Irene Szijan

Gene expression profiling of the hedgehog signaling pathway in human meningiomas

The Hedgehog (Hh) signaling pathway has an important role during embryogenesis and in adult life, regulating proliferation, angiogenesis, matrix remodeling and stem-cell renewal. Deregulation of the Hh pathway is involved in tumor development, since mutations in several components of this pathway were found in patients with basal cell carcinoma, medulloblastoma and other tumors; however, the role of Hh in meningiomas has not been studied yet. Meningiomas represent 30% of primary cranial tumors, are mostly benign and prevail in the second half of life. Novel therapies for meningiomas such as targeted molecular agents could use Hh pathway components. To provide information concerning molecular alterations, by use of real-time RT-PCR, we studied expression at the mRNA level of 32 Hh pathway and target genes in 36 meningioma specimens of different grades. mRNA levels of 16 genes, involved mainly in Hh pathway activation and cell proliferation, increased in meningiomas in comparison with normal tissue, whereas those of 7 genes, mainly related to Hh pathway repression, decreased. The most significant changes occurred in signal transduction (SMO) and GLI-transcription factor genes, and the target FOXM1 mRNA attained the highest values; their overexpression was found in aggressive and in benign tumors. Some proliferation-related genes (SPP1, IGF2) were overexpressed in higher meningioma grades. A correlation in expression between genes with a similar function was also found. Our results show a marked activation of the Hh pathway in meningiomas, which may be important for their biological and clinical characterization and would be useful for gene therapy.

Hormonal regulation of brain development. IV. Effect of neonatal thyroidectomy upon incorporationin vivo ofl-[3H]phenylalanine into proteins of developing rat cerebral tissues and pituitary gland

Abstract After the subcutaneous administration of l -[3H]phenylalanine, the pattern of changes in the entry of the amino acid into tissues and its incorporation into proteins has been studied comparatively in the cerebral cortex, cerebellum, hypothalamus and pituitary gland from developing rats. During normal development, the changes in the uptake of the amino acid and its rate of disappearance in cerebral tissues markedly contrasted with those occuring in the pituitary gland. Whereas in the former, such processes increased progressively with age, in the pituitary gland they remained unchanged throughout the entire experimental period. Striking differences were also observed in relation to the protein-synthetic ability, which decreased markedly with age in the cerebral tissues, and increased up to 15 days without further changes in the pituitary gland. Neonatal thyroidectomy produced a significant increase in the entry of the amino acid into all the tissues studied; this condition also led to a decreased rate of disappearance in the cerebral tissues, but did not affect such process in the pituitary gland. The RSA of proteins in cerebral tissues was significantly decreased in hypothyroid rats, suggesting an impairment of protein synthesis. The observation that protein synthesis was also depressed in the pituitary gland by neonatal thyroidectomy, suggests that thyroid hormone might play a critical role in pituitary metabolism during early postnatal life.

Localization of RC3 (neurogranin) in rat brain subcellular fractions

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.

Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion

We report a Becker muscular dystrophy (BMD) family with one 5‐year‐old affected patient and a 69‐year‐old asymptomatic grandfather. Dystrophin gene multiplex polymerase chain reaction and multiplex ligation‐dependant probe amplification analysis showed that both males carried an in‐frame deletion of exons 45–55. Segregation analysis revealed two additional asymptomatic boys in this family. Our finding supports previous predictions that exons 45–55 are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype. Muscle Nerve, 2008

Genotypic and phenotypic characterization of the histoblood group ABO(H) in primary bladder tumors

The ABO(H) histoblood group genes have been mapped by linkage analysis to 9q34.1‐34.2, an area of common deletions in bladder cancer. Lack of ABO(H) antigen expression in bladder tumors is a frequent and well‐documented event. In bladder neoplasms the loss of A and B transferase activity is due to down‐regulation of the ABO gene transcripts. Our study was undertaken in order to determine the presence of structural alterations of the ABO(H) gene‐encoding locus in primary bladder tumors, to estimate the extent of allelic deletions and to characterize further the pattern of histoblood group antigen expression. Fifty‐three primary bladder tumors were analyzed by immuno‐histochemistry (IHC) using a panel of well‐characterized antibodies and fresh frozen tissue sections. Normal and tumor DNA also were analyzed by PCR coupled with restriction enzyme analysis in order to establish the ABO genotype. Results obtained from these analyses were then compared to allelotyping data at the 9q34.1‐4 region by Southern blotting. IHC data showed undetectable levels of antigen expression on neoplastic cells in 59% of informative cases. PCR‐based genotypic results revealed allelic losses in 27% of heterozygous cases. Four of the 16 pheno‐ and/or genotypically altered cases (25%) presented loss of heterozygosity at D9S10 or D9S7 loci. Our data indicate that the lack of ABO antigen expression in certain bladder tumors is due to the allelic loss of the ABO glycosyltransferase‐encoding genes and that in some of these tumors the loss involves the surrounding chromosomal region at 9q34.1‐4. Int. J. Cancer 75:819–824, 1998.

