Haploinsufficient Cells Research Articles

Murine and Human NF1 Haploinsufficient Mast Cells Promote Alterations in Fibroblast Function and Organization of the Extracellular Matrix in Three-Dimensional Collagen Lattices and this Gain in Function Is Abrogated by the Addition of STI-571.

Cutaneous neurofibromas are a hallmark of neurofibromatosis type 1 (NF1), a common genetic disorder that is caused by mutations in the NF1 gene, which functions as a GAP for p21ras. Though the pathogenesis of neurofibroma formation is not completely known, haploinsufficiency of the nonneuronal lineages (fibroblasts, mast cells and endothelial cells) in the tumor microenvironment are required for neurofibroma formation (Zhu, Science 2002). These tumors are characterized by a high concentration of degranulating mast cells (MC) closely associated with fibroblasts, endothelial cells and Schwann cells. We have recently shown that Nf1−/− Schwann cells secrete kit-ligand to recruit Nf1+/− MCs to the tumor microenvironment via hyperactivation of a p21ras-PI3K-Rac dependent pathway (Yang, JCI 2003). Further, Nf1+/− MC also promote Schwann cell invasion and proliferation. Given that collagen is synthesized by fibroblasts and is approximately 80% of the weight of neurofibromas, we tested whether Nf1+/− MC promote the proliferation and collagen synthesis of fibroblasts. Strikingly, the proliferation of Nf1+/− fibroblasts in response to Nf1+/− MC conditioned media (MCCM) was 3 fold higher than any other group tested. In a wound healing assay Nf1+/− MCCM provided potent stimulus for the migration of Nf1+/− but not WT fibroblasts. Similarly, MCCM from primary human NF1+/− MC stimulated the proliferation, migration, and collagen synthesis of human NF1+/− fibroblasts, validating that our observations in Nf1+/− murine cells faithfully phenocopy the biology of human NF1 heterozygous cells. We next established three dimensional collagen lattices containing MC and fibroblasts of the respective genotypes to evaluate extracellular matrix (ECM) reorganization given that remodeling of the ECM by inflammatory cells promotes tumorigenesis. Histological examination revealed that while MC and fibroblasts of both genotypes localized to each other, there was a 2–3 fold quantitative increase in the localization or attachment of Nf1+/− MC to Nf1+/− fibroblasts. Further, Nf1+/− MC preferentially promoted a 2–3 fold increase in the lattice contraction indicative of alteration of the ECM in lattices containing either Nf1+/− or WT fibroblasts. Given that c-kit/kit-ligand interactions between MC and fibroblasts contribute to MC-fibroblast interactions, c-kit blocking antibodies or Gleevec, an antitumor drug that inhibits both BCR/ABL and c-kit tyrosine kinases, were added to MC-fibroblast cultures. Both of these agents blocked the activity of Nf1+/− MC on fibroblast proliferation, collagen remodeling and fibroblast migration. Collectively, these studies demonstrate that murine and human NF1 (Nf1) haploinsufficient MC stimulate the proliferation, migration, collagen synthesis of Nf1+/− fibroblasts as well as remodeling the ECM. This study provides strong evidence that Gleevec may be a candidate therapy for the treatment or prevention of neurofibromas.

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation.

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

Neodymium-YAG laser iridotomy in angle closure glaucoma: preliminary study.

A prospective short-term preliminary clinical study to evaluate the efficacy and immediate complications of Q-switched Nd-YAG laser iridotomy in the treatment of acute and chronic angle closure glaucoma is described. The follow-up period ranged from four to 10 months. Of 40 eyes treated 36 (90%) required a single lasing session for patency (19, one application; 17, two applications), and four eyes (10%) required two sessions. Closure of the iridotomy site following Nd-YAG lasing due to pigment epithelium proliferation occurred in 10% of eyes, an incidence remarkably lower than that of argon laser iridotomy. Transitory closure or diminution of a prior patent iridotomy during the first hour after lasing was observed in 6.7% of eyes. Patency was again noted up to three weeks later and remained unchanged. Immediate postoperative complications included a marked increase in ocular pressure (42% of eyes), minimal transitory bleeding, and transitory localised corneal oedema at the lasing site. Persistent iridocorneal adhesion at the lasing site was noted in three eyes, and localised lenticular opacities, of non-progressive type, were observed in one eye.