Re: X-force Fusion Connect 2006 Keygen
Pompilio Intindola
IBM Security maintains X-Force Cyber Range facilities in both Cambridge, Massachusetts and Bangalore, India. Each facility provides an immersive, stimulating setting for organizations to experience true-to-life cyber response scenarios, in a full-scale security operations center (SOC) based on a fusion team model.
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Identification, isolation, and cloning of fused cells. Rat intestinal epithelial cells (IEC-6 cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (green) or carboxylic acid acetate succinimidyl ester (SNARF-1) (red) and fused using 50% polyethylene glycol (PEG). Fused and nonfused cells identified based on size, nuclear contents, and fluorescence were isolated and single-cell sorted by fluorescence activated cell sorting (FACS). A: Schematic illustrating isolation of fused cells. B: Identification and sorting by FACS of fused and nonfused cells. Cells with a relatively large postfusion size were gated using forward scatter (FSC)-A versus side scatter (SSC)-A plots (left panel). After eliminating doublets on FSC-A versus FSC-W plots (not shown), fused cells emitting both CFSE and SNARF-1 were single-cell sorted by FACS and placed into individual wells (right panel). C: Typical image of fused cell sorted by FACS and visualized by fluorescence microscopy. The fused cell emits both CFSE and SNARF-1 fluorescence; the image represents at least 50 cells examined per experiment. D: Mitosis of fused cells. Images are of a fused cell undergoing initial two cell divisions are representative of 20 cells per experiment. Green, CFSE; red, SNARF-1; blue, DAPI. E: Localization of p53 protein in clones of nonfused and fusion-derived IEC-6 cells. Early passage cells from nonfused clones and fusion-derived clones were stained with monoclonal antibodies specific for p53. Representative images of p53 staining at low cell density (left panel) or high cell density (right panel). Scale bars: 5 μm (C and D); 10 μm (E).
To determine whether admixture of chromosomal DNA and formation of synkaryons requires mitosis or might instead reflect fusion of nuclei, we tested whether genistein, which arrests cell cycle progression at G2/M,46 would impede synkaryon formation. Fused cells treated with genistein maintained intact nuclei and did not form synkaryons (Figure 2C). The experiments also indicated that the proliferative potential of fusion-derived cells depended on the number of nuclei. Synkaryons could proliferate and initiate clones; however, fusion-derived cells with more than two nuclei rarely underwent mitosis, and the nuclear contents remained separate (Figure 2D).
We next asked whether cell fusion engenders aneuploidy by analyzing karyotypes of newly established clones. Of 79 fusion-derived (IEC-6) clones studied, 32 (41%) were aneuploid, 44 (56%) near diploid (modal chromosome numbers 40 to 44), and 3 (4%) tetraploid (modal chromosome number 84) (Figure 3A). In contrast, nonfused clones were predominantly diploid [62 (86%) of 72 clones], with only eight (11%) being aneuploid and one tetraploid and one hypodiploid (Figure 3A). Fusion-derived clones also exhibited a greater difference between the maximum and the minimum numbers of chromosomes than nonfused clones (P
Chromosome number and cell morphology during serial passage of fusion-derived clones and nonfused clones. A: Range versus modal numbers of chromosomes in newly established nonfused and fusion-derived clones. IEC-6 cells have a stable mode of 2N = 42 (not shown). At least 15 metaphase spreads were evaluated for each clone. The range of chromosome numbers was taken as the difference between the maximum and minimum chromosome numbers of 90% of metaphase chromosome spreads in a clone. Blue circles represent nonfused clones (n = 72). Red dots represent fusion-derived clones (n = 79). B: Changes in chromosome number during repeated passage of fusion-derived clones and nonfused clones. Chromosome numbers at passages 1 to 3 (P1, P2, or P3) and passages 10 or 11 (P10 or P11) are shown. The red dashed line denotes 2N (42). Twenty metaphase spreads were analyzed for each clone. Chromosome numbers >95 are not shown in the plots. C: Unequal segregation during the second mitotic division of a fused-cell clone. Box denotes region shown at higher magnification. Green, carboxyfluorescein diacetate succinimidyl ester (CFSE); red, carboxylic acid acetate succinimidyl ester (SNARF-1); blue DAPI. D: Variation in size and morphology of cells in fusion-derived clones. At passage 2 (P2), cells from fusion-derived clone 75 exhibit three distinct sizes and morphologies; large cells on the left appear separated, smaller cells in the middle are closely clustered, and cells of intermediate size and spindle shape are on the right. At passage 9 (P9), the cells exhibit uniform size and appearance. Scale bars: 5 μm (C); 100 μm (D). Original magnification: 866 (C, boxed area); 72 (D).
