Cytotoxic activity of lymphocytes from F 344 rats bearing intraocular tumor derived from human adenovirus 12-induced retinoblastoma-like cell line.

Lymphocyte cytotoxicity using 51Cr releasing assay was investigated in 10 F344 rats bearing intraocular tumor derived from human adenovirus 12 (Ad 12)-induced retinoblastoma -like cell line (EXP-5). Lymphocytes obtained from tumor bored animal (1 X 10(6)/well) incubated with 51Cr-labelled EXP-5 cells (1 X 10(5)/well) for 24 hours, and counted by beta-scintillation counter. The cytotoxic activity of lymphocytes of transplanted animals was higher in 3 out of 10 subjected rats (24-30%) than in 10 rats of control (3-5%). The results support the view that the correspond animal model in its resemblance and suggested that the rats with retinal tumor have concomitant cell-mediated immunity in the early stage of tumor bearing.

Cell-mediated immunity in F 344 rats bearing intraocular tumors derived from human adenovirus 12-induced retinal tumor.

The eyes of 10 F344 rats were inoculated with retinal tumor cells (EXP-5 cell line) induced by human adenovirus 12. The animals were killed at 4 weeks thereafter, and the cytotoxicity of their lymphocytes was investigated by using 51Cr-releasing assay. The percentage of EXP-5 cells killed in vitro by lymphocytes was higher in 10 rats with ocular tumors (24.6% +/- 6.1%, mean +/- SD) than in 10 control rats (6.2% +/- 1.8%). Morphologic investigation using syngeneic spleen cells confirmed the presence of lymphoid cells, resembling T-lymphocytes, adhering to EXP-5 cells. The influence of subcutaneous injection of EXP-5 cells on the growth of intravitreously injected tumor cells was investigated. Cells injected subcutaneously prior to intravitreous injection elicited an immune response that was capable of controlling vitreous tumor growth. These findings suggest that the rats with transplanted retinal tumors develop a cell-mediated immune response in the early stage of tumor bearing, and that a state of pre-existing specific immunity can overcome so-called "immunologic privilege" of the vitreous body.

Chemotherapeutic response of tumor derived from human adenovirus 12--induced retinal tumor cell line in syngeneic CDF (F 344) rats.

The effect of two anticancer agents, vincristine (VCR) and cyclophosphamide (CTX), on an established cell line (EXP-5) derived from human adenovirus serotype 12 (Ad 12)--induced retinal tumor was studied in vitro and in vivo. VCR at a concentration of 5 and 10 micrograms/ml of culture medium and CTX at 50 and 100 micrograms/ml suppressed growth in vitro. EXP-5 cells were transplanted into the vitreous of 56 inbred CDF (F 344 strain) rats. The implants grew almost exclusively as intravitreous tumors within one month. When the tumor was full grown in the vitreous, VCR and CTX were administered intravenously, singly or in combination, on a schedule based on the protocol CCG-961 for localized unilateral retinoblastoma, Reese-Ellsworth group 5. At a dosage of 0.05 mg/kg, VCR was effective in reducing tumor size; at a dosage of 5 mg/kg, CTX did not reduce tumor size. Combined VCR/CTX therapy induced reduction of about two thirds in tumor size in 2 of 10 treated animals; in all 10 animals, the tumor became morphologically less distinct during the course of treatment although some characteristic features remained. Cytotoxic tumor changes (necrosis, fibrous proliferation, cell transformation, and bizarre giant cells) were observed in all treated animals. This model used the EXP-5 cell line grown in the vitreous, thereby providing a potential tool for evaluating growth and chemotherapeutic responsiveness of retinoblastoma.

Effects of cyclic AMP on growth and differentiation of rat retinoblastoma-like tumor cells in vitro.

