Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

To demonstrate regional activation in the rat cerebral cortex related to stress-evoked neuroendocrine response, Fos expression in both the cerebral cortex and hypothalamic paraventricular nucleus (PVN) was immunohistochemically examined in two experimental groups; a lipopolysaccharide (LPS) intraperitoneally injected group for inflammatory stress and a restraint group for emotional stress. The LPS injection (100 microg/100 g b.w.) and restraint (for 30 min) had similar effect on Fos-like immunoreactivity (Fos-LI) in PVN with regard to the number of immunoreactive nuclei and their distribution pattern, while the times to maximize Fos-LI were different. Numerical analysis of cortical Fos-LI in untreated rats showed a distinct region-specific pattern. Statistical analysis revealed no significant increase in Fos-LI density in any cortical regions in the LPS group, but restraint resulted in a dramatic and region-specific increase. A significant increase was detected in the prefrontal cortex (the cingulate, orbital and agranular insular cortex), the frontal area 2, the agranular retrosplenial cortex, the parietal cortex, and the medial and lateral occipital area 2. These results indicate that cortical activation relevant to specific functions may be involved in stress-specific neural circuitry.     

Exon scanning for mutation of the NF2 gene in schwannomas

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.     

Regulation of the neurofibromatosis type 2 tumor suppressor protein, merlin, by adhesion and growth arrest stimuli

The neurofibromatosis type 2 tumor suppressor gene is inactivated in the development of familial and sporadic schwannomas and meningiomas. The encoded protein, Merlin, is closely related to the Ezrin, Radixin, and Moesin family of membrane/cytoskeletal linker proteins. Examination of Merlin in several cell lines revealed that the protein migrates as two distinct species near 70 kDa. Phosphatase treatment and orthophosphate labeling demonstrated that the species with decreased mobility is phosphorylated. Given Merlin's localization to cortical actin structures, we examined the effect of cell-cell contact or other forms of growth arrest on Merlin expression and post-translational modification. Under conditions of confluency or serum deprivation, the levels of phosphorylated and unphosphorylated Merlin species increased significantly. Cells arrested in G1 by other methods or other phases of the cell cycle did not show changes in Merlin levels. Furthermore, loss of adhesion resulted in a nearly complete dephosphorylation of Merlin, which was reversed upon re-plating of cells, suggesting Merlin phosphorylation may be responsive to cell spreading or changes in cell shape. Thus, the tumor suppressor function of Merlin may involve the regulation of cellular responses to cues such as cell-cell contact, growth factor microenvironment, or changes in cell shape.     

On the involvement of calpains in the degradation of the tumor suppressor protein p53

A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (delta GP), were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JPMOP, and delta GP was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable JPMOP of 0.24 +/- 0.06 mmol ATP l-1.s-1 and a midpoint potential, (delta GP)0.5, of 58.1 +/- 1.2 kJ/mole in the muscle cells.     

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.     

The product of the NF2 tumour suppressor gene localizes near the plasma membrane and is highly expressed in muscle cells

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.     

Allelic status of 1p, 14q, and 22q and NF2 gene mutations in sporadic schwannomas

Watson is a fully developed suburb of some 30 years in Canberra (the capital city of Australia). A plunge dip using arsenical pesticides for tick control was operated there between 1946 and 1960. Chemical investigations revealed that many soil samples obtained from the study area contained levels of arsenic exceeding the current health-based investigation levels of 100 mg kg-1 set by the National Healthy and Medical Research Council in Australia. For the speciation study, nine composite samples of surface and sub-surface soils and a composite samples of rocks were selected. ICP-MS analysis showed that arsenic levels in these samples ranged from 32 to 1597 mg kg-1. Chemical speciation of arsenic showed that the arsenite (trivalent) components were 0.32-56% in the soil and 44.8% in the rock composite samples. Using a rat model, the absolute bioavailability of these contaminated soils relative to As3+ or As5+ ranged from 1.02 to 9.87% and 0.26 to 2.98%, respectively. An attempt was made to develop a suitable leachate test as an index of bioavailability. However, the results indicated that there was no significant correlation between the bioavailability and leachates using neutral pH water or 1M HC1. Our results indicate that speciation is highly significant for the interpretation of bioavailability and risk assessment data; the bioavailability fractions of arsenic in soils from Watson are small and therefore the healthy impact upon the environment and humans due to this element is limited.     

