A potential role for NF1 mRNA editing in the pathogenesis of NF1 tumors.

Neurofibromatosis type I (NF1) is a common disorder that predisposes to neoplasia in tissues derived from the embryonic neural crest. The NF1 gene encodes a tumor suppressor that most likely acts through the interaction of its GTPase-activating protein (GAP)-related domain (GRD) with the product of the ras protooncogene. We have previously identified a site in the NF1 mRNA, within the first half of the NF1 GRD, which undergoes base-modification editing. Editing at that site changes a C to a U, thereby introducing an in-frame stop codon. NF1 RNA editing has been detected in all cell types studied, to date. In order to investigate the role played by editing in NF1 tumorigenesis, we analyzed RNA from 19 NF1 and 4 non-NF1 tumors. We observed varying levels of NF1 mRNA editing in different tumors, with a higher range of editing levels in more malignant tumors (e.g., neurofibrosarcomas) compared to benign tumors (cutaneous neurofibromas). Plexiform neurofibromas have an intermediate range of levels of NF1 mRNA editing. We also compared tumor and nontumor tissues from several NF1 individuals, to determine the extent of variability present in the constitutional levels of NF1 mRNA editing and to determine whether higher levels are present in tumors. The constitutional levels of NF1 mRNA editing varied slightly but were consistent with the levels observed in non-NF1 individuals. In every case, there was a greater level of NF1 mRNA editing in the tumor than in the nontumor tissue from the same patient. These results suggest that inappropriately high levels of NF1 mRNA editing does play a role in NF1 tumorigenesis and that editing may result in the functional equivalent of biallelic inactivation of the NF1 tumor suppressor.

RNA processing and clinical variability in neurofibromatosis type I (NF1).

Neurofibromatosis type 1 (NF1) is a common genetic disorder which predisposes affected individuals to a variety of clinical features including tumors of the central and peripheral nervous systems. The product of the NF1 gene, neurofibromin, is a tumor suppressor which most likely acts through the interaction of its GTPase activating protein (GAP) related domain (GRD) with RAS to regulate cellular growth. Two intriguing features of NF1 are the wide range of potentially affected tissues and the great variation in expressivity of disease traits across those affected. To date, the underlying source of this variation remains somewhat unclear, but evidence suggests that aberrations in normal NF1 RNA processing may be involved. This evidence includes: (i) differences in the relative ratios of the type I and type II splice variants in NF1 tumors compared with nontumor tissues; (ii) unequal expression of mutant and normal NF1 alleles in cultured cells derived from NF1 patients; (iii) the existence of NF1 tumors which display NF1 mRNA editing levels that are greater than that seen in non-NF1 tumors; and (iv) tissue-specific and developmental stage-specific expression of particular alternative NF1 transcripts. These findings suggest that the classical 2-hit model for tumor suppressor inactivation used to explain NF1 tumorigenesis can be expanded to include the post-transcriptional mechanisms which regulate NF1 gene expression. Aberrations in these mechanisms may lead to the pathogenesis of NF1 and may play a role in the observed clinical variability.

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.

The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing.

A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.

Genetic and epigenetic mechanisms in the pathogenesis of neurofibromatosis type I.

Neurofibromatosis type I (NF1) is a common genetic disease which leads to a variety of clinical features affecting cells of neural crest origin. In the period since the NF1 gene was isolated in 1991, our understanding of the genetics of NF1 has increased remarkably. One of the most striking aspects of NF1 genetics is its complexity, both in terms of gene organization and expression. The gene is large and, when mutated, gives rise to diverse manifestations. A growing body of data suggests that mutations in the NF1 gene alone may not be responsible for all of the features of this disease. Epigenetic mechanisms, those which affect the NF1 transcript, play a role in the normal expression of the NF1 gene. Therefore, aberrations in those epigenetic processes are most likely pathogenic. Herein we summarize salient aspects of the vast body of NF1 literature and provide some insights into the myriad of regulatory mechanisms that may go awry in the genesis of this common but complex disease.

Viral oncoprotein binding to pRB, p107, p130, and p300.

The purpose of this review is to bring attention to some additional work in the tumor virus/tumor suppressor field which may have been overshadowed by reports describing adenovirus, SV40, and HPV oncoprotein binding to pRB and p53. The data reviewed herein provide further support for the model that a common mechanism by which DNA tumor viruses transform cells involves inactivation of cellular proteins which function as negative regulators of cell growth.

The neurofibroma in von Recklinghausen neurofibromatosis has a unicellular origin.

von Recklinghausen neurofibromatosis (NF1) is the most common hereditary syndrome predisposing to neoplasia. NF1 is an autosomal dominant disease caused by a single gene which maps to chromosome 17q11.2. The most common symptomatic manifestation of NF1 is the benign neurofibroma. Our previous studies of tumors in NF1, studies which detected a loss of heterozygosity for DNA markers from the NF1 region of chromosome 17 in malignant tumors, did not detect a loss in neurofibromas. We report here that a more extensive study, including the analysis of neurofibromas from 19 unrelated NF1 patients by using seven probes, failed to detect a single instance of loss of heterozygosity. This finding suggests that neurofibromas are either polyclonal or monoclonal in origin but arise by a mechanism different from that of NF1 malignancies. In order to investigate the first possibility, we analyzed neurofibromas from female NF1 patients by using an X chromosome-specific probe, from the phosphoglycerokinase (PGK) gene, which detects an RFLP. The detected alleles carry additional recognition sites for the methylation-sensitive enzyme HpaII, so that the allele derived from the active X chromosome is digested by HpaII while the one from the hypermethylated, inactive X chromosome is not. We analyzed neurofibromas from 30 unrelated females with NF1. Eight patients were heterozygous for the PGK RFLP. By this assay, neurofibromas from all eight appeared monoclonal in origin. These results suggest that benign neurofibromas in NF1 arise by a mechanism that is different from that of malignant tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Transposition of the oncogene ets-1 in t(11;19) translocation in acute leukemia.

