FIGURE 17. 1 Clonal Variation FIGURE 17.1 Clonal Variation. Variation in tyrosine aminotransferase activity among four subclones of clone 18 of a rat minimal-deviation hepatoma cell strain, H-4-II-E-C3. Cells were cloned; clone 18 was isolated, grown up, and recloned; and the second-generation clones were assayed for tyrosine aminotransferase activity, with and without pretreatment of the culture with dexamethasone. Light gray bars, basal level; dark gray bars. induced level. (Data J. Somerville.)

FIGURE 17. 2 Chromosome Aberrations FIGURE 17.2 Chromosome Aberrations. Examples of aberrant recombinations. (a) P2 cells, a clone of L929 mouse fibroblasts, showing multiple telomeric fusions, with two marked by arrows. (b) Recombination event between two dissimilar chromosomes of the larger group in Y5 Chinese hamster cells. This cell would be unlikely to survive.

FIGURE 17. 3 Transformation Foci FIGURE 17.3 Transformation Foci. A monolayer of normal, contact-inhibited NIH 3T3 mouse fibroblasts left at confluence for 2 weeks. (a) 75-cm2 flask stained with Giemsa. (b) Phase-contrast image of focus of transformed cells overgrowing normal monolayer (10x objective). (See also Plate 14c.)

FIGURE 17.4 Cumulative Population Doublings (PD) of hTERTImmortalized Cells. Cumulative PD were calculated for cultures of human mesenchymal stem cells (see Protocol 17.2) and plotted against time in culture for cells with ectopic expression of hTERT after retroviral transduction (circles) and control (nontransduced) cells (squares). (Data courtesy of N. Serakinci).

FIGURE 17. 5 Density Limitation of Cell Proliferation FIGURE 17.5 Density Limitation of Cell Proliferation. The difference in plateaus (saturation densities) attained by cultures from normal brain (circles, solid line) and a glioma (squares, broken line). Cells were seeded onto 13-mm coverslips, and 48 h later the coverslips were transferred to 9-cm Petri dishes containing 20 mL of growth medium, to minimize exhaustion of the medium.

FIGURE 17. 6 Chick Heart Assay. Tumor spheroids (see Protocol 25 FIGURE 17.6 Chick Heart Assay. Tumor spheroids (see Protocol 25.2) are cocultured with healed fragments of 8-day embryonic chick heart. (1) The spheroid adheres to the heart after a few hours; (2) after 24 to 48 h it starts to penetrate the chick heart. (3) It spreadswithin the heart fragment; (4) by 8 to 10 days it has completelyreplaced the heart tissue. (After Mareel et al., 1979).

FIGURE 17. 7 Filter Well Invasion FIGURE 17.7 Filter Well Invasion. Cells plated on the underside of the filter migrate through the filter into growth factor-depleted Matrigel in the well of the filter insert, encouraged by the addition of a chemoattractant, such as fibroblast-conditioned medium, to the upper side of the Matrigel . (After Brunton et al. [1997].) ™ ™

FIGURE 17. 8 In vitro Angiogenesis Assay FIGURE 17.8 In vitro Angiogenesis Assay. Endothelial cells grown on Cytodex beads induced to form tubes. (a) Endothelial cells forming a monolayer on the surface of the Cytodex beads (control) stained with DAPI. (b) Endothelial cells grown on beads and induced to form tubes by treatment with 10 ng/mL FGF. Stained with DAPI. (Courtesy of V. Subramanian. See also Plate 15e). coated Cytodex-3 beads is reliable, easy to perform and the results can be easily quantified as the endothelial tubes radiate out from a focal point—the Cytodex3 bead (see Plate 15e)[Crabtree and Subramanian, 2007].

FIGURE 17. 9 Plasminogen Activator FIGURE 17.9 Plasminogen Activator. PA produced by tumor cells in vitro (the units are arbitrary). The PA activities of the four gliomas—JPT, ATA, RAT, and VAG—were all higher than cells cultured from normal brain (NMB-C, GDU-T). It was also found that the only cells to produce the differentiated glial marker glial fibrillary acidic protein—CCM and C6—had the lowest PA of all. (Data courtesy of M. Frame).