Expression of the retinoblastoma tumor suppressor gene (RB-1) in acute leukemia

In this report we review current studies concerning the RB-1 gene expression in acute leukemias. The RB-1 gene was analyzed in several studies by protein-, RNA and DNA-techniques in acute lymphoblastic leukemia (ALL) as well as in acute myelogenous leukemia (AML). The frequency of RB-1 inactivation in ALL-patients ranged between 30% and 64% in several studies. Structural abnormalities of the RB-1 gene were reported in 18% of ALL-patients and in 27% of Philadelphia chromosome-positive ALL, respectively. The proportion of AML-patients with absent RB-1 protein expression ranged between 19% and 55%. Structural RB-1-abnormalities in AML were predominantly reported in leukemias with monocytic differentiation. Furthermore, the prognostic value of an abnormal RB-1 gene expression was also estimated in some studies. In childhood ALL RB-1 inactivation was reported to have prognostic significance while in contrast, in another study on adults no prognostic value of RB-1 was found. In 4 out of 5 documented studies AML-patients with RB-1 inactivation generally had a poorer prognosis. In conclusion, RB-1 inactivation is frequently observed in acute leukemia. The prognostic value of low RB-1 expression is controversial but the majority of published studies found low RB-1 expression to be a negative prognostic predictor, in acute leukemia.     

Mutational analysis of the tumor suppressor Smad2 in acute lymphoid and myeloid leukemia

Smad4 is a tumor suppressor that is inactivated in about 50% of pancreatic carcinomas. Mutations in this gene have also been found with variable, yet much lower frequency in other tumor types and were absent from a large number of samples from patients with hematological malignancies. Smad2 shows considerable sequence similarity with Smad4 and cooperates with it in the growth inhibitory TGF-beta pathway. Smad2 mutations have been found in a fraction of colon carcinomas and have been shown to impair the function of the corresponding proteins. However, only a few other tumor types have been screened for Smad2 mutations so far. Therefore, we analyzed 50 primary tumor samples from patients with acute lymphoid or myeloid leukemia (ALL or AML) and five cell lines of hematopoietic origin for alterations in the Smad2 gene. None of the specimens tested carried mutations in the conserved MH1 or MH2 domains of Smad2.     

Immunohistoselective sequencing (IHSS) of p53 tumor suppressor gene in human oesophageal precancerous lesions

Accumulation of p53 protein occurs in human oesophageal precancerous lesions and even in near-normal oesophageal epithelium. In some instances, p53 gene mutations have been detected. In many of the cases of p53 protein accumulation in early lesions, however, p53 mutations were not detected due to either the lack of mutation or the low abundance of cells with a mutation. In order to enrich p53 immunostain-positive cells for single strand conformation polymorphism (SSCP) analysis and DNA sequencing, an immunohisto-selective sequencing (IHSS) method was developed. Anti-p53 antibody-peroxidase stained oesophageal tissue sections were subjected to ultraviolet (UV) irradiation to damage the DNA in p53 immunostain-negative cells. The immunostain protected p53 immunostain-positive cells from the UV light and thus preserved the DNA in those cells for PCR amplification. Comparison of the SSCP results from sections with and without UV treatment showed that the IHSS method selectively enriched p53 immunostain-positive cells. With this method, we could analyse mutations in samples with as few as 30 p53 immunostain-positive cells per tissue section. Analysis was carried out on tissues with precancerous lesions from six surgically-resected oesophageal specimens and 13 oesophageal biopsies from symptom-free subjects. The results of mutation analysis for some of the samples were confirmed by microdissection to enrich the p53-positive cells. The mutations in tissues with precancerous lesions were compared with those in the corresponding squamous cell carcinomas. The IHSS method is shown to be a simple and effective way to analyse mutations in p53 immunostain-positive cells. IHSS may also be a general method for molecular analysis of biological specimens after immunohistochemical staining.     

