Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity

Abstract

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 protein in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2 −/− mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth-suppressive merlin isoform 3.

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“…Recently, a point mutation has been identified in the NF2 gene of the HEI-193 cells, resulting in a nonfunctional Merlin with attenuated tumor-suppressive function (31). Although the HEI-193 cells have been noted to have increased proliferative capacity in vitro, they do not grow as in vivo xenografts.…”

Mammalian Ste20-like Kinase (Mst2) Indirectly Supports Raf-1/ERK Pathway Activity via Maintenance of Protein Phosphatase-2A Catalytic Subunit Levels and Consequent Suppression of Inhibitory Raf-1 Phosphorylation

“…Cell Lines and Cell Culture-HEI-193 cells are an immortalized, patient-derived NF2 schwannoma line (35). akt2/3 Ϫ/Ϫ ; akt-1 flox/flox primary mouse embryonic fibroblast (MEF) cells, kindly provided by Philip Tsichlis, are a mouse embryo fibroblast cell line in which both akt2 and akt3 have been disrupted and endogenous akt1 has been replaced with a floxed allele.…”

Combining Curcumin (Diferuloylmethane) and Heat Shock Protein Inhibition for Neurofibromatosis 2 Treatment: Analysis of Response and Resistance Pathways

“…2D) is a truncated protein, identical to the normally occurring merlin protein designated isoform III ($60 kD). Isoform III retains some growth suppressive properties, albeit to a limited extent; hence, the presence of this isoform in the absence of isoform I (70 kD) is associated with the development of a mild form of NF2 (33)(34)(35). SK-N-AS neuroblastoma cells contain mRNA for wild-type merlin (isoforms I, II, and III; ref.…”