Cell proliferation in the rat pituitary gland: A mechanism of control in prolactin cells

We investigated the relationship between prolactin content and DNA replication in the anterior pituitary gland. Thymidine incorporation in pregnant rats is significantly lower than in virgin controls. This is accompanied by a decreased activity of DNA polymerase. Sulpiride administration to pregnant rats enhances thymidine incorporation to levels similar to virgin controls. The results indicate a negative feedback between prolactin content and DNA synthesis in the rat anterior pituitary gland.

Expression of c-myc and c-fos oncogenes in different rat brain regions during postnatal development

We have studied the expression of c‐myc and c‐fos proto‐oncogenes in various areas of the central nervous system during postnatal development, c‐myc mRNA levels increased during the first 5 days and then decreased over the next 15 days in all nervous regions studied, c‐fos mRNA levels changed in a different way in four brain areas. While in cerebral cortex and cerebellum there was a sharp decrease during the first 10 days, in white matter and hypothalamus c‐fos transcript levels remained high during the same period, decreasing at a later stage.

Detection of germline mosaicism in two duchenne muscular dystrophy families using polymorphic dinucleotide (CA)n repeat loci within the dystrophin gene

AbstractBackground: Approximately one-third of new cases of Duchenne muscular dystrophy (DMD) can be attributed to sporadically arising new mutations, however in the majority of cases the DMD mutation has been inherited from the mother. These female carriers can have either a constitutive or mosaic mutation. Aim: The aim of this study was to determine the segregation of the at-risk haplotype and to find a deletion in the dystrophin gene of patients. Method: We analyzed individuals from two families with a history of DMD in order to predict the carrier status of related females. In one of these cases the mother had two affected sons, while in the other one son and two grandchildren were affected; therefore we predict that the mother would be an obligatory carrier. Results: Haplotype analysis of the DMD loci revealed that in the two families both the healthy and affected brothers had inherited the same X maternal chromosome. However, the affected brother carried a deletion, which was absent in the unaffected sibling. Conclusion: These findings suggested that the mothers in the two families were germline mosaics for the DMD gene. The results of this study demonstrate the usefulness of the methodology that combine the haplotype analysis with the identification of the mutation in order to detect hidden germline mosaicisms and, thus, improve genetic counseling.

Gene Expression Profiling of ErbB Receptors and Ligands in Human Meningiomas

ErbB family receptors mediate major cellular functions implied in tumorigenesis, though their role in meningiomas was not thoroughly studied. Meningiomas represent 30% of primary cranial tumors, are mostly benign, and prevail in the second half of life. Tumor therapy requires information about molecular alterations, thus we studied expression of ErbB receptor and ligand genes by real-time RT-PCR in different meningioma grades. Receptors were overexpressed (ErbB1, ErbB2) or underexpressed (ErbB3, ErbB4). Ligands EGF, TGFA, AREG, DTR, BTD were underexpressed and the neuregulins were overexpressed or underexpressed. A strong ErbB1–ErbB2 correlation was found. These data might be useful for gene therapy.

Expression of p16 INK4A gene in human pituitary tumours

Pituitary adenomas comprise 10–15% of primary intracranial tumours but the mechanisms leading to tumour development are yet to be clearly established. The retinoblastoma pathway, which regulates the progression through the cell cycle, is often deregulated in different types of tumours. We studied the cyclin-dependent kinase inhibitor p16INK4A gene expression at mRNA level in human pituitary adenomas. Forty-six tumour specimens of different subtypes, 21 clinically non-functioning, 12 growth hormone-secreting, 6 prolactin-secreting, 6 adrenocorticotropin-secreting, and 1 thyrotropin-secreting tumours were studied. All clinically non-functioning and most of the hormone-secreting tumours were macroadenomas (38/46). The RT–PCR assay and electrophoresis of the PCR-products showed that p16INK4A mRNA was undetectable in: 62% of non-functioning, 8% of growth hormone-secreting, 17% of prolactin-secreting and 17% of adrenocorticotropin-secreting adenomas. Forty percent of all macroadenomas and 25% of microadenomas had negative p16INK4A mRNA, the latter results suggest that the absence of p16INK4A product might be an early event in tumours with no expression of this suppressor gene. Within the non-functioning adenomas 63% were “null cell” and 37% were positive for some hormone, both subgroups showed similar percentage of cases with absence of p16INK4A mRNA. Our results show that clinically non-functioning macroadenomas have impaired p16INK4A expression in a clearly higher proportion than any other pituitary tumour subtype investigated. Other regulatory pathways may be implicated in the development of tumours with positive p16INK4A expression.