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Fig. 5. Efficacy of A 24 against human gliomas. (Left) Dose-dependent assay. Cells of each cell line were plated and treated with increasing doses of A 24 (0-10 multiplicity of infection [MOI]). Cells were stained with crystal violet to assess viability. A 24 produced significant killing of tumor cells in a dose-dependent fashion with each cell line tested. (Right) Viability assay. Cells were plated and then infected with A 24 or UV-inactivated A 24 at 10 MOI. Cells were counted in triplicate 7 to 24 d after infection. The growth of cells treated with A 24 (hatched bars) compared with UV-inactivated A 24 (solid bars) is shown (Reproduced with permission from ref. 19.)

Fig. 5. Efficacy of A 24 against human gliomas. (Left) Dose-dependent assay. Cells of each cell line were plated and treated with increasing doses of A 24 (0-10 multiplicity of infection [MOI]). Cells were stained with crystal violet to assess viability. A 24 produced significant killing of tumor cells in a dose-dependent fashion with each cell line tested. (Right) Viability assay. Cells were plated and then infected with A 24 or UV-inactivated A 24 at 10 MOI. Cells were counted in triplicate 7 to 24 d after infection. The growth of cells treated with A 24 (hatched bars) compared with UV-inactivated A 24 (solid bars) is shown (Reproduced with permission from ref. 19.)

is also accumulating that A 24 is effective against other tumor types, including sarcomas, lung cancer, ovarian cancer, and prostate cancer (21-24). A 24 is expected to be a universal anticancer agent as inactivation of the Rb pathway is generally accepted to be an obligate alteration underlying the cancer cell phenotype.

3.2.2. Effects of A 24 on Normal Cells

Consistent with the hypothesis that mutation of E1A abrogates the capacity of normal cells to support adenoviral replication, Fueyo et al. initially showed that lung fibroblast cell lines are resistant to the cytotoxic effects of A 24 (17). Important for

Fig. 6. Fueyo Dissemination of A 24 with glioma. (Top) Diagram showing predicted spreading of virus from point of injection. Three zones are evident: a central zone of necrotic cells that have been lysed by A 24, an intermediate zone of infected cells (inset shows viral inclusions in cells), and an outer zone of cells not yet infected. (Bottom) Photomicrograph of human xenograft taken from the brain of a mouse treated with A 24. The three zones are evident: T, uninfected tumor; V, cells infected with virus; N, necrotic lysed cells.

Fig. 6. Fueyo Dissemination of A 24 with glioma. (Top) Diagram showing predicted spreading of virus from point of injection. Three zones are evident: a central zone of necrotic cells that have been lysed by A 24, an intermediate zone of infected cells (inset shows viral inclusions in cells), and an outer zone of cells not yet infected. (Bottom) Photomicrograph of human xenograft taken from the brain of a mouse treated with A 24. The three zones are evident: T, uninfected tumor; V, cells infected with virus; N, necrotic lysed cells.

brain tumor therapy, similar studies performed on human astrocytes have confirmed the inability of A 24 to replicate in normal brain cells (18). In contrast with tumor cells, treatment of these fibroblasts or astrocytes with A 24 resulted in no increase in progeny viral titer. These results did not result from the inability of the virus to infect the cells, but were specifically the result of the reduced capacity of the virus to replicate in these cells. The most convincing evidence for the capacity of A 24 to replicate only in Rb-inactivated cells comes from Rb-transfer studies. Specifically, using an Ad-Rb construct (replication incompetent adenoviral vector carrying the Rb gene) Fueyo et al. restored Rb function in Saos-2 cells (harbor an inactive Rb gene) (17). They then showed that A 24 was only able to replicate in and kill the parental Saos-2 cell line (Rb inactive), but not the isogenetic Ad-Rb-infected Saos-2 cells (see Fig. 7). Thus, restoration of Rb was able to rescue the Saos-2 cells from the cytopathic effects of A 24, confirming the dependence of A 24 replication on the presence of an inactivated Rb pathway.

A caveat regarding A 24 is that dividing normal cells are potentially vulnerable to the cytopathic effects of the virus (17-19). Because normal initiation of S-phase requires attenuating the activity of Rb (via cdk phosphorylation of Rb, see above) dividing cells o

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