We next determined whether chromosome number remained stable over time. Nonfused clones generally exhibited a normal karyotype at early (passages 1 to 3) and later (passages 10 to 11) passages (Figure 3B), like unmanipulated IEC-6 cells. Fusion-derived clones that were near diploid at early passage generally remained so during 10 to 11 passages (Figure 3B). In contrast, the number of chromosomes in fusion-derived clones near triploid or tetraploid at early passage usually decreased with repeated passage, with 6 of 15 becoming near diploid (Figure 3B), 9 of 15 having >2N chromosomes but fewer than the number at early passage (Figure 3B). These results indicate that cell fusion generates karyotypic instability, with the changes sometimes resolving toward near diploidy but often persisting. Whether reversion of the karyotype toward diploidy reflects selection or ongoing plasticity is not clear.
Chromosome missegregation could underlie the transition from near tetraploidy to aneuploidy after cell fusion.40,42,47,48 Consistent with this possibility, chromosomes in fusion-derived cells did not always segregate evenly into daughter cells. Figure 3C shows an example of unequal segregation during the second division of a fused cell: one daughter cell inherited most of the chromosomes, forming a much larger nucleus, whereas the other inherited only a few loosely packed chromosomes.
Aneuploidy in tumors is often associated with DNA damage,48,51 which could presage rearrangement. To test whether fusion-derived clones sustained DNA damage, we determined the frequency and extent of double-strand DNA breaks in fusion-derived clones with modal chromosome numbers of 42, 44, and 75 at early passage. Phosphorylated (Ser 139) γ-H2AX, which clusters at the sites of double-strand DNA breaks, was localized using monoclonal antibodies.52 Multiple clusters of γ-H2AX were detected in the nuclei of 35% to 42% of cells from fusion-derived clones, whereas only 4% to 9% of cells of nonfused clones had such clusters (P 2-tail unit), representing migration beyond the nucleus, as against only 5% of nonfused cells. The comet tails were more frequent in fusion-derived clones, and the comet tail lengths were on average 2.3-fold longer than the lengths measured in nonfused clones (P
Twelve (32%) of 38 fusion-derived clones, after 12 passages at low cell density, lost cell contact inhibition, assayed by focus formation, forming discrete foci of stacked cells (Figure 5). Similarly, 11 (29%) of 38 exhibited anchorage-independent growth, assayed by colony formation in soft agar. In contrast, only 2 (3%) of 60 nonfused clones formed foci (P
Cell fusion and tumor formation. A: Frequency of tumor formation after injection of 2 106 cells from fused or nonfused clones in immunodeficient mice. Unmanipulated IEC-6 cells, nonfused clones, a pool of fused cells, and fusion-derived clones that did or did not form colonies in soft agar were inoculated subcutaneously into flanks and axillae of immunodeficient (NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac) mice, and the frequency of tumors, identified by palpation and confirmed by histology, within 12 weeks was noted. Each dot represents at least four injection sites and indicates the percentage of injection sites at which tumors developed. Results for nonfused cells and clones are shown in blue. Results for fused cells and clones are shown in red. Results represent two independent experiments. B: Tumor (arrow) 7 weeks after injection of 2 106 cells from fusion clone 15. No tumor formed in the opposite flank injected with of 2 106 nonfused clone 28 cells (arrowhead). C: Cytogenetic analysis of cells isolated from the tumor shown in B. The number indicates chromosome counts. The arrow denotes a Robertsonian translocation. Chromosomes from 20 cells at metaphase were analyzed. D: Frequency of tumor formation as a function of number of cells injected. Bars represent the percentage of injection sites at which tumors formed within 12 weeks after injection of various numbers of cells from fusion clone 15. At least four sites were injected for each number of cells tested. E: Rate of growth of tumors formed from various clones of fused and nonfused cells. Labeled IEC-6 cells were treated with polyethylene glycol, and fused and nonfused cells were isolated and cloned by fluorescence activated cell sorting. Samples of each clone (2 106 cells) were injected into each of at least four sites, and the size of the ensuing tumors was measured weekly. Tumor volumes were estimated as 1/2 (length width2) and depicted as means SD. F: Histology and invasiveness of tumors originated by cell fusion. Tumors formed after implantation of fusion-derived clones 15 (Fc 15), 20 (Fc 20), and 82 (Fc 82) were sectioned and stained with hematoxylin and eosin. Upper panels show red blood cells in each tumor and moderate focal glandular differentiation in the tumor formed by Fc 82. Arrowheads indicate mitotic cells. Lower panels show invasiveness of tumors. Note a clear border (arrow) between muscle fibers in tumor derived from Fc 15: invasiveness of Fc 20 and Fc 82 tumor cells into muscle layers. MS, muscle fibers; TM, tumor cells.
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