We examined the effects of cyclic AMP (cAMP) on the growth and differentiation of RAO 188 cells, a cultured cell line derived from a retinoblastoma-like tumor induced in an inbred rat by intravitreous inoculation with human adenovirus serotype 12. After adding cAMP analogs (dibutyryl cAMP and 8-bromo cAMP) and phosphodiesterase inhibitors (theophylline, amino-phylline, and 1-methyl-3-isobutyl xanthine) to the RAO 188 cell culture medium, we measured changes in cell incorporation of the DNA and RNA precursors 14C-thymidine and 3H-uridine, and we observed the morphologic alterations of RAO 188 by phase-contrast and transmission and scanning electron microscopy. Incorporation of the labeled precursors decreased with increased concentrations of cAMP analogs and phosphodiesterase inhibitors. Incorporation of the labeled precursors was inhibited shortly after the addition of dibutyryl cAMP to the culture medium. The effect was maximal at 8 hr and was sustained for up to 48 hr. Reversibility of cAMP effects on incorporation gradually decreased for 10 days; at 10 days these effects were essentially irreversible. Neuritelike processes developed shortly after cAMP analog treatment and formed a network after 24 hr. Transmission electron microscopy disclosed changes in the cell membrane and cytoplasm of cells treated with 8-bromo cAMP and theophylline: perturbation of the cell membrane and the appearance of intercellular junctional devices and microfilaments. The activity of glutamate decarboxylase, which is involved in the biosynthesis of gamma-aminobutyric acid, was increased in treated cells. These results show that cAMP decreases DNA and RNA synthesis and cell proliferation and facilitates morphologic and biochemical differentiation of RAO 188 cells.

A highly predictable animal model of retinoblastoma.

A new animal model of retinoblastoma was developed in newborn inbred CDF rats by intravitreous inoculation of retinal tumor cells (5 X 10(4)/5 microliter) derived from the cultured tumor cell line EXP-5. The retinal tumor from which the cell line originated was induced by a single intravitreous inoculation of human adenovirus serotype 12 (5 microliter of 10(8) TCID 50/0.1 ml) in syngeneic rats. Within 1 month after intravitreous inoculation of EXP-5 cells, a clinically recognizable ocular tumor was obtained in all 39 rats. Ad 12-specific T-antigens were demonstrated in the cultured tumor cells using immunofluorescent techniques. Morphologically these tumor cells closely resembled retinoblastoma, with poorly differentiated intracytoplasmic organelles, solitary cilia with a 9 + 0 tubule pattern, and abnormal nuclear membrane associated with a set of basal bodies. The significance of this highly manipulable retinal tumor cell line is the capability of providing a full-fledged intravitreous tumor in 1-month-old CDF rats, whose actual life span is known to be 42 months. Transplantable retinal tumors described to date are reviewed briefly and compared with the presently reported cell line.

Complement-dependent cytotoxicity in rats bearing human adenovirus type 12-induced primary retinoblastoma-like tumor in the eye.

Using an animal model of retinoblastoma in inbred rats and cultured human adenovirus type 12-induced retinoblastoma-like tumor cells (RAO 188), complement-dependent cytotoxicity was determined by measuring release of 3H-uridine labelled RNA. Sera from rats in which tumors did not grow after adenovirus type 12 inoculation had higher cytotoxicity against RAO 188 cells than sera from rats bearing primary adenovirus type 12-induced retinoblastoma-like tumor. These results showed that the rat which could raise antibodies against adenovirus type 12-induced retinoblastoma-like tumor cells did not allow the tumor growth in the eye after virus inoculation.

The relationship of a monoamine fiber system to a somatosensory tectal projection in the salamander Ambystoma tigrinum.

After hemisection of the spinal cord and medulla oblongata, a projection has been traced to the inner half of the tectal white of the tiger salamander, using Fink-Heimer degeneration staining. By microelectrode recording it was found that the tectal projection forms a topographic somatosensory map of the contralateral half of the body. This map is in register with the overlying retino-tectal visual projection. Using the Falck-Hillarp technique, it was found that the somatosensory tectal input is associated with yellow-fluorescing 5-hydroxytryptamine fibers.