The specific chromosomal rearrangement t(11;19)(q23;p13) has been identified as a nonrandom chromosomal rearrangement in acute leukemia. The breakpoint, 11q23, coincides with the ets-1 oncogene locus. However, only very few studies have been done to verify the genomic alteration and transposition of ets-1 in the t(11;19) chromosomal rearrangement. In the present study, we identified the t(11;19)(q23;p13) translocation in two acute leukemic cases. One of the cases, biphenotypic leukemia, has been followed thoroughly. An abnormal karyotype was identified in the patient's blood and marrow samples at diagnosis and at relapse, while only normal karyotypes were identified at remission. In situ hybridization of chromosomal preparations with the ets-1 probe pHE5.4 resulted in silver grains nonrandomly localized to 19p13 in the metaphase spreads prepared from the blood sample taken at relapse, while no detectable grains were found on chromosome 19p13 in a sample taken at remission. To determine if genomic alterations of ets-1 are associated with this translocation, Southern blot hybridizations with the pHE5.4 probe were performed on deoxyribonucleic acid (DNA) isolated from blood or marrow samples of the patient at remission and relapse as well as on DNA from a disease-free normal control. Any DNA digested with AvaII, SstI, XbaI, and Bam HI, followed by hybridization with pHE5.4, demonstrated no genomic alterations or amplification of the ets-1 oncogene. Our study indicates that the ets-1 oncogene is transposed in the t(11;19) translocation without detectable alteration at the DNA level. The absence of ets-1 amplification in t(11;19) and its presence in the t(4,11) and t(9;11) translocations demonstrated by others suggests the possible existence of different molecular mechanisms involving the ets-1 oncogene in the pathogenesis of these leukemias.

Two identical active X chromosomes in human mammary carcinoma cells.

Chromosome G-banding analysis of two human mammary carcinoma cell lines, Elco and MCF-7, showed the existence of two X chromosomes in both cell lines. To determine the state of activity of the X chromosomes, a methylation-sensitive restriction endonuclease, HpaII, was used to distinguish the active X from the hypermethylated, inactive X chromosome with a probe for the phosphogalactokinase locus by Southern blot hybridization. DNA digested with the restriction enzymes PstI and BstXI showed a band at either 1.05 or 0.9 kilobases. After HpaII digestion, a 50% reduction in intensity was observed in the female controls, whereas total reduction of the band was observed for the tumor cell lines and the male control. This indicates the absence of an inactive X and the presence of only active X chromosomes in the mammary carcinoma cell lines and the male control. To investigate the mechanisms involved in the alteration of the X chromosome composition and activity, restriction fragment length polymorphism analyses of seven additional X chromosome markers (L1.28, DX13, p52A, pX65H7, L782, pA13.RI, and pXG-12) were performed on the DNA isolated from the tumor cells and controls. Heterozygosity for at least one of the seven markers was detected in the six female controls whereas only homozygosity was detected for each marker in the tumor cell lines and the male control. These results indicate that the two active X chromosomes identified in each of the two tumor cell lines are identical, resulting from duplication or nondisjunction of the active X and loss of the inactive X chromosome.

Molecular genetic analysis of tumors in von Recklinghausen neurofibromatosis: loss of heterozygosity for chromosome 17.

The most common inherited syndrome in man predisposing to neoplasia is neurofibromatosis-1 (von Recklinghausen disease) (NF1). We investigated the hypothesis that affected individuals carry a single inactive allele at the NF1 locus in the germline and that a tumor arises from a cell in a susceptible tissue in which the remaining normal allele has been lost or inactivated. DNA from tumor and nontumor tissue from 27 NF1 patients was analyzed with three markers closely linked to the NF1 locus and two additional markers from chromosome 17. No loss of heterozygosity was observed in neurofibromas, plexiform or not. For other tumor types analyzed, seven of 14 showed a loss. A loss of heterozygosity was observed in six of 11 of the malignant peripheral nerve tumors analyzed. Of the seven malignancies demonstrating a loss, five involved a neurofibrosarcoma. These findings suggest that the pathogenesis of neurofibrosarcoma in NF1 involves a deficiency of the NF1 gene product. In any given patient, loss of heterozygosity was detected at some marker loci but not others. Thus the mutations demonstrated in these tumors comprise a set of overlapping mutations, which may facilitate more precise localization of the NF1 gene.

Oncogene expression in myelopoiesis.

Oncogenes are a class of genes hypothesized to be causally related to neoplasia. To date, specific oncogenes have been recognized chiefly by their ability to transform test cells to a neoplastic phenotype. This has been accomplished largely through mutational analysis of the genotype of retroviruses or through the analysis of tumor cell DNA by in vitro transfection of rodent fibroblasts. Oncogenes are believed to arise by some genetic alteration from normal cellular genes called proto-oncogenes. Although the normal function of most proto-oncogenes is unknown, it has been proposed that they may function as tissue-specific and temporally specific regulators of differentiation. The role of oncogenes in lymphoid malignancies has been extensively analyzed. Less is known about their role in myeloid leukemias and especially in normal myelopoiesis. Space limitations permit discussion of only salient features of a limited number of oncogenes; we have arbitrarily selected myc, myb, fos, fms, fes, sis, and abl.

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)