Recommendations for Measurement of Attention Outcomes in Preschoolers With Neurofibromatosis.

Children with neurofibromatosis type 1 (NF1) are at increased risk for attention problems. While most research has been conducted with school-aged cohorts, preschool-aged children offer a novel developmental window for clinical studies, with the promise that treatments implemented earlier in the developmental trajectory may most effectively modify risk for later difficulties. Designing research studies around the youngest children with NF1 can result in intervention earlier in the developmental cascade associated with NF1 gene abnormalities. Furthermore, clinical trials for medications targeting physical and psychological aspects of NF1 often include individuals spanning a wide age range, including preschool-aged children. In a prior report, the REiNS Neurocognitive Subcommittee made recommendations regarding performance-based and observer-rated measures of attention for use in clinical trials and highlighted the need for separate consideration of assessment methods for young children. The observer-rated Attention-Deficit/Hyperactivity Disorder Rating Scale-Preschool version is recommended as a primary outcome measure. The NIH Toolbox Flanker, Dimensional Change Card Sort, and List Sort Working Memory tasks and Digits Forward from the Differential Ability Scales-2nd Edition (performance-based measures) are recommended as secondary outcome measures. Specific methodologic recommendations for inclusion of preschoolers in clinical trials research are also offered.

Tranilast inhibits the expression of genes related to epithelial-mesenchymal transition and angiogenesis in neurofibromin-deficient cells.

Neurofibromatosis type 1 (NF1) is caused by germline mutations in the NF1 gene and is characterized by café au lait spots and benign tumours known as neurofibromas. NF1 encodes the tumour suppressor protein neurofibromin, which negatively regulates the small GTPase Ras, with the constitutive activation of Ras signalling resulting from NF1 mutations being thought to underlie neurofibroma development. We previously showed that knockdown of neurofibromin triggers epithelial-mesenchymal transition (EMT) signalling and that such signalling is activated in NF1-associated neurofibromas. With the use of a cell-based drug screening assay, we have now identified the antiallergy drug tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) as an inhibitor of EMT and found that it attenuated the expression of mesenchymal markers and angiogenesis-related genes in NF1-mutated sNF96.2 cells and in neurofibroma cells from NF1 patients. Tranilast also suppressed the proliferation of neurofibromin-deficient cells in vitro more effectively than it did that of intact cells. In addition, tranilast inhibited sNF96.2 cell migration and proliferation in vivo. Knockdown of type III collagen (COL3A1) also suppressed the proliferation of neurofibroma cells, whereas expression of COL3A1 and SOX2 was increased in tranilast-resistant cells, suggesting that COL3A1 and the transcription factor SOX2 might contribute to the development of tranilast resistance.

A novel genetic- and cell-based tool for assessing the efficacy and toxicity of anticancer drugs in vitro.

AIMS: To develop an in vitro tool for assessing the efficacy and toxicity of anticancer drugs using mixed culture containing both tumor and non-tumor cells. Such in vitro tool should have high application potential in drug-screening and personalized cancer care. METHODS: Fibroblasts were spiked as non-tumor cells into tumor cells of an established line. The mixed culture was treated with a test drug at various concentrations. After the treatment, DNA was prepared directly from the survived adhesive cells in the wells of the 96-well plates using a simple and inexpensive method, and subjected to digital PCR for measuring relative copy numbers of a target gene NF1 to that of a reference gene RPP30. The NF1 gene is known to be heterozygously deleted in these tumor cells while the RPP30 gene has two copies in both tumor and non-tumor cells. Using the NF1/ RPP30 ratios resulting from the dual digital PCR assay, the proportions of tumor cells were calculated for each drug concentration. RESULTS: Digital PCR confirmed that the tumor cells have only one copy of the NF1 gene while the non-tumor fibroblasts have two copies. By contrast, both types of cells have two copies of the reference gene RPP30. Using the ratio of the two genes, we successfully calculated the proportion of tumor cells which decreased as the dose of the test drug increased up to a certain concentration, indicating that the drug is more effective for the tumor cells than for the non-tumor cells in this dose-range. At the highest dose, we observed a slight increase in the proportion of tumor cells, likely reflecting the toxic effect of the drug on both tumor and non-tumor cells. CONCLUSION: This pilot study demonstrated the feasibility of a genetic- and cell-based tool for testing efficacy and toxicity of anticancer drugs in vitro. The promising results suggest that additional efforts are merited, for further development since such a tool will likely have high application potential (1) in drug discovery where it enables simultaneously assessing therapeutic effect on target cells and toxic effect on non-target cells, and (2) in personalized adjuvant chemotherapy where multiple drugs can be tested in primary cultures derived from surgically removed tumor.

PACAP and VIP affect NF1 expression in rat malignant peripheral nerve sheath tumor (MPNST) cells.

In our previous study we have identified PACAP, VIP and their receptors in rat malignant peripheral nerve sheath tumor (MPNST) cells, thus showing anti-apoptotic roles. Recently it has been shown that the tumor suppressor neurofibromin, encoded by the Neurofibromatosis type I (NF1) gene, promotes MPNST cells sensitivity to apoptosis after serum withdrawal. In the present study we investigated whether PACAP or VIP negatively regulate NF1 expression under normal or serum-dependent pro-apoptotic culture conditions. Results indicated that serum itself significantly influenced gene and protein levels. In fact, the low NF1 levels of cells cultured in normal serum-containing medium were remarkably increased in cells switched to low- or no-serum after 24h and 48 h. Treatment with 100 nM PACAP or VIP did not affect NF1 expression when using normal amounts of serum, whereas it significantly inhibited transcript and protein levels both in low- or no-serum cultured cells. In particular, PACAP reduced NF1 levels already after 24h in low-serum cultured cells, while VIP showed a similar effect only after serum deprivation. However, both PACAP and VIP downregulated gene and protein levels within 48 h either in low-dose and serum-starved cells. Results were confirmed by fluorescence microscopy, showing that 100 nM PACAP or VIP attenuated neurofibromin cytoplasmic localization only in low- or no-serum cultured cells. The present study provides a comprehensive analysis of both neuropeptides effect on NF1 expression in normal, low- or serum-starved MPNST cells, ameliorating the hypothesis that resistance to apoptosis in serum-deprived cells might be correlated to PACAP-/VIP-induced NF1 inhibition.

NF1 mRNA isoform expression in PC12 cells: modulation by extrinsic factors.

The rat neurofibromatosis type I (NF1) gene expresses several transcript isoforms which differ by the alternative splicing of exons 23a and 23b in the region encoding the GTPase-activating protein-related domain. The significance of this alternative splicing event is unclear and the factors which influence isoform expression are largely unknown. Here we show that a variety of factors can modulate the expression of these isoforms in PC12 cells. Nerve growth factor and dexamethasone lead to an increase in the type I isoform concurrent with a decrease in cellular proliferation. Upregulation of the type I isoform by dexamethasone occurs in an RNA synthesis-dependent manner. Cycloheximide treatment leads to the detection of an additional species identified as the murine type III transcript. These results suggest that the NF1 alternative splicing event can respond to environmental cues. The changes in the type of NF1 transcript expressed may be important in the normal physiological regulation of neurofibromin and may modulate its role in differentiation and proliferation.