Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-cc (TGF-a), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and aT3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta -estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas

Common Variants Near ZIC1 and ZIC4 in Autopsy‐Confirmed Multiple System Atrophy

Background: Multiple System Atrophy is a rare neurodegenerative disease with alpha-synuclein aggregation in glial cytoplasmic inclusions and either predominant olivopontocerebellar atrophy or striatonigral degeneration, leading to dysautonomia, parkinsonism, and cerebellar ataxia. One prior genome-wide association study in mainly clinically diagnosed patients with Multiple System Atrophy failed to identify genetic variants predisposing for the disease. Objective: Since the clinical diagnosis of Multiple System Atrophy yields a high rate of misdiagnosis when compared to the neuropathological gold standard, we studied only autopsy-confirmed cases. Methods: We studied common genetic variations in Multiple System Atrophy cases (N = 731) and controls (N = 2898). Results: The most strongly disease-associated markers were rs16859966 on chromosome 3, rs7013955 on chromosome 8, and rs116607983 on chromosome 4 with P-values below 5 × 10-6 , all of which were supported by at least one additional genotyped and several imputed single nucleotide polymorphisms. The genes closest to the chromosome 3 locus are ZIC1 and ZIC4 encoding the zinc finger proteins of cerebellum 1 and 4 (ZIC1 and ZIC4). Interpretation: Since mutations of ZIC1 and ZIC4 and paraneoplastic autoantibodies directed against ZIC4 are associated with severe cerebellar dysfunction, we conducted immunohistochemical analyses in brain tissue of the frontal cortex and the cerebellum from 24 Multiple System Atrophy patients. Strong immunohistochemical expression of ZIC4 was detected in a subset of neurons of the dentate nucleus in all healthy controls and in patients with striatonigral degeneration, whereas ZIC4-immunoreactive neurons were significantly reduced inpatients with olivopontocerebellar atrophy. These findings point to a potential ZIC4-mediated vulnerability of neurons in Multiple System Atrophy. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. Keywords: ZIC1; ZIC4; autopsy-confirmed; genome-wide association study; multiple system atroph

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion

Meningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells. © 1999 Cancer Research Campaig

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ‘ruler’ that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements

ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells

International audienceICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factors/cytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4/Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17β-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smadl physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type β transforming growth factor family have important roles.Facultad de Ciencias ExactasInstituto de Investigaciones Bioquímicas de La Plat

Serum neuronal exosomes predict and differentiate Parkinson's disease from atypical parkinsonism

ObjectiveParkinson's disease is characterised neuropathologically by α-synuclein aggregation. Currently, there is no blood test to predict the underlying pathology or distinguish Parkinson's from atypical parkinsonian syndromes. We assessed the clinical utility of serum neuronal exosomes as biomarkers across the spectrum of Parkinson's disease, multiple system atrophy and other proteinopathies.MethodsWe performed a cross-sectional study of 664 serum samples from the Oxford, Kiel and Brescia cohorts consisting of individuals with rapid eye movement sleep behavioural disorder, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, corticobasal syndrome and controls. Longitudinal samples were analysed from Parkinson's and control individuals. We developed poly(carboxybetaine-methacrylate) coated beads to isolate L1 cell adhesion molecule (L1CAM)-positive extracellular vesicles with characteristics of exosomes and used mass spectrometry or multiplexed electrochemiluminescence to measure exosomal proteins.ResultsMean neuron-derived exosomal α-synuclein was increased by twofold in prodromal and clinical Parkinson's disease when compared with multiple system atrophy, controls or other neurodegenerative diseases. With 314 subjects in the training group and 105 in the validation group, exosomal α-synuclein exhibited a consistent performance (AUC=0.86) in separating clinical Parkinson's disease from controls across populations. Exosomal clusterin was elevated in subjects with non-α-synuclein proteinopathies. Combined neuron-derived exosomal α-synuclein and clusterin measurement predicted Parkinson's disease from other proteinopathies with AUC=0.98 and from multiple system atrophy with AUC=0.94. Longitudinal sample analysis showed that exosomal α-synuclein remains stably elevated with Parkinson's disease progression.ConclusionsIncreased α-synuclein egress in serum neuronal exosomes precedes the diagnosis of Parkinson's disease, persists with disease progression and in combination with clusterin predicts and differentiates Parkinson's disease from atypical parkinsonism

The 3′–5′ proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases

The Role of Sulfur Dioxide in Stratospheric Aerosol Formation Evaluated Using In Situ Measurements in the Tropical Lower Stratosphere

Stratospheric aerosols (SAs) are a variable component of the Earth's albedo that may be intentionally enhanced in the future to offset greenhouse gases (geoengineering). The role of tropospheric-sourced sulfur dioxide (SO2) in maintaining background SAs has been debated for decades without in-situ measurements of SO2 at the tropical tropopause to inform this issue. Here we clarify the role of SO2 in maintaining SAs by using new in-situ SO2 measurements to evaluate climate models and satellite retrievals. We then use the observed tropical tropopause SO2 mixing ratios to estimate the global flux of SO2 across the tropical tropopause. These analyses show that the tropopause background SO2 is about 5 times smaller than reported by the average satellite observations that have been used recently to test atmospheric models. This shifts the view of SO2 as a dominant source of SAs to a near-negligible one, possibly revealing a significant gap in the SA budget