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1)

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.     

The Saccharomyces cerevisiae SOP1 and SOP2 genes, which act in cation homeostasis, can be functionally substituted by the Drosophila lethal(2)giant larvae tumor suppressor gene

By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, we cloned the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids. Cells deleted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Following exposure of sop1Delta cells to NaCl stress, the intracellular Na+ level and the Na+/K+ ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produced only slight Na+ sensitivity. Cells carrying a sop1Deltasop2Delta double deletion became, however, hypersensitive to Na+ and exhibited increased sensitivity also to Li+ and K+, suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fractionation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1Deltasop2Delta strain partially restored the Na+ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.     

Expression of the bcl-2 oncoprotein in meningiomas

The purpose of this study was to assess the expression of the bcl-2 oncoprotein in meningiomas and to compare it with the phenotype, the Ki-67 proliferative index and the sex hormone receptor status of the tumors. The expression of the bcl-2 oncoprotein was studied by Western blotting and immunohistochemistry. A quantitative study of the Ki-67 proliferative index and the expression of estrogen and progesterone receptors was performed. Western blot detected the bcl-2 oncoprotein in nearly all meningiomas. Immunohistochemistry detected the oncogene in only 43.5% of the cases. Expression of bcl-2 was essentially by spindle cells of transitional and fibrous meningiomas expressing neural cell adhesion molecule. There was neither correlation between the expression of bcl-2 and Ki-67 proliferative index of meningiomas nor statistical concordance between the expression of bcl-2 oncoprotein by meningiomas and their sex hormone receptor protein status. Inhibition of apoptosis could be involved in the growth of meningiomas with a mesenchymal differentiation.     

Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor

Brains from human neurofibromatosis type 1 (NF1) patients show increased expression of glial fibrillary acidic protein (GFAP), consistent with activation of astrocytes (M.L. Nordlund, T.A. Rizvi, C.I. Brannan, N. Ratner, Neurofibromin expression and astrogliosis in neurofibromatosis (type 1) brains, J. Neuropathol. Exp. Neurology 54 (1995) 588-600). We analyzed brains from transgenic mice in which the Nf1 gene was targeted by homologous recombination. We show here that, in all heterozygous mice analyzed, there are increased numbers of astrocytes expressing high levels of GFAP in medial regions of the periaqueductal gray and in the nucleus accumbens. More subtle, but significant, changes in the number of GFAP positive astrocytes were observed in the hippocampus in 60% of mutant mice analyzed. Astrocytes with elevated GFAP were present at 1 month, 2 months, 6 months and 12 months after birth. Most brain regions, including the cerebellum, basal ganglia, cerebral cortex, hypothalamus, thalamus, cortical amygdaloid area, and white matter tracts did not show any gliotic changes. No evidence of degenerating neurons was found using de Olmos' cupric silver stain. We conclude that Nf1/nf1 mice provide a model to study astrogliosis associated with neurofibromatosis type 1.     

Structures and functions of the tumor suppressor p53

The tumour suppressor p53 plays a crucial role in the cellular response to DNA damage. The p53 protein is able both to detect sites of DNA damage and to interact with DNA in a sequence-specific manner and function in the regulation of target gene expression. These two properties map to discrete functional domains of the protein, the C-terminus and the central core domain respectively. They are essential for integration of a normal cellular response to DNA damage, with initiation of either G1 cell cycle arrest or apoptosis. This review considers the domain structure of p53 in relation to the protein's various functions, together with the importance of tertiary structure and conformational flexibility. The precise regulation of p53 function remains to be established, although the protein is known to be phosphorylated/de-phosphorylated by a number of specific protein kinases/phosphatases. A recent discovery indicates that p53 may be activated by autoproteolysis and that proteolytic cleavage is induced by direct interaction with sites of DNA damage. This process is reminiscent of the bacterial Lex A system and would provide one mechanism for activation of p53 in response to cellular DNA damage.