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.

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Affinity purification of the photoreceptor cGMP-gated cation channel.

The cGMP-gated cation channel is a member of a new family of channel proteins that appear to be directly regulated by cyclic nucleotides. A protein with a subunit molecular mass of 78 kDa that exhibits cGMP-gated calcium flux when reconstituted into phospholipid-containing vesicles has been purified using 8-bromo-cGMP-agarose affinity chromatography. This channel activity is sensitive to the inhibitor l-cis-diltiazem. Treatment of the reconstituted channel with trypsin abolishes the l-cis-diltiazem sensitivity. Apparent endogenous proteolysis can also result in smaller molecular weight polypeptides that exhibit cGMP-gated channel activity but are insensitive to l-cis-diltiazem. These results show that the channel can bind cGMP and that it contains a l-cis-diltiazem inhibitory domain that is distinct from the cGMP-binding domain.

Expression of the functional cone phototransduction cascade in retinoblastoma.

Retinoblastoma is a malignant intraocular tumor that primarily affects small children. These tumors are primitive neuroectodermal malignancies, however some of them show morphologic evidence of differentiation into photoreceptors. Phototransduction cascades are a series of biochemical reactions that convert a photon of light into a neural impulse in rods and cones. The components of these cascades are uniquely expressed in photoreceptors and, although functionally similar, distinct components of these cascades are expressed in rods and cones. Using HPLC anion exchange chromatography, Western blot analysis, and specific monoclonal and polyclonal antibodies, we found that the cone but not the rod cGMP phosphodiesterase is functionally expressed in all six primary retinoblastomas examined and in three continuous retinoblastoma cell lines. Morphologic evidence of differentiation did not correlate with the expression of the enzyme. Furthermore, GTP analogues could activate the phosphodiesterase activity suggesting that an intact phototransduction cascade is present in the tumors. The presence of the cone phototransduction cascade in retinoblastoma confirms that this tumor has biochemically differentiated along the cone cell lineage.

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.

Monoclonal antibodies to the guanine-nucleotide binding proteins of adenylate cyclase.

Monoclonal antibodies (Mabs) to the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide regulatory proteins associated with adenylate cyclase have been developed. Two Mabs (2A3 and 5G12), which are of the IgG2b subclass, recognize the beta-subunits (beta) of Ns, Ni and transducin. Iodinated beta can be immunoprecipitated by either Mab coupled to Affi-Gel 10 and this can be decreased by prior incubation of the Mabs with excess unlabelled beta. The Mabs stabilize the activated state of Ns while decreasing the rate of deactivation of activated Ns in the presence of beta.

The organization of retinal maps within the dorsal and ventral lateral geniculate nuclei of the rabbit.

The cytoarchitectonic subdivisions in the rabbit's dorsal and ventral lateral geniculate nuclei have been related to the several retinal maps that can be defined in terms of the distribution of retinal axons within these nuclei. Destruction of different retinal sectors was combined with intravitreal injections of 3H-proline, so that the distribution of fiber degeneration and autoradiographic label in the geniculate nuclei could be used to define the retinal maps in each nucleus, and to compare the two nuclei with each other. The two nuclei show surprisingly similar patterns of organization. Each is made up of a laminated alpha sector that curves around a relatively cell-sparse beta sector. Two morphologically distinct layers of each alpha sector receive contralateral retinal afferents and between these there is a small region in receipt of ipsilateral afferents. In each nucleus, the lines of projection that represent single points in visual space pass perpendicular to the layers of the alpha sector and continue an almost straight course into the beta sector. Quantitative comparisons of the retinal maps show that the relative volumes devoted to the representation of segments of the visual field are approximately the same in the two nuclei.

The laminar origin and distribution of the crossed tectoreticular pathways.

The superior colliculus is the source of a prominent descending pathway which crosses the midline in the mesencephalon and projects to the paramedian pontine reticular formation. The primary goal of the present study was to identify the cells in the superior colliculus of the grey squirrel which give rise to this pathway by using a combination of anterograde and retrograde tracing techniques. Results from the anterograde studies demonstrated the course and terminal distribution of this pathway and suggested that its laminar origin is the intermediate grey layer, stratum griseum intermediale. The retrograde studies were used to confirm the results of the anterograde experiments and to provide a more quantitative estimate of the laminar distribution of the cells which give rise to this pathway. In most cases, over 90% of the cells retrogradely labeled following injections of horseradish peroxidase along the course of this pathway were located in the intermediate grey lamina. This origin is in contrast to that of the ipsilateral tectoreticular pathway which originates primarily in stratum griseum profundum (Holcombe, V., and W. C. Hall (1981) Neuroscience 6: 255-260) and suggests that these two grey layers of the deep tectum are functionally distinct.