Metastatic and nonmetastatic models of retinoblastoma.

To generate animal models of retinoblastoma that closely resemble metastatic and nonmetastatic human disease for the purposes of examining tumor biology and developing alternate treatments, human retinoblastoma cell lines were injected into the vitreal cavities of immunodeficient mice. Two reproducible animal models with contrasting biological behaviors analogous to human retinoblastoma have been developed. The Y79 retinoblastoma model demonstrated specific tumor evolution similar to that seen in human invasive and metastatic disease. Y79 retinoblastoma cells formed intraocular tumors that were initially confined to the vitreal cavity. Tumors progressively invaded the retina, subretinal space, choroid, optic nerve head, and anterior chamber of the eye. Tumors progressed into the subarachnoid space and focally invaded the brain. Metastases were detected in the contralateral optic nerve. Large tumors developed extraocular extensions. The histology of the tumors showed a poorly differentiated pattern with high mitotic rate, foci of necrosis, and calcification. The WERI-Rb model more closely resembled nonmetastatic human retinoblastoma. WERI- Rb tumors were localized in the eye with only anterior choroidal invasion at late stages. To examine potential biological differences in vitro, the retinoblastoma cell lines were cocultured with adherent choroid cells or adherent glioma cells which represent the targets of invasive retinoblastoma in vivo. Consistent with the in vivo observations, Y79 cells but not WERI-Rb cells adhere specifically to both the choroidal and the glioma cell lines.

Induction of a calcium/calmodulin-dependent phosphodiesterase during phytohemagglutinin-stimulated lymphocyte mitogenesis.

A calmodulin (CaM)-dependent phosphodiesterase activity that hydrolyzes both cGMP and cAMP was observed in anion exchange high performance liquid chromatography (HPLC) profiles from phytohemagglutinin-stimulated mononuclear cells but not in profiles from unstimulated cells. A single polypeptide was detected by an antibody to the calmodulin-dependent phosphodiesterases on a Western blot of homogenates of stimulated mononuclear cells. The phosphodiesterase activity was immunoadsorbed in a calcium-dependent manner by an antibody to calmodulin but not by an antibody to the 61-kDa bovine brain phosphodiesterase. The mononuclear cell enzyme eluted from the HPLC column in the same fractions as the 63-kDa calmodulin-dependent isozyme from bovine brain and appeared to have the same subunit molecular weight when probed on a Western blot. The electrophoretic mobility of proteolytic fragments derived from the mononuclear cell phosphodiesterase were identical to those from the 63-kDa brain isozyme. The enzyme could be detected in mononuclear cells by activity assays and on a Western blot 14 h after stimulation with mitogen. The enzyme remained elevated for at least 100 h after stimulation. A dose-response experiment with phytohemagglutinin demonstrated that similar concentrations of mitogen could induce both mitogenesis and the phosphodiesterase. The induction of this enzyme requires mRNA as well as protein synthesis but not DNA synthesis. An enzyme similar to the 63-kDa phosphodiesterase found in brain seems to demonstrate a regulatory interface for the metabolism of calcium and cyclic nucleotides during lymphocyte mitogenesis.