or commercial ginseng preparations, may alter the biological activity of ginseng.     

Toxoplasma gondii does not persist in goldfish (Carassius auratus)

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.     

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.     

Immune responses of mice intraduodenally infected with Toxoplasma gondii KI-1 tachyzoites

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Infections in mice with tachyzoites and bradyzoites of Neospora caninum (Protozoa: Apicomplexa)

Tachyzoites of 2 isolates of Neospora caninum (NC-1 and NC-2) were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or orally into mice to compare the effects of route of inoculation on pathogenicity. Mice developed more severe disease, and disease occurred sooner when inoculated with the NC-1 isolate compared to the NC-2 isolate. Deaths occurred earlier in mice inoculated i.p. with either isolate. Mice inoculated orally or s.c. with tachyzoites responded similarly to infection. Tissue cysts of the NC-2 isolate produced infections in mice following oral or s.c. inoculation. Lesions seen in mice inoculated with tachyzoites or bradyzoites were primarily acute pneumonia, myositis, encephalitis, ganglioradiculoneuritis, and pancreatitis. In vitro studies demonstrated that tachyzoites of both isolates were killed by incubation in pepsin-HCl solution but not 1% trypsin solution. Bradyzoites of the NC-2 isolate were able to withstand treatment with pepsin-HCl solution.     

Toxoplasma gondii: Effect of maternal infection in the development of lymphoid organs of BALB/c neonates

Toxoplasmosis is one of the worldwide parasitic zoonoses. Alterations in the lymphopoietic system are still poorly studied. We analyzed lymphoid organs of BALB/c mice neonates from Toxoplasma gondii-intraperitoneally-infected mothers on 19th day of gestation, with 30 tachyzoites of strain RH. Normal non-infected pregnant females were used as controls. At 7 days after birth, animals were classified as neonates from infected (NIM) and neonates from non-infected mothers (NNIM). Weight of the thymus and number of thymic cells in NIM were decreased, percentage of apoptosis was significantly increased. Decrease in lymphocytes and monocytes and an increase of plasma cells were observed in bone marrow of NIM. Peripheral blood of NIM showed an increase of monocytes and neutrophils and a decrease in lymphocytes. Infection of the mother during the last day of gestation provokes in the neonates changes in the lymphoid organs that could explain survival of 75% of them.     

Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils

Neosporosis is an infectious disease primarily of dogs and cattle which has been found in many countries around the world. Neospora caninum causes an important immune response (cellular and humoral) in animals that it infects. Since the participation of the cholinergic system in the immune response is well documented, the aim of this study was to evaluate the relationship between N. caninum infection and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) during the acute and chronic phase of infection. For that, tachyzoites of N. caninum (Nc-1 strain) were inoculated intraperitoneally in gerbils (Meriones unguiculatus), which were separated in two experiments, I and II, differing in infective doses of tachyzoites, aiming to reach an acute phase as well as chronic phase, respectively. Samples were collected on day 7 post infection (PI) for Experiment I and on days 15 and 30 PI for Experiment II. AChE activity was evaluated on whole blood and brain, while BChE was evaluated in plasma. On day 7 a reduction of AChE in total blood and brain was observed, along with reduction of BChE in plasma of infected animals when compared with non-infected. In Experiment II, AChE activity increased in total blood on day 30 PI; however, maintaining, during the same period, the AChE activity has a reduced in brain tissue. BChE activity was significantly increased on day 30 PI. Based on the results obtained, it was possible to observe a response of the cholinergic system, providing different grades of AChE and BChE activities, in response to the acute and chronic infection of gerbils experimentally infected with N. caninum. These results will serve as initial points to further studies of our research group about the relationship between the infection/disease and the cholinergic system.     

Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species

The antimicrobial activity of 35 indigenous South African Helichrysum species was determined against six microorganisms. Seven of the 36 chloroform:methanol (1:1) extracts (leaf and stem extracts for all plants and an additional flower extract for H. rugulosum) exhibited minimum inhibitory concentration (MIC) values lower than 0.1 mg/ml against Bacillus cereus and/or Staphylococcus aureus. The in vitro cytotoxicity [against transformed human kidney epithelial (Graham) cells, MCF-7 breast adenocarcinoma and SF-268 glioblastoma cells] of these extracts was also determined at a concentration of 0.1 mg/ml using the sulforhodamine B (SRB) assay. For seven species less than 25% growth was observed for the Graham and MCF-7 cell lines at the test concentration.     

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.     

MCF-7/VD(R): a new vitamin D resistant cell line

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (M?rk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

A methylene chloride fraction of Saururus chinensis induces apoptosis through the activation of caspase-3 in prostate and breast cancer cells

The aerial parts of Saururus chinensis (SC) have been used for the treatment of edema, fever, jaundice, and inflammatory diseases in Korean folk medicine for centuries. However, the mechanism by which SC exerts these anti-tumorigenic activities in human prostate and breast cancer cells has not yet been fully understood. In this study, we report on the methylene chloride fraction from SC exerting cytotoxicity against prostate and breast cancer cells in a dose-dependent manner. Specifically, SC exerted the most potent cytotoxicity in LNCaP and MCF-7 cells. SC was shown to down-regulate various angiogenetic (VEGF), proliferative (Cyclin D₁), anti-apoptotic (Bcl-2) gene products in these cells. SC also increased the number of annexin V-positive apoptotic bodies and the sub-G1 DNA contents of the cell cycle undergoing apoptosis through caspase-3 activation in both LNCaP and MCF-7 cells. We further confirmed that caspase-3 plays an important role in SC-induced apoptosis in LNCaP and MCF-7 cells through the use of the caspase-3 inhibitor. Moreover, we observed that SC potentiated paclitaxel-induced apoptosis in MCF-7 cells and sauchinone is a major active constituent of SC, which could induce apoptosis in the cells. Taken together, our data provide the evidence that SC induces apoptosis depending on caspase-3 activation and overcomes the natural biological resistance to chemotherapy found in human prostate and breast cancer cells.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

Functional activation of T cells by dendritic cells and macrophages exposed to the intracellular parasite Neospora caninum

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion.