Varied manifestations of persistent hyperplastic primary vitreous with graded somatic mosaic deletion of a single gene.

PURPOSE: Persistent hyperplastic primary vitreous (PHPV) represents a developmental eye disease known to have diverse manifestations ranging from a trivial remnant of hyaloid vessels to a dense fibrovascular mass causing lens opacity and retinal detachment. PHPV can be modeled in mice lacking individual genes, but certain features of such models differ from the clinical realm. For example, mice lacking the Arf gene have uniformly severe disease with consistent autosomal recessive disease penetrance. We tested whether the graded somatic loss of Arf in a subset of cells in chimeric mice mimics the range of disease in a non-heritable manner. METHODS: Wild type ↔ Arf(-/-) mouse chimeras were generated by morulae fusion, and when the mice were 10 weeks old, fundoscopic, slit-lamp, and histological evaluations were performed. The relative fraction of cells of the Arf(-/-) lineage was assessed with visual, molecular genetic, and histological analysis. Objective quantification of various aspects of the phenotype was correlated with the genotype. RESULTS: Sixteen chimeras were generated and shown to have low, medium, and high contributions of Arf(-/-) cells to tail DNA, the cornea, and the retinal pigment epithelium (RPE), with excellent correlation between chimerism in the tail DNA and the RPE. Phenotypic differences (coat color and severity of eye disease) were evident, objectively quantified, and found to correlate with the contribution of Arf(-/-) cells to the RPE and tail-derived DNA, but not the cornea. CONCLUSIONS: Generating animals composed of different numbers of Arf(-/-) cells mimicked the range of disease severity observed in patients with PHPV. This establishes the potential for full manifestations of PHPV to be caused by somatic mutations of a single gene during development.

p18Ink4c and p53 Act as tumor suppressors in cyclin D1-driven primitive neuroectodermal tumor.

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Expression of the Arf tumor suppressor gene is controlled by Tgfbeta2 during development.

The Arf tumor suppressor (also known as Cdkn2a) acts as an oncogene sensor induced by ;abnormal' mitogenic signals in incipient cancer cells. It also plays a crucial role in embryonic development: newborn mice lacking Arf are blind due to a pathological process resembling severe persistent hyperplastic primary vitreous (PHPV), a human eye disease. The cell-intrinsic mechanism implied in the oncogene sensor model seems unlikely to explain Arf regulation during embryo development. Instead, transforming growth factor beta2 (Tgfbeta2) might control Arf expression, as we show that mice lacking Tgfbeta2 have primary vitreous hyperplasia similar to Arf(-/-) mice. Consistent with a potential linear pathway, Tgfbeta2 induces Arf transcription and p19(Arf) expression in cultured mouse embryo fibroblasts (MEFs); and Tgfbeta2-dependent cell cycle arrest in MEFs is maintained in an Arf-dependent manner. Using a new model in which Arf expression can be tracked by beta-galactosidase activity in Arf(lacZ/+) mice, we show that Tgfbeta2 is required for Arf transcription in the developing vitreous as well as in the cornea and the umbilical arteries, two previously unrecognized sites of Arf expression. Chemical and genetic strategies show that Arf promoter induction depends on Tgfbeta receptor activation of Smad proteins; the induction correlates with Smad2 phosphorylation in MEFs and Arf-expressing cells in vivo. Chromatin immunoprecipitation shows that Smads bind to genomic DNA proximal to Arf exon 1beta. In summary, Tgfbeta2 and p19(Arf) act in a linear pathway during embryonic development. We present the first evidence that p19(Arf) expression can be coupled to extracellular cues in normal cells and suggest a new mechanism for Arf control in tumor cells.