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Tumor suppressor genes encode proteins involved in growth regulation in differentiating and proliferating cells. Previous work from our laboratory has demonstrated that the neurofibromatosis 1 (NF1) tumor suppressor gene is dramatically upregulated in astrocytes stimulated with dibutyryl cyclic AMP and proinflammatory cytokines. To explore the possibility that the NF1 gene product, neurofibromin, plays a role in the reactive gliosis seen in response to cerebral ischemia, expression of NF1 was examined in both focal and global models of rat cerebral ischemia. In this report, we demonstrate the increased expression of both neurofibromin and glial fibrillary acidic protein (GFAP) in astrocytes surrounding areas of focal ischemia. Similar increases in neurofibromin and GFAP immunoreactivity were also observed in reactive astrocytes in the hippocampal region in a global model of ischemia. These results suggest a novel role for the NF1 tumor suppressor gene in growth regulatory pathways involved in cellular remodeling and in response to injury.

The neurofibromatosis type 1 (NF1) gene encodes a tumor-suppressor protein termed neurofibromin, which, in adults, is expressed predominantly in neurons, Schwann cells, and the adrenal medulla. Loss of NF1 gene expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 as well as in malignant melanomas and neuroblastomas from patients without NF1. Previously, we demonstrated the lack of neurofibromin expression in six pheochromocytomas from patients with NF1, supporting the idea that neurofibromin might be an essential regulator of cell growth in these cells. To determine whether NF1 gene expression is similarly altered in pheochromocytomas from patients without NF1, we examined 20 pheochromocytomas for the presence of NF1 RNA and neurofibromin by reverse-transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Reduced or absent NF1 gene expression was documented in 7 of these 20 tumors (35%) including 1 of 4 sporadic tumors, 3 of 10 tumors from patients with multiple endocrine neoplasia (MEN) 2A, 2 of 4 tumors from patients with MEN2B, and 1 of 2 tumors from patients with von Hippel-Lindau syndrome. In addition, most of these tumors expressed predominantly the type 1 NF1 isoform (75% type 1 NF1 isoform expression) as opposed to other neural crest-derived tissues such as adrenal gland and Schwann cells, which express predominantly type 2 NF1. This type 1 isoform predominance was also observed in the rat pheochromocytoma PC12 cell line, suggesting that this change in isoform expression may be associated with the genesis of these tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

The neurofibromatosis 1 (NF1) gene product, neurofibromin, is a tumor suppressor gene product capable of inhibiting the growth of cells in culture. If neurofibromin suppresses cell growth by arresting cells in G0 or G1, its expression might be regulated in a cell cycle-dependent fashion. In this study, we demonstrate that RAT-1A fibroblasts arrested in G0/G1 by serum starvation and then released to progress through the cell cycle do not demonstrate significant changes in NF1 expression. However, when arrested in G0/G1 by contact inhibition, NF1 expression in these cells is reversibly upregulated within 72 h, suggesting that NF1 expression is a late event associated with cell growth arrest which may contribute to the maintenance of the differentiated state.

The neurofibromatosis type 1 (NFl) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed pre- dominantly in neurons, Schwann cells, oligoden- drocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alter- native use of exons 23a and 48a. Previously we described the identification of an NFl mRNA iso- form containing an additional 54 nucleotides from exon 48a (type 3 NFl) in human skeletal, cardiac and smooth muscle tissues by reverse-transcribed (RT)-PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle-specific neurofibromin isoform. An NF1 cDNA was gener- ated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle-specific neurofibromin antibodies de- tected this exon 48a fusion protein by Western im- munoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat mus- cle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in liver, brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NFl RNA dur- ing rat embryonic development was detected at high levels in El5 heart, tongue, and limb bud. In addition, using type 2 neurofibromin-specific an- tibodies, the existence of a fourth isoform of neu- rofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demon- strated in rat heart muscle tissues. The identifica- tion of two muscle-specific isoforms of neurofibro- min expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. o 1995 WiIey-Liss, Inc.

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss of NF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation of NF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression of NF1 RNA was found in this tumor by in situ hybridization using an NF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression of NF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